Está en la página 1de 216

UNIVERSIDAD DE OVIEDO

Departamento de Biologa Funcional


rea de Microbiologa

Perfiles de susceptibilidad/resistencia a antibiticos


en bacterias del cido lctico y bifidobacterias.
Caracterizacin molecular de genes de resistencia.

Ana Beln Flrez Garca

TESIS DOCTORAL
Oviedo-2007
UNIVERSIDAD DE OVIEDO
Departamento de Biologa Funcional

Perfiles de susceptibilidad/resistencia a antibiticos


en bacterias del cido lctico y bifidobacterias.
Caracterizacin molecular de genes de resistencia.

Memoria presentada por Ana Beln Flrez Garca

Para optar al grado de Doctor por la Universidad de Oviedo

Este trabajo ha sido realizado en el


Instituto de Productos Lcteos de Asturias (IPLA)

Oviedo 2007
AGRADECIMIENTOS

Deseo expresar mi ms sincero agradecimiento:

Al Dr. Baltasar Mayo Prez y el Dr. Abelardo Margolles Barros como directores
de esta tesis doctoral por su asesoramiento cientfico y su dedicacin y apoyo
en la realizacin de este trabajo.

Al Dr. Juan Carlos Bada, director del Instituto de Productos Lcteos de


Asturias (IPLA), por darme la oportunidad de hacer esta tesis en el centro.

Al Dr. Juan Evaristo Surez, mi tutor en la Universidad de Oviedo, por su


ayuda en la revisin de esta memoria.

A la Unin Europea, por la financiacin de una beca Predoctoral asociada al


proyecto ACEART (FP6-506214).

A todos los que han colaborado de una forma u otra en el desarrollo de esta
Tesis, y en especial a mis compaeros del IPLA por todos los buenos
momentos que compartimos.

A Santi, por estar ah cuando necesitaba desconectar y por todo el cario que
me da da a da.

Y en especial, a mis Padres y a mi familia por su apoyo incondicional y su


motivacin constante para seguir adelante.
NDICE
_______________________________________
_______________________________________
___________________________________________
ndice

I.-INTRODUCCIN

I.1- LOS ANTIBITICOS...........................................................................................................1


I.1.1.- INTRODUCCIN.....................................................................................................1
I.1.2.- MODO DE ACCIN ................................................................................................2
I.1.3.- RESISTENCIA A ANTIBITICOS .........................................................................5
I.1.3.1.- Modificacin del blanco molecular de accin del antibitico ...................6
I.1.3.2.- Detoxificacin por modificacin y/o inactivacin del antibitico ...........7
I.1.3.3.- Ausencia o cambios en el transporte del antibitico ..................................7
I.1.3.4.- Transporte inespecfico del antibitico al exterior de la bacteria. ............8
I.1.4.- BASES GENTICAS DE LA RESISTENCIA .........................................................8
I.1.4.1.- Mutacin...........................................................................................................9
I.1.4.2.- Transferencia horizontal.................................................................................9
I.1.5.- EL PROBLEMA DE LA RESISTENCIA A ANTIBITICOS.............................10
I.2.- BACTERIAS DEL CIDO LCTICO (BAL) ................................................................11
I.2.1.- INTRODUCCIN...................................................................................................11
I.2.2.- TIPOS Y GNEROS PRINCIPALES.....................................................................12
I.2.3.- PERFILES DE SUSCEPTIBILIDAD Y GENES DE RESISTENCIA A
ANTIBITICOS EN BAL .................................................................................................15
I.2.4.- TRANSFERENCIA GNICA EN BAL ................................................................21
I.2.4.1.- Transformacin y transfeccin ....................................................................21
I.2.4.2.- Conjugacin ...................................................................................................22

II.-OBJETIVOS

II.1.-OBJETIVOS DEL TRABAJO ..............................................................................................25

III.-TRABAJO EXPERIMENTAL

III.1.-CAPTULO I.-Perfiles de susceptibilidad/resistencia en BAL y bifidobacterias


aisladas de quesos artesanos y del tracto gastrointestinal humano mediante
microdilucin. .............................................................................................................................27
III.2.-CAPTULO II.-Ensayo de susceptibilidad a antibiticos en distintas cepas tipo de
BAL y bifidobacterias mediante Etest. Determinacin de nuevos puntos de corte en
Lactococcus lactis y Lactobacillus plantarum frente a seis antibiticos.................................... 45
III.3.-CAPTULO III.-Caracterizacin molecular de la resistencia adquirida a
antibiticos en BAL y bifidobacterias...................................................................................... 71

i
_______________________________________
ndice _______________________________________
___________________________________________

III.4.-CAPTULO IV.-Caracterizacin molecular de la resistencia intrnseca en BAL y


bifidobacterias mediada por transportadores inespecficos (MDR) ..................................113

IV.-DISCUSIN GENERAL..................................................................................................133

V.-CONCLUSIONES...............................................................................................................145

VI.-BIBLIOGRAFA ................................................................................................................149

VII.-ANEXOS............................................................................................................................159

ii
INTRODUCCIN
________________________________________
_______________________________________
_____________________________________
Introduccin

I.-INTRODUCCIN

I.1.- LOS ANTIBITICOS

I.1.1.- INTRODUCCIN

De forma emprica, el hombre ya utilizaba en la antigedad sustancias


antimicrobianas como vino, mirra y sales inorgnicas para el tratamiento de heridas e
infecciones cutneas (Mandell et al., 1997). Sin embargo, hasta el descubrimiento del
salvarsan en 1913 y el posterior descubrimiento accidental de la penicilina en 1929 por
Alexander Fleming no se produjo una revolucin en el tratamiento de las
enfermedades infecciosas de origen bacteriano. La aplicacin de los antibiticos es uno
de los logros ms importantes de la medicina moderna, puesto que se constituye una
herramienta con la que se previene, cura o reduce la transmisin de determinadas
enfermedades. Su empleo consigui una disminucin significativa de la morbilidad y
mortalidad asociadas a enfermedades devastadoras hasta entonces (Cohen, 2000). Los
antibiticos son imprescindibles tambin en otros aspectos de la medicina como la
oncologa o los transplantes.
Adems de en la prctica mdica, los antibiticos se utilizan abundantemente
en veterinaria, tanto para el tratamiento de las enfermedades de los animales (Chopra y
Roberts, 2001) como con funcin profilctica en la prevencin de enfermedades (Levy,
1997; Wegener, 2003). Se utilizan tambin en agricultura para el control de algunas
enfermedades de plantas (Mc Manus et al., 2002).
El trmino antibitico deriva de la palabra antibiosis creada en 1889 por Jean
Paul Vuillemin en su trabajo Symbiose et Antibiose y literalmente significa contra la vida.
En 1942 Waksman defini el trmino como aquella sustancia que generada por un
microorganismo tiene la capacidad de inhibir el crecimiento e incluso destruir a otros
microorganismos. Posteriormente la definicin se extendi para incluir cualquier
sustancia producida por un organismo vivo que, en bajas concentraciones, fuera capaz
de inhibir el crecimiento o anular la vida de una o ms especies microbianas. En la
actualidad en el trmino se incluyen no solo las sustancias naturales, sino tambin los
derivados semisintticos y los quimioterpicos que se obtienen por sntesis qumica.

1
________________________________________
Introduccin _______________________________________
_____________________________________

La era de los antibiticos comienza en realidad en los aos 30 con la


introduccin de las sulfonamidas, y se contina con la aplicacin de la penicilina en los
aos 40. Al xito clnico e industrial de este ltimo antibitico sigui una bsqueda
intensiva de nuevos compuestos. As pues, durante los aos 40 y hasta comienzos de
los 70, el desarrollo y la produccin de nuevos antibiticos se increment de forma
exponencial. Entre los compuestos con mayor xito antimicrobiano destacamos los -
lactmicos, los glicopptidos, los aminoglicsidos, el cloranfenicol, las tetraciclinas y
los macrlidos. A partir de esta fecha se produjo un declive en la bsqueda de nuevos
compuestos. De esta forma, pasaron casi 30 aos hasta la aparicin de un nuevo grupo
de antimicrobianos: las oxazolidinonas, compuestos completamente sintticos que se
empezaron a comercializar en el ao 2000 (FDA, 2000; Moellering, 2003). Los
antibiticos semi-sintticos y sintticos persiguen incrementar la potencia antibitica,
aumentar el espectro antimicrobiano, reducir la toxicidad para el paciente y/o
combatir la resistencia.
A modo de resumen, en la Figura 1 puede verse un diagrama de las fechas
aproximadas de la utilizacin prctica de diversos grupos de antibiticos.

Oxazolidinonas
Oxazolidonas
Trimetroprim
Trimetropin
Estreptograminas
Quinolonas
Lincosamidas
Cloranfenicol
Tetraciclinas
Macrlidos
Glicopptidos

Aminoglicsidos
Penicilinas
Penicilinas

Sulfonamidas

1930 1940 1950 1960 1970 2000

Figura 1.- Fecha de introduccin de diferentes antibiticos en la prctica clnica (Sjlund, 2004).

I.1.2.- MODO DE ACCIN

La accin de los antibiticos afecta a componentes esenciales de la


multiplicacin celular o a estructuras necesarias para el mantenimiento de la vida, pero

2
________________________________________
_______________________________________
_____________________________________
Introduccin

mientras que unos inhiben el crecimiento de manera reversible (bacteriostticos), otros


causan la muerte celular (bactericidas). Una de las formas ms coherentes de clasificar
a los antibiticos es de acuerdo a la diana molecular sobre la que actan. Con este
criterio, Edwards (1980) establece cuatro grupos principales, tal y como se recoge en la
Tabla 1 de la pgina siguiente. Estos cuatro grupos se describen de forma sumaria a
continuacin:
1.- Inhibidores de la sntesis de la pared celular. Los antibiticos de este grupo
actan interfiriendo con diversos pasos de la biosntesis del peptidoglicano de la pared
celular, bien intracelularmente como la fosfomicina y la D-cicloserina, o bien
extracelularmente como ocurre con los -lactmicos que interaccionan con las
protenas encargadas del ensamblaje del polmero. En ltimo trmino siempre se
provoca la lisis celular.
2.- Inhibidores de la sntesis proteica. El blanco molecular de accin de estos
antibiticos son los ribosomas. La unin al ribosoma ocasiona una parada en la
traduccin, inhibindose, en consecuencia, la sntesis proteica. La interferencia puede
producirse con la subunidad ribosmica 30S (aminoglicsidos y tetraciclinas) o con la
subunidad 50S (cloranfenicol, macrlidos, lincosamidas y oxazolidinonas).
3.- Inhibidores de la sntesis de cidos nucleicos. Los antibiticos de esta clase
inhiben alguno de los componentes esenciales que participan en la sntesis de los
cidos nucleicos. As, las quinolonas actan inhibiendo la accin de las topoisomerasas
II y IV, encargadas de controlar el superenrollamiento y desenrollamiento del ADN. La
rifampicina, por su parte, inhibe a la ARN polimerasa, impidiendo la formacin de
nuevo ARN.
4.- Antibiticos que afectan a la membrana. Los antibiticos de este grupo afectan a
la organizacin estructural de la membrana, desorganizndola o aumentando su
permeabilidad. Como antibitico caracterstico de este grupo se puede citar a la
polimixina E o colistina.

3
________________________________________
Introduccin _______________________________________
_____________________________________

Tabla 1.- Clasificacin de los antibiticos segn su modo de accin.

Grupo antimicrobiano Ejemplos Diana molecular

Inhibidores de la pared celular

Penicilinas Penicilina G
-lactmicos

Cefalosporinas Ceftriaxona
Protenas de unin a penicilina
Carbapenmicos Imipenem

Monobactmicos Aztreonam

Glicopptidos Vancomicina Precursores de la pared celular

Fosfonatos Fosfomicina enolpiruvato-transferasa

Inhibidores de la sntesis proteca

Aminoglicsidos Gentamicina
Estreptomicina Subunidad ribosomal 30S
Kanamicina

Tetraciclinas Doxiciclina Subunidad ribosomal 30S

Macrlidos Eritromicina Subunidad ribosomal 50S

Lincosamidas Clindamicina Subunidad ribosomal 50S

Cloranfenicol Cloranfenicol Subunidad ribosomal 50S

Inhibidores de los cidos nucleicos

Sulfonamidas Sulfonamida Sntesis de cido flico

Trimetoprima Trimetroprima Sntesis de cido flico

Quinolonas Ciprofloxacino ADN girasa, topoisomerasas

Rifampicinas Rifampicina ARN polimerasa

Antibiticos que afectan a la integridad de la membrana

Polimixinas Polimixina E Membrana celular

4
________________________________________
_______________________________________
_____________________________________
Introduccin

I.1.3.- RESISTENCIA A ANTIBITICOS

En los ltimos aos se estima que se han aportado a la biosfera entre una y diez
millones de toneladas de antibiticos (European-Commission, 2005). La presencia
continuada de estos compuestos en diversos hbitats es un poderoso agente selectivo
que promueve la multiplicacin de los microorganismos resistentes. La correlacin
entre la utilizacin de antibiticos y el aislamiento de bacterias resistentes ha sido
puesta de manifiesto por diversos autores (Barbosa y Levy, 2000; Granizo et al., 2000;
Bronzwaer et al., 2002). De este modo, la introduccin de nuevas drogas va seguida, en
un periodo ms o menos corto, de la aparicin de microorganismos resistentes. De
hecho, la aparicin de las primeras cepas resistentes de Staphylococcus aureus a
penicilina G se detectaron tras un breve periodo de utilizacin del antibitico (en el ao
1944) y en 1970 la mayora de los aislados clnicos de S. aureus eran resistentes a ella.
De modo inverso, el abandono del empleo de un antibitico va acompaado de una
reduccin significativa de microorganismos resistentes al mismo (Aarestrup et al.,
2001).
Las bacterias pueden presentar una resistencia intrnseca o natural a
determinados antibiticos o pueden adquirir dicha resistencia por medio de
mutaciones o mediante la adquisicin de material gentico que aporte nuevas
funcionalidades bioqumicas (Normark y Normark, 2002). La aparicin de resistencia a
antibiticos es un fenmeno natural que puede ser debido a la diseminacin de los
sistemas de defensa que presentan las cepas productoras (Hawkey, 2000), o a cambios
en genes cuyas protenas codificadas modifican su actividad hasta ser capaces de
proteger o modificar el blanco de accin o de modificar los antibiticos (Davies, 1994).
Parece claro que ambos tipos de procesos han contribuido a la enorme diversidad y
variabilidad de los mecanismos de resistencia (Walsh, 2000).

1.- Resistencia intrnseca (natural). Esta resistencia es la que presentan, por


ejemplo, los microorganismos productores de antibiticos como autodefensa frente a
su efecto inhibidor. Se incluye, adems, la resistencia de los microorganismos
insensibles a los que no afectan determinados antibiticos, sin que ello conlleve
ningn tipo de alteracin. La ausencia de la diana molecular de accin es una clara
forma de resistencia intrnseca. As por ejemplo, la resistencia a vancomicina de
muchas especies de Lactobacillus y Leuconostoc es consecuencia de la ausencia del

5
________________________________________
Introduccin _______________________________________
_____________________________________

dipptido D-alanina-D-alanina que en estos microorganismos se sustituye de forma


natural por D-alanina-D-lactato (Elisha y Courvalin, 1995).
2.- Resistencia adquirida. La resistencia adquirida, como su nombre indica, se
adquiere en un determinado momento del ciclo vital del organismo, transmitindose
con posterioridad a la descendencia. La resistencia puede adquirirse por mutacin, o
estar mediada por fenmenos de transferencia y adquisicin gnica (conjugacin,
transformacin, transduccin).

Sean intrnsecas o adquiridas, las bases bioqumicas de la resistencia son


extremadamente variadas, aunque la mayora pueden agruparse en alguna de las
categoras que se relacionan a continuacin (Figura 2):

Figura 2.- Base bioqumica de la resistencia a antibiticos (A) Modificacin y/o inactivacin del
antibitico, (B) Modificacin del blanco molecular de accin, (C) Ausencia o cambios en el
transporte, (D) Transporte inespecfico al exterior (Putman et al., 2000b).

I.1.3.1.- Modificacin del blanco molecular de accin del antibitico

Una alteracin en la diana del antibitico acarrea la incapacidad de unin del


antimicrobiano y, consecuentemente, la resistencia al mismo. Encontramos este
mecanismo de resistencia en aquellas modificaciones estructurales que afectan a las
protenas de unin a penicilina (PBPs) presentes en la pared bacteriana de algunas
cepas resistentes a este antibitico. Las alteraciones de las PBPs son adquiridas y casi

6
________________________________________
_______________________________________
_____________________________________
Introduccin

siempre se deben a mutaciones cromosmicas (Walsh, 2000). Otro ejemplo lo


constituyen las modificaciones ribosomales que hacen que una bacteria sea
invulnerable a la accin de aminoglicsidos, macrlidos o tetraciclinas. En algunas
cepas, la resistencia a eritromicina est mediada por una metiltransferasa que incorpora
un grupo metilo en el residuo de adenina situado en la posicin 2058 del ARNr 23S o
en posiciones adyacentes (Bussiere et al., 1998). De igual forma, las cepas se convierten
en resistentes cuando se sustituye por mutacin el residuo de adenina de esta posicin
por guanina (Lucier et al., 1995).

I.1.3.2.- Detoxificacin por modificacin y/o inactivacin del antibitico

Muchas bacterias son capaces de sintetizar enzimas que modifican de forma


especfica e irreversible los antibiticos. La actuacin puede llevarse a cabo de dos
formas claramente diferentes. El enzima puede inactivar el antibitico como
consecuencia de la ruptura de enlaces covalentes de su estructura, o modificarlo
qumicamente por adicin o sustitucin de radicales de su molcula. La destruccin
enzimtica es uno de los mecanismos de resistencia ms frecuente a los -lactmicos
(Jacoby y Archer, 1991). Se conoce un gran nmero de -lactamasas que hidrolizan el
anillo -lactmico de penicilinas y cefalosporinas, originando derivados
biolgicamente inactivos. Entre los mecanismos de inactivacin por sustitucin o
modificacin del antibitico, podemos mencionar la resistencia al cloranfenicol en la
que el enzima cloranfenicol acetil transferasa acetila los grupos hidroxilos del mismo. De
igual forma, las enzimas adenil transferasas (AMP), fosforil transferasas (-PO3) o acetil
transferasas modifican los aminoglicsidos de manera que reducen o pierden su
afinidad por el ribosoma (Shaw et al., 1993). En este mismo sentido, la adicin de un
grupo glutatin a la fosfomicina por una glutation S-transferasa inactiva el antibitico
(Surez y Mendoza, 1991).

I.1.3.3.- Ausencia o cambios en el transporte del antibitico

La interferencia con el transporte del antibitico, asociada al establecimiento de


una barrera de permeabilidad al mismo, es un mecanismo habitual de resistencia. A
causa de su tamao, los macrlidos encuentran dificultades para salvar la barrera que

7
________________________________________
Introduccin _______________________________________
_____________________________________

supone la membrana externa. La penetracin de tetraciclina en las clulas se ve


reducida cuando las porinas disminuyen o se modifican (Thanassi et al., 1995). La
ausencia de transporte mediada por los citocromos es causa de resistencia natural a los
aminoglicsidos en organismos anaerobios como las bifidobacterias (Bryan y Kwan,
1981a).
En otra categora podramos situar la secrecin del antibitico al exterior celular
una vez que ha sido incorporado. De esta forma, la expulsin de tetraciclina por
protenas de membrana reduce la concentracin intracelular (Chopra y Roberts, 2001).

I.1.3.4.- Transporte inespecfico del antibitico al exterior de la bacteria

Adems de mecanismos especficos, en las clulas existen sistemas generales de


detoxificacin como los sistemas de resistencia mltiple a drogas (Multi-Drug
Resistance), capaces de expulsar numerosos sustratos txicos. Los MDR estn presentes
en todos los seres vivos y son capaces de expulsar numerosos compuestos citotxicos
no relacionados estructuralmente entre si, incluyendo diversos antibiticos (Thanassi et
al., 1995; Putman et al., 2000a). La activacin de los sistemas MDR ha sido relacionada
con la resistencia a diferentes antibiticos, como fluoroquinolonas y cloranfenicol
(Piddock, 2006).

I.1.4.- BASES GENTICAS DE LA RESISTENCIA

Los avances realizados en las ltimas dcadas en los campos de la fisiologa y


gentica bacterianas han sido imprescindibles para desentraar los mecanismos
gentico-moleculares de la resistencia a antibiticos. Este conocimiento se pretende
utilizar, en ltimo trmino, para el desarrollo de nuevos antibiticos insensibles a los
mecanismos de resistencia, as como para el diseo de estrategias que, en lo posible,
frenen su diseminacin.

Muchas resistencias intrnsecas (ausencia de diana molecular o incapacidad de


transporte del antibitico) carecen de una base gentica especfica; base gentica que s
presentan las resistencias adquiridas. stas pueden deberse a mutaciones o a la
adquisicin de nuevos genes por transferencia horizontal.

8
________________________________________
_______________________________________
_____________________________________
Introduccin

I.1.4.1.- Mutacin

La mutacin espontnea aporta una subpoblacin resistente que ser


seleccionada en presencia del antibitico. La tasa de mutacin se incrementa en
presencia del agente selectivo, siendo las mutaciones ms frecuentes aquellas que
afectan a la estructura molecular del sitio de accin del antibitico (protenas
ribosomales, ARN polimerasa, etc.). Tambin existen mutaciones que modifican la
pared o la membrana de modo que el antibitico no se transporta al interior de la
clula. Otras mutaciones, finalmente, inducen la superproduccin de protenas de
membrana encargadas de expulsar el antibitico al exterior de la clula (Normark y
Normark, 2002; Piddock, 2006).
La mutacin espontnea depende del ndice de mutacin gnica (relacionado a
su vez con la longitud y las caractersticas fsicas del gen) y de los sistemas bacterianos
de reparacin de ADN (Miller, 1996). En general, el sistema de reparacin tiene una
importancia mayor, ya que una alta tasa de mutacin se relaciona casi siempre con
sistemas defectuosos (Falker et al., 2005).

I.1.4.2.- Transferencia horizontal

La transferencia horizontal permite a las bacterias adquirir material gentico de


otras clulas o del entorno y con ello aumentar sus capacidades bioqumicas. ste es,
sin duda, el mecanismo principal para la adquisicin de determinantes genticos de
resistencia a antibiticos. En ocasiones, por este mecanismo se adquieren bloques
gnicos dedicados a la resistencia a muchos antibiticos distintos. La transferencia
gnica entre bacterias se puede llevar a cabo por tres mecanismos naturales: la
conjugacin, la transduccin y la transformacin (Alanis, 2005):
La transduccin se produce con la colaboracin de un bacterifago capaz de
infectar cepas distintas. De forma errnea, los virus que infectan cepas resistentes
pueden incorporar los determinantes de resistencia. stos pueden transferirse a una
nueva cepa infectada y, ocasionalmente, integrarse en su genoma. La transduccin est
limitada por el tamao de la cabeza del fago y por el rango de husped del fago.
La transformacin consiste en la adquisicin de ADN libre del medio por un
microorganismo. Aunque es posible que en determinadas circunstancias todos los

9
________________________________________
Introduccin _______________________________________
_____________________________________

microorganismos sean capaces de incorporar ADN, solo unos pocos presentan


transformacin natural.
La conjugacin es el mecanismo ms importante y comn para la transmisin de
determinantes genticos entre bacterias, incluyendo la transferencia de genes de
resistencia. La conjugacin se produce entre una clula que acta de donadora y otra
que recibe una copia del elemento transferido y se denomina receptora. Normalmente,
el proceso est mediado por plsmidos o transposones que codifican todos los
elementos necesarios para que ste tenga lugar. En principio no hay una limitacin
terica a la cantidad de ADN que puede incorporarse a la clula receptora. As, en las
cepas Hfr de Escherichia coli se puede transferir todo el genoma bacteriano. La
conjugacin es un proceso de intercambio gnico muy poderoso puesto que puede
llevarse a cabo, incluso, entre bacterias no relacionadas.
La transmisin de las resistencias depender de la naturaleza de los elementos
en que se codifica (plsmidos, transposones, etc.), de la concentracin y proximidad de
las cepas donadora y receptora, y de la presencia de agentes selectores en el medio que
favorezcan la multiplicacin de la progenie resistente (Teuber et al., 1999).

I.1.5.- EL PROBLEMA DE LA RESISTENCIA A ANTIBITICOS

Durante las ltimas dcadas, el uso excesivo e inapropiado de antibiticos ha


propiciado la aparicin y diseminacin de bacterias resistentes. La presin selectiva
que los antibiticos ejercen en determinados hbitats favorece la adquisicin de
sistemas de resistencia como respuesta adaptativa de los microorganismos. La
resistencia es, pues, un proceso evolutivo inevitable y predecible. En estas condiciones,
son frecuentes las mutaciones espontneas; primer paso en el desarrollo de altos
niveles de resistencia (Mazel y Davies, 1999). Sin embargo, como se ha comentado, el
principal mecanismo por el que las bacterias obtienen genes de resistencia a
antibiticos es la adquisicin de ADN exgeno (Levy, 1997).
La transmisin de los factores de resistencia a los microorganismos patgenos
complica o impide el tratamiento de las enfermedades que stos causan. En ambientes
hospitalarios es habitual la presencia de microorganismos resistentes a muchos
antibiticos distintos, fruto de procesos de concentracin de genes dedicados a este fin.
De esta forma, han ido surgiendo cepas resistentes y multiresistentes de estafilococos,
neumococos y enterococos responsables de graves infecciones, difciles de tratar dado

10
________________________________________
_______________________________________
_____________________________________
Introduccin

su nivel de resistencias (Baquero, 1997; Williams, 2001). Adems, algunos


microorganismos son capaces de transmitir en bloque las resistencias a otras cepas,
incluso de especies distintas.
La resistencia a antibiticos tambin presenta un peligro potencial cuando se
encuentra en los microorganismos comensales o beneficiosos, ya que estos pudieran
convertirse en reservorios desde dnde los determinantes podran transferirse a los
microorganismos oportunistas y a los patgenos (Figura 3; Teuber et al., 1999; Salyers
et al., 2004).

Otras bacterias Bacterias ingeridas


intestinales

Bacteria
Genes

Bacterias intestinales Transmisin


resistentes Fecal-Oral

Figura 3.- Hiptesis del reservorio de genes de resistencia a antibiticos (Salyers et al., 2004).

I.2- BACTERIAS DEL CIDO LCTICO (BAL)

I.2.1.- INTRODUCCIN

Las bacterias del cido lctico (BAL) son un grupo taxonmicamente diverso de
bacterias Gram-positivas, anaerobias estrictas o aerotolerantes, generalmente
inmviles, no esporuladas ni pigmentadas, con la facultad de convertir los
carbohidratos en cido lctico, provocando la acidificacin del medio. Adems, tienen
una limitada capacidad biosinttica, por lo que son muy exigentes nutricionalmente y
requieren numerosos factores de crecimiento, incluyendo aminocidos, vitaminas y

11
________________________________________
Introduccin _______________________________________
_____________________________________

precursores de los cidos nucleicos (Dellaglio et al., 1994). Por esta causa se encuentran
en nichos ecolgicos ricos en nutrientes como la superficie de las plantas y el material
vegetal en descomposicin (Stiles, 1996). Son tambin componentes destacados del
tracto gastrointestinal y genitourinario de los vertebrados, donde cumplen un
importante papel en el mantenimiento del equilibrio microbiano necesario para la
salud (Guarner y Malagelada, 2003).
Estas bacterias tienen gran importancia aplicada porque se utilizan como
cultivos iniciadores en la elaboracin de alimentos fermentados como quesos, yogures,
embutidos, encurtidos, etc., a los que imparten caractersticas sensoriales deseables y
una buena capacidad de conservacin (Stiles, 1996). En los ltimos tiempos, diversas
especies de este grupo se utilizan tambin como probiticos en hombres y animales
para mantener el estado de salud o combatir las enfermedades intestinales
(Ouwenhand et al., 2002).
El hombre ha consumido grandes cantidades de BAL con los productos
fermentados desde tiempo inmemorial sin que aparecieran efectos adversos, por lo que
estas bacterias se consideran seguras [Generally Recognized As Safe (GRAS)] desde el
punto de vista de la salud. A excepcin de los enterococos (Asahara et al., 2003), que no
se tratan en esta Tesis, las BAL raramente producen infecciones y cuando lo hacen se
debe a que, por alguna causa subyacente, la inmunidad est severamente disminuida
(Gasser, 1994; Cannon et al., 2005).

I.2.2.- TIPOS Y GNEROS PRINCIPALES

Las BAL ms tpicas se encuadran en los gneros Lactococcus, Lactobacillus,


Leuconostoc y Pediococcus y la especie Streptococcus thermophilus. Con motivo de
encontrarse en los mismos hbitats y tener una aplicacin industrial similar, dentro de
este grupo se han incluido tradicionalmente los gneros Propionibacterium y
Bifidobacterium. Desde el punto de vista filogentico, sin embargo, propionibacterias y
bifidobacterias no estn emparentadas con las bacterias lcticas verdaderas (Figura 4).
Por el contrario, las especies del gnero Enterococcus que si pueden considerarse
bacterias lcticas verdaderas y estn presentes en los mismos hbitats, no se han
considerado en este trabajo por la controversia que causa su capacidad infectiva
(Foulqui Moreno et al., 2006).

12
________________________________________
_______________________________________
_____________________________________
Introduccin

Lactococcus
Tetragenococcus Streptococcus

Alloiococcus
Weisella Leuconostoc
Aerococcus

Enterococcus

Vagococcus
Lactobacillus
Pediococcus
Carnobacterium

Escherichia coli
Bifidobacterium

10%
Propionibacterium

Figura 4.- rbol filogentico basado en la secuencia del ARNr 16S del grupo de las bacterias
del cido lctico (Schleifer et al., 1995).

En los prrafos siguientes se enumeran las caractersticas ms significativas de


los gneros ms importantes y se presentan las especies ms representativas.

1.- Lactococcus. Los lactococos son bacterias Gram-positivas, mesfilas,


homofermentadoras con morfologa cocoide que producen cido lctico de tipo L(+). El
gnero Lactococcus incluye las especies: Lactococcus lactis (con tres subespecies lactis,
cremoris y hordniae), Lc. garviae, Lc. piscium, Lc. plantarum y Lc. raffinolactis. En la
industria quesera la nica especie empleada es Lc. lactis, y en especial la subespecie
cremoris. Un tipo particular de Lc. lactis subsp. lactis lo constituye la biovariedad
diacetylactis, capaz de metabolizar el citrato de la leche produciendo diacetilo,
compuesto caracterstico de la mantequilla y de varios tipos de quesos.

2.- Lactobacillus. Los lactobacilos son bacilos regulares Gram-positivos, no


esporulados, capaces de colonizar hbitats tan diversos como el material vegetal, los
productos lcteos y la piel y las mucosas del hombre y los animales (Klander y Weiss,

13
________________________________________
Introduccin _______________________________________
_____________________________________

1986). En el gnero se incluyen ms de 100 especies con propiedades muy


heterogneas. Esta diversidad se ve reflejada genotpicamente, ya que la proporcin de
G+C de las distintas especies vara entre un 32% y un 52%. Las especies se clasifican
como mesfilas o termfilas, de acuerdo a su temperatura ptima de crecimiento, y en
homofermentadoras, heterofermentadoras facultativas o heterofermentadoras estrictas,
dependiendo de sus caractersticas fermentativas.
Grupo I. Especies homofermentadoras estrictas. Comprende el grupo acidophilus
formado por las especies: Lactobacillus acidophilus, Lb. amilovorus, Lb. crispatus, Lb.
gallinarum, Lb. gasseri y Lb. johnsonii. Tambin se incluyen en este grupo otras
especies como Lb. delbrueckii, Lb. helveticus, Lb. jensenii, Lb. leichmanii y Lb.
salivarius.
Grupo II. Especies heterofermentadoras facultativas. Las especies principales de
este grupo son Lb. casei, Lb. curvatus, Lb. plantarum, Lb. pentosus y Lb. sakei.
Grupo III. Especies heterofermentadoras estrictas. Entre otras, el grupo incluye
las especies Lb. brevis, Lb. buchneri, Lb. cellobiosus, Lb. fermentum, Lb. reuteri y Lb.
viridescens.

3.- Leuconostoc. Los leuconostocs son bacterias lcticas heterofermentadoras


obligadas que producen cido lctico de tipo D(-) y cantidades equimoleculares de
CO2, etanol y cido actico. Se agrupan en cadenas como los lactococos, aunque se
diferencian de stos por la produccin de gas y la incapacidad de hidrolizar la
arginina. El gnero Leuconostoc comprende actualmente ocho especies, de las que slo
tres se utilizan en la industria alimentaria: Leuconostoc mesenteroides subsp. cremoris, Ln.
citreum y Ln. lactis. El inters tecnolgico de los leuconostoc viene dado por su
capacidad para producir diacetilo y otros compuestos aromatizantes.

4.- Streptococcus thermophilus. Esta especie homofermentadora est emparentada


con la bacteria comensal de la boca Streptococcus salivarus. Se utiliza junto con Lb.
delbrueckii en la fermentacin del yogurt y es un componente habitual de los fermentos
de los quesos de pasta cocida junto a otras especies de lactobacilos.

5.- Pediococcus. Los pediococos son bacterias Gram-positivas, anaerobias


aerotolerantes que se agrupan en ttradas y que poseen un metabolismo
homofermentativo por el que transforman los carbohidratos en DL-lactato. El gnero

14
________________________________________
_______________________________________
_____________________________________
Introduccin

est constituido por ocho especies de las que solo dos, Pediococcus acidilactici y P.
pentosaceus, se aslan de quesos y productos crnicos fermentados, en los que tambin
se utilizan como cultivos iniciadores.

6.- Bifidobacterium. Las bifidobacterias son microorganismos Gram-positivos,


anaerobios estrictos, inmviles, no esporulados y, en su mayora, catalasa negativos.
Presentan una gran variedad de formas (cocoide, con bifurcaciones, con cadenas
estrelladas o extremos espatulados). Su nombre hace referencia a las formas con dos
ramas en Y o V que muestran en ocasiones. El gnero est compuesto por 29 especies,
de las que unas 12 se aslan del tracto gastrointestinal humano y el resto proceden de
ambientes tan diversos como alimentos, cavidad oral, tracto gastrointestinal animal,
intestino de insectos y aguas residuales (Tannock, 2005).
Las bifidobacterias se distinguen de las BAL por su alta proporcin de G+C
(>50%). Tambin es caracterstica del gnero la presencia del enzima fructosa-6-fosfato
fosfocetolasa, que conduce a la formacin de cido lctico y cido actico en una
proporcin 2:3 sin generacin de gas.

7.- Propionibacterium. El gnero Propionibacterium incluye varias especies con


morfologa pleomrfica al igual que las bifidobacterias. Son bacterias Gram-positivas,
inmviles, no formadoras de esporas y quimioorganotrofas. Los productos de la
fermentacin de las propionibacterias incluyen grandes cantidades de cido propinico
y actico y cantidades menores de cido iso-valrico, frmico, succnico y lctico.
Producen tambin una pequea cantidad de CO2, responsable de la formacin de ojos
en el queso. Solo unas pocas (Propionibacterium freudenreichii y P. jensenii) se utilizan
como fermento en los quesos tipo suizos para la produccin de los caractersticos ojos.

I.2.3.- PERFILES DE SUSCEPTIBILIDAD Y GENES DE RESISTENCIA


A ANTIBITICOS EN BAL

Tanto las metodologas como los perfiles de susceptibilidad/resistencia a


antibiticos estn muy establecidas para la mayora de los microorganismos patgenos.
Existen medios y protocolos muy estandarizados y claros valores de corte para
distinguir cepas sensibles de resistentes (CLSI, 2004).

15
________________________________________
Introduccin _______________________________________
_____________________________________

Menor atencin, sin embargo, se ha dedicado a otros grupos bacterianos, entre


los que podemos contar las bacterias lcticas (Teuber et al., 1999). Adems, las BAL y
las bifidobacterias no crecen bien en los medios y las condiciones de ensayo
establecidas para los microorganismos de importancia clnica. Los estudios realizados
hasta la fecha se han llevado a cabo utilizando una multiplicidad de mtodos: difusin
en disco (Herreros et al., 2005), dilucin en agar (Herrero et al.., 1996), Etest (Danielsen
y Wind, 2003), microdilucin (Flrez et al., 2005), etc., lo que dificulta la comparacin
de los resultados obtenidos (Huys et al., 2002). Por estas razones, para la mayora de
los antibiticos no hay claros puntos de corte con los que separar cepas resistentes de
susceptibles. Dada su enorme distribucin, el inters por conocer los niveles de
resistencia a antibiticos en las bacterias lcticas y en otros grupos de microorganismos
comensales ha aumentado en los ltimos tiempos. Adems, como se ha comentado,
existe la preocupacin de que estas bacterias se conviertan en reservorios de
resistencias, desde donde los determinantes puedan transferirse a los microorganismos
perjudiciales (Teuber et al., 1999).
Recientemente, se han establecido puntos de corte para las cepas utilizadas en
alimentacin animal (European Commission, 2005). En los prrafos que siguen,
trataremos de resumir los conocimientos generales sobre la resistencia a antibiticos en
las BAL, indicando, cuando se conocen, las resistencias intrnsecas, las adquiridas, los
genes responsables, de los que puede verse un resumen en la Tabla 2 y una descripcin
ms detallada en el Anexo 4, y los elementos en que se codifican (para revisin vase
Teuber et al., 1999; Mathur y Singh, 2005).

1.- Lactococos

Dada su importancia como componentes de los fermentos del queso, se han


analizado gran variedad de cepas de Lc. lactis frente a los antibiticos ms usuales
(Orberg y Sandine, 1985; Mayo et al., 1990; Herrero et al., 1996; Temmerman et al.,
2003). Los lactococos son sensibles en general frente a macrlidos, bacitracina,
lincomicina, novobiocina, teicoplanina y vancomicina, as como, frente a antibiticos de
amplio espectro como aminoglicsidos (gentamicina y kanamicina), tetraciclinas y
cloranfenicol. La mayora de las cepas se muestran resistentes a metronidazol,
cefoxitina, trimetoprima, rifampicina y a los antibiticos activos sobre bacterias Gram-
negativas (cido fusdico, cido nalidxico, polimixina E). La resistencia a rifampicina,

16
________________________________________
_______________________________________
_____________________________________
Introduccin

en particular, se ha utilizado para su aislamiento selectivo. Se sospecha que, aunque


por distintas causas, la baja susceptibilidad a todos estos antibiticos es intrnseca.
De los estudios efectuados parece claro que los lactococos no presentan
demasiadas resistencias adquiridas. Sin embargo, en la cepa Lc. lactis K214, procedente
de un queso fresco elaborado con leche cruda, se encontr un plsmido (pK214) que
porta tres genes de resistencia a antibiticos (estreptomicina, cloranfenicol (cat) y
tetraciclina [tet(S)] (Perreten et al., 1997a). Estos genes son idnticos a otros
caracterizados con anterioridad en cepas de S. aureus, Streptococcus pyogenes y Listeria
monocytogenes. pK214 codifica adems un sistema general de exportacin de
compuestos txicos [mdt(A)] que confiere resistencia a distintos tipos de antibitios
(Perreten et al., 2001). Recientemente, en otra cepa, procedente del intestino de un pollo
sano, se ha caracterizado un plsmido (pAJ01) que confiere resistencia a eritromicina
(Raha et al., 2002).

2.-Lactobacilos

En general, los lactobacilos son sensibles a las penicilinas y otros -lactmicos,


aunque muestran una ligera resistencia a la oxacilina y a las cefalosporinas (Danielsen
y Wind, 2003; Coppola et al., 2005). Adems, algunas especies poseen resistencia
intrnseca frente a glicopptidos (vancomicina) debido, como ya se ha comentado, a
una modificacin en el peptidoglicano.
Respecto al grupo de antimicrobianos inhibidores de la sntesis proteica, los
lactobacilos suelen ser sensibles a cloranfenicol, eritromicina, clindamicina y
tetraciclina y ms resistentes a los aminoglicsidos (kanamicina, gentamicina,
neomicina y estreptomicina) (Charteris et al., 1998; Coppola et al., 2005; Zhou et al.,
2005). La ausencia de transporte mediada por la cadena de citocromos parece ser la
principal causa de resistencia a aminoglicsidos (Condon, 1983).
Finalmente, parecen ser resistentes tambin a la mayora de los inhibidores de
la sntesis de cidos nucleicos, tales como enoxacina, pefloxacina, norfloxacino, cido
nalidxico, trimetroprim y metronidazol (Charteris et al., 1998; Coppola et al., 2005).
En distintas cepas de lactobacilos se han caracterizado genes plasmdicos que
codifican resistencia al cloranfenicol, tetraciclinas y a los antibiticos del grupo de los
macrlidos, lincosamidas y estreptogramina B (fenotipo MLS), como, por ejemplo, en
una cepa de Lb. reuteri aislada de muestras de pollo (Tannock et al., 1994). El plsmido

17
________________________________________
Introduccin _______________________________________
_____________________________________

pTC82 de 7Kb, caracterizado en una cepa de Lb. reuteri, lleva un gen que codifica una
cloranfenicol acetil-transferasa similar al codificado en el plsmido pC194 de S. aureus
(Lin et al., 1996). Adems, una cepa de Lb. fermentum aislada de heces de cerdo posee
un plsmido (pLEM3) con un gen erm que presenta una homologa del 98,2% con el
gen erm identificado en el transposn Tn1545 de Streptococcus pneumoniae (Fons et al.,
1997).
Distintas cepas de lactobacilos resistentes a tetraciclina de las especies Lb.
acidophilus (Cataloluk y Gogebakan, 2004), Lb. alimentarius (Gevers et al., 2003), Lb. casei
(Cataloluk y Gogebakan, 2004), Lb. crispatus (Cataloluk y Gogebakan, 2004), Lb.
curvatus (Gevers et al., 2003), Lb. gasseri (Cataloluk y Gogebakan, 2004) y Lb. plantarum
(Cataloluk y Gogebakan, 2004) poseen el gen tet(M), que codifica una protena de
proteccin ribosomal. Adems de tet(M), en cepas de lactobacilos se han identificado
tambin otros genes como, tet(K), tet(S), tet(W), tet(36), tet(O) y tet(Q) que codifican
protenas secretoras o de proteccin ribosomal (Roberts, 2005; Chopra y Roberts, 2001;
Villedieu et al., 2003).

3.- Estreptococos

St. thermophilus es la nica especie del gnero Streptococcus con relevancia


tecnolgica. St. thermophilus parece ser susceptible a cloranfenicol, tetraciclina,
eritromicina, cefalotina, quinupristina/dalfopristina y ciprofloxacino (Katla et al., 2001;
Temmerman et al., 2003; Aslim et al., 2004). Por otro lado, la resistencia parece ser
variable a los aminoglicsidos (gentamicina, kanamicina y estreptomicina),
trimetoprim y sulfadiazina (Katla et al., 2001; Temmerman et al., 2003; Aslim et al.,
2004). Tambin se ha detectado gran variabilidad en la susceptibilidad frente a
antibiticos como penicilina G, ampicilina y vancomicina (Katla et al., 2001;
Temmerman et al., 2003; Aslim et al., 2004), aunque los niveles de resistencia son tan
bajos que las cepas se consideran sensibles.
De forma muy reciente, se ha detectado la presencia del gen erm(B) en cuatro
cepas de St. thermophilus resistentes a eritromicina de una coleccin de 70 (Berruti et al.,
2005).

18
________________________________________
_______________________________________
_____________________________________
Introduccin

4.- Leuconostoc

Las distintas especies que constituyen el gnero Leuconostoc son resistentes a


vancomicina y teicoplanina, as como frente a cefoxitina y metronidazol. La resistencia
a los glicopptidos es intrnseca y viene determinada por la presencia del dipptido D-
alanina-D-lactato como componente del pptidoglicano (Elisha y Courvalin, 1995).
Adems, los leuconostoc muestran menor susceptibilidad que otras BAL a amoxicilina,
imipenem, nitrofurantoina, sulfodiazina y trimetoprim (Swenson et al., 1990; Herrero
et al., 1996; Katla et al., 2001; Herreros et al., 2005). Por el contrario, la mayora de las
cepas son muy sensibles a eritromicina, clindamicina, cloranfenicol y tetraciclina
(Swenson et al., 1990). A estos dos ltimos antibiticos, sin embargo, se han detectado
algunas cepas resistentes (Swenson et al., 1990; Herreros et al., 2005), aunque la
naturaleza molecular de las resistencias est todava por determinar.

5.- Pediococos

Los datos de resistencia a antibioticos en los pediococos son escasos. Estos


microorganismos son sensibles a penicilina G, imipenem, gentamicina, netilmicina,
eritromicina, clindamicina, rifampicina, cloranfenicol y otros muchos (Swenson et al.,
1990; Tankovic et al., 1993; Temmerman et al., 2003; Zarazaga et al., 1999). Sin embargo,
son resistentes intrnsecos a los glicopptidos (vancomicina y teicoplanina), a
estreptomicina, kanamicina, tetraciclina, ciprofloxacin y sulfametoxazol (Swenson et
al., 1990). Se han encontrado cepas resistentes a los -lactmicos, los aminoglicsidos,
los macrlidos y las tetraciclinas (Tankovic et al., 1993; Temmerman et al., 2003).
En este gnero se han caracterizado algunos genes de resistencia, entre los que
cabe mencionar un gen de Pediococcus acidilactici que codifica la resistencia MLS
[erm(AM)] (Tankovic et al., 1993; Zarazaga et al., 1999) localizado en un plsmido de 46
kpb no transmisible (Tankovic et al., 1993). Ms recientemente, se ha caracterizado un
gen de resistencia a tetraciclina [tet(L)] y dos de resistencia a aminoglicsidos [aac(6)-
aph(2) y ant(6)] en cepas de P. pentosaceus y P. parvulus aisladas de vinos (Rojo-Bezares
et al., 2006).

19
________________________________________
Introduccin _______________________________________
_____________________________________

6.- Bifidobacterias

Las bifidobacterias son sensibles a la mayora de los antibiticos activos sobre


bacterias Gram-positivas como los macrlidos, la bacitracina, la lincomicina, la
novobiocina y la vancomicina. Tambin son sensibles a la rifampicina, la
espectinomicina, la tetraciclina, el cloranfenicol y los -lactmicos (penicilina,
ampicilina, amoxicilina, piperacilina, ticarcilina e imipinem) (Matteuzzi et al., 1983;
Lim et al., 1993; Teuber et al., 1999; Moubareck et al., 2005). Resistencias atpicas en
cepas de este gnero frente a vancomicina y cefoxitina han sido descritas por Charteris
et al. (1998). Por el contrario, la mayora de las especies de Bifidobacterium son
resistentes a metronidazol, as como a los antibiticos neomicina, gentamicina,
kanamicina y estreptomicina (Matteuzzi et al., 1983; Lim et al., 1993; Charteris et al.,
1998; Moubareck et al., 2005; Zhou et al., 2005). La susceptibilidad parece ser muy
variable a tetraciclina, cefalotina, nitrofurantoina y cefotetn (Lim et al., 1993;
Moubareck et al., 2005), de forma que algunas cepas pudieran ser resistentes.
Como en otros organismos anaerobios, la resistencia a aminoglicsidos est
determinada por la ausencia de transporte ligado a la cadena de citocromos (Bryan y
Kwan, 1981a). Sin embargo, se han caracterizado determinantes de resistencia para
tetraciclina. En particular se ha detectado la presencia de tet(W) y tet(M). El primero
parece estar abundantemente distribuido entre las cepas intestinales y las que se
incluyen en productos probiticos (Scott et al., 2000; Moubareck et al., 2005; Masco et
al., 2006).

7.- Propionibacterias

No existen datos de los niveles de resistencia a antibiticos en propionibacterias


de productos lcteos. Sin embargo, son corrientes las cepas de Propionibacterium acnes
con resistencia a eritromicina y clindamicina, quiz como consecuencia del tratamiento
del acn con estos antibiticos (Swanson, 2003).

20
________________________________________
_______________________________________
_____________________________________
Introduccin

Tabla 2.- Resumen de los genes de resistencia caracterizados en las bacterias del cido lctico.

Gneros Determinantes genticos Resistencia

Lactococcus tet (M), tet (S) Tetraciclina


erm (T) Eritromicina
cat Cloranfenicol
str Estreptomicina
mdt (A), Lmr (A), Lmr (P), Lmr (CD) Transportadores MDR

tet (M), tet (K), tet (S), tet (W), tet (36),
Lactobacillus Tetraciclina
tet (O), tet (Q)
erm (B), erm (T), erm (LF) Eritromicina
cat Cloranfenicol
aac (6'), aph (2''), aph (3')-IIIa, ant (6) Aminoglicsidos
vat (E-1) Estreptograminas

Streptococcus erm (B) Eritromicina

Pediococcus tet (L) Tetraciclina


erm (B) Eritromicina
aac (6'), aph (2''), ant (6) Aminoglicsidos

Bifidobacterium tet (W), tet (M) Tetraciclina


Bbm (R) Transportador MDR

I.2.4.- TRANSFERENCIA GNICA EN BAL

Las BAL presentan, en general, pocas resistencias adquiridas. La mayora de las


que se han caracterizado son idnticas a las descritas en otros grupos bacterianos, por
lo que es fcil pensar que estas resistencias se han adquirido de forma muy reciente
(Perreten et al., 1997a; Teuber et al., 1999). Los procesos de intercambio gnico en las
bacterias del cido lctico son los mismos que presentan otros grupos microbianos:
transformacin, transduccin y conjugacin.

I.2.4.1.- Transformacin y transfeccin

La transformacin natural se ha descrito de forma reciente en, al menos, una


cepa de Ln. carnosum (Helmark et al., 2004). Sin embargo, no parece ser una propiedad
extendida entre las bacterias lcticas. Con todo, en el genoma de Lc. lactis se ha
encontrado el juego completo de los genes tardos de competencia (Bolotin et al., 2001).
Parte de esta maquinaria aparece tambin en el genoma de St. thermophilus (Bolotin et

21
________________________________________
Introduccin _______________________________________
_____________________________________

al., 2004). La transduccin, por su parte, fue la primera tcnica utilizada en la


transferencia de genes con inters tecnolgico entre cepas distintas de Lc. lactis
(Sandine et al., 1962). Sin embargo, se piensa que la diseminacin de genes de
resistencia por este mtodo es escasa debido a la especificidad de los bacterifagos.

I.2.4.2.- Conjugacin

El proceso de conjugacin no se conoce con exactitud en las bacterias Gram-


positivas pero, para que tenga lugar, es necesario que exista un contacto ntimo entre
clula donadora y receptora (Gasson y Fitzgerald, 1994). La maquinaria bioqumica
consta de 18-20 protenas ubicadas en 15-20 kb de ADN (Dougherty et al., 1998). Esta
maquinaria est codificada en plsmidos y en transposones conjugativos. Adems, en
Lc. lactis se ha detectado la existencia de un factor sexual que parece mediar en el
proceso de conjugacin (Gasson et al., 1995). Plsmidos conjugativos se han detectado
en diversas cepas de lactococos, leuconostoc y algunas especies de lactobacilos
(Davidson et al., 1996), sin embargo, no se han descrito en el gnero Bifidobacterium ni
en St. thermophilus (Sgorbati et al., 1982; Mercenier et al., 1993). En todo caso, ninguno
de los plsmidos conjugativos de las bacterias lcticas caracterizados hasta la fecha
incluye genes de resistencia a antibiticos. Estos plsmidos son corrientes en bacterias
de gneros prximos como Enterococcus, Streptococcus y Staphylococcus. Entre los ms
estudiados, podemos mencionar pAM1, procedente de una cepa de Enterococcus
faecalis (Clewell et al., 1974), y pIP501, que procede de una cepa de Streptococcus
agalactiae (Evans y Marciana, 1983). El primero codifica resistencia a eritromicina y el
segundo a eritromicina y cloranfenicol. Ambos plsmidos presentan una enorme
promiscuidad y son capaces de transferirse por conjugacin a numerosos gneros de
bacterias Gram-positivas, incluyendo los de las bacterias lcticas (Vescovo et al., 1983;
Langella y Chopin, 1989).
Otra categora de plsmidos son aquellos no conjugativos pero que pueden ser
transferidos entre clulas utilizando la maquinaria de conjugacin de otros plsmidos o
la de factores sexuales. stos se denominan plsmidos movilizables. Para su
movilizacin los plsmidos pueden codificar protenas auxiliares que intervengan en el
proceso o nicamente el sitio fsico de reconocimiento en el que se inicia el proceso
(sitio oriT). De este tipo es el plsmido pK214 de Lc. lactis subsp. lactis K214 (Perreten et
al., 1997a). Como se ha comentado anteriormente, este plsmido es el paradigma de la
concentracin de determinantes genticos de resistencia en las bacterias lcticas, puesto

22
________________________________________
_______________________________________
_____________________________________
Introduccin

que en l se agrupan cuatro genes de resistencia de procedencias diversas. Secuencias y


elementos de movilizacin se encuentran tambin en los plsmidos pLME300,
procedente de la cepa Lb. fermentum ROT1 y que confiere resistencia a eritromicina y
estreptogramina A (Gfeller et al., 2003), y pMD5057, detectado en la cepa Lb. plantarum
5057 y que codifica resistencia a tetraciclina (Danielsen, 2002). Lo mismo ocurre con el
plsmido de resistencia a cloranfenicol pCaT de Lb. plantarum TC2R, en el que se ha
comprobado experimentalmente su capacidad de movilizacin por el plsmido
conjugativo pAM1 (Ahn et al., 1992).
Los transposones conjugativos son elementos genticos mviles que codifican
la maquinaria necesaria para promover su propia transferencia. En general, tienen gran
capacidad de infeccin y en algunos gneros bacterianos se han visto claramente
involucrados en la diseminacin de resistencias a antibiticos (Whittle et al., 2002). A
diferencia de los plsmidos, no poseen sistemas de replicacin autnoma, por lo que
requieren de la integracin en el cromosoma o en un plsmido. En las bacterias del
cido lctico se han descrito una gran variedad de transposones conjugativos con
amplio rango de husped. Se conoce desde hace tiempo la presencia de transposones
conjugativos en Lc. lactis que movilizan la produccin y resistencia a nisina, y la
capacidad de utilizacin de sacarosa (Fitzgerald y Gasson, 1988; Rauch et al., 1994).
Transposones que portan genes de resistencia a antibiticos se han descrito en
distintas cepas de Enterococcus, pero no en las bacterias lcticas que estamos
considerando. La mayora pertenecen a la familia Tn916-Tn1545 y confieren resistencia
a tetraciclina, eritromicina y/o cloranfenicol (Huys et al., 2004 y Perreten et al., 1997b).
Estos transposones s se transfieren entre las bacterias lcticas. As, TnFO1 fue
transferido mediante conjugacin desde la especie original E. faecalis a un buen nmero
de especies distintas, incluyendo Lc. lactis subsp. lactis y Ln. mesenteroides. Su completa
funcionalidad en Lc. lactis se comprob mediante una nueva transferencia a Listeria
innocua (Perreten et al., 1997b).

23
OBJETIVOS
______________________________________
_____________________________________
_____________________________________
Objetivos

II.- OBJETIVOS

II.1.- OBJETIVOS DEL TRABAJO

La aparicin, diseminacin y concentracin de resistencias a antimicrobianos en


microorganismos oportunistas y patgenos representa un problema grave para la
salud humana y animal, puesto que complica el tratamiento y, por tanto, la
recuperacin de las enfermedades que causan. Esto es ya una realidad para las
neumonas por cepas multiresistentes de Streptococcus pneumoniae (Garau, 2002) y las
infecciones causadas por cepas de Staphylococcus aureus resistentes a vancomicina
(Pfeltz y Wilkinson, 2004).
Los tipos bacterianos en los que se ha trabajo en esta Tesis, sin embargo, no
causan enfermedades. Muy al contrario, una gran mayora se considera capaz de
prolongar el estado de salud e, incluso, de participar en la recuperacin de diversas
enfermedades intestinales (Bernardeau et al., 2006). Las bacterias del cido lctico
procedentes de alimentos fermentados forman parte de la dieta del hombre y de otros
animales desde tiempo inmemorial. En los alimentos fermentados elaborados con leche
cruda, estos microorganismos conviven con muchos otros tipos, incluyendo
representantes patgenos. Especies de lactobacilos y bifidobacterias colonizan las
mucosas del hombre y los animales superiores poco despus del nacimiento,
participando en el mantenimiento del equilibrio microbiano necesario para la salud. Las
bifidobacterias, por ejemplo, constituyen la poblacin dominante en el intestino del
recin nacido y se mantienen en concentraciones elevadas a lo largo de toda la vida.
Ahora bien, estas bacterias conviven en las mucosas con microorganismos que pueden
actuar como patgenos y oportunistas. Los microorganismos beneficiosos pueden
convertirse en reservorios de genes de resistencias y transferirlos a los perjudiciales.
La problemtica de la resistencia a antibiticos en poblaciones comensales o
beneficiosas no est bien estudiada (Teuber et al., 1999; Salyers et al., 2004; Mathur y
Singh, 2005). De hecho, para muchas de estas bacterias no existen medios de cultivo
apropiados en los que llevar a cabo los ensayos, ni procedimientos estandarizados que
faciliten la comparacin de los datos. Tampoco existen puntos de corte reconocidos para
diferenciar los organismos resistentes de los sensibles. Finalmente, los flujos gnicos que
diseminan las resistencias entre microorganismos beneficiosos y patgenos se
desconocen. Por todas estas razones, en la actualidad, hay una preocupacin creciente

25
______________________________________
Objetivos _____________________________________
_____________________________________

de que las bacterias comensales de los alimentos y el tracto digestivo de animales y el


hombre se conviertan en reservorios de genes de resistencia a antibiticos, desde donde
pudieran transferirse a los microorganismos patgenos (Teuber et al., 1999; Salyers et
al., 2004).
Con estos antecedentes, los objetivos que nos hemos marcado en esta Tesis son
los siguientes:

1.- Contribuir a la estandarizacin de medios y mtodos para el ensayo de los


niveles de susceptibilidad/resistencia a antibiticos en las bacterias lcticas.

2.- Establecer puntos de corte fiables entre cepas sensibles y resistentes para
grupos bacterianos de inters y antibiticos representativos.

3.- Diferenciar resistencias intrnsecas de resistencias adquiridas y estudiar la base


molecular que subyace a ambos tipos de mecanismos.

4.- Evaluar la capacidad de transferencia horizontal de los determinantes de


resistencia a otros tipos microbianos mediante conjugacin.

26
TRABAJO EXPERIMENTAL
CAPTULO I.-Perfiles de susceptibilidad/resistencia en BAL y
bifidobacterias aisladas de quesos artesanos y del tracto gastrointestinal
humano mediante microdilucin.
Artculo I.- Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese
environment.
Canadian Journal. Microbiol. 51:51-58 (2005).
Artculo II.- Antibiotic susceptibility of Lactobacillus and Bifidobacterium species from the
human gastrointestinal tract.
Current Microbiology. 50:202-207 (2005).

Objetivos: En este trabajo se analizaron los perfiles de susceptibilidad/resistencia a


antibiticos en un numeroso grupo de bacterias mediante el sistema de microdilucin
comercial Sensititre Anaero 3. Los antibiticos del sistema Sensititre Anaero 3 incluyen
inhibidores de la sntesis proteica, inhibidores de la sntesis de la pared celular e
inhibidores de la sntesis de cidos nucleicos. La identificacin de cepas con
resistencias atpicas excluye su incorporacin en cultivos iniciadores o probiticos,
evitando la transferencia de genes a travs de la cadena alimentaria.

Resultados: En general, las BAL y bifidobacterias presentaron resistencia intrnseca al


metronidazol, lo que parece deberse a la ausencia en estos microorganismos de la
cadena de citocromos. Mostraron tambin una elevada resistencia a la cefoxitina,
debido a la impermeabilidad de su membrana y/o a otros mecanismos no especficos,
la resistencia fue mucho menos acusada en las bifidobacterias. Todos los aislados
fueron susceptibles a la penicilina, la amoxicilina, el imipinem, la piperacilina y a la
piperacilina ms tazobactam. Los leuconostocs y lactobacilos heterofermentativos se
mostraron resistentes a altas concentraciones de vancomicina, como consecuencia de la
composicin de su peptidoglicano. La mayora de los aislados de BAL y bifidobacterias
se mostraron susceptibles a los inhibidores de la sntesis proteica (cloranfenicol,
eritromicina, clindamicina y tetraciclina), unos pocos mostraron resistencia a
antibiticos concretos.

Conclusiones: El nmero de BAL y bifidobacterias del queso y del tracto


gastrointestinal con resistencias fue muy reducido, ya que solo una minora mostr
resistencia frente alguno de los siguientes antibiticos: tetraciclina, eritromicina y
clindamicina. Las resistencias mltiples a antibiticos no parecen ser comunes en estos
tipos bacterianos, ya que muy pocas cepas presentaron resistencias a ms de un
antibitico. En todo caso, la presencia de cepas resistentes justifica el anlisis de
aquellos microorganismos que puedan tener una aplicacin industrial.

29
Antimicrobial susceptibility of lactic acid bacteria
isolated from a cheese environment
Ana Belitn Florez, Susana Delgado, and Baltasar Mayo

Abstract: In the production of the Spanish traditional blue-veined Cabrales cheese, lactic acid bacteria strains free of
antibiotic resistance that have a transferrable capacity are necessary as components of a specific starter. To select for
these bacteria, the minimurn inhibitory concentsation (MIC) of 12 antibiotics and 2 mixtures (containing $-lactamase
inhibitor and penicillin) were determined by microbroth and agar dilution techniques in 146 strains belonging to the
genera Lacrococcus, Enterococcrcs, Luctobncillus, and Leuconostoc. The antibiotic-resistance protiles of I~c:tococcirs
and E~rterococcusspecies were different from those of Loctobacillris and Leuconostoc, but clear genus- or species-
associated patterns were not observed. Cefoxitin and metronidazole were not effective against bacteria of these genera.
The MICs of $-lactam antibiotics for lactobacilli and leuconostoc isolates were higher than those for lactococci and
enterococci. but no strain was clinically resistant. All lactobacilli and leuconostoc isolates were resistant to high levels
of vancomycin, i type of resistance not seen among the tested members of the genera Iacfococcus and Enterococcrrs.
The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of
Lacrococcus lactis, Enterococcus spp., and Lactobncillus spp. were resistant to antibiotics, such as chloraniphenicol,
erythromycin, clindamycin, or tetracycline.
Key words: Lnctococcris, Lnctohacillirs, Leuconostoc, lactic acid bacteria, antibiotic resistance. antimicrobials.
RCsumf : Afin de ~Clectionnerdes souches de bactEries lactiques depourvues de resistance anx antibiotiques ayant une
capacitt de transfert en tant que de composantes d'un ferment spCcifique de fromage traditionnel espagnol Carbales
bleu veinC, la concentration minimale inhibitrice (CMI) de 12 antibiotiques et de 2 mClanges (un inhibiteur de la p-lac-
talnase et la pCnicilline) a CtC dCterminCe par microdilution en milieu liquide et sur agar chez 146 souches appartenant
aux genres Luctococcus, Enterococcus, Lncrobacilltrs et Leuconostoc. Les profils de resistance aux antibiotiques des es-
ptces de Lnctococcus et d'Enterococcrrs diffkraient de ceux de Lnctobncillrrs et de Leuco~lostoc,mais aucun profil as-
socii: au genre ou 2 I'espbce n'a CtC observe. La cefoxitine et le metronidazole furent inefficaces contre les bactiries de
ces genres. Les CMI des antibiotiques p-lactam chez les isolats de lactobacilles et de leuconostocs Ctaient supCrieures j.
celles des lactocoques et des endrocoqucs, mais aucune souche n'Ctait cliniquenlent resistante. Tous les isolats de lac-
tobacilles et de leuconostocs Ctaient rksistants B des niveaux ClevCs de vancomycine, un type de resistance qui n'a pas
CtC rencontri chez les membres analys6s des genres Lactococcus et Enterococctts. La majorit6 des resistances obsewtes
semblaient Ctre soit int~instqueou non-sptcifique, bien que certaines souches de Lactococcus lnctis, Enrerococcus spp.
et Lactohucillus spp. Ctaient resistantes a des antibiotiques cotlime le chloramphCnicol, I'Crythromycine, la clindarnycine
ou la tttracycline.
Mots cl6.s : ktctococcirs, Luctohrtcil1u.s. Lerrconosroc, batteries lactiques, resistance aux antibiotiques, antimicrobiens.
[Traciuit par la RCdaction]

Introduction ferred to pathogens, either in the food matrix or, more


importantly, in the gastrointestinal tract. T h e production of
Antimicrobial agents are of enormous value for combating fermented dairy products from raw milk in antibiotic-
infectious diseases. but their efficacy has been threatened by challenged environments may select antibiotic-resistant LAB
microbial resistance. Currently, there is concern over the harbouring transmissible resistance genes (Perreten et al.
possible spread of resistance determinants (from the food 1997). In fact, horizontal gene transfer is essential for bacte-
chain) to antimicrobials (Teuber e t al. 1999). Lactic acid ria t o survive and adapt to new environments (Kurland et al.
bacteria (LAB) from fermented products may act as a reser- 2003). Strains intended for the use in rood systems as start-
voir of antimicrobial-resistance genes that could b e trans- ers or vrobiotics should therefore b e carefully examined for
antimicrobial susceptibility, especially those isolated during

I
the s o called "post-antibiotic era" (Teuber et al. 1999).
Received 25 June 2004. Revision received 13 October 3,004.
Accepted 16 October 2004. Published on the NRC Research However, there is still a lack of agreement on the resistance-
Press Web site at http:I/cjm.nrc.ca on 18 March 2005. susceptibility breakpoints for most antimicrobials in LAB
(Felten et al. 1999; Charteris et al. 2001; Katla et al. 2001;

I1 A.R. Fibrez, S. Delgado, and R. Mayo.' Instituto de


Productos LBcteos de Asturias (CSIC). Carretera de
Infiesto sln, 33300-Villaviciosa, Astu~ias,Spain.
'Corresponding author (e-mail: baltasar.n~ayo@ipla.csic.es).
Danielsen and Wind 2003). Distinguishing between intrinsic,
nonspecific, and acquired resistance is difficult and requires
that the antimicrobial-rcsistance patterns of many LAB spe-
cies from different sources b e compared (Teuber et al.
Can. J. Microbial. 51: 51-58 (2005) doi: 10. W04-I 14 O 2005 NRC Canada

31
a Can. J. Microbial. Vol. 51. 2005

1999). This is a very inlportant task since genes conferring termined using the Sensititre Anaero3 kit (Trek Diagnostic
resistance to several antimicrobials (i.e., chloramphenicol, Systems, East Grinstead, England) following the manufac-
erythromycin, streptomycin, tetracycline, and vancomycin) turer's instructions. Briefly, colonies from solid media were
located on transferable genetic elements (plasmids or used to make an even suspension (equivalent to the 0.5
transposons) have already been charactcrizcd in lactococci McFarland turbidity standard) in Brucclla broth (Oxoid Ltd.,
(Petl-eteo et al. 1997), lactobacilli (Axelsson et al. 1988; Basingsoke, Hamphire, England) and 100 ILL of this was
Danielsen 2002; Gevers el al. 2003; Gfeller et al. 2003), and transferred to the same medium containing 0.2 glL of
enterococci (Eaton and Gasson 2001; Ozawa et al. 2002; haemin (Sigma Chemical Co., St. Louis, Missouri) and
Schwartz et al. 2002; Huys et al. 2004) from foods. Further- 10 pg/L of vitamin Kt (Merck). The final bacterial concen-
more, under certain circumstances, LAB strains themselves tration was 1 x lo6 CFU/mL. One-hundred-microlitre
have been reported to cause infections in humans (Gasser aliquots of this suspension were inoculated into each well of
1994; Husni el al. 1997). the Sensititre Anaero3 plate. The antibiotics utilized and
This work reports the susceptibility patterns of a number their range of dilutions arc indicated (Table I). This system
of LAB species (belonging to the genera Luctococcri.s, does not allow a sufficiently wide range of concentrations to
Enterococcus, Lucrobocillus, and Leuconostoc) isolated from properly test some antibiotics; the upper MIC limits in these
different batches of blue-veined Cabrales cheese, a tradi- cases were, therefore, assessed using the standardized agar
tional protected designation of origin (PDO) Spanish cheese dilution technique of the National Committee for Clinical
made from raw milk without the addition of starter cultures. Laboratory Standards (NCCLS) in Mueller-Hinton agar
This work was performed to select strains that do not con- (Merck) platcs containing 1% glucose (NCCLS 2000). The
tain antibiotic transferable resistances among those with de- antibiotics analyzed included inhibitors of cell-wall synthesis
sirable technological characteristics. In addition, the analysis (the P-lactams penicillin G, amoxycillin. amoxycillin plus
could also indicate the types and degrees of antimicrobial re- clawlanic acid, piperacillin, piperacillin plus tazobactam, and
sistance already present among the LAB community of this hnipcneln; the cephalosporin ccfoxitin; and the glycopeptide
cheese environment. vancomycin), protein synthesis (chloramphenicol, clindamycin,
erythromycin, and tetracycline), and nucleic-acid synthesis
(the fluoroquinolone moxifloxacin; and rnetronidazole).
Materials and methods
Bacterial strains, media, and culture conditions
The LAB strains studied were isolated during the manu-
Results and discussion
facturing and ripcning of 4 batches of traditional bluc-veined In this study, the MIC of 12 antibiotics and 2 antibiotic
Cabrales cheese (farmhouse made from raw milk without mixtures for 146 LAB strains isolated from Cabrales cheese
starters at 2 different farmhouses (F16rez et al. 2005)). The was analyzed (Tables 2 and 3). In duplicate experiments, us-
isolates belonged to the dominant bacterial populations and
were isolated on agar plates of either media M I 7 (Scharlau,
-
ing inde~endentinocula. the differences in MIC results
never exceeded I order of dilution. For the sake of clarity,
Scharlau Chemie SA, Barcelona, Spain) (Lactococcus lactis E. fuecium and E. durnns strains and L. mesenteroi~le.s
(71), Enterococcrrs rlurui7.s (lo), E~zterococcusfueciunz (3)), and L. pseudonlesenteroides strains were included as single
Man, Rogosa, and Sharpe (MRS) (Merck. VWR Interna- groups (despite the different species showing small differ-
tional. Da~mstadt, Gennany) (I~ctol~acillus plantar~~ird cnccs with rcspcct to thc MICs of some antibiotics). The
LuctoOacillus paraplantarrcnz (23), Lnctobacillus cusei/ phenotypic and molecular classification utilized, failed to
Lactobacillus paracasei (12)), or Mayeux, Sandine, and distinguish any more than the 2 remaining groups in Ta-
Elliker (MSE) (Scharlau) (Le~tcorzostocrne.serzteroides (1 9), bles 2 and 3 (L. plaiztarum/l. pc~raplairtar~~mand L. casein.
lkuco~zostoc p.sendon~e.seizteroide.r (2), and Leuconostoc paracasei).
citreuin (6)). They were first grouped by phenotypic criteria The Lactococcus and Etlterococcus spp. showed antibiotic-
and then the groups wcre classified by polymerase chain re- resistance profiles different from those of Lactobacillus and
action (PCR) amplification, sequencing, and analysis of the Lerrconostoc. Lactococcal and enterococcal species could
VI and V2 regions of their 16s rRNA gene. Approximately not be distinguished on the basis of their MIC patlerns. Nev-
10% of the isolates probed were found to be replicates by ertheless, enterococci isolates (mainly E. faeciuln) showed
the random amplification of polymorphic DNA (RAPD) more resistance than did the lactococci, but in both cases, no
technique (data not shown), w l ~ c l iagrecd well with a high genus- or species-specific antibiotic-resistance pattern was
phenotypic and genetic variability reported for LAB from observed.
Spanish raw milk cheeses (Sanchez et al. 2000; Delgado and As cxpectcd, all analyzed strains were resistant to
Mayo 2004). Lactococcus lactis and Brterococclls spp. metronidazole (MIC 2 32 pg/mL) since LAB have no
strains were grown in M17 at 32 "C for 18-24 h, and hydrogenase activity (Church et al. 1996). However, all
Lactobucillus and Lcuconostoc spp. were grown in MRS at strains were susceptible to the lowest concentration of
30 "C in a 5% C02-enriched chamber (Precision, Pacisa y piperacillin and piperacillin plus tazobactam tested (all
Giralt SL, Madrid, Spain) for 24-48 h. MICs 5 16 pg11nL). The MICs of the remaining antibiotics
showed a certain degree of variability. For all antibiotics,
Determination of the minimum inhibitory concentration strains with clinical resistance were found (Lennette et al.
For these LAB isolates, the MICs of 12 antibiotics and 2 1985). except for amoxycillin and moxifloxacin, for which,
mixtures of a P-lactamase inhibitor and a penicillin were de- breakpoints remain to be defined. MICs for the amoxycillin -

O 2005 NRC Canada

32
Florez et al.

Table 1. Antimicrobials includcd in the Sensititre Anacro3 and their range of dilutions used to
determine the minimum inhibitory concentrations (MICs) of lactic acid bacteria (LAC).
Antibiotic Typc of antibiotic Range of dilutions WglmL)
Inhibitors of cell-wall synthesis
Penicillin G Penicillin 0.06-8
Amoxicillin Penicillin 0.25-32
A~noxicillin- clavulanic acid* Penicillin - p-lactamase inhibitor 0.25-16
Piperacillin Penicillin 16-128
Piperacillin-tazobactani Penicillin - P-lactamase inhibitor 1614-12814'
Imipenem Carbapenems
Cefoxitin Ccphalosporin
Vancomycin Glycopeptide
Inhibitors of prntein synthesis
Chlora~nphenicol 2-16
Tetracycline 2-16
Erythromycin Macrolide 1-128
Clindamycin Lincosamide 0.5-64
Inhibitors of DNA synthesis
Moxifloxacin Fluoroquinolone 0.12-8
Metronidazol 0.5-32
"The source of the amoxicillin - cla\'ulanic acid mixture was aupmentine, which is 500 mg of amoxicillin
and 125 mg of clavulanic acid per gram.
'The concentration of tazobacram in the piperacillin-tazohacram mixture was 4 pg11nLin all dilutions.

clavulanic acid mixture were always lower than those of antibiotics of this group (chloramphenicol, erythro~nycin,
amoxycillin alone, but followed the same pattern. Thus, clindamycin, and tetracycline), most strains were clearly
again for the sake of clarity, only MICs for the lattcr were susceptible, although a few moderate to strongly resistant
inclodcd in the tablcs. strains were seen (Table 3). Some strains proved resistant to
MICs for the different LAB groups for cell-wall-synthesis more than I of these antibiotics. For instance, 2 E. ,fizeciurn
inhibi~orsare summarized in Table 2. The highest MTCs for isolates were resistant to chloramphenicol (MIC =
penicillin were shown by strains of the L. plantarum/l,. 32 pg/mL), erythromycin (MIC 2 128 pg/mL), clindamycin
paraplantarurn group (116 pg/mL in 4 strains). The highest (MIC 2 128 pglmL), tetracycline (MIC = 64 and
for amoxycillin and imipenem were shown by Lcrrcorzostoc 128 pg/mL), and vancomycin (MIC 2 16 pg/mL). One
spp. (18 pg/mL for both antibiotics). The MICs for all cell- E. dirr-ans isolate showed resistance to erythromycin (MIC
wall-synthesis inhibiting antibiotics were not very high. 32 pg/mL) and clindamycin (MTC 64 pg/mL). In con-
Similar MTC values for thcse LAB species havc been re- trast, L. 1acii.s strains resistant to more than I antibiotic were
ported elsewherc (Kim et al. 1982; Chartcris ct al. 1998). never found. However, 3 L. 1acti.r isolates were found to be
The resistance of LAB species to high levels of cefoxitin resistant to high lcvcls of tetracycline (MIC 2 256 pg11nL);
(most MTCs 2 30 pg/mL) have been repeatedly observed the 2 presumed resistant strains (Table 3; see footnote) were
(Croco et al. 1994; Charteris et al. 1998; Goldstein et al. confirmed by an E-test assay (not shown).
2001). Cell-wall imperlneability seems to be the main mech- Seven L. plantarrrnl isolates were also resistant to this antibi-
anism of rcsistancc to inhibitors of cell-wall synthesis (peni- otic (MTC 2 256 yg/mL), 4 of which were moderately resis-
cillins and cephalosporins), since LAB species lack tant to chloramphenicol (MTC = 32 pg/mL). As mentioned
cytochrome-mediated electron transport (Condon 1983). above, potentially transferable genes conferring resistance to
However, the cooperation of nonspecific mechanisms, such I or Inore of these 'antibiotics have been characterized in
as multi-drug transporters (Putman et al. 2001) and defective several LAB species (Axelsson et al. 1988; Perreten et al.
cell wall autolytic systems (Kim et al. 1982), may also ac- 1997; Eaton and Gasson 2001; Danielsen 2002; Ozawa et al.
count for the differences between strains. 2002; Schwartz et al. 2002; Gevers et al. 2003; Gfeller et al.
All Lnctobacillus and Leuconostoc spp. were resistant to 2003: Huys el al. 2004).
high concentrations of vancomycin (MICs 2 256 mg/mL), The MTCs for different antibiotics seen1 to be strain-
whcreas all Luctococcus and Elzterococcus isolatcs wcre specific. However, differences may also be a result of the
very susceptible (MTCs of 5 2 mg/mL), except for 1 L. lactis different methods being used, such as the E-test (Croco et al.
strain and 2 enterococci strains (MIC 2 8 pg/mL). The resis- 1994; Herra et al. 1995; Felten et al. 1999; Charteris et al.
tance of Lartobncillus and Leuconostoc spp. lo vancomycin 2001; Katla et al. 2001; Danielsen and Wind 2003), agar di-
may be due to the presence of D-Ala-D-Lac as the normal di- lution (Mayo et al. 1990; Herrero et al. 1996; Goldstein et
peptide in their peptidoglycan (Klein et al. 2000). al. 2000), disk diffusion (Sozzi and Smiley 1980; Chartcris
With the exception of vancomycin, the MTCs of antibiot- et al. 1998), and microbroth methods (Klein et al. 2000).
ics affecting the synthesis of proteins showed the greatest The location of a resistance gene at different sites (in the
variation between species and strains (Table 3). For all other chromosome or in a plasmid) (Gevers et al. 2003) or the par-

O 2005 NRC Canada

33
Can. J. Microbiol. Vol. 51, 2005

O 2005 NRC Canada

34
Table 3. Distribution of MICs to several antimicrobials inhibiting protein synthesis and to the fluoroquinolone rnoxifloxacin inhibiting the synthesis of DNA for LAB species
from Cabrales cheese.
No. of isolates with the following MfCs @g/mL)
No. of
Antibiotic Species strains a.12 0.25 0.5 1 2 4 8 16 32 64 128 >I28 256 >256
Chlorarnphenicol Lnctococcus lactis 71 17 21 31 2
E~ztemcoccrrsduranslEntemcoccu.s faeci~cm 13 8 2 1 2
Luctob~rcillusplcrntur~u~d 23 4 3 9 3 4
Lactobacillus paraplanrar?rtn
Lactobacillus crrsei/lactobacillus paramsei 12 5 7
Lc~lconostoccifrcum 6 3 2 1
Leuconostoc t~resenteroides/ 21 2 4 15
Leltconostoc pseudomesentemides
Tetracycline L. lactis 71
E. durans/E. fmcium 13
L. planrar~rndL.paraplanfar.um 23
L. casei/L. parucasci 12
L. citr-eum 6

35
L. mesenreroidcs/L. pscrrdomesteroidcs 21
Erythromycin L. luctis 71
E. duruns/E. faeciunr 13

L. rilesenteruides/L. pseudonresmteroides 21
Clindamycin L. lucris 71
E. durons/E. .fuecium 13

L. mescnteroidcs/L. p,scudomesctreroidcs 21 20 1
Moxifloxacin L. lrrctis 71 13 24 23' 9 1 1
E. drrrairs/E, fiteciunr 13 5 5 1 2
L plar~rarr~m/L..purclplr~rztar~tm 23 2 3 8 4 1 2 1 2
L, casei/L. parclcusei 12 3 6 3
0 L. citreum 6 3 2 1
g
Cn
L. tilcsenteroirles/L. pseudornesenteroides 21 14 3 3 I
*These strains were nor examined for higher concentrations.
3n
56 Can. J. Microbial. Vol. 51. 2005

Table 4. Microbiological breakpoints for LAB species from Cabriles cheese.


Proposed breakpoint. MTC (pg/mL)
Antibiotic Species This work NCCLS* SCAN'
Penicillin G All I 24 2-8
Lnctococccr.~lacris/Er~~erococcus spp. 2
Lactobacil1u.s plantarurn/lnctobocillcr.s pnraplarttarzrm 32 R
Anioxicillin All - 21 6
Enterococcus spp. 0.5
L. luctis/~~ctohncilhrs spp. 2

Lerrconosfoc rrresenferoides/Leuco,,ostoc pse~rdomesenreroicies 16


Piperacillin All <I6 21 28
Imipenem All 2 >I6
L. citreurn 4
L. mesenteroides/ L. pseudonzesenteroides
Cefoxitin All
Erlterococcus spp.
Leucortostoc spp.
Vancomycin All
Lactococczrs lactis
Enierncoccus spp.
Lactobacillus spp.lLeuconostoc spp.
Chlorarnphenicol All
L. lactis/Entcrococc14s spp.
L. plan~urzmdL.purcpluntur~tm
Lacrohacill~tscmsei/Lactohacillusparacasei
Lr~lcomostocspp.
Tetracycline All
Etlterucoccus sppfL. cclsei/ L. purucusei
Leucortostoc spp.
Erythromycin All
Clindaniycin All
L. kuctis
L. plarztarzmtL. paruplarzrarrrm
Moxifloxacin L. lucris/Enrerococcus spp.
L. plantaruna. paruplanfarun~
L. cusei/l. purucusei I
Le~co~~osroc spp. 1
Metronidazole All >32 232
Note: --. antibiotics for which a general breakpoint for LAB is not proposed.
*NCCLS, National Committee for Clinical Laboratory Standards, the clinical resistant breakpoints are indicated.
'SCAN, Scientific Committee of Animal Nutrition. R indicates that certain species are inherently resistant.

ticipation of other unspecific mechanisms, such as multi- sistant (Olsson-Liljequist et al. 1997). Microbiological
drug transporters (Putman et al. 2001), may also account for breakpoints are thought to be more relevant than clinical
strain-specific differences. breakpoints for the purpose of identifying bacterial strains
The determination of well-defined breakpoints for LAB with acquired and potentially transferable antibiotic
species could be of clinical and biological importance and resistances. In this papcr, we have defined the rnicrobiologi-
may even be helpful in strain identification (Elliot and cal breakpoints as the MICs immediately above the apparent
Facklam 1996; Herrero et al. 1996: Husni ct al. 1997). Ef- normal range for a given antibiotic and a given species. Fol-
forts should, therefore, be made to examine a large number lowing this definition, the microbiological breakpoints for
of strains from different origins using the same methodology antibiotics and species studied in this work arc presented
to clearly define such breakpoints for LAB. (Table 4). Reference values for some antibiotics were
Besides the traditional clinical breakpoint, which may included for comparison, such as the clinical breakpoints re-
help clinicians in the choice of antibiotics, the term microbi- ported by the NCCLS (NCCLS 2000) and the microbiologi-
ological breakpoint has recently been defined. Microbiologi- cal breakpoints proposed by the Scientific Committee on
cal breakpoints are set by studying the distribution of MICs Animal Nutrition (SCAN) (European Commission 2001).
in bacterial populations and the part of the population that In conclusion, multi-resistant LAB species were not com-
clearly deviates from a susceptible majority is considered re- mon in this Cabrales cheese environment; only a few

O 2005 NRC Canada

36
Florez et al.

Enterococcus isolates showed this characteristic. Species of Condon. S. 1983. Aerobic metabolis~nof lactic acid bacteria. Tr. J.
this genus are usually present in high numbers in cheeses Food Sci. Technol. 7: 15-25.
made of raw milk and other fermented foods (Giraffa et al. Croco, J.L.. Erwin, M.E., Jennings, J.M., Putnam, L.R.: and Jones.
1997). Their metabolic activities (acidification, proteolytic R.N. 1994. Evaluation of the E-test for anti~nicrobialspectrum
activity, and production of anti-Listerin bactcriocins) could and potency determinations of anaerobes associated with bacte-
have desirable technological roles and a number of strains rial vaginosis and peritonitis. Diagn. Microbiol Infect. Dis. 20:
have been assayed as starter or adjunct cultures (Giraffa et 213-219.
al. 1997; Franz et al. 1999). However, the presence of these Danielsen, M. 2002. Characterization of the tetracycline resistance
species in food systems is a matter of controversy due to plasmid pMD5057 from Lncfobacillus plantarum 5057 reveals a
their potential pathogenicity (toxigenicity, production of composite structure. Plasmid, 48: 98-103.
biogenic amines, presence of adhesins and other cell aggre- Danielsen, M., and Wind, A. 2003. Susceptibility of Lacrobacillrrs
gation proteins, and ailtimicrobial resistance) (Eaton and spp, to antimicrobial agents. Int. J. Food Microbiol. 82: 1-1 1.
Gasson 2001; Franz et al. 3,001). Antimicrobial rcsistaiices Delgado, S.. and Mayo, B. 2004. Molecular classification and
alone cannot be considered virulence factors, but they can phenotypic and genetic diversity of Lnctococcus luctis and
Enterococcus spp. strains isolated from Northern Spain starter-
complicate the treatment of opportunistic infections. The
free farmhouse cheeses. Int. J. Food Microbiol. 90: 309-319.
presence of effective gene transfer mechanisms in members
Eaton, T.J., and Gasson, M.J. 2001. Molecular screening of
of this genus (such as conjugation and conjugative transposi-
Enterococcus virulence determinants and potential for ~cnetic
tion) also has to be taken into consideration (Eaton and
exchange between food and medical isolates. Appl. Environ.
Gasson 2001; Franz et al. 2001). Characterization of the ob-
Microbiol. 67: 1628-1635.
served high vancornycin resistance in E. fneciunl isolates is
Elliot. J.A.. and Facklam, R.R. 1996. Antimicrobial susceptibilities
currently underway.
of Lactococcus lactis and Lactococcus garviae and a proposed
Only a ininority of the normal starter LAB strains showecl method to discriminate between them. J. Clin. Microbiol. 34:
antibiotic resistance. This small fraction, however, justifies 1296- 1298.
performing antibiotic-susceptibility assays to avoid includ- European Counmission. 3001. Opinion of the Scientific Committee
ing antibiotic-resistant strains in starter cultures. Indeed, sev- on Animal Nutrition on the criteria for assessing the safety of
eral of the types and levels of resistance found are micro-organisms resistant to antibiotics of human clinical and
compatible with transmissible genes. Compared with the re- veterinary importance. Available from http://www.eoropa.eu.int/
sults of surveys of strains from the pre-antibiotic cra, in conumlfoodlfs/sc/scan/out64~en.pdf[accessed on I8 April
which no resistance was found at all (Cogan 1972: Orberg 20011.
and Sandine 1985: Katla et al. 2001), the present findings Felten, A,, Barreau. C.. Bizet, C., Lagrange, P.H., and Philippon.
suggest that the antibiotic pressure on LAB from the wide A. 1999. Luctol~crcillusspecies identification. H20z production.
use of antibiotics, in veterinary medicine and agriculture for and antibiotic resistance and correlation with human clinical sta-
example. could be contributing to the dissemination of tus. J. Clin. Microbiol. 37: 729-733.
resistances into cheese-related ecological niches. Fldrez, A.R., Zbpez-Diaz, T.M.. and Mayo, B. 2005. New bio-
chemical and microbiological characterisations of the traditional
Spanish blue-veined Cahrales cheese: identification of its tech-
Acknowledgements
nologically-relevant microbiota. Int. J. Food Microbiol. In press.
This work was supported in part by a Strategic Action Franz, C.M.A.P.. Holzappel, W.H., and Stiles, M.E. 1999.
("ConservaciBn de 10s Recursos Geneticos de Inter& Enterococci at the crossroad of food safety. Int. J. Food
Agroalimentario") of the "Prograrna Nacional de Recirsos y Microbiol. 47: 1-24.
Tecnologias Agroalimentarias", INIA (reference RF02-019), Franz, C.M.A.P., Muscholl-Silberhorn, A.B., Yousif, N.M.K.,
and by an EU pro,ject (ACE-ART, reference 506214). The Vancanneyt, M., Swings, J., and Holzappel, W.H. 2001. Inci-
skillrul Lech~icalassistance of M.J. Gonziilez is fully ac- dence of virulence factors and antibiotic resistance among
knowledged. enterococci isolated from food. Appl. Environ. Microbiol. 67:
43854389.
Gasser, F. 1994. Safety of lactic acid bacteria and their occurrence
References in human clinical infections. Bull. Inst. Pasteur, 92: 45-67.
Axelsson, L.T., AhrnB, S.E.. Andersson, M.C., and Stahl, S.R. Gevers, D., Danielsen, M., Huys, G., and Swings, J. 2003. Molecu-
1988. Identification and cloning of a plasmid-encoded lar characterization of rsr(M) genes in Lacrobacillus isolates
erythromycin resistance determinant from Lactobacillus reuferi. from different types of fermented dry sausages. Appl. Environ.
Plasmid, 20: 171-174. Microbiol. 69: 1270-1275.
Charteris. W.P.. Kelly, P.M., Morelli, L., and Collins, J.K. 1998. Gfeller, K.Y., Roth, M., Meile. L., and Teuber, M. 2003. Sequence
Antibiotic susceptibility of potentially probiotic Lacfobucillra and genetic organization of the 19.3-kh erythromycin- and
species. J. Food Prot. 61: 1636-1643. dalfopristin-resistance plasmid pLME300 from Lnctobacillrrs
Charteris, \V.P.., Kelly, P.M., Morelli, L., and Collins, J.K. 2001. fermenturn ROTI. Plasmid, 50: 190-20 1.
Gradient diffusion antibiotic susceptibility testing of potentially Giraffa, G., Carminati, D., and Neviani, E. 1997. Enterococci iso-
probiotic lactobacilli. J. Food Prot. 64: 2007-2014. lated from dairy products: a review of risks and potential tech-
Church, D.L., Bryant, R.D., Sim, V., and Laishley, E.J. 1996. nological use. J. Food Prot. 60: 732-738.
Metronidazole susceptibility and the presence of hydrogenase in Goldstein, E.J.. Citron, D.M.. Merriam, C.V., Warren, Y.A., Tyrrell.
pathogenic bacteria. Anaerobe, 2: 147-153. K., and Fernandez, H. 2001. Colnpdrative in vitro activity of
Cogan, T.M. 1972. Susceptibility of cheese and yoghurt starter ertapeneln and 1 I other anti~nicrobialagents against aerobic and
bacteria to antibiotics. Appl. Environ. Microbiol. 23: 960-965. anaerobic pathogens isolated from skin and soft tissue animal

O 2005 NRC Canada

37
Can. J. Microbiol. Vol. 51, 2005

and hunian bite wound infections. J. Antimicrob. Chemother. Mayo. R.,Hardisson. C., and Braca, A.F. 1990. Characterization of
48: 641-651. wild strains of Lactococcrrs lactis isolated from Cabrales cheese.
Herra. C.M., Cafierkey, M.T.. and Keane. C.T 1995. The in-vitro J. Dairy Res. 57: 125-134.
susceptibilities of vaginal lactobacilli to four broad- spectrum NCCLS. 2000. Methods for dilution antimicrobial susceptibility
antibiotics, as determined hy thc agar dilution and E test meth- testing on bacteria that grow aerobically. In Approved Standard
ods. J. Antimicrob. Chemother. 35: 775-783. M7-A5., 5th ed. National Committee for Clinical Laboratory
Herrero, M.. Mayo. B., Gonzilez. B., and Sukeii, J.E. 1996. Eval- Standards, Wayne. Pa.
uation of technologically important traits in lactic acid bacteria Olsson-Liljeqoist, B., Larsson, P., Walder. M., and Miorner, N.
isolated from spontaneous fermentations. J. Appl. Bacteriol. 81: 1997. Antimicrobial susceptibility testing in Swedcn. TIT. Meth-
565-570. odology ior susceptibility testing in Sweden. Scand. J. Infect.
Husni. R.N.. Gordon. S.M., Washington, J.A.. and Longworth. Dis. 105s: 13-23.
D.L. 1997. Lnctobacillr~sbacterie~niaand endocarditis: review Orberg, P.K., and Swdine, W.E. 1985. Survey of antimicrobial re-
of 45 cases. Clin. Infect. Dis. 25: 1048-1055. sistance in lactic strcptococci. Appl. Environ. Microbiol. 49:
Huys. G., D'Haene, K., Collard, J.-C.. and Swings, J. 2004. Preva- 538-542.
lence and lnolecular characterization of tetracycline resistance in Ozawa, Y., Tanimo~o.K., Nornura, T., Yoshinaga, M.. Arakawa. Y.,
Enteracoccus isolates from food. Appl. Environ. Microbiol. 70: and Ike, Y. 2002. Vancomycin-resistant enterococci in humans
1555-1562. and i~ilportedchickens in Japan. Appl. Environ. Microbiol. 68:
Katla. A.K., Kruse, H., Johnsen, G., and Herikstad. H. 2001. 6457-646 1.
Antimicrobial susceptibility of starter culture bacteria used in Perreten. V., Schwarz, F., Cresta, L., Boeglin. M., Dasen, G., and
Norwegian dairy products. Int. J. Food Microbiol. 67: 147-152. Teuber. M. 1997. Antibiotic resistance spread in food. Nature
Kim. K.S., Morrison, J.O., and Bayer, A.S. 1982. Deficient (London), 389: 801-802.
autholytic enzyme activity in antibiotic-tolerant lactobacilli. In- Putman, M.. van Veen, H.W., Degener, J.E.. and Konings, W.N.
fect. Immun. 36: 582-585. 2001. The lactococcal secondary rnultidrug transporter LmrP
Klein, G., Hallmann, C.. Casas, L.A., Abad, J.. Louwers, J., and confers resistance to lincosarnides, unacrolides, streptogramins
Rcuter, G. 2000. Exclusion of v a d . vrmR, and vanC type and tetracyclines. Microbiology, 147: 2873-2880.
glycopeptide resistance in strains of Lnctobacill~rsreuteri and Shchez, M.M., Delgado, T.. Alonso, L., and Mayo, B. 2000.
Lactobacilllrs rhnrnnosus used as probiotics by polymerase Phenotypic and genetic characterization of a selected set of
chain reaction and hybridizatio~imethods. J. Appl. M~crobiol. Lncrococcus lactis strains isolated from a starter-free farmhouse
89: 8 15-824. cheese. Food Microbiol. 17: 449-460.
Kurland, C.G., Canback, B., and Berg, O.G. 2003. Horizontal gene Sozzi, T., and Smiley, M.B. 1980. Antibiotic resistances of yogurt
transfer: a critical review. Proc. Natl. Acad. Sci. USA. 100: starter culttires Streptococcus thcrnroj?hilrrs and Lactobacillus
9658-9662. Dulgaricus. Appl. Environ. Microbiol. 40: 862-865.
Lcnnette, E.H., Balows, A.L., Hausler, W.J., Jr.. and Shadomy, H.J. Tcuber, M., Meile, L., and Schwarz, F. 1999. Acquired antibiotic
1985. Manual of clinical microbiology, 4th ed. American Soci- resistance in lactic acid bacteria from food. Antonie
ety for Microbiology. Washington, DC. Leeuwenhoek, 76: 1 15-137.

0 2005 NRC Canada

38
CURRENT MICROBIOLOGY Vol. 50 (2005), pp. 202207
DOI: 10.1007/s00284-004-4431-3 Current
Microbiology
An International Journal
Springer Science+Business Media, Inc. 2005

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species


from the Human Gastrointestinal Tract
Susana Delgado, Ana Beln Flrez, Baltasar Mayo
Instituto de Productos L$cteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300 Villaviciosa, Asturias, Spain

Received: 30 July 2004 / Accepted: 19 October 2004

Abstract. One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been
tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3;
Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were
completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol
and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were
susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the
Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to
four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas
a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium
longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and
Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resis-
tances seemed to be intrinsic, but some others could be compatible with transmissible determinants.

Lactic acid bacteria (LAB) species, including lactoba- In recent years several studies concerning antibiotic
cilli and bifidobacteria, are indigenous members of the susceptibility of intestinal LAB species from humans
gastrointestinal microbiota of humans and enjoy a time- have been undertaken [3, 4, 13, 15, 16, 20]. However,
honored reputation as health promoters. In fact, these due to the multiplicity of methods used and the unre-
bacteria have a generally regarded as safe (GRAS) latedness of the strains, there is still a lack of agreement
status and are frequently uses as probiotics [12]. Bac- in the resistance-susceptibility breakpoints for most
terial strains intended to be used as probiotics in food antibiotics in LAB [4, 7].
systems have to be systematically examined for antibi- In this paper we report on the level of susceptibility
otic susceptibility in order to avoid the spread of anti- to several antimicrobial agents of some Lactobacillus
biotic-resistant determinants by the food chain [19]. The and Bifidobacterium species isolated from the human
presence of several resistance genes in many LAB GIT of healthy individuals, which could eventually be
strains from the human gastrointestinal tract (GIT) has used as probiotics. Antibiotic resistance was used as a
been firmly stated [2, 14, 18]. Some determinants have negative criterion in the selection process of the strains.
been found to be transferred even into the GIT of At the same time, the survey was regarded as an over-
mammals [17]. Furthermore, it should also be consid- view of the level of antibiotic resistance in LAB from
ered that, although rarely, LAB species, including pro- the human GIT.
biotic strains, have been reported to cause infections in
humans [9], thus antibiotic resistance hampering treat-
ment of the diseases. Materials and Methods
Bacterial strains, media, and culture conditions. One hundred and
twenty-two strains of Bifidobacterium and Lactobacillus species,
isolated from the feces of eight individuals, were screened for
Correspondence to: B. Mayo; email: baltasar.mayo@ipla.csic.es antibiotic susceptibility in this study. They have been isolated as part

39
S. Delgado et al.: Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species 203

of the dominant LAB populations on MRS agar (VWR International, Sweden) and found to vary between 0.032 and 0.50 lg/
Darmstadt, Germany) containing 0.25% cysteine (Sigma Chemical, St. mL (data not shown).
Louis, MO). The strains were grouped by their carbohydrate
fermentation profiles using a commercial kit (PhenePlate system,
All bifidobacteria and lactobacilli strains assayed
PhP, Stockholm, Sweden) and classified by amplification, sequencing, were susceptible to penicillin and amoxicillin (MICs
and comparison of a stretch of their 16S rDNA gene, using two lower than 4 lg/mL), except for those of L. plantarum
universal primers based on prokaryotic conserved regions of the 16S and L. brevis strains, and in clinical terms none of the
rRNA gene, as previously described [21]. Around 10% of the isolates strains should be considered resistant (clinical resistant
proved to be replicates by RAPD (data not shown). The strains were
assigned to the following species: 47 Bifidobacterium longum, 16
breakpoint by the NCCLS 4 lg/mL). Cefoxitin was
Bifidobacterium bifidum, 11 Bifidobacterium pseudocatenulatum, 2 the only b-lactam for which a high number of resistant
Bifidobacterium catenulatum, 20 Lactobacillus gasseri, 8 strains were found. All strains of L. casei/L. paracasei
Lactobacillus delbrueckii, 7 Lactobacillus casei/Lactobacillus (7), L. rhamnosus (5), L. acidophilus (2), L. plantarum
paracasei, 5 Lactobacillus rhamnosus, 2 Lactobacillus acidophilus, (1), L. brevis (1), L. vaginalis (1), and a few L. gasseri
and single strains of Lactobacillus plantarum, Lactobacillus
parabuchneri, Lactobacillus brevis, and Lactobacillus vaginalis.
strains (4 out of 20). The resistance of the lactobacilli to
Incubations were performed at 37C in an anaerobic chamber this antibiotic has been repeatedly reported [3, 7, 15]. On
(Mac500, Down Whitley Scientific, West Yorkshire, UK) containing the contrary, only two B. longum isolates showed MICs
an anoxic atmosphere (10% H2, 10% CO2, 80% N2). of 32 and 64 g/mL, respectively. This result differs from
Determination of the Minimum Inhibitory Concentration those of Charteris et al. [4], who found most of the
(MIC). MICs to 12 antibiotics and two antibiotic mixtures were bifidobacteria isolates resistant by an overlay disc dif-
tested with the Sensititre Anaero3 kit (Trek Diagnostic Systems, East fusion test. Cell wall impermeability seems to be the
Grinstead, UK) following the recommendations of the supplier. In principal mechanism of resistance to inhibitors of the
short, colonies from solid media were used to make a 0.5 McFarland
cell wall synthesis (penicillins and cephalosporins), as
suspension in Brucella standard broth, and 100 lL of this suspension
was transferred to the same medium supplemented with haemin and anaerobic microorganisms lacks cytochrome-mediated
vitamin K1, given an approximate concentration of 1 106 CFU/mL. electron transport [6]. But the cooperation of nonspecific
One hundred microliters of this suspension was inoculated into each mechanisms (multidrug transporters, general stress-in-
well of the Sensititre Anaero3 plate. Because this system does not have duced response, mutation on penicillin binding proteins)
an adequate range of concentrations for some antibiotics, the upper
may also account for differences between strains.
limits of the MICs were determined by the standardized NCCLS agar
dilution technique in MRS-cystein agar (VWR International) plates All tested bifidobacteria were very susceptible to
[1], although a higher inoculum was needed to obtain enough growth. vancomycin, as were all isolates of L. gassseri, L. del-
The antibiotics analyzed include inhibitors of the cell wall synthesis brueckii, and L. acidophilus. However, resistance at a
(b-lactams: penicillin G, amoxicillin, amoxicillin plus clavulanic high level was found in strains of L. casei/L. paracasei,
acid, piperacillin, piperacillin plus tazobactam and imipenem;
L. rhamnosus, L. plantarum, L. parabuchneri, L. brevis,
cephalosporins: cefoxitin; and glycopetides: vancomycin), protein
synthesis (chloramphenicol, clindamycin, erythromycin, and and L. vaginalis, which showed MICs 256 lg/mL. The
tetracycline), and nucleic acid synthesis (metronidazole and level of resistance found in these species is in accor-
moxifloxacin). dance with previous observations [7, 10]. Intrinsic
resistance in lactobacilli to this glycopeptide is attrib-
uted to the synthesis of modified cell wall peptidoglycan
Results and Discussion
precursors that terminate in lactate [11]. This type of
The distribution of MICs to the several antibiotics resistance does not seem to pose a problem since it is
inhibiting cell wall synthesis (b-lactams: penicillins, different from the inducible, transferable mechanism
cephalosporins, and carbapenems; and the glycopeptide observed in other bacteria.
vancomycin) are summarized in Table 1. All tested Table 2 summarizes the distribution of MICs to
strains were susceptible to the lower concentration of several antibiotics inhibiting the synthesis of proteins.
piperacillin and piperacillin plus tazobactam (all None of the assayed strains studied showed resistance to
MICs 16 lg/mL). MICs to all other antibiotics chloramphenicol (most MICs of 4 lg/mL). Although
showed variability. MICs to the amoxicillinclavulanic most of the isolates of both bifidobacteria and lactoba-
acid mixture were always lower and followed the same cilli had a low MIC to tetracycline, several resistant
pattern as those for amoxicillin alone. Thus, for reasons strains appeared. The 15 isolates that were only exam-
of clarity, only MICs to the latter were included in Ta- ined for a concentration of 16 lg/mL (Table 2), were
ble 1. For the same reason, MICs to imipinem were not further confirmed as highly resistant by an E-test assay
summarized in the table as all were lower than 0.25 lg/ (data not shown). The two L. acidophilus isolates and
mL. The real MIC to piperacillin was further evaluated the single L. plantarum one had the highest MICs to this
in a set of 10 different bifidobacteria and lactobacilli antibiotic: over 256 lg/mL. Intermediate levels of
isolated by the E-test technique (AB Biodisk, Solna, resistance were encountered in 11 Bifidobacterium

40
204 CURRENT MICROBIOLOGY Vol. 50 (2005)

Table 1. Distribution of MICs to several antibiotics inhibiting cell wall synthesis (b-lactams: penicillins and cephalosporins and the glycopeptide
vancomycin) in Bifidobacterium and Lactobacillus species of the human gastrointestinal tract

Number of isolates for which the MIC (lg/mL) was as follows:

Number of
Antibiotic Species strains 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128 256

Penicillin G B. longum 47 6 21 17 2 1
B. bifidum 16 14 2
B. pseudocatenulatum 11 6 1 3 1
B. catenulatum 2 2
L. gasseri 20 13 7
L. delbrueckii 8 8
L. casei/L. paracasei 7 4 1 1 1
L. rhamnosus 5 1 2 2
L. acidophilus 2 1 1
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
0.25 0.5 1 2 4 8 16 32 64
Amoxicillin B. longum 47 35 10 1 1
B. bifidum 16 16
B. pseudocatenulatum 11 7 4
B. catenulatum 2 2
L. gasseri 20 9 11
L. delbrueckii 8 8
L. casei/L. paracasei 7 5 2
L. rhamnosus 5 3 2
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
0.5 1 2 4 8 16 32 64
Cefoxitin B. longum 47 2 17 9 9 7 1 1 1
B. bifidum 16 10 6
B. pseudocatenulatum 11 2 1 2 3 3
B. catenulatum 2 1 1
L. gasseri 20 1 1 1 7 7 1 3
L. delbrueckii 8 2 2 1 3
L. casei/L. paracasei 7 7
L. rhamnosus 5 5
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
2 4 8 16 32 64 128 256
Vancomycin B. longum 47 46 1
B. bifidum 16 4 12
B. pseudocatenulatum 11 10 1
B. catenulatum 2 2
L. gasseri 20 10 4 6
L. delbrueckii 8 6 2
L. casei/L. paracasei 7 7
L. rhamnosus 5 5
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1

41
S. Delgado et al.: Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species 205

Table 2. Distribution of MICs to several antimicrobials inhibiting protein synthesis for Bifidobacterium and Lactobacillus species of the human
gastrointestinal tract

Number of isolates for which the MIC (lg/ml)1)


was as follows:

Number of
Antibiotic Species strains 2 4 8 16 32 64

Chloramphenicol B. longum 47 45 2
B. bifidum 16 16
B. pseudocatenulatum 11 9 2
B. catenulatum 2 2
L. gasseri 20 1 13 3 3
L. delbrueckii 8 4 2 2
L. casei/L. paracasei 7 5 1 1
L. rhamnosus 5 3 1 1
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
2 4 8 16 32 64 128 256 256
Tetracycline B. longum 47 28 2 2 8* 1 3 3
B. bifidum 16 8 1 3* 4
B. pseudocatenulatum 11 9 2*
B. catenulatum 2 1 1
L. gasseri 20 5 12 3
L. delbrueckii 8 7 1
L. casei/L. paracasei 7 7
L. rhamnosus 5 5
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1*
L. vaginalis 1 1*
1 2 4 8 16 32 64 128 256 512 1024
Erythromycin B. longum 47 31 2 1 4 4 4 1
B. bifidum 16 16
B. pseudocatenulatum 11 8 2 1
B. catenulatum 2 1 1
L. gasseri 20 19 1
L. delbrueckii 8 8
L. casei/L. paracasei 7 5 2
L. rhamnosus 5 3 2
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
0.5 1 2 4 8 16 32 64 128 256
Clindamycin B. longum 47 34 3 2 2 3 3
B. bifidum 16 16
B. pseudocatenulatum 11 10 1
B. catenulatum 2 2
L. gasseri 20 1 4 9 6
L. delbrueckii 8 8
L. casei/L. paracasei 7 6 1
L. rhamnosus 5 3 2
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1

*These strains were found to be highly resistant (MICs between 128 and 256 l g/ml)1) by an E-test assay (not shown).

42
206 CURRENT MICROBIOLOGY Vol. 50 (2005)

Table 3. Distribution of MICs to the DNA synthesis inhibitor metronidazole and the fluoroquinolone moxifloxacin for Bifidobacterium and
Lactobacillus species of the human gastrointestinal tract

Number of isolates for which the MIC (lg/ml)1)


was as follows:

Number of
Antibiotic Species strains 0.50 1 2 4 8 16 32 64

Metronidadozole B. longum 47 4 19 6 2 3 1 12
B. bifidum 16 6 7 3 1
B. pseudocatenulatum 11 2 4 3 1 1
B. catenulatum 2 1 1
L. gasseri 20 20
L. delbrueckii 8 8
L. casei/L. paracasei 7 7
L. rhamnosus 5 5
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1
0.12 0.25 0.50 1 2 4 8 16
Moxifloxacin B. longum 47 1 8 20 5 8 2 3
B. bifidum 16 7 1 4 4
B. pseudocatenulatum 11 2 1 1 2 2 3
B. catenulatum 2 1 1
L. gasseri 20 1 5 8 6
L. delbrueckii 8 1 1 4 2
L. casei/L. paracasei 7 1 4 2
L. rhamnosus 5 3 2
L. acidophilus 2 2
L. plantarum 1 1
L. parabuchneri 1 1
L. brevis 1 1
L. vaginalis 1 1

isolates (MICs between 64 and 256 lg/mL). Our results also found in several bifidobacterial isolates, one of
confirm the findings of Matteuzzi et al. [16], who which displayed resistance to tetracycline (MIC 16 lg/
showed that tetracycline has a very variable effect mL). These results differs from those published else-
against bacteria of this genus, while all were susceptible where [4, 13, 16], in which bifidobacteria of human and
to chloramphenicol. The number of tetracycline-resis- commercial origin were shown to be sensitive to these
tant bacteria in the GIT has been correlated with the use two broad spectrum antibiotics.
of this antibiotic [18], suggesting that the increasing Metronidazole is an antibiotic frequently used in
antibiotic pressure posed by the wide use of antibiotics cases of anaerobic infections of the digestive tract, being
in veterinary and human medicine is certainly contrib- the agent of choice for Clostridium difficile-induced
uting to the dissemination of resistances into intestinal pseudomembranous colitis [8]. In accordance with other
bacteria. surveys [3, 7], all lactobacilli strains were found to be
The findings for tetracycline should apply also to resistant to metronidazole (MIC 32 lg/mL) (Table 3),
erythromycin and clindamycin, for which most MICs indicating that this antibiotics is not active in this genus.
were very low but a few high resistant strains were Resistance of lactobacilli to metronidazole might be, as
found. Among the 46 strains of lactobacilli analyzed, in other LAB species, because of the absence of
two of five L. rhamnosus isolates and the two L. aci- hydrogenase activity [5]. Susceptibility to metronidazole
dophilus strains, which were resistant to tetracycline, was variable in bifidobacterial strains. Some of them
showed high resistance to both erythromycin and clin- were very sensitive whereas others had intermediate to
damycin (MIC 1024 and 256 lg/mL, respectively). high resistance, a result which has been reported before
High resistance to erythromycin and/or clindamycin was [13, 20]. The MICs to the fluoroquinolone moxifloxacin

43
S. Delgado et al.: Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species 207

were randomly distributed from less than 0.12 to 16 5. Church DL, Bryant RD, Sim V, Laishley EJ (1996) Metronidazole
lg/mL, and no clear pattern could be seen (Table 3). susceptibility and the presence of hydrogenase in pathogenic
bacteria. Anaerobe 2:147153
Multiple resistances are not common in intestinal 6. Condon S (1983) Aerobic metabolism of lactic acid bacteria. Ir J
lactobacilli and bifidobacteria and were only observed in Food Sci Technol 7:1525
a couple of L. acidophilus strains (resistant to tetracy- 7. Danielsen M, Wind A (2004) Susceptibility of Lactobacillus spp.
cline, erythromycin, and clindamycin). Only a minority to antimicrobial agents. Int J Food Microbiol 82:111
of intestinal lactobacilli and bifidobacterial species 8. Freeman CD, Klutman NE, Lamp KC (1997) Metronidazole: A
therapeutic review and update. Drugs 54:679708
showed antibiotic resistances (against tetracycline, 9. Gasser F (1994) Safety of lactic acid bacteria and their occurrence
erythromycin, and/or clindamycin). However, for the in human clinical infections. Bull Inst Pasteur 92:4567
results to be confident more strains should be analyzed 10. Hamilton-Miller JM, Shah S (1994) Susceptibility patterns of
for several species. Some of the observed resistances vaginal lactobacilli to eleven oral antibiotics. J Antimicrob Che-
(cefoxitin, vancomycin, metronidazole) seemed to be mother 33:10591060
11. Handwerger S, Pucci MJ, Volk KJ, Liu J, Lee MS (1994)
intrinsic and the level is genus- or species-dependent. Vancomycin-resistant Leuconostoc mesenteroides and Lactoba-
This small antibiotic-resistant fraction justifies an anti- cillus casei synthesize cytoplasmic peptidoglycan precursors that
biotic-susceptibility assay to avoid the inclusion of terminate in lactate. J Bacteriol 176:260264
resistant strains in the formulation of probiotics. A 12. Klaenhammer TR, Muller MJ (1999) Selection and design of
phenotypic test can not confirm the presence or absence probiotics. Int J Food Microbiol 50:4557
13. Lim KS, Huh CS, Baek YJ (1992) Antimicrobial susceptibility of
of transferable resistance genes, but some of the ob- bifidobacteria. J Dairy Sci 76:21682174
served levels are compatible with common transmissible 14. Liu CF, Fung ZF, Wu CL, Chung TC (1996) Molecular charac-
determinants. terization of a plasmid-borne (pTC82) chloramphenicol resistance
determinant (catTC) from Lactobacillus reuteri G4. Plasmid
ACKNOWLEDGMENTS 36:116124
15. Mandar R, Lijvukene K, Huftt P, Karki T, Mikelsaar M (2001)
This work was supported by projects from the Spanish Ministry of Antibacterial susceptibility of intestinal lactobacilli of healthy
Education and Science (ref. AGL2000-1474) and from the EU children. Scand J Infect Dis 33:344349
(ACEART, ref. 506214). S.D. was the recipient of a fellowship from 16. Matteuzzi D, Crocciani F, Brigidi P (1983) Antimicrobial sus-
the FPI Program. ceptibility of Bifidobacterium. Ann Microbiol Inst Pasteur
134A:339349
17. Netherwood T, Bowden R, Harrosin P, ODonnel AG, Parker DS,
Literature Cited Gilbert HJ (1999) Gene transfer in the gastrointestinal tract. Appl
1. Anonymous (1997) Approved standard M11-A4. Methods for Environ Microbiol 65:51395141
antimicrobial susceptibility testing of anaerobic bacteria, 4th 18. Scott KP, Melville CM, Barbosa TM, Flint HJ (2000) Occurrence
edn. National Committee for Clinical Laboratory Standards, of the new tetracycline resistance gene tet(W) in bacteria from the
Villanova human gut. Antimicrob Agents Chemother 44:775777
2. Axelsson LT, Ahrn SE, Andersson MC, Stahl SR (1988) Identi- 19. Teuber M, Meile L, Schwarz F (1999) Acquired antibiotic resis-
fication and cloning of a plasmid-encoded erythromycin resistance tance in lactic acid bacteria from food. Antonie van Leeuwenhoek
determinant from Lactobacillus reuteri. Plasmid 20:171174 76:115137
3. Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic 20. Yazid AM, Ali AM, Shuhaimi M, Kalaivaani V, Rokiah MY,
susceptibility of potentially probiotic Lactobacillus species. J Food Reezal A (2000) Antimicrobial susceptibility of bifidobacteria.
Prot 61:16361643 Lett Appl Microbiol 31:5762
4. Charteris WP, Kelly PM, Morelli L, Collins JK (1998) Antibiotic 21. Young JPW, Downer HL, Eardly BD (1991) Phylogeny of the
susceptibility of potentially probiotic Bifidobacterium isolates prototrophic Rhizobium strain BTAil by polymerase chain reac-
from the human gastrointestinal tract. Lett Appl Microbiol tion-based sequencing of a 16S rRNA segment. J Bacteriol
26:333337 173:22712277

44
Captulo II.- Ensayo de susceptibilidad a antibiticos en distintas cepas
tipo de BAL y bifidobacterias mediante Etest. Determinacin de nuevos
puntos de corte en Lactococcus lactis y Lactobacillus plantarum frente a
seis antibiticos.
Artculo III.- Antimicrobial susceptibility levels to twelve antibiotics of lactic acid bacteria
and bifidobacteria type strains.

Objetivo: El objetivo que perseguamos en este trabajo era conocer los niveles de
susceptibilidad a antibiticos en cepas tipo pertenecientes a los gneros Bifidobacterium,
Lactococcus, Lactobacillus y Streptococcus thermophilus.

Resultados: Se ensay la susceptibilidad a antibiticos de 29 cepas tipo mediante el


mtodo Etest utilizando 12 antibiticos distintos. Los valores de CIMs obtenidos para
los antibiticos azitromicina, claritromicina, eritromicina y cloranfenicol mostraron
cierta variacin entre cepas y entre especies, an as los valores fueron tan bajos que
todas las cepas se consideraron susceptibles. La polimixina B y los aminoglicsidos no
parecen ser eficaces contra las BAL y bifidobacterias. Por su parte, la resistencia a
vancomicina y rifampicina es considerada resistencia intrnseca en los lactobacilos
heterofermentativos y en Lactococcus lactis, respectivamente. Las cepas Lactobacillus
gasseri LMG9203T con una CIM para clindamicina de 32 g/ml, Bifidobacterium
thermophilum LMG21813 con un valor de CIM de 64 y 48 g/ml para tetraciclina y
minociclina y Bifidobacterium pseudolongum subsp. globossum con una CIM de 16 g/ml
para tetraciclina se consideraron resistentes.

Conclusiones: Estos ensayos demuestran que algunas de las cepas tipo poseen valores
de CIM compatibles con la existencia de determinantes de resistencia adquiridos. Se
sugiere la posibilidad de reemplazar estas cepas por otras susceptibles.

47
Antimicrobial susceptibility levels to twelve antibiotics of lactic acid
bacteria and bifidobacteria type strains

Abstract

The antimicrobial susceptibility of 28 type and reference strains belonging to species


and subspecies of the genus Bifidobacterium, Lactococcus, and Lactobacillus, as well as
the type strain of Streptococcus thermophilus, to 12 antibiotics was assayed by the Etest
method. The minimum inhibitory concentrations (MICs) to azithromycin, clarithromycin,
erythromycin, and chloramphenicol varied among species and strains but MIC values
were all low. MICs to minocycline, clindamycin, and rifampicin were also low in all except
a few strains. MICs to vancomycin presented a clear bimodal distribution; MIC values
were low in approximately half of the species, and were high in all heterofermentative
lactobacilli strains. Finally, MICs to polymyxin B, kanamycin, streptomycin, and
tetracycline varied widely from low to high values between species and strains.
Comparison of the MICs to values in previous surveys, showed the strains to be either
susceptible or intrinsically resistant to most antibiotics. Atypical MICs to three
antibiotics compatible with the presence of acquired resistance determinants were
scored in at least two strains: Lactobacillus gasseri LMG9203T, to clindamycin (32 g ml-
1
), and in Bifidobacterium thermophilum LMG21813T, to both tetracycline and
minocycline (64 and 48 g ml-1, respectively).

Introduction Besides desirable technological properties,


strains to be used in food systems need to
At present, there is great concern about the meet several safety criteria, among which the
possibility of food-borne and commensal absence of acquired antibiotic resistance
intestinal populations to become reservoirs determinants is crucial (Saarela et al., 2002).
for antibiotic resistance determinants from This is especially important because
which they could be transferred to transferable or mobilizable antibiotic resistant
opportunistic and pathogenic bacteria, thus genes have already been characterized in
hampering the treatment of infections (Srum strains of LAB and bifidobacteria (for a review
and LAbe-Lund, 2002; Salyers et al., 2004). see Teuber et al., 1999; Mathur and Singh,
The possibility of transfer is related to the 2005). However, while standard procedures
genetic basis of the resistance. Intrinsic and and breakpoints for antibiotic susceptibility
acquired resistance by a mutation are testing of bacteria with clinical significance
presumed to present a low risk of horizontal are well established (CLSI, 2004), test
spread, while risk of transfer is maximum if methods for LAB and bifidobacteria (Klein et
acquired resistance is mediated by added al., 2000; Charteris et al., 2001; Danielsen
genes (European Commission, 2005). and Wind 2003;), growth media (Huys et al.
Lactic acid bacteria (LAB) and 2002; Klare et al., 2005; Mtt et al., 2006),
bifidobacteria are of outstanding importance and microbiological breakpoints
among commensal populations, as species of discriminating intrinsic from acquired
these two groups are largely found in raw resistances (Felten et al., 1999; Charteris et
materials and fermented food products, and al., 2001; Katla et al., 2001; Danielsen and
some others are normal inhabitants of the Wind 2003) are currently being standardised.
gastrointestinal tract (GIT) of human and Type strains are frequently used as a
animals. Because of this, selected strains are control in many antibiotic surveys, although
used as starters in feed and food their antibiotic resistance status is not
fermentations (Desmazeaud and de Roissart, previously known. In this work, the MICs of
1994) and as probiotics in foods and dietary 12 different antibiotics have been addressed
supplements (Ouwehand et al., 2002). by the Etest method in 29 type and reference
strains of LAB and bifidobacteria. Results

49
were then compared to other in previous anoxic atmosphere (10% H2, 10% CO2, 80%
surveys allowing to differentiate among N2).
susceptible strains, intrinsically resistant
strains, and resistant strains suspected of Determination of MICs by Etest. The
harbouring acquired resistance determinants. determination of MICs by the Etest method
was performed according to the
Material and Methods manufacturers instructions (AB Biodisk,
Solna, Sweden). Antimicrobial agents
Bacterial strains and growth conditions. assayed and the antibiotic concentration
Freeze dried bacterial cultures were gradients in the strips are summarized in
recovered in appropriate agar plates, either Table 1. Lactococcus, Lactobacillus and S.
M17 (Oxoid, Oxoid Ltd., Basingstoke, thermophilus strains were cultured on the
Hamphire, UK) (Lactococcus and S. recently described LSM medium (90%
thermophilus strains), de Man, Rogosa, and Isosensitest, 10% MRS; both from Oxoid)
-1
Sharpe (MRS) (Merck, VWR International, (Klare et al., 2005). Cysteine (0.3 g l )
Darmstadt, Germany) (Lactobacillus strains), (Merck) was added to LSM for
or MRS added with 0.3 g l-1 of cysteine Bifidobacterium strains.
(Sigma Chemical Co., St. Louis, Miss., USA) Colonies from 48 h cultures were
(Bifidobacterium strains). Lactococci were suspended in a saline solution (0.85% NaCl)
grown overnight at 32C. Mesophilic (BioMerieux, Marcy lEtoile, Lyon, France) to
lactobacilli were cultivated at 28C in a 5% a density approaching that of McFarland
CO2-enriched (5%) chamber (Precission; standard 1. The suspension was evenly
Pacisa-Giralt, Madrid, Spain). Thermophilic spread on the agar plates with a cotton swab.
lactobacilli, bifidobacteria and S. The agar was allowed to dry for 15 min
thermophilus were grown at 37C in an before applying the Etest strips. Readings
anaerobic chamber (Mac500; Down Whitley were all done after 48 h incubation.
Scientific, West Yorkshire, UK) containing an
Range of dilution
Antibiotic Type -1
(g ml )

Inhibitor of cell wall synthesis


Vancomycin Glycopeptide 0.016-256

Disorganizer of the cell membrane


Polymyxin B Polypeptide 0.064-1024

Inhibitors of protein synthesis 0.016-256


Binding to the 30S ribosomal subunit:
Kanamycine Aminoglycoside 0.016-256
Streptomycin Aminoglycoside 0.016-256
Minocycline Tetracycline 0.016-256
Tetracycline Tetracycline 0.016-256
Binding to the 50S ribosomal subunit:
Azithromycin Macrolide 0.016-256
Clarithromycin Macrolide 0.016-256
Erythromycin Macrolide 0.016-256
Clindamycin Lincosamide 0.016-256
Chloramphenicol - 0.016-256

Inhibitor of RNA synthesis


Rifampicin - 0.002-32

Table 1.- Antibiotics studied in this work and range of dilution in their Etest strips.

Results and discussion chloramphenicol (<0.016-0.75 g ml-1) varied


quantitatively but val es were rather low,
MICs of the 12 different antibiotics assayed to suggesting strains are susceptible to all four
29 type and reference strains are antibiotics. In contrast, MICs to all other
summarized in Table 2. MICs values to all antibiotics also varied qualitatively, but high
antibiotics varied widely between species and MIC values were seen in one or more strains,
strains, although two types of variations could which strongly suggests the presence of
be considered. MICs of azithromycin (0.032 resistant strains. However, no clear cut-off
to 6 g ml-1), clarithromycin (0.032-4 g ml-1), values have been defined for LAB and
erythromycin (<0.016-1 g ml-1), and bifidobacteria species, making difficult the
distinguishing of susceptible from resistant

50
strains. Further, different cut-off values have intrinsic and acquired resistances, is usually
been considered in distinct assays, made by comparison of MICs from different
hampering a direct comparison of the results surveys (Katla et al., 2001; Danielsen and
between surveys (Katla et al., 2001; Wind, 2003; Delgado et al., 2005; Flrez et
Danielsen and Wind, 2003; Delgado et al., al., 2005). Further, the study of the MIC
2005). distributions between species and strains is
Based on the body of research published also of much help to separate resistant
and from national and European monitoring strains from a majority of susceptible bacteria
programmes, the SCAN Panel established (Murray et al., 2003). Comparison of the
microbiological breakpoints for some MICs to vancomycin (from 0.19 to >256 g
antibiotics of human and veterinary ml-1) (for which the FEEDAP Panel did not
importance for the safety risk assessment of considered breakpoints in heterofermentative
bacteria used in animal feed, including most lactobacilli), polymyxin B (from 4 to 1024 g
LAB species. These breakpoints have been ml-1), minocycline (0.023 to 48 g ml-1), and
recently updated by the FEEDAP Panel rifampicin (from 0.004 to 32 g ml-1) through
(European Commission, 2005). Taking these species and strains of this work showed
breakpoints as cut-off values, a majority of bimodal distributions, indicative of a part of
the strains analysed should be considered the population being resistant. Resistance of
resistant to kanamycin (24 strains) and heterofermentative lactobacilli species to
streptomycin (16 strains), and a few to vancomycin has been reported elsewhere
tetracycline (5 strains) and clindamycin (2 (Katla et al., 2001; Danielsen and Wind,
strains) (in grey in Table 2). High resistance 2003; Delgado et al., 2005; Flrez et al.,
of LAB and bifidobacteria species to 2005), and is attributed to the presence of D-
aminoglycosides, such as kanamycin and alanine-D-lactate as the normal component in
streptomycin, has been repeatedly reported their peptidoglycan (Klein et al., 2000). This
(Lim et al., 1993; Charteris et al., 1998; Katla type of resistance does not seem to pose a
et al., 2001; Danielsen and Wind, 2003), problem since it is different from the
suggesting inherent resistance. In general, inducible, transferable mechanism present in
the lack of cytochrome-mediated transport is enterococci (DeLisle and Perl, 2005).
responsible for the resistance of anaerobic Likewise, polymyxin B is inactive against
and facultative bacteria to aminoglycosides most Gram-positive bacteria (Hermsen et al.,
(Bryan and Kwan, 1981). In the experimental 2003), suggesting intrinsic resistance in
conditions of this study, MIC values to species and strains with high MICs. B.
kanamycin and streptomycin were quite thermophilum LMG21813 showed a MIC to
different to those of the FEEDAP Committee minocycline of 48 g ml . This strain, which
-1
(European Commission, 2005). Thought was also considered resistant to tetracycline
more strains of these genera have to be (MIC of 64 g ml-1), was suspected do
analysed, a revision of the microbiological harbour an acquired resistance determinant.
breakpoints to these antibiotics could be Resistance genes conferring resistance to
needed. Tetracycline showed the greatest these two antibiotics have been reported in
variability of MICs. Most strains were clearly many LAB (for a review see Roberts, 2005).
susceptible. However, strains showing Indeed, the presence of tet(W) has been
moderate to high resistance (MICs between 4 recently reported in several Bifidobacterium
to 64 g ml ) were scored. Of these, five
-1
species (Scott et al., 2000; Moubareck et al.,
have MIC values above the FEEDAP 2005; Masco et al., 2006). By PCR using
breakpoints (Table 2), including the specific primers, the tet(W) gene was also
Bifidobacterium pseudolongum type strain. detected in the B. thermophilum type strain
MICs to clindamycin higher than the (data not shown). Most LAB and
breakpoints proposed by FEEDAP were also bifidobacteria type strains were susceptible to
observed in Lactobacillus acidophilus rifampicin (MICs between 0.004 to 0.75 g
LMG9433 (6 g ml-1) and L. gasseri -1
ml ), except for the two Lactococcus lactis
LMG9203 (32 g ml-1). Although higher, strains (MICs of >32 g ml-1). Rifampicin
some of these MIC figures were close to interacts in a specific manner with the -
FEEDAP values, and differences may be subunit of the bacterial RNA polymerase
attributed to parameters such as the test encoded by the rpoB gene (Wehrli, 1983).
assay, inoculums size, and/or media utilized Lactococci and enterococci are intrinsically
(Huys et al., 2002; Klare et al., 2005; Mtt et resistant to this antibiotic (Orberg and
al., 2006). Sandine, 1985; Teuber et al., 1999).
Distinguishing between susceptible and
resistant strains, and distinction between

51
Minimum inhibitory concentration (g ml-1)
LMG* code
Species number VA PO KM SM MC TC AZ CL EM CM CH RI

Lactococci
Lc. lactis subsp. cremoris LMG6897 0.19 48 1 3 0.064 0.25 0.094 1.5 0.094 0.19 < 0.016 >32
Lc. lactis subsp. lactis LMG6890 0.5 256 4 12 0.064 0.125 0.38 4 0.125 0.25 0.094 >32
Streptococci
St. thermophilus LMG6896 0.75 6 16 2 0.023 0.032 0.5 1 0.094 0.023 0.047 0.004
Heterofermentative lactobacilli
Lb. brevis LMG6906 >256 128 >256 128 0.5 6 0.75 1 0.094 4 0.023 0.38
Lb. brevis subsp. gravesensis LMG7934 >256 4 24 24 2 12 0.75 3 0.19 0.094 0.064 0.016
Lb. casei LMG6904 >256 384 >256 24 0.032 0.125 0.38 1.5 0.064 1.5 0.032 0.006
Lb. fermentum LMG6902 96 64 64 16 1.5 4 0.38 3 0.125 < 0.016 0.064 0.094
Lb. paracasei subsp. paracasei LMG13087 >256 >1024 >256 96 0.125 0.38 1 2 0.19 0.125 0.094 0.047
Lb. paracasei subsp. tolerans LMG9191 >256 >1024 >256 48 0.047 0.125 0.75 1 0.064 0.125 0.047 0.064
Lb. pentosus LMG10755 >256 >1024 64 32 0.75 3 3 2 0.5 0.032 0.25 0.19
Lb. plantarum LMG6907 >256 >1024 >256 96 3 12 6 3 1 2 0.75 0.75
Lb. reuteri LMG9213 >256 768 6 2 0.75 16 4 1 0.75 0.094 0.5 0.023
Lb. rhamnosus LMG6400 >256 >1024 48 6 0.25 0.75 0.5 4 0.125 0.38 0.094 0.064
Lb. sakei subsp. sakei LMG9468 >256 24 12 24 0.125 1 1.5 1.5 0.125 0.38 0.047 0.5

52
Homofermentative lactobacilli
Lb. acidophilus LMG9433 1 >1024 16 8 0.125 0.75 1 3 0.125 6 0.064 0.38
Lb. amylovorus LMG9496 0.75 256 >256 24 0.125 0.75 4 3 0.38 0.064 0.064 0.19
Lb. delbrueckii subsp. bulgaricus LMG6901 0.5 8 48 6 0.023 0.125 0.032 0.75 < 0.016 0.023 < 0.016 0.064
Lb. delbrueckii subsp. delbrueckii LMG6412 1 384 96 8 0.25 0.38 1 2 0.25 0.094 0.032 0.012
Lb. delbrueckii subsp. lactis LMG7942 0.5 96 6 2 0.5 0.75 0.064 0.032 0.023 2 < 0.016 0.064
Lb. gasseri LMG9203 1.5 >1024 64 16 0.094 0.75 1 3 0.094 32 0.064 0.004
Lb .helveticus LMG6413 0.5 12 48 1.5 0.125 0.38 0.38 1 0.064 0.75 0.032 0.5
Lb. johnsonii LMG9436 2 >1024 >256 24 1.5 16 1.5 4 0.25 0.75 0.125 0.094
Bifidobacteria
B. adolescentis LMG10502 0.5 96 >256 96 0.25 0.5 0.75 1 0.094 0.047 0.032 0.094
B. animalis LMG10508 0.75 768 >256 96 0.38 0.75 1 1.5 0.125 2 0.047 0.19
B. longum LMG13197 0.75 64 >256 96 0.38 0.75 1 1.5 0.094 < 0.016 0.032 0.032
B. pseudolongum subsp. pseudolongum LMG11571 0.38 256 >256 48 0.125 0.38 0.75 0.5 0.094 0.064 0.032 0.064
B. pseudolongum subsp. globossum LMG11569 0.5 256 >256 >256 1 16 0.064 0.75 0.032 1 < 0.016 0.125
B. thermophilum LMG21813 0.75 96 >256 >256 48 64 0.75 0.38 0.094 0.064 0.023 0.19

*LMG, Laboratorium voor Microbiologie, University of Gent, Belgium; obtained from the Belgian Co-ordinated Collections of Micro-organisms (BCCM), Belgium.
Key of antibiotics: VA, vancomycin, PO, polymyxin B; KM, kanamycin; SM, streptomycin; MC, minocycline; TC, tetracycline; AZ, Azithromycin; CL, clarithromycin; EM, erythromycin; CM, clindamycin; CH, chloramphenicol; and
RI, rifampicin.
In grey, antibiotics for which microbiological breakpoints have been recently defined by the FEEDAP Panel (except for vancomycin in heterofermentative lactobacilli) (European Commission, 2005), and MIC values surpassing
the corresponding breakpoints; strains to which these MICs belong should be considered resistant.
Boxed, qualitatively higher MIC values of antibiotics without breakpoints.

Table 2.- MICs of twelve different antibiotics for type strains of Lactococcus, Lactobacillus, and Bifidobacterium species and Streptococcus thermophilus.
In conclusion, polymyxin B and H2O2 production, and antibiotic resistance and correlation
with human clinical status. J Clin Microbiol 37:729-733.
aminoglycosides do not seem to act properly Flrez, A. B., Delgado, S., and Mayo, B. 2005. Antimicrobial
against LAB and bifidobacteria species. susceptibility of lactic acid bacteria isolated from a cheese
environment. Can J Microbiol 51:51-58.
Intrinsic resistance to vancomycin and Hermsen, E. D., Sullivan, C. J., and Rotschafer, J. C. 2003.
rifampicin was observed for Polymyxins: pharmacology, pharmacokinetics,
pharmacodynamics, and clinical applications. Infect Dis
heterofermentative lactobacilli and L. lactis Clin North Am 17:545-62.
strains, respectively. Resistance levels to Huys G, K. DHaene and J. Swings. 2002.Influence of the
tetracycline and clindamycin compatible with culture medium on antibiotic susceptibility testing of food-
associated lactic acid bacteria with the agar overlay disc
acquired determinants were encountered in a diffusion method. Lett Appl Microbiol 34:402406.
few strains. Indeed, the presence of tet(W) in Katla, A.-K., Kruse, H., Johnsen, G. and Herikstad, H. 2001.
Antimicrobial susceptibility of starter culture bacteria used
B. thermophilum LMG21813 was in Norwegian dairy products. Int J Food Microbiol 67:147
demonstrated by PCR. Care should be taken 152.
Klare I, Konstabel C, Muller-Bertling S, Reissbrodt R, Huys
in the interpretation of the results if such G, Vancanneyt M, Swings J, Goossens H, and Witte W.
strains were used as a control in antibiotic 2005. Evaluation of new broth media for microdilution
surveys. antibiotic susceptibility testing of lactobacilli, pediococci,
lactococci, and bifidobacteria. Appl Environ Microbiol.
71:8982-8986.
Acknowledgements Klein, G., Hallmann, C., Casas, I. A., Abad, J., Louwers, J.,
and Reuter, G. 2000. Exclusion of vanA, vanB and vanC
type glycopeptide resistance in strains of Lactobacillus
reuteri and Lactobacillus rhamnosus used as probiotics by
This work was supported by an EU project polymerase chain reaction and hybridization methods. J
within the VI Frame Program (ACE-ART, ref. Appl Microbiol 89:815-824.
CT-2003-506214). M. S. Ammor was Lim, K. S., Huh, C. S., Baek, Y. J. 1993. Antimicrobial
susceptibility of bifidobacteria. J Dairy Sci 76:2168-74.
awarded a postdoctoral fellowship from the Masco L, Van Hoorde K, De Brandt E, Swings J, Huys G.
Secretara de Estado de Universidades e 2006. Antimicrobial susceptibility of Bifidobacterium strains
from humans, animals and probiotic products. J Antimicrob
Investigacin of the Spanish Ministry of Chemother 58:85-94.
Education and Science (ref. SB2004-0165). Mathur, S., and Singh, R. 2005. Antibiotic resistance in food
LMG/BCCMTM, University of Gent, Belgium, lactic acid bacteria. A review. Int J Food Microbiol 105:281-
295.
is acknowledged for providing LAB and Mtt, J., Suihko, M. L., and Saarela, M. 2006. Comparison of
bifidobacteria type strains. three test media for the antibiotic susceptibility testing of
bifidobacteria with the Etest method. Int J Antimicrob
Agents 28:42-48.
References Moubareck, C., F. Gavini, L. Vaugien, M. J. Butel, and
Doucet-Populaie, F. 2005. Antimicrobial susceptibility of
bifidobacteria. J Antimicrob Chemother 55:38-44.
Bryan, L. E., and Kwan, S. 1981. Mechanisms of Murray, P. R., Baron, E. J., Jorgensen, J. H., Pfaller, M. A.,
aminoglycoside resistance of anaerobic bacteria and and Yolken, R. H. 2003. Manual of Clinical Microbiology.
facultative bacteria grown anaerobically. J Antimicrob American Society for Microbiology, Washington DC.
Chemother 8:S1-S8. Orberg PK and WE Sandine. 1985. Survey of antimicrobial
Charteris, W.P., Kelly, P.M., Morelli, L., and Collins, J.K. resistance in lactic streptococci. Appl Environ Microbiol
1998. Antibiotic susceptibility of potentially probiotic 49:538-542.
Bifidobacterium isolates from the human gastrointestinal Ouwehand A., Salminen S., and Isolauri E. 2002. Probiotics:
tract. Lett Appl Microbiol 26:333-337 an overview of beneficial effects. Anthonie van
Charteris, W. P., Kelly, P. M., Morelli, L., and Collins, J. K. Leeuwenhoek, 82:279-289.
2001. Gradient diffusion antibiotic susceptibility testing of Roberts, M. C. 2005. Update on acquired tetracycline resistance
potentially probiotic lactobacilli. J Food Prot 64:2007-2014. genes. FEMS Microbiol Lett 245:195-203.
Danielsen, M., and Wind, A. 2003. Susceptibility of Saarela, M., Mtt, J. and Mattila-Sandholm, T. 2002. Safety
Lactobacillus spp. to antimicrobial agents. Int J Food aspects of Lactobacillus and Bifidobacterium species
Microbiol 82:1-11. originating from human oro-gastrointestinal tract or from
Delgado, S., Florez, A. B., and Mayo, B. 2005. Antibiotic probiotic products. Microbiol Ecol Health Dis 14:233-240.
susceptibility of Lactobacillus and Bifidobacterium species Salyers, A. A., Gupta, A., and Wang, Y. 2004. Human intestinal
from the human gastrointestinal tract. Curr Microbiol bacteria as reservoirs for antibiotics resistance genes.
50:202-207. Trends Microbiol. 12:412-416.
DeLisle, S., and Perl, T. M. 2005. Vancomycin-resistant Scott, K. P., Melville, C. M., Barbosa, T. M, and Flint, H. J.
enterococci. A road map on how to prevent the emergence 2000. Occurrence of the new tetracycline resistance gene
and transmission of antimicrobial resistance. Chest tet(W) in bacteria from the human gut. Antimicrob Agents
123:s504-S518. Chemother 44:775-777.
Desmazeaud, M. J., de Roissart, H. 1994. Metabolisme general Srum, H., and LAbe-Lund, T. M. 2002. Antibiotic resistance
des bactries lactiques, pp. 169-207. In Bactries in food-related bacteria. A result of interfering with the
Lactiques, Vol. 1, de Roissart, H., and Luquet, F. M. (eds.). global web of bacterial genetics. Int J Food Microbiol 78:43-
Lorica, Uriage, France. 56.
European Commission. 2005. Opinion of the FEEDAP Panel Teuber M, Meile L, Schwarz F. 1999. Acquired antibiotic
on the updating of the criteria used in the assessment of resistance in lactic acid bacteria from food. Antonie van
bacteria for resistance to antibiotics of human or veterinary Leeuwenhoek 76:115-137.
importance. EFSA J 223:1-12. Wehrli, W. 1983. Rifampin: mechanisms of action and
Felten, A., Barreau, C., Bizet, C., Lagrange, P. H., and resistance.Rev Infect Dis 5:S407-S411.
Philippon, A. 1999. Lactobacillus species identification,

53
Artculo IV.- Antibiotic survey of Lactococcus lactis strains to six antibiotics by Etest, and
establismente of new susceptibility-resistance cut-off values.
Journal of Dairy Research (Aceptado).
Artculo V.- Susceptibility of Lactobacillus plantarum strains to six antibiotics and
definition of new susceptibility-resistance cut-off values.
Microbial Drug Resistance (Aceptado).

Objetivos: El objetivo de este trabajo fue analizar un grupo grande de cepas de


Lactococcus lactis y Lactobacillus plantarum procedentes de orgenes y periodos diversos
frente a seis antibiticos mediante el sistema Etest con el fin de establecer nuevos
puntos de corte microbiolgicos para estas especies a seis antibiticos distintos. Los
nuevos puntos de corte se espera que sean de utilidad para discriminar resistencias
intrnsecas de resistencias adquiridas.

Resultados: Los puntos de corte se establecieron tras el anlisis de las distribuciones


de CIMs a los antibiticos ensayados. Los puntos de corte propuestos para la especie
Lc. lactis son los siguientes: tetraciclina (2 g/ml), eritromicina (1 g/ml), clindamicina
(1 g/ml), estreptomicina (64 g/ml), cloranfenicol (12 g/ml) y vancomicina (6
g/ml). Segn las distribuciones de CIMs obtenidas para la especie L. plantarum se han
fijado los siguientes puntos de corte: ampicilina (4 g/ml), clindamicina (16 g/ml),
eritromicina (4 g/ml), gentamicina (16 g/ml) y tetraciclina (64 g/ml). En general,
los puntos de corte propuestos en este trabajo fueron superiores a los definidos
previamente por el Panel FEEDAP de la Comisin Europea en el ao 2005.
Basndonos en los nuevos puntos de corte, todas las cepas de Lc. lactis fueron
susceptibles a eritromicina, cloranfenicol, clindamicina y vancomicina; tan slo 11
cepas mostraron resistencia a tetraciclina. Por su parte, la mayora de las cepas de Lb.
plantarum resultaron susceptibles a los antibiticos ensayados, a excepcin de a
estreptomicina, con valores de CIM iguales o superiores a 64 g/ml en todos los casos,
y a tetraciclina, para el que cuatro cepas presentaban una CIM igual o superior a 128
g/ml.

Conclusiones: La distribucin de CIMs en cepas de Lc. lactis y Lb. plantarum de distinto


origen y geogrficamente alejadas nos han permitido establecer puntos de corte
microbiolgicos para la mayora de los antibiticos estudiados. La definicin de estos

55
puntos de corte es de gran importancia, ya que no se dispone de valores
estandarizados con los que discriminar resistencias intrnsecas de adquiridas.

56
Antibiotic survey of Lactococcus lactis strains to six antibiotics by Etest, and

establishment of new susceptibility-resistance cut-off values

Abstract

In order to establish cut-off values in Lactococcus lactis to six antibiotics for distinguishing
susceptible and intrinsically resistant strains from those having acquired resistances, the
minimum inhibitory concentration (MIC) of tetracycline, erythromycin, clindamycin,
streptomycin, chloramphenicol and vancomycin was determined in 93 different L. lactis
strains using the Etest. These bacterial strains were originally isolated from dairy and animal
sources in widely separated geographical locations. Cut-offs were defined on the basis of
the distribution of the MICs frequency of the studied antibiotics, which in the absence of
acquired determinants should approach to a normal statistical distribution. In general, the
new cut-off values proposed in this study are higher than previously defined (European
Commission, 2005. The EFSA Journal 223, 1-12). Based on these new values, all the strains
tested were susceptible to erythromycin, chloramphenicol and vancomycin, and 79
susceptible to all six antibiotics. However, 11 strains (around 12%) were considered
resistant to tetracycline (six of which had been identified after screening of a large collection
of lactococci strains for tetracycline resistance) and five (5.4%) resistant to streptomycin. Of
these, two fish isolates proved to be resistance to both tetracycline and streptomycin. tet(M)
and mosaic tet(L/S) genes were amplified by PCR from the tetracycline resistant strains,
demonstrating they harboured acquired antibiotic resistance determinants.

Introduction expression of heterologous proteins, the


synthesis of food-grade additives and
Lactococcus lactis is a lactic acid bacterium nutraceuticals, and in vaccine delivery (Hols et
commonly dominant in natural niches such us al. 1999; Nouaille et al. 2003).
spontaneous milk fermentations, on cattle, and Food and certain food supplements can
on plant material (Mundt, 1986). Recently, it introduce large numbers of live L. lactis into the
has also been reported as a dominant human and animal gastrointestinal tract (GIT),
microorganism in the intestinal tract of a complex ecosystem containing hundreds of
freshwater fish (Hagi et al. 2004; Ringo, 2004). different types of resident and transient
Strains of this species are also the main microorganisms including opportunistic and
component of starters used in the economically pathogenic bacteria (Guarner & Malagelada,
important fermentation of milk into cheese (Fox 2003). There is therefore concern about the
et al. 2000). In this process, the major function possibility of commensal bacteria acquiring
of L. lactis is to produce lactic acid, which antibiotic resistance genes from their
promotes rennet activity and whey drainage environment and acting as a reservoir of such
and prevents the growth of undesirable determinants (Teuber et al. 1999; European
bacteria (Fox et al. 2000). Lactococci are also Commission, 2005). Resistance could
responsible for flavour formation through their ultimately be transferred to pathogenic species,
proteolytic and amino acid conversion which would hamper the treatment of infections
pathways (Smit et al. 2005). In addition, these (Hamilton-Miller, 2004). Indeed, the transfer of
bacteria have recently found a use in other resistance into food matrices and the GIT of
biotechnological applications, such as the mammals has been shown possible in a

57
number of experiments (Netherwood et al. Warsaw, Poland; and UKU, University of
1999). Kuopio, Kuopio, Finland.
Antibiotic resistant lactococci may be
selected for in environments where they come Bacterial strains, growth media and culture
into contact with antibiotics, such as the udder conditions. Ninety three L. lactis isolates from
of antibiotic-treated cows and, therefore, the industrial starter cultures (28), cows milk (12),
products derived from their raw milk (Bennish, artisanal starter-free cheeses (41), cat (4) and
1999), or in the ponds for fish culturing (Chopra fish (5) intestines, and culture collections (3)
& Roberts, 2001). The need for new were surveyed for antimicrobial resistance. The
lactococcal strains to complement those isolates were geographically well separated, as
currently used as starters (Klijn et al. 1995; they were collected in Spain (31), Denmark
Salama et al. 1995) demands that, as well (15), Belgium (18), Poland (7), Finland (5) and
technological characterization studies, other European countries (17). Most were
antibiotic surveys be performed to avoid isolated in the 1980s and 1990s (73), but
selecting those harbouring acquired, several were collected before the generalized
transferable resistance determinants (Teuber et use of antibiotics (20), the so-called pre-
al. 1999). This need is all the more evident antibiotic era (Teuber et al. 1999).
given that several antibiotic resistance genes Enterococcus faecalis LMG 8222 (=ATTC
have been successfully transferred to L. lactis 29212), Lactococcus lactis LMG 8520, and two
from related bacteria under laboratory well-known laboratory strains (L. lactis MG
conditions (Gasson & Fitzgerald, 1994; Clewell 1363; L. lactis IL 1403), all from the LMG
et al. 1995), and since a multiple resistance collection of the Belgian Co-ordinated
TM
plasmid has been discovered in an isolate from Collections of Microorganisms, BCCM ,
a raw-milk cheese (Perreten et al. 1997). University of Ghent, Belgium, were used as
Antibiotic resistance cut-off values for L. lactis controls throughout the study.
have only been defined for strains used in Cryopreserved cultures of L. lactis strains
animal feed (European Commission, 2005). in glycerol were first recovered on Mueller-
These have been based on antibiotic surveys Hinton agar (Oxoid, Basingstoke, UK). Isolated
using different methodologies (microdilution, colonies were then streaked onto Mueller-
plate dilution, Etest), media and culture Hinton plates and incubated for 16-18 h at
conditions, and sometimes a small number of 28C. The isolates from fish intestines,
related strains (Cogan, 1972; Orberg & however, did not grow well at 28C; these
Sandine, 1985; Elliot & Facklam, 1996; Teuber incubations were therefore performed at 25C.
et al., 1999).
This paper reports the determination of the Classification and characterisation of
MICs of six antibiotics with activity against isolates. The isolates were phenotypically
Gram positive bacteria, inhibiting either the grouped by their carbohydrate fermentation
synthesis of proteins (tetracycline, profiles using the API50 CHL system
erythromycin, clindamycin, streptomycin, and (bioMrieux, Marcy l'Etoile, France) and later
chloramphenicol) or the synthesis of the cell identified by PCR using either universal primers
wall (vancomycin) in a large collection of (Young et al. 1991) or species-specific primers
lactococcal strains. These strains were isolated (Pu et al. 2002), all based on conserved
from different geographical locations over many sequences of 16S rRNA genes. Amplicons
years (from 1950 to the present), and from were then sequenced and the sequences
different dairy products and animal compared to those in public databases.
environments. Genetic fingerprinting was performed by
RAPD-PCR and/or by PFGE as previously
Material and Methods described (Delgado & Mayo, 2004). Clustering
calculations were performed using GelCompar
The following analyses were performed at the II v. 2.5 software (Applied Maths BVBA, Sint-
different laboratories involved in this research: Martens-Latem, Belgium).
CSIC, Instituto de Productos Lcteos de
Asturias (CSIC), Villaviciosa, Spain; CH, Minimum inhibitory concentrations
Christian Hansen A/S, Hrsholm, Denmark; determined by the Etest method. Individual
IBB, Institute of Biochemistry and Biophysics, colonies from the Mueller-Hinton plates were
suspended in a sterile glass or plastic tube

58
containing 2-5 ml of sterile saline (Oxoid) until a Ma., USA) and double-stranded sequenced in
density corresponding to McFarland standard 1 an ABI 370 Genetic Analyzer (Applied
or its spectrophotometric equivalent Biosystems, Warrington, UK). Sequences were
(corresponding to around 3x108 cfu/ml) was then compared to other in the EMBL datalibrary
obtained. by using the online BLASTN resource
A sterile cotton swab was dipped into the (http://www.ncbi.nlm.nih.gov/blast/).
above McFarland suspension and used to
inoculate the Mueller-Hinton agar plates. The
agar surface was then allowed to dry for Results and Discussion
approximately 15 min before applying the Etest
strips (AB Biodisk, Solna, Sweden). Readings All strains grew well on plain Mueller-Hinton
were recorded after 48 h of incubation at 28C without the need for frequent additions of
(or 25C for fish isolates). The MICs for the glucose and yeast extract (Katla et al. 2001) or
bacteriocidal antibiotics (streptomycin, lysed horse blood (Green et al. 1990; Elliot &
chloramphenicol and vancomycin) studied were Facklam, 1996), thus allowing the
taken as the first value on the Etest strips at determination of MICs by the Etest method.
which growth did not occur, as per the Etest modal MIC values of the analysis
manufacturers recommendations. For the performed in the different laboratories (in
bacteriostatic antibiotics (tetracycline, g/ml) for the internal control strain
erythromycin and clindamycin), the MIC was Enterococcus faecalis ATCC 29212 of
taken as the point where growth was tetracycline (8), streptomycin (256),
significantly inhibited (around 80%, as erythromycin (3), clindamycin (16),
estimated by visual inspection). chloramphenicol (8), and vancomycin (2)
compared well to the Etest manufacturer
Measurement of MICs by the standard guidelines.
NCCLS agar dilution method. To check the
performance of the Etest as compared to the MICs of the different antibiotics assayed
standard plate agar dilution technique (CLSI,
2004), the MICs of these antibiotics for 25 In this study, MICs of six antibiotics with action
selected strains were also determined by this on Gram-positive bacteria were evaluated by
last procedure using Mueller-Hinton agar. the Etest method in 93 L. lactis isolates from
different environments, locations and periods of
PCR amplification of tetracycline resistance time. Table 1 shows the range of MICs to the
genes. Total DNA was isolated from antibiotics analysed plus the MICs that inhibited
tetracycline resistant strains by using a the growth of 50% and 90% of the strains.
commercial kit (GenElute bacterial genomic Differences in ranges converged when the
DNA kit; Sigma-Altdrich Co., St. Louis, Mo., MIC50% and MIC90% values recorded at the
USA). DNA dilutions were used as template in different laboratories are compared. Moreover,
PRC reactions with universal primers of differences were always within one or two
ribosomal protection genes (DI, 5- dilutions of the range, the normal variation in
GA(T/C)ACICCIGGICA(T/C)(A/G)TIGA(T/C)TT inter-laboratory assays. Similar differences
-3, and DII, 5- were observed for MICs obtained in the same
GCCCA(A/G)(T/A)IGG(A/G)TTIGGIGGIAC(T/C strains by either Etest or the standard CLSI
)TC-3) (Clermont et al., 1997). Following PCR agar dilution method. Grouping the strains by
conditions as described by Gevers et al. their year of isolation, their geographical origin
(2003), specific primers for tet(K) (TetK-FW1 or their environment of origin appeared to have
5-TTATGGTGGTTGTAGCTAGAAA-3 and no influence on the distribution of the MICs.
TetK-RV1 5- Early antibiotic surveys involving L. lactis
AAAGGGTTAGAAACTCTTGAAA-3), tet(L) showed this species to show little in the way of
(TetL-FW3 5-GYMHYYHVHVHVYSYSYYVV- antibiotic resistance (Cogan, 1972; Reinbold &
3 and TetL-RV3 5- Reddy, 1974; Orberg & Sandine, 1985). The
GTGAAMGRWAGCCCACCTAA-3), and tet(M) present results agree well with those of other
(DI and tetMR, 5- authors who found lactococcal species to be
CACCGAGCAGGGATTTCTCCAC-3) were susceptible to broad and Gram positive
also used. Amplicons obtained were purified spectrum antibiotics and -lactams (Elliot &
using Microcon PCR filters (Millipore, Bedford, Facklam, 1996; Katla et al. 2001; Delgado et al.

59
2002; Flrez et al. 2005). Variations in the MIC addition to antibiotic resistance genes,
for streptomycin similar to those obtained in the differences in the activity of general
present work have been reported by other detoxification systems such as those of multi-
authors (Orberg & Sandine, 1985; Katla et al. drug resistance (MDR) transporters might
2001). In the present work, the standardised contribute towards enhancing the MICs of
protocol probably reduced the differences that otherwise susceptible lactococcal strains
might arise in microdilution and the Etest (Perreten et al. 2001; Putman et al. 2001). The
assays through the use of different culture MICs for 79 lactococci strains showed them to
media (Huys et al. 2002) or inoculum sizes (M. be susceptible to all six antibiotics.
Egervrn & S. Lindgren, unpublished). In

LAB (no. of Antimicrobial agent MIC (g/ml)


isolates) (no. of isolates) Range 50% 90%

CSIC (66) Tetracycline 0.064-96 0.125 0.38


Streptomycin 0.19-32 8 24
Erythromycin 0.023-0.5 0.094 0.25
Clindamycin <0.016-0.5 0.094 0.25
Chloramphenicol 0.5-6 2 6
Vancomycin 0.125-3 0.38 0.5

CH (15) Tetracycline 0.094-0.19 0.125 0.19


Streptomycin 1.5-24 12 24
Erythromycin 0.094-0.19 0.125 0.19
Clindamycin 0.047-0.19 0.094 0.125
Chloramphenicol 2-4 3 3
Vancomycin 0.25-0.5 0.38 0.5

IBB (7) Tetracycline 0.25-256 - -


Streptomycin 6-12 - -
Erythromycin 0.064-0.125 - -
Clindamycin 0.047-0.25 - -
Chloramphenicol 1.5-2 - -
Vancomycin 0.25-0.5 - -

UKU (5) Tetracycline 0.094-96 - -


Streptomycin 96-128 - -
Erythromycin 0.38-0.5 - -
Clindamycin 0.125-0.5 - -
Chloramphenicol 1-2 - -
Vancomycin 0.125-0.25 - -

Total (93) Tetracycline 0.064-256 0.125 0.25


Streptomycin 0.19-128 12 24
Erythromycin 0.023-0.5 0.125 0.38
Clindamycin 0.016-0.5 0.125 0.25
Chloramphenicol 0.5-6 2 4
Vancomycin 0.125-3 0.38 0.5

Abbreviation key of collections and laboratories: CSIC, Instituto de Productos Lcteos de Asturias (CSIC), Villaviciosa, Spain; CH, Christian Hansen A/S, Hrsholm,
Denmark; IBB, Institute of Biochemistry and Biophysics, Warsaw, Poland; and UKU, University of Kuopio, Kuopio, Finland.

50% and 90%, MIC50 and MIC90, respectively.

Too few isolates for percentual MIC determinations

Excluding the six tetracycline resistant strains from IBB.

Table 1.- MICs of the different antibiotics assayed against Lactococcus lactis isolates.

Distribution of MICs among the L. lactis showed bimodal curves. The curves for
strains tetracycline, chloramphenicol and vancomycin
were very narrow, while those for erythromycin,
Figure 1 shows the distributions of the MIC clindamycin and streptomycin were wider. The
values frequencies for the antibiotics over the distribution of some tetracycline and
range of strains. Most frequency distributions streptomycin MICs strongly suggests that
followed a rather normal curve; only certain isolates posses active resistance
tetracycline and streptomycin differed, which mechanisms
.

60
Proposition of new microbiological Liljequist et al. 1997). In the present work,
susceptibility-resistance cut-off values however, a microbiological term more related to
risk assessment is used: the resistance-
The term microbiological breakpoint was susceptibility cut-off value. The cut-off for each
introduced to replace the concept of the clinical antibiotic was considered as the second MIC
breakpoint for the purpose of identifying above the apparently normal range of the MICs
bacterial strains with acquired and potentially (Fig.1).
transferable resistance determinants (Olsson-
Tetracycline Streptomycin

35 35

30 30
25 25
20 20

15 15

10 10

5 5

0 0

<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

Erythromycin Clindamycin

35
Number of strains

35
30 30

25 25

20 20
15 15
10 10
5 5
0 0
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192
Chloramphenicol Vancomycin

35 35

30 30

25 25

20 20

15 15

10 10
5 5
0 0
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

MICs (g/ml)
Figure 1.- Distribution of MICs frequency of the Lactococcus lactis strains analysed for the six antibiotics. Arrows point to the
new cut-off MIC values proposed in this study.

The large number of strains examined allows additives and products or substances used in
the proposition of the resistance-susceptibility animal feed (FEEDAP) (European
cut-offs shown in Table 2, which are based on Commission, 2005), and others, which are also
both the MIC ranges and their unimodal or summarized in the table for comparison. To
bimodal distribution. In the experimental validate the new cut-offs analysis of resistant
conditions of this study, some of the cut-offs strains to all antibiotics will be necessary. In
were quite different to previously published especial, a carefully inspection of the strains
breakpoints, such us those of the European contributing to the higher MIC levels of the
Commission Scientific Panel Committee on curves will be needed to exclude the presence

61
of dedicated (acquired) mechanisms of ribosome protection genes and primers specific
resistance. for tet(K), tet(L), and tetM. An amplification
Strains with a MIC equal to or higher than product of around 1.5-kbp was obtained by
the cut-off values were considered resistant. In using specific primers for tet(M) when DNA
this sense, 11 strains were found resistant to from two resistant strains from a farmhouse
tetracycline (six of which had been selected by cheese (Spain) and a resistant strain from a cat
TM
their tetracycline resistance phenotype after tonsil (BCCM , Belgium) was used as a
screening of a large collection of dairy template. The same gene was also amplified
lactococci for resistance to this antibiotic) (J. from the two tetracycline resistant fish isolates.
Zycka, J. B. Lampkowska & J. Bardowski, The partial sequences of all these genes
unpublished). Tetracyclines continue to be proved to be 100% identical to each other and
important as therapeutic antibiotics, and are also to the tet(M) gene from Tn916 of
still employed in stockbreeding and aquaculture Enterococcus faecalis (Su et al., 1992).
in many countries (Chopra & Roberts, 2001), Further, two different tetracycline resistance
which may favour the selection of tetracycline genes were detected among the Polish
resistant bacteria. Furthermore, five strains resistant strains, a mosaic tet(L/S) gene along
resistant to streptomycin were also with tet(M). This demonstrated the usefulness
encountered among the fish intestinal tract of the tetracycline resistance cut-offs and the
isolates; of these, two proved to be resistance presence of acquired tetracycline resistance
to both tetracycline and streptomycin. determinants among the isolates of this work.
Proposed susceptibility-resistance cut-off MICs The analysis of genes involved in streptomycin
resistance is currently underway.
ANTIBIOTIC (g/ml)
Clinical Conclusion
FEEDAP This work
breakpoints
The Etest is a convenient method that
Tetracycline 8 4 2
accurately determines MICs and suggests the
Streptomycin - 16 64 presence of atypical resistances which may be
encoded by acquired determinants. These
Erythromycin 0.25 4 1 added genes are considered to have the
greatest risk for horizontal dissemination
Clindamycin 1 4 1
(European Commission, 2005). The analysis of
Chloramphenicol 4 8 12 strains from different ecotypes may be useful
for precisely defining the cut-off values
Vancomycin 1 4 6 separating acquired resistances from all other

types, while at the same time revealing


Breakpoints by the Clinical and Laboratory Standards Institute (CLSI,
2004), former NCCLS, as defined for Streptococcus spp. other than acquired resistance that has already spread
Streptococcus pneumoniae.

among the L. lactis populations. Owing to the


FEEDAP, Scientific Panel on additives and products or substances
used in animal feed, Breakpoints as related on a recent report current low incidence of resistance in L. lactis,
(European Commission, 2005).

Breakpoint as defined by the Socit Franaise de Microbiologie


in which multi-resistance is practically absent,
(http://sfm.asso.fr). this species could be useful for studying the
Table 2.- Microbiological susceptibility-resistance cut- acquisition and spread of antibiotic
off values proposed for Lactococcus lactis to the six determinants among commensal organisms. In
antibiotics under assay. this work, a few strains harbouring tetracycline
resistance genes identical to those present in
Antimicrobial resistance genes in L. lactis other bacteria have been identified.

Few antimicrobial resistance determinants Acknowledgements


have been found in strains of L. lactis, although
plasmid-encoded genes have been described This work was supported by an EU project
for tetracycline [tet(S)], macrolides [mdt(A) and within the VI Frame Programme (ACE-ART,
erm(T)], chloramphenicol (cat) and Reference CT-2003-506214). BCCMTM,
streptomycin (str) (Perreten et al. 1997; Raha University of Gent, Gent, Belgium, and EFFCA,
et al. 2002). The tetracycline resistant strains Paris, France, are acknowledged for providing
were screened for tetracycline resistance some of the strains in this work.
determinants by using universal primers for

62
lactic acid bacteria with the agar overlay disc diffusion method.
References Letters in Applied Microbiology 34 402-406.
Katla AK, Kruse H, Johnsen G & Herikstad H 2001 Antimicrobial
Bennish M L 1999 Animals, humans, and antibiotics: implications susceptibility of starter culture bacteria used in Norwegian dairy
of the veterinary use of antibiotics on human health. Advances in products. International Journal of Food Microbiology 67 147-
Pediatric Infection Diseases 14 269-290. 152.
Charteris WP, Kelly PM, Morelli L & Collins JK 2001 Gradient Klijn N, Weerkamp AH & de Vos WM 1995 Detection and
diffusion antibiotic susceptibility testing of potentially probiotic characterization of lactose-utilizing Lactococcus spp. in natural
lactobacilli. Journal of Food Protection 64 2007-2014. ecosystems. Applied and Environmental Microbiology 61 788-
Chopra I Roberts M 2001 Tetracycline antibiotics: mode of action, 792.
applications, molecular biology, and epidemiology of bacterial Mundt JO 1986 Lactic acid streptococci. Bergeys Manual of
resistance. Microbiology and Molecular Biology Reviews 66 Systematic Bacteriology, Vol. 2 (Sneath PHA, Mair NS, Sharpe
232-260. ME & Holt JG, eds), pp. 1065-1068. Williams and Wilkins,
Clermont D, Chesneau O, De Cespds G & Horaud T 1997 New Baltimore, USA.
tetracycline resistance determinants coding for ribosomal Netherwood T, Bowden R, Harrosin P, ODonnel AG, Parker DS
protection in streptococci and nucleotide sequence of tet(T) & Gilbert HJ 1999 Gene transfer in the gastrointestinal tract.
isolated from Streptococcus pyogenes A498. Antimicrobial Applied and Environmental Microbiology 65 5139-5141.
Agents and Chemotherapy 41 112-116. Nouaille S, Ribeiro LA, Myoshi A, Pontes D, Le Loir Y, Oliveira
Clewell DB, Flannagan SE & Jaworsky DD 1995 Unconstrained SC, Langella P & Azevedo V 2003 Heterologous protein
bacterial promiscuity: the Tn916-Tn1545 family of conjugative production and delivery systems for Lactococcus lactis.
transposon. Trends in Microbiology 3 229-236. Genetics and Molecular Research 2 102-111.
CLSI (Clinical and Laboratory Standards Institute) 2004 Olsson-Liljequist B, Larsson P, Walder M & Miorner H 1997
Methods for Antimicrobial Susceptibility Testing of Anaerobic Antimicrobial susceptibility testing in Sweden. III. Methodology
Bacteria: Approved Standard-six edition. M11-A6, Vol. 24, n 2. for susceptibility testing. Scand. Journal of Infectious Diseases
Wayne, Pennsylvania, USA. Supplement 105 13-23.
Cogan TM 1972 Susceptibility of cheese and yogurt starter bacteria Orberg PK & Sandine WE 1985 Survey of antimicrobial resistance
to antibiotics. Applied Microbiology 23 960-965. in lactic streptococci. Applied and Environmental Microbiology
Delgado S, Delgado T & Mayo B 2002 Technological performance 49 538-542.
of several Lactococcus and Enterococcus strains of dairy origin Perreten V, Schwarz F, Cresta L, Boeglin M, Dasen G & Teuber
in milk. Journal of Food Protection 65 1590-1596. M 1997 Antibiotic resistance spread in food. Nature 389 801-
Delgado S & Mayo, B 2004 Phenotypic and genetic diversity of 802.
Lactococcus lactis and Enterococcus spp. strains isolated from Perreten V, Schwarz FV, Teuber M & Levy SB 2001 Mdt(A), a
Northern Spain starter-free farmhouse cheeses. International new efflux protein conferring multiple antibiotic resistance in
Journal of Food Microbiology 90 309-319. Lactococcus lactis and Escherichia coli. Antimicrobial Agents
Elliot JA & Facklam RR 1996 Antimicrobial susceptibilities of and Chemotheraphy 45 1109-1114.
Lactococcus lactis and Lactococcus garviae and a proposed Pu ZY, Dobos M, Liwsowtin GK & Pobell IB 2002 Integrated
method to discriminate between them. Journal of Clinical polymerase chain reaction-based procedures for the detection
Microbiology 34 1296-1298. and identification of species and subspecies of the Gram-
European Commission 2005 Opinion of the Scientific Panel on positive bacterial genus Lactococcus. Journal of Applied
Additives and Products or Substances used in Animal Feed Microbiology 93 353-361.
(FEEDAP) on the updating of the criteria used in the Putman M, van Veen HW, Degener JE & Konings WN 2001 The
assessment of bacteria for resistance to antibiotics of human lactococcal secondary multidrug transporter LmrP confers
and veterinary importance. The EFSA Journal 223 1-12. resistance to lincosamides, macrolides, streptogramins and
Flrez AB, Delgado S & Mayo B 2005 Antimicrobial susceptibility tetracyclines. Microbiology 147 2873-2880.
of lactic acid bacteria isolated from a cheese environment. Raha AR, Ross E, Yusoff K, Manap MY & Ideris A 2002
Canadian Journal of Microbiology 51 51-58. Characterisation and molecular cloning of an erythromycin
Fox PF, Guinee TP, Cogan TM & McSweeney PLH 2000 resistance plasmid of Lactococcus lactis isolated from chicken
Fundamentals of Cheese Science. AN Aspen Publishers Inc., cecum. Journal of Biochemistry, Molecular Biology and
Gaithersburg, Ma., USA. Biophysics 6 7-11.
Gasson MJ & Fitzgerald GF 1994 Gene transfer systems and Reinbold GW & Reddy MS 1974 Sensitivity or resistance of dairy
transposition. Genetics and Biotechnology of Lactic Acid starter and associated microorganisms to selected antibiotics.
Bacteria, (Gasson MJ & de Vos WM, eds), pp. 1-51. Blackie Journal of Milk and Food Technology 37 517-521.
Academic and Professional, London. Ringo E 2004 Lactic acid bacteria in fish and fish farming. Lactic
Gevers D, Danielsen M, Huys G & Swings J 2003 Molecular Acid Bacteria. Microbiological and Functional Aspects
characterization of tet(M) genes in Lactobacillus isolates from (Salminen S, von Wright A & Ouwehand A, eds), pp.581-610.
different types of fermented dry sausage. Applied and Marcel Decker Inc., Ney York, USA.
Environmental Microbiology 69 1270-1275. Salama MS, Musafa-Jeknic T, Sandine WE & Giovannioni SJ
Green M, Wadowsky RM & Barbadora K 1990 Recovery of 1995 An ecological study of lactic acid bacteria: isolation of new
vancomycin-resistant Gram-positive cocci from children. Journal strains of Lactococcus including Lactococcus lactis subspecies
of Clinical Microbiology 28 484-488. cremoris. Journal of Dairy Science 78 1004-1017.
Guarner F & Malagelada JR 2003 Gut flora in health and disease. Smit G, Smit BA & Engels WJM 2005 Flavour formation by lactic
Lancet 360 512-519. acid bacteria and biochemical flavours profiling of cheese
Hagi T, Tanaka D, Iwamura Y & Hoshimo T 2004 Diversity and products. FEMS Microbiology Reviews 29 591-610.
seasonal changes in lactic acid bacteria in the intestinal tract of Su YA, He P & Clewell DB 1992. Characterization of the tet(M)
cultured freshwater fish. Aquaculture 234 335-346. determinant of Tn916: evidence for regulation by transcription
Hamilton-Miller JM 2004 Antibiotic resistance from two attenuation. Antimicrobials Agents and Chemotheraphy 36 769-
perspectives: man and microbe. International Journal of 778.
Antimicrobial Agents 23 209-212. Teuber M, Meile L & Schwarz F 1999 Acquired antibiotic
Hols P, Kleerebezem M, Schanck AN, Ferain T, Hugenholtz J, resistance in lactic acid bacteria from food. Antonie van
Delcour J & de Vos WM 1999 Conversion of Lactococcus Leeuwenhoek 76 115-137.
lactis from homolactic to homoalanine fermentation through Young JPW, Downer HL & Eardly BD 1991 Phylogeny of the
metabolic engineering. Nature Biotechnology 17 588-592. prototrophic Rhizobium strain BTAil by polymerase chain
Huys G, DHaene K & Swings J 2002 Influence of the culture reaction-based sequencing of a 16S rRNA segment. Journal of
medium on antibiotic susceptibility testing of food-associated Bacteriology 173 2271-2277.

63
Susceptibility of Lactobacillus plantarum strains to six antibiotics and

definition of new susceptibility-resistance cut-off values

Abstract

The minimum inhibitory concentrations (MICs) of six antibiotics with activity against
Gram positive bacteria (ampicillin, clindamycin, erythromycin, gentamicin, streptomycin,
and tetracycline) were determined by microdilution and the Etest in 121 Lactobacillus
plantarum strains of plant and dairy origin. MIC values for all antibiotics varied widely
between strains. The analysis of both absolute MICs and their distribution was used to
define new susceptibility-resistance cut-off values for all antibiotics, except for
streptomycin. Based on these new cut-offs, the studied strains were nearly all identified as
either susceptible (ampicillin, clindamycin, erythromycin, and gentamicin) or intrinsically
resistant (streptomycin). The exceptions were four strains with MICs for tetracycline higher
than the cut-off point (64 g mL-1); these were suspected to harbour acquired resistance
determinants.

Introduction e.g., the Etest (Charteris et al., 2001; Danielsen


& Wind, 2003), disk diffusion (Charteris et al.,
Lactobacillus plantarum is a lactic acid 1998), and microdilution (Flrez et al., 2005)
bacterium (LAB) able to colonise habitats as are a source of confusion since they cannot be
diverse as fresh and fermented plant materials, directly compared. In addition, variations in the
meat products, fish, dairy, sourdoughs, cation content and the concentration of thymine
fermented beverages, and the gastrointestinal or other critical compounds in the test medium
tract of animals and humans (Vescovo et al., can further influence antibiotic susceptibility
1993). Strains of this species are increasingly results (Huys et al., 2002; Danielsen et al.,
used as a starter for the fermentation of feed 2004; Klare et al., 2005). Even the inoculum
and foods of plant origin, but also as a silage size and the temperature and incubation period
inoculant and as a human probiotic (Caplice & can be the source of differences.
Fitzgerald, 1999; Haller et al., 2001). This paper reports the MICs of six
At present there is a concern that microbial antibiotics for 121 L. plantarum strains from
cultures used for food production or naturally different geographical locations and fermented
occurring in food can be vehicles for products. MICs were determined by the Etest
transmission of antibiotic resistance genes and microdilution methods. Analyses were
(Teuber et al., 1999; European Commission, conducted at different laboratories following
2005). Owing to their ubiquitous nature, L. standardised protocols, thus allowing a
plantarum strains may face antimicrobial comparison between methods and the
selection in numerous habitats (e.g., in definition of new susceptibility-resistance cut-
animals, consumers or the environment), off values for this species.
potentially allowing their acquired resistance
determinants to be passed to pathogens. Material and methods
Antibiotic resistance cut-off values for L.
plantarum have only been defined for strains Bacterial strains, growth media and culture
used in animal feed (European Commission, conditions. One hundred and twenty one L.
2005). This renders distinguishing between plantarum isolates from different dairy (n=28)
intrinsic, non-specific, and acquired resistance and plant fermented products (n=93), isolated
rather difficult. The results provided by the between 1981 and the present, were tested for
different methods used in antibiotic surveys, their susceptibility to six antibiotics. Forty five of

65
these strains were provided by the NFA control, a tube of non-inoculated test medium
(National Food Administration, Sweden), 28 was incubated under the same conditions.
came from the IPLA-CSIC (Instituto de
Productos Lcteos de Asturias-CSIC, Results
Villaviciosa, Spain), 27 from the UCSC
(Universit Cattolicca del Sacro Cuore, The susceptibility of 121 L. plantarum
Piacenza, Italy), and 21 from Chr. Hansen strains to ampicillin, clindamycin, erythromycin,
(Chr. Hansen A/S, Hrsholm, Denmark). gentamicin, streptomycin, and tetracycline was
assayed by both Etest and broth microdilution
Susceptibility testing using Etests. on LSM medium. Table 1 shows the range of
Determination of MICs by the Etest method MICs of the six antibiotics obtained by both the
was performed according to the preparation of Etest and broth microdilution methods. Etest
the samples and reading conditions MICs falling between two-fold dilution steps
recommended by the supplier (AB Biodisk, were rounded to the higher concentration on
Solna, Sweden). In short, individual colonies Table 1.MICs of several antibiotics for some
were suspended in a sterile 0.85%-ClNa isolates were identical with both methods.
solution (Oxoid, Basinsgtoke, Hampshire, UK) Although this, MICs determined by
until a density corresponding to McFarland microdilution were higher for tetracycline,
standard 1 (3x108 cfu mL-1) was obtained. A streptomycin and gentamicin. The contrary was
sterile cotton swab was dipped into the seen for erythromycin, whose Etest MICs
-1
suspension and used to inoculate LSM agar showed a wider range (0.064-2 g mL ) than
plates (90% IsoSensitest, 10% MRS, both from that obtained by microdilution (0.25-1 g mL-1).
Oxoid), a newly defined medium which has The MICs obtained for the same strains in
been recently proposed for susceptibility testing different laboratories by either of the two
of lactic acid bacteria (Klare et al., 2005). The methods were always within two dilutions of the
agar surface was allowed to dry for range, as were those obtained for repeat tests
approximately 15 min before applying the Etest of a single strain (data not shown). In particular,
strips. Readings were recorded after 48 h MIC results agree well for the E. faecalis and L.
incubation at 28C. plantarum internal control strains. Table1).
All strains were tested in their parent Distribution of the MICs obtained by the
laboratory, but several exchanges were made Etest method is graphically presented in Figure
to assess inter-laboratory reproducibility. 1. Statistical unimodal curves could be
Lactobacillus plantarum ATCC 14917T (= LMG observed for MICs of all antibiotics, except for
6907T) and Enterococcus faecalis ATCC MIC values of clindamycin, which followed a
29212 (= LMG 8222) were included as control distribution with no clear maximum (also
strains in every susceptibility assay. obtained by broth microdilution; Table 1), and
for MICs of tetracycline, of which four strains
Susceptibility testing by microdilution. clearly deviated from the majority. Grouping of
Determination of the MICs by the microdilution the strains by year of isolation, geographical
method was accomplished using custom-made origin, or the sample type from which they were
plates (VetMICTM, National Veterinary Institute isolated had no influence on MIC distribution
of Sweden, Uppsala, Sweden) containing serial (data not shown).
two-fold dilutions of the selected antibiotics.
Cells suspensions to McFarland standard 1 Based on both the Etest and microdilution
prepared as above were diluted 1:1000 with results and their distribution, new susceptibility-
LSM broth for the inoculation of the VetMICTM resistance cut-off values for five antibiotics
plates (final concentration 3x105 cfu mL-1). One were defined (Table 2). Compared to the
hundred microlitres of this inoculum were microbiological breakpoints recently proposed
added to each well within 30 min of by the FEEDAP Committee (European
preparation, and incubated at 28C for 48 h. Commission, 2005), our values are equal for
Bacterial growth was detected as a pellet in the ampicillin and erythromycin, higher for
bottom of the well. The MIC was defined as the clindamycin and tetracycline, and lower for
lowest antibiotic concentration at which no gentamicin.
visual growth was observed. As a negative

66
N of
Distribution of MICs (g mL-1)
Antibiotic Method strains
0.12 0.25 0.5 1 2 4 8 16 32 64 128 256

Etest 3 39 19 3 2 66
Ampicillin
Microdilution 6 12 27 7 2 54

Etest 15 15 7 13 11 4 8 73
Clindamycin
Microdilution 10 3 13 9 19 54

Etest 7 18 38 42 16 121
Erythromycin
Microdilution 4 31 46 81

Etest 3 3 2 13 31 14 66
Gentamicin
Microdilution 5 9 15 19 6

Etest 1 2 7 20 18 27 31 6 121
Streptomycin
Microdilution 3 13 24 31 6 81

Etest 1 13 44 51 8 1 3 121
Tetracycline
Microdilution 54 24 1 2 81

-1
Etest/Microdilution modal MIC values (in g mL ) for the internal control strain Enterococcus faecalis ATCC 29212 were ampicillin (0.5/0.5), clindamycin (16/>8), erythromycin (3/4), gentamicin (16/32),
T
streptomycin (256/256) and tetracycline (8/16), and those for Lactobacillus plantarum ATCC 14917 were ampicillin (0.25/0.5), clindamycin (1/1), erythromycin (1/0.5), gentamicin (8/4), streptomycin
(32/32) and tetracycline (8/16)

Table 1.- Distribution of MICs for Lactobacillus plantarum strains to six antibiotics by Etest and broth microdilution.

Discussion Proposed susceptibility-resistance cut-off MICs (g mL-1) and number of resistant strains
Antibiotic
No. of resistant strains No. of resistant strains
There have been few studies on antibiotic This work FEEDAPa
using this works cut-offs using FEEDAPs cut-offs
susceptibility-resistance in L. plantarum
involving large numbers of strains (Felten et al., Ampicillin 4 0 4 0

1999; Zarazaga et al., 1999; Danielsen & Wind, Clindamycin 16 0 4 12 (10%)


2003; Flrez et al., 2005). A majority of strains
Erythromycin 4 0 4 0
has been found susceptible to most antibiotics
except for vancomycin, cefoxitin and Gentamicin 16 0 64 0
ciprofloxacin, to which intrinsic resistance may
Streptomycin - ? 64 73 (60.3%)
exist. In spite of this, antibiotic-resistant strains
from different fermented products have been Tetracycline 64 4 (3.3%) 32 14 (11.6%)
identified (Jewell & Collins-Thompson, 1989; a
Breakpoints by the FEEDAP Committee as recently reported (European Commission, 2005).
Gevers et al., 2000). Further, several genetic
Table 2.- Susceptibility-resistance cut-off values proposed in this work for
determinants encoding resistance to Lactobacillus plantarum and comparison to those defined by the FEEDAP
chloramphenicol (Jewell & Collins-Thompson, Committee, and estimated number of resistant strains using each of the cut-
offs.
1989) or tetracycline (Danielsen, 2002; Gevers
et al., 2003) have already been characterised. The distinction between natural and
acquired determinants is of great importance,
In the present work, the MICs of six since only the latter have a serious chance of
antibiotics were evaluated in a series of 121 being transferred (European Commission,
strains from different origins. MICs obtained by 2005). Analysis of the MIC distributions could
either of the two methods were equal or similar help to differentiate between intrinsic and
for most strains. Differences between the two acquired resistance, since the distribution of
methods were of 1 log2 dilution, which is the MICs for a number of strains in the absence
thought to be the normal standard deviation of of acquired mechanisms approaches a
MIC dilution tests (Murray et al., 2003; CLSI, statistical normal curve (Murray et al., 2003).
2004). Similar results for both the absolute MIC The distribution of MICs for streptomycin
values and MIC ranges for this species have indicates that most strains are able to resist
been reported by other authors (Zarazaga et antibiotic concentrations of 64 g mL or
-1

al., 1999; Danielsen & Wind, 2003). higher. More assays with concentrations above
256 g mL-1 will therefore be needed to define
a clear cut-off for this antibiotic. The wide

67
distribution of MICs for clindamycin should also those defined by FEEDAP (Table 2). Strains
be further investigated. with a MIC equal to or higher than the cut-off
were considered resistant. Consequently, only
In this study, the cut-off for each antibiotic four strains showing a tetracycline MIC of 128
was a critical value defined as the second MIC g mL-1 were suspected to possess acquired
above the apparent normal range (see arrows resistance to this antibiotic. This small
in Figure 1). The large number of strains percentage contrasts with the high number of
examined allowed the proposition of new resistant strains if FEEDAPs microbiological
susceptibility-resistance cut-offs for these six breakpoints were used (Table 2).
antibiotics in L. plantarum, which for
tetracycline and clindamycin were higher than
Tetracycline Streptomycin

35 35
30 30
25 25
20 20
15 15
10 10
5 5
0 0
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192
Erythromycin Clindamycin

35 35
30 30
Number of strains

25 25

20 20

15 15

10 10

5 5

0 0
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

Ampicillin Gentamicin

35 35

30 30
25 25

20 20

15 15

10 10

5 5

0 0
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192
<0.016
0.023
0.047
0.094
0.19
0.38
0.75
1.5
3
6
12
24
48
96
192

MICs (g/ml)
Figure 1.- Distribution of MICs among the Lactobacillus plantarum strains for the six antibiotics in this study. The real Etest MIC distribution values are indicated, while
data were grouped in classes in Table 1. Arrowheads point to the susceptibility-resistance cut-off MIC values proposed in this work.

Representative strains are currently been precisely defining the cut-off values separating
examined for antibiotic resistance determinants acquired resistances from all other types, while
by gene-specific PCR and DNA microarray revealing acquired resistances that have
hibridization. already spread among populations. By the use
As a conclusion, the analysis of strains of a standardized protocol, thus reducing
from different ecotypes may be useful for differences attributable to the culture medium

68
or inoculums size, and the analysis of a large Felten, A., C. Barreau, C. Bizet, P. H. Lagrange, and A.
Philippon. 1999. Lactobacillus species identification, H2O2
series of L. plantarum strains, new cut-offs for production, and antibiotic resistance and correlation with human
five antibiotics were defined in this species. clinical status. J. Clin. Microbiol. 37:729-733.
Flrez, A. B., S. Delgado, and B. Mayo. 2005. Antimicrobial
susceptibility of lactic acid bacteria isolated from a cheese
Acknowledgements environment. Can. J. Microbiol. 51:51-8.
Gevers, D., M. Danielsen, G. Huys, and J. Swings. 2003.
Molecular characterization of tet(M) genes in Lactobacillus isolates
This work was supported by an EU project from different types of fermented dry sausage. Appl. Environ.
Microbiol. 69:1270-1275.
within the VI Frame Program (ACE-ART, Gevers, D., G. Huys, F. Devlieghere, M. Uyttendaele, J.
Reference 506214). BCCMTM, University of Debevere, and J. Swings. 2000. Isolation and identification of
Gent, Belgium, and EFFCA, Paris, France, are tetracycline resistant lactic acid bacteria from pre-packed sliced
meat products. Syst. Appl. Microbiol. 23:279-284.
acknowledged for providing some of the strains Haller, D., H. Colbus, M. G. Ganzle, P. Scherenbacher, C. Bode,
in this work. and W. P. Hammes. 2001. Metabolic and functional properties of
lactic acid bacteria in the gastro-intestinal ecosystem: a
comparative in vitro study between bacteria of intestinal and
References fermented food origin. Syst. Appl. Microbiol. 24:218-226.
Huys, G., K. DHaene, and J. Swings. 2002. Influence of the
culture medium on antibiotic susceptibility testing of food-associated
Caplice, E., and G. F. Fitzgerald. 1999. Food fermentations: role lactic acid bacteria with the agar overlay disc diffusion method. Lett.
of microorganisms in food production and preservation. Int. J. Food Appl. Microbiol. 34:402-406.
Microbiol. 50:131-49. Jewell, B., and D. L. Collins-Thompson. 1989. Characterization of
Charteris, W.P., P. M. Kelly, L. Morelli, and J. K. Collins. 1998. chloramphenicol resistance in Lactobacillus plantarum caTC2. Curr.
Antibiotic susceptibility of potentially probiotic Lactobacillus species. Microbiol. 19:343-346.
J. Food Prot. 61:1636-1643. Klare, I., C. Konstabel, S. Mller-Bertling, R. Reissbrodt, G.
Charteris, W.P., P. M. Kelly, L. Morelli, and J. K. Collins. 2001. Huys, M. Vancanneyt, J. Swings, H. Goossens, and W. Witte.
Gradient diffusion antibiotic susceptibility testing of potentially 2005. Evaluation of new broth media for microdilution antibiotic
probiotic lactobacilli. J. Food Prot. 64:2007-2014. susceptibility testing of lactobacilli, lactococci, pediococci, and
CLSI (Clinical and Laboratory Standards Institute) (former bifidobacteria. Appl. Environ. Microbiol. 71:8982-8986.
NCCLS). 2004. Methods for Antimicrobial Susceptibility Testing of Murray, P. R., E. J. Baron, J. H. Jorgensen, M. A. Pfaller, and R.
Anaerobic Bacteria: Approved Standard, 6th ed. M11-A6, Vol. 24, H. Yolken. 2003. Manual of Clinical Microbiology. American Society
No. 2. Paikka, USA. for Microbiology, Washington DC.
Danielsen, M. 2002. Characterization of the tetracycline resistance Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic
plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a resistance in lactic acid bacteria from food. Antonie van
composite structure. Plasmid 48:98-103. Leeuwenhoek 76:115-137.
Danielsen, M., and A. Wind. 2003. Susceptibility of Lactobacillus Vescovo, M., S. Torriani, F. Dellaglio, and V. Bottazzi. 1993.
spp. to antimicrobial agents. Int. J. Food Microbiol. 82:1-11. Basic characteristics, ecology and application of Lactobacillus
Danielsen, M., H. S. Andersen, and A. Wind. 2004. Use of folic plantarum: a review. Ann. Microbiol. Enzimol. 43:261-284.
acid casei medium reveals trimethoprim susceptibility of Zarazaga, M., Y. Senz, A. Portillo, C. Tenorio, F. Ruiz-Larrea,
Lactobacillus species. Lett. Appl. Microbiol. 38:206-210. R. del Campo, F. Baquero, and C. Torres. 1999. In vitro activities
European Commission. 2005. Opinion of the Scientific Panel on of ketolide HMR3647, macrolides, and other antibiotics against
Additives and Products or Substances used in Animal Feed on the Lactobacillus, Leuconostoc, and Pediococcus isolates. Antimicrob.
updating of the criteria used in the assessment of bacteria for Agents Chemother. 43:3039-41.
resistance to antibiotics of human and veterinary importance. The
EFSA J. 223:1-12.

69
Captulo III.- Caracterizacin molecular de la resistencia adquirida a
antibiticos en BAL y bifidobacterias
Artculo VI.- Molecular characterization of specific and non-especific antibiotic resistance
in lactic acid bacteria and bifidobacteria.
Journal of Molecular Microbiology and Biotechnology (Aceptado).

Objetivo: El objetivo del trabajo fue estudiar los patrones generales de resistencia a
antibiticos en una gran coleccin de BAL y bifidobacterias y la caracterizacin de los
determinantes genticos implicados en la resistencia, hacer una primera estimacin del
papel de los transportadores MDR en la resistencia.

Resultados: Las CIMs obtenidas para la mayora de los antibiticos muestran una
distribucin unimodal, tratndose por tanto de cepas susceptibles o con resistencias
intrnsecas. Sin embargo, la distribucin bimodal para alguno de los antibiticos
sugiere la presencia de resistencias adquiridas. La bsqueda de los genes de resistencia
se llev a cabo mediante PCR e hibridacin de microarrays. Con estos mtodos se
detectaron en algunas cepas genes que confieren resistencia a tetraciclina [tet(M),
tet(W), tet(O) y tet(O/W)], a eritromicina y clindamicina [erm(B) y mef(A)] y a
estreptomicina [aph(E) y sat(3)]. Los determinantes genticos se localizaron en todos los
casos en el cromosoma bacteriano, a excepcin del gen tet(M) que pareca codificado en
un plsmido de Lc. lactis. La clonacin y expresin de dos transportadores MDR de
Bifidobacterium breve en Lc. lactis incrementa la resistencia de esta especie a macrlidos,
aminoglicsidos, polimixina B y nisina.

Conclusin: La resistencia a antibiticos en las BAL y bifidobacterias no es comn, sin


embargo, se encontraron varias cepas resistentes. En muchas cepas resistentes se
comprob la presencia de genes especficos de resistencia, localizados
mayoritariamente en el cromosoma. Adems, los sistemas inespecficos estudiados en
este trabajo podran contribuir a una mayor tolerancia a los antibiticos.

73
Molecular characterization of specific and non-specific antibiotic
resistance in lactic acid bacteria and bifidobacteria

Abstract

The minimum inhibitory concentrations (MICs) of six different antibiotics


(chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and
vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria
using the E-test. Different MICs were found for different species and strains. Based on
the distribution of these MIC values most of the strains were either susceptible or
intrinsically resistant to these antibiotics, however, the MIC range of some of these
antibiotics showed a bimodal distribution. This type of distribution suggests that some
of the tested strains possess acquired antibiotic resistance. Screening for resistance
genes was performed by PCR using specific primers, or using a DNA microarray with
around 300 nucleotide probes representing seven classes of antibiotic resistance genes.
The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O), and
tet(O/W)], erythromycin and clindamycin [erm(B) and mef(A)], and streptomycin [aph(E)
and sat(3)]. Internal portions of some of these determinants were sequenced and found
to be identical to genes described in other bacteria from the same environments. All
resistance determinants were located on the bacterial chromosome, except for tet(M)
which was identified on plasmids in Lactococcus lactis. The contribution of non-specific
multidrug transporters to the level of antibiotic resistance was investigated by cloning
and measuring the expression of Bifidobacterium breve genes in L. lactis.

Introduction products that are not heat-treated before


consumption provide a vehicle for
The resistance of bacteria to antibiotics is transmission from the indigenous microbiota
an increasingly important public health of animals to the bacteria of the human
problem worldwide. There is a pressing need gastrointestinal tract (GIT) [Bates et al., 1994;
to limit the spread of resistance genes since Nikolich et al., 1994].
these could be transferred to opportunistic Either added as a starter or present in
and pathogenic bacteria [Blzquez et al., raw materials, many lactic acid bacteria
2002]. Antibiotic resistance can be intrinsic (LAB) species participate in the manufacture
or acquired [European Commission, 2005]. and preservation of fermented foods and feed
Intrinsic or natural resistance is that products. LAB are also commonly found,
inherent to a bacterial species and involves together with bifidobacteria, LAB&B, among
the absence of the target or the presence of the resident microbiota of the GIT of humans
low affinity targets, low cell permeability, or and animals. LAB&B have considerable
the presence of efflux mechanisms. The potential as probiotics based on their long
acquisition of antibiotic resistance occurs via history of safe use and a growing body of
the mutation of pre-existing genes or by evidence supporting their positive health-
horizontal transmission. With some promoting effects [Ouwehand et al., 2002].
exceptions, intrinsic resistance and However, attention is currently being paid to
resistance by mutation are unlikely to be commensal LAB&B with respect to their
disseminated; horizontally transferred genes, potential role in the spread and transmission
particularly those carried on mobile genetic of antibiotic resistance determinants in food
elements, are those most likely to be matrices and the GIT [Teuber et al., 1999].
transmitted [Normark and Normark, 2002]. This interest is strengthened by the fact that a
The food chain has been recognized as large number of these bacteria are
one of the main routes of transmission of extensively used as functional
antibiotic resistance from animal to human microorganisms. Further, the number of
bacterial populations [Witte, 2000; Teuber et papers reporting the isolation of antibiotic-
al., 1999]. More specifically, fermented resistant LAB&B strains is increasing [Ahn et

75
al., 1992; Tannock et al., 1994; Fons et al., published elsewhere [Zarazaga et al., 1999;
1997; Perreten et al., 1997; Scott et al., 2000; Danielsen and Wind, 2003; Flrez et al.,
Danielsen, 2002; Scott et al., 2002]. 2005; Delgado et al., 2005; Zhou et al.,
Therefore, there is a need to establish clear 2005]. With respect to the CLSI (formerly
cut-off values for separating susceptible and NCCSL) clinical breakpoint of this drug (32
resistant bacteria, to distinguish between g mL-1) [CLSI, 2004], none of the strains
intrinsic and acquired forms of resistance, could be considered resistant. However,
and to study the molecular mechanisms according to the microbiological breakpoints
responsible for the spread of resistance of the FEEDAP Panel Report [European
among the LAB&B community. Commission, 2005], and those of CLSI [CLSI,
The aim of this study was to assess the 2004], at least four lactobacilli could be thus
antibiotic resistance patterns of a large considered (one strain each of Lb. plantarum,
collection of LAB&B strains from dairy and Lb. johnsonii, Lb. rhamnosus, and Lb.
intestinal sources, to characterize the genetic vaginalis). In spite of this, the unimodal
determinants responsible for the resistances distribution of the MICs does not support the
and to estimate their transmission capability. presence of acquired resistance.
The role of multidrug resistance (MDR) The MICs of erythromycin and
transporters in antibiotic resistance was also clindamycin for most of the LAB&B strains
addressed. assayed (Table 1) were within the normal
range of susceptibility [CLSI, 2004].
Results and Discussions Variations in the MICs of erythromycin and
clindamycin similar to those obtained in the
MICs of different antibiotics for different present work have been reported by other
LAB&B strains. Tables 1 and 2 show the authors [Danielsen and Wind 2003; Delgado
MIC ranges of six antibiotics for 143 LAB and et al., 2005]. Moderate levels of clindamycin
BB strains isolated from different resistance (8-32 g mL-1) have previously
environments when clustered into seven been observed in Lb. gasseri [Danielsen and
groups: Lactococcus lactis (50), Lactobacillus Wind, 2003]. Based on these results, the
plantarum (29), Lb. acidophilus-Lb. raising of the cut-off value for clindamycin in
delbrueckii (1 Lb. acidophilus, 1 Lb. this species should be considered. However,
amylovorus, 7 Lb. delbrueckii, 3 Lb. gasseri, the bimodal distribution of MICs strongly
and 2 Lb. johnsoni), other lactobacilli (3 Lb. suggests that certain intestinal isolates
brevis, 1 Lb. casei, 1 Lb. fermentum, 1 Lb. possess acquired resistance mechanisms.
helveticus, 2 Lb. paracasei, 1 Lb. pentosus, 1 Resistance to both antibiotics was usually
Lb. reuteri, 3 Lb. rhamnosus, 1 Lb. sakei, and observed in the same strain, suggesting a
1 Lb. vaginalis), Bifidobacterium longum (17 common resistance mechanism (that
B. longum), B. bifidum (6), and other provided by the macrolide-lincosamide-
bifidobacteria (1 B. adolescentis, 2 B. streptogramin B [MLS] phenotype).
animalis, 1 B. breve, 4 B. The LAB&B strains showed a wide range
pseudocatenolatum, 2 B. pseudolongum, and of streptomycin MICs (2->256 g mL-1) (Table
1 B. thermophilus). 2), although the same as that reported by
The distinction between natural and other authors [Delgado et al., 2005; Katla et
acquired resistance is of great importance al., 2001]. However, clear cut-off MICs for the
since only the latter has a serious chance of LAB and BB strains is still to be defined.
being transferred [European-Commission, Indeed, the highest MICs for this
2005]. Analysis of MICs and their aminoglycoside were moderate compared to
distributions helps differentiate between the high NCCLS breakpoint for enterococci
these two resistance mechanisms. The MIC (1024 g mL-1) [CLSI, 2004]. All
distribution of a given antibiotic for a single Bifidobacterium bifidum isolates showed a
bacterial species in the absence of resistance streptomycin MIC of >256 g mL-1. Such
mechanisms should approach statistical resistance might be intrinsic in this species.
normality [Murray et al., 2003]. Figure 1 In addition, several other strains showing a
shows the MIC distributions of erythromycin MIC higher than 256 g mL-1 (Table 2) were
(normal distribution) and tetracycline (bimodal suspected of possessing acquired resistance
distribution, thus suggesting the possession to this drug (two strains of B.
of acquired resistance) for the Lc. lactis pseudocatenolatum, two of Lb. rhamnosus,
strains. and one strain each of B. longum, B.
Both the LAB&B were susceptible to pseudolongum subsp. globosum and B.
chloramphenicol (MIC range 0.032-12 g mL- thermophilus).
1
; Table 1). These results agree with data

76
-1
a No. of No. of isolates with the following MICs (g mL )
Antibiotic Species
strains <0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256

Cm Lc. lactis 50 3 30 12 5
Lb. plantarum 29 6 16 6 1
Lb. acidophilus/Lb. delbrueckii 15 1 1 5 7 1
Other lactobacilli 15 4 4 5 2
B. longum 17 2 10 5
B. bifidum 6 2 4
Other bifidobacteria 11 3 6 2

Ery Lc. lactis 50 3 10 30 4 3


Lb. plantarum 29 2 11 13 2 1
Lb. acidophilus/Lb. delbrueckii 15 3 2 2 6 1 1

77
Other lactobacilli 15 3 4 2 2 2 2
B. longum 17 1 2 6 1 2 5
B. bifidum 6 1 3 2
Other bifidobacteria 11 1 1 6 2 1

Clin Lc. lactis 50 4 10 14 18 4


Lb. plantarum 29 4 2 5 3 6 7 2
Lb. acidophilus/Lb. delbrueckii 15 4 1 2 1 1 2 2 1 1
Other lactobacilli 15 2 1 4 2 1 2 1 1 1
B. longum 17 3 2 1 4 7
B. bifidum 6 2 2 2
Other bifidobacteria 11 4 4 1 1 1

a
Cm, chloramphenicol; Ery, erythomycin; Clin, clindamycin

Table 1. Distribution of MICs of chloramphenicol, clindamycin and erythromycin (antibiotics that inhibit protein synthesis; target 50S ribosomal subunit) for LAB&B species from
different environments.
-1
a No. of No. of isolates with the following MICs (g mL )
Antibiotic Species
strains <0.032 0.064 0.125 0.25 0.5 1 2 4 8 16 32 64 128 256

Tc Lc. lactis 50 30 14 3 2 1
Lb. plantarum 29 3 9 10 6 1
Lb. acidophilus/Lb. delbrueckii 15 1 4 4 4 1 1
Other lactobacilli 15 2 3 3 2 1 4
B. longum 17 3 3 6 5
B. bifidum 6 1 5
Other bifidobacteria 11 7 1 1 1 1

Str Lc. lactis 50 5 6 8 20 11


Lb. plantarum 29 2 10 13 3 1
Lb. acidophilus/Lb. delbrueckii 15 4 7 2 2

78
Other lactobacilli 15 2 1 2 5 1 2 2
B. longum 17 4 3 3 5 1 1
B. bifidum 6 6
Other bifidobacteria 11 3 1 3 4

Van Lc. lactis 50 2 9 35 3 1


Lb. plantarum 29 29
Lb. acidophilus/Lb. delbrueckii 15 3 6 6
Other lactobacilli 15 1 1 13
B. longum 17 1 13 3
B. bifidum 6 6
Other bifidobacteria 11 7 4

a
Tc, tetracycline; Str, streptomycin; Van, vancomycin

Table 2. Distribution of MICs of streptomycin and tetracycline (antibiotics that inhibit protein synthesis; target 30S ribosomal subunit), and of the peptidoglycane synthesis inhibitor
vancomycin, for LAB&B species from different environments.
30 both of the primer pairs used (DI-DII and tet1-
tet2) was obtained for 11 B. longum, 5 B.
25 bifidum, 3 Lc. lactis, 1 B. animalis, and 1 Lb.
johnsonii strains (Table 3). More particularly,
N u m b e r o f s tra in s

20 the sequences from the amplicons of Lc.


lactis proved to be 100% identical to the
15 tet(M) gene present in the Tn916 transposon
of Enterococcus faecalis [Herzog-Velikonja et
10 al., 1994]. A section of the nucleotide
sequence of the amplicon obtained from Lb.
5 johnsonii showed 99% homology with tet(W)
genes, while another region matched the
0 sequence of tet(O) genes, suggesting the
< 0 .0 1 6
0 .0 3 2
0 .0 6 4

determinant for this strain may be mosaic


0 .1 2 5
0 .2 5
0 .5
1
2
3
4

(data not shown). A single PCR product of


6
8
12
16
24
32
48

around 1250 bp was obtained for all the 17


64

MICs (g mL-1)
96
128
>256 tetracycline resistant bifidobacteria, which
Figure 1. Distribution of tetracycline MICs for the
Lactococcus lactis strains (in black), showing a clear was identical to the recently reported tet(W)
bimodal distribution, and of erythromycin MICs (in grey), gene [Barbosa et al., 1999; Scott et al.,
showing a unimodal distribution. 2000]. On the contrary, no positive
amplification was obtained with Lb. plantarum
Although most strains were susceptible to and Lb. vaginalis when using specific primers
tetracycline (Table 2), the MIC distributions for tet(K), tet(L), tet(M), tet(O), tet(S) and
suggested 11 B. longum, 5 B. bifidum, 3 Lc. tet(W). Preliminary results suggested the
lactis, and one strain each of B. animalis, B. possibility of this apparent resistance being
pseudolongum subsp. globosum, Lb. an effect of the size of the inoculum (data not
johnsonii, Lb. plantarum, and Lb. vaginalis, shown).
possessed potentially acquired resistance Total DNAs of the MLS-resistant isolates
mechanisms. Indeed, resistance to were amplified with primers specific for the
tetracycline has been widely reported erm(A), erm(B), erm(C), erm(F) and mef(A)
[Danielsen and Wind, 2003; Flrez et al., genes [Chung et al., 1999; Luna et al., 2000].
2005; Delgado et al., 2005; Temmerman et Among the eleven previously detected
al., 2003; Moubareck et al., 2005]. resistant isolates, a positive amplification
Heterofermentative lactobacilli were product of 639 bp was obtained when using
intrinsically resistant to vancomycin (Table 2), the DNA of the Lb. johnsonii strain as a
as reported by other authors [Hamilton-Miller template plus specific primers for erm(B). The
and Shah, 1998]. The obligate nucleotide sequence of the amplicon showed
homofermentative lactobacilli, lactococci and 99% homology with the plasmid-encoded
bifidobacteria, however, proved to be very erm(B) gene of a Lactobacillus fermentum
susceptible. The resistance of some strain (GenBank Acession No. U48430). No
lactobacilli species to vancomycin is intrinsic, amplification was obtained with any other
due to the presence of D-Ala-D-Lactate in erythromycin or clindamycin resistant strains,
their peptidoglycan instead of the normal indicating they might harbour other MLS
dipeptide D-Ala-D-Ala [Klein et al., 2000]. resistance genes.
The two Lb. rhamnosus and one Lb.
johnsonii strains which showed resistance to Screening for resistance genes using DNA
two (erythromycin and clindamycin) and three microarrays. To corroborate the results
(erythromycin, clindamycin, and tetracycline) obtained by PCR screening, and to try to
different antibiotics respectively are worthy of identify previously undetected genes, some
note. isolates were analyzed using a DNA
microarray containing nearly 300
Screening for resistance genes by PCR oligonucleotide probes specific for the
and sequencing. Isolates on the right side of majority of antibiotic resistance gene classes.
the tetracycline MIC distribution curves were Table 3 shows a summary of the results
all screened for tetracycline resistance obtained by this technique and a comparison
determinants using universal primers for with those obtained by PCR. In general, the
genes encoding ribosome protection proteins results of the two methods correlated well.
[Clermont et al., 1997; Barbosa et al., 1999]. Indeed, the presence of tet(W) and erm(B) as
A single PCR product of the same size with determined by PCR was confirmed by the

79
microarray analysis. Further, the DNA of tet(W), tet(O) and mef(A) in these susceptible
three susceptible strains used as controls LAB&B strains. The phenomenon of unstable
(two of B. longum and one of B. bifidum was resistance determinants has also been
found to hybridize with tet(O) (two strains) reported in other species [Strman et al.,
and tet(W) (one strain). In this last strain, 2003]
fragments of the tet(W) were found by PCR, The microarray analysis also detected
however, no amplification was obtained with aph(E) and sat(3) genes coding for
primers encompassing the whole tet(W) gene aminoglycoside resistance in two
[Barbosa et al., 1999], suggesting the streptomycin-resistant isolates of B. longum,
existence of a non-functional (interrupted) in one B. bifidum, and in two streptomycin-
tet(W) gene. Moreover, the Lb. johnsonii susceptible B. pseudocatenulatum strains.
strain resistant to erythromycin and Microarray hybridization was found to be a
tetracycline was shown to harbour a mef(A) powerful screening technique; in a single step
gene in addition to erm(B) and tet(O/W). it was able to screen for hundreds of genes
However, no amplification was obtained belonging to the most common antibiotic
when PCR for mef(A) was performed. Our resistance families, although the results
findings suggest the presence of partial require confirmation by sequencing.

No. of Genes detected by


Relevant
Species strains
phenotypea PCR DNA microarrays
examined

B. animalis 1 Strr, Tetr tet(W) Nd

B. bifidum 1 Strr - aph(E), tet(O)


B. bifidum 5 Strr, Tetr tet(W) tet(W)

B. longum 2 Susceptible - tet(O)


B. longum 4 Clinr, Eryr, Str tet(W)b tet(W)
B. longum 3 Clinr, Eryr, Str, Tetr tet(W) tet(W)
B. longum 8 Strr, Tetr tet(W) tet(W)

B. pseudocatenulatum 1 Susceptible - -
B. pseudocatenulatum 2 Susceptible - aph(E)
B. pseudocatenulatum 1 Clinr, Strr - -

Lc. lactis subsp. lactis 3 Tetr tet(M) Nd

Lb. brevis 1 Susceptible - -

Lb. johnsonii 1 Eryr, Strr, Tetr erm(B), tet(W/O) erm(B), mef(A), tet(W)

Lb. plantarum 1 Susceptible - -


Lb. plantarum 2 Tetr - -

Lb. rhamnosus 1 Susceptible - -


Lb. rhamnosus 2 Eryr, Strr - -

Lb. vaginalis 1 Tetr - -

a
Clinr, Eryr, Strr, and Tetr, erythromycin, streptomycin and tetracycline resistance, respectively.
b
The tet(W) gene detected in these strains was found to be incomplete.
Nd, not done.

Table 3.- Results of screening selected LAB and BB strains for antibiotic resistance genes by PCR and DNA
microarray analysis.

80
Genetic location of resistance genes. Antibiotic resistance mediated by
Among the erythromycin- and tetracycline- membrane transporters in
resistant isolates, only four strains were Bifidobacterium. As well as the possession
found to harbour plasmids (the 3 Lc. lactis of dedicated antibiotic resistance genes,
strains and a single B. bifidum strain). several non-specific mechanisms have been
Consequently, most resistance traits were suggested to contribute to increased
deemed to be encoded on the bacterial antibiotic resistance in bacteria, including the
chromosome. Internal segments of the tet(M) thickness and compactness of the cell wall
(1.5 kb), tet(W) (1.2 kb) and erm(B) (639 bp) [Cui et al., 2003], defective cell wall autolytic
genes obtained by PCR were digoxigenin- systems [Kim et al., 1982], and multi-drug
labelled and hybridized against total and transporters (MDRs) [Putman et al., 2001]. In
plasmid DNA digested with the restriction the present study, the involvement of two
enzymes EcoRI and HindIII. Southern blot MDR transporters of B. breve in antibiotic
assays confirmed the chromosomal location resistance was analyzed since recent studies
of the tet(W) in the bifidobacteria, as well as have reported this bacterium to have MIC
the erm(B) and tet(O/W) genes in Lb. values for several antibiotics that are higher
johnsonii. These assays showed a plasmid than those of other Bifidobacterium species
location for the tet(M) gene in all three Lc. [Moubareck et al., 2005].
lactis strains (results not shown).

A BbmR BbmA BbmB


G G
K F
L
V C
C
P
Y
R Out
I Y
P I A G
V S G
G G TS S SY K QQK A E V
L G
V N VM Y I T F
S I T M P
A K M GL Q P
L A
VD G
R
E A
V L E A SD In
WG K P L I L K T
K S L
L A G D K P
Out A
S
T
Y
G
S
L S
V
I
G
F I V
I
S G F
F
L
A
T
A P Y H G M Q G S D
V M L F I I L H C V A L S F A
F N Y I G V CA L TS L V I G L I L GYC N S P
F V L A C L I L I D R
A F G V A A P A I I G N H A A
W V M I F P V L V
I G L L P M I F L V V I V P S R
V I T M P V A V T
V L T I I L L V K A N P I F F G
T F L F M V G A S
In K
W D
D
H
A
Q
L
A
N
P
M L
S
S
V W
N
H
G
T F K T F
L I L L A I A GD P DA D
W K L V H
T A F F S V D D
D R
A G S L G N SGK K N
F I A E
V Q S S K K
A L G
K I Y E I H WD A N
T N
N- MEK
V
Q
M
V V V
S P
L
S D
KHE I SD
A
T
P L V
S
W
D
H
E
V K
AD
NBD NBD
W N
R G

B S. typhimurium (AAL23019)
LmrD (L. lactis, AAK04409)
EfrB (E. faecalis, AAO82607)
S. oenidensis (AAN57483) BbmB (B. breve, DQ486860)
P. aeruginosa (AAG06652) MD2 (H. hominis, AAO039422)
B. suis (AAN30588) LmrC (L. lactis, AAK04408)
N. meningitidis (AAF41122)
EfrA (E. faecalis, AAO82608)
B. subtilis (CAB13827)
MD1 (M. hominis, AAO039421)
S. aureus (AAW37143)
BbmA (B. breve, DQ486860)
C. glutamicum (CAF19961)
N-Pgp (H. sapiens, AAA59576)
B. breve BbmR (AAZ23954)
B. longum Ctr (NP696274) C-Pgp (H. sapiens, AAA59576)

B. adolescentis (ZP_01028851) MsbA (E. coli, CAA77839)


S. thermophilus (AAV62871) BmrA (B. subtilis, D70031)
M. musculus IBAT (AAH27440) HorA (L. brevis, BAD80897)
H. sapiens IBAT (I38655) OmrA (O. oeni, AAO64432)
LmrA (L. lactis, AAB49750)

Figure 2.- Topology models of the transporter proteins of Bifidobacterium breve and their phylogenetic relationships to
other MDR proteins. 2A. Topology model for the MDR protein BbmR (left) and the ABC transporter BbmAB (right) from
B. breve. The sodium/bile acid family signature of BbmR is represented in grey. Conserved residues in BbmA and
BbmB and the motif DIExxxxGxxxxRxxxD (x=variable residue), located within the first cytoplasmid loop and common to
bacterial ABC-multidrug transporters, are also shown in grey. NBD, Nucelotide-binding domain. 2B. Left: Phylogenetic
tree of bacterial homologs of BbmR and ileal sodium/bile acid transporters (IBAT) from Homo sapiens and Mus
musculus. Right: Phylogenetic tree of bacterial ABC multidrug tranporters with a proven function and the N-terminal and
C-terminal parts of P-glycoprotein. Protein accession numbers are given in brackets. Trees were constructed by the
neighbor-joining algorithm and clustered by the UPGMA method. The distances represent percentages of different
residues.

81
Our group has recently characterized a Streptococcus thermophilus. Homologous
membrane protein (BbmR) from B. breve proteins from other bacteria fell into a
UCC2003 which shows characteristics different cluster. The ileal bile acid transporter
reminiscent of proton motive force (pmf)- (IBAT) proteins of mice and humans,
driven MDR proteins [Margolles et al., 2005]. responsible for the co-transport of sodium
Hydropathy profile analysis of BbmR and bile across the plasma membrane in the
revealed the presence of eight putative ileum [Hagenbuch and Dawson 2004], were
transmembrane segments showing the phylogenetically more distant (Fig. 2B, left).
typical sodium/bile acid family signature The heterologous expression of BbmR in Lc.
(Figure 2A, left). This motif is common to lactis was shown to confer resistance to
prokaryotic and eukaryotic bile acid macrolides (Table 4). Its homolog in B.
transporters [Altenberg, 2004]. BbmR was longum, Ctr, has been also found to confer
shown to be phylogenetically related to Ctr resistance to erythromycin [Price et al.,
from B. longum (Fig. 2B, left), which was also 2006]. Therefore, all these proteins may be
shown to transport the bile salt cholate [Price involved in combating hydrophobic noxious
et al., 2006], and to the hypothetical bile acid compounds, including antibiotics [Nikaido and
transporters of B. adolescentis and Zgurskaya, 2001].
a
Antibiotic class
-lactams Tetracyclines Aminoglycosides Macrolides Membrane-acting Others
Control - - - - - -
BbmR - - +/-a + - +/-b
BbmAB - - - - + -
a
-lactams (ampicillin, benzylpenicillin, ceftazidime, cephalothin, and meropenem); tetracyclines (tetracycline, doxycycline, and minocycline); aminoglycosides
(kanamycin, gentamicin, and streptomycin); macrolides (erythromycin, azithromycin, dirithromycin, and clarithromycin); membrane-acting agents (polymyxin and nisin
A); others (clindamycin, quinupristin-dalfopristin, rifampicin, trimethoprim-sulfamethoxazole, vancomycin, and ciprofloxacin).

Resistance levels were determined by the E-test method (except for nisin A). Key: +, cells with more than 3-fold
increase in MIC compared with the control; +/-, cells in which the MIC increased between 2.5 and 3 fold compared
with the control; a, only for streptomycin; b, only for vancomycin.
Table 4. Resistance to different antibiotic groups of Lc. lactis cells expressing the membrane proteins BbmR or
BbmAB, and of Lc. lactis cells not expressing the proteins (control).

Most bacterial MDRs use the pmf to (BbmA/BbmB)) conferred resistance to nisin
expel cytotoxic compounds. However, a few and polymyxin in Lc. lactis (Table 4).
drug efflux systems are powered by the
hydrolysis of ATP [Putman et al., 2000]. The Experimental procedures
ATP-dependent drug efflux proteins are
members of the ATP-binding cassette Bacterial strains, growth media and
superfamily (ABC transporters) [Altenberg, culture conditions. A total of 143 LAB&B
2004]. Homodimeric [van Veen et al., 2000; strains were surveyed for antimicrobial
Ravaud et al., 2006] and heterodimeric [Lee resistance. The 31 Lc. lactis and 22 Lb.
et al., 2003; Lubelski et al., 2004] ABC- plantarum strains were isolated as part of the
transporters have all seen involved in drug dominant populations of artisanal starter-free
resistance. Recently, two genes encoding a cheeses [Flrez et al., 2005]. The 12
heterodimeric transporter (BbmA and BbmB) intestinal lactobacilli and 27 bifidobacteria
were identified in the B. breve UCC2003 belonged to the dominant populations of the
genome (Figure 2A, right). This transporter faeces and intestinal mucosa of healthy
has the typical architecture of all ABC individuals [Delgado et al., 2005]. Nineteen L.
transporters, with an N-terminal membrane lactis, 18 lactobacilli, and seven bifidobacteria
domain containing six membrane-spanning were provided by the Belgian Co-ordinated
segments plus a nucleotide binding domain Collections of Microorganisms (BCCMTM),
located in the cytoplasm. Phylogenetic University of Ghent, Ghent, Belgium. Finally,
analysis grouped the heterodimeric ABC seven Lb. plantarum strains were obtained
transporters into a separate cluster from the from the Coleccin Espaola de Cultivos Tipo
homodimeric types (Figure 2B, right), (CECT), University of Valencia, Valencia,
suggesting they have a different evolutionary Spain.
origin. The B. breve ABC transporter

82
Culture media were all purchased from was checked for by PCR with two pairs of
Oxoid (Basingstoke, Hampshire, UK). degenerate primers: DI-DII [Clermont et al.,
Cryopreserved cultures in glycerol were first 1997] and tet1 and tet2 [Barbosa et al.,
recovered on Mueller-Hinton agar (for L. 1999]. Additional PCR assays were
lactis), MRS agar (for dairy lactobacilli and performed with primers tetWF and tet2,
lactobacilli of vegetable origin), or MRS agar specific for tet(W) [Scott et al., 2000], and
supplemented with 0.3 gL-1 cysteine-HCl primers DI and TetMR, specific for tet(M)
(Merck, Darmstadt, Germany) (for intestinal [Clermont et al., 1997]. The set of primers
lactobacilli and bifidobacteria). Isolated included specific oligomers for tet(K), tet(L),
colonies were then streaked onto Mueller- tet(M), tet(O), tet(S) and tet(W) [Scott et al.,
Hinton plates (for Lc. lactis) or onto lactic acid 2000; Gevers et al., 2003]. The presence of
bacteria susceptibility test medium (LSM; macrolide, lincosamine and streptogramin
90% IsoSensitest and 10% MRS [Klare et al., resistance genes was checked for with
2005]) (for lactobacilli), and incubated for 24 primers specific for the erm(A), erm(B),
h at 30C. Colonies of intestinal lactobacilli erm(C), erm(F) and mef(A) genes [Chung et
and bifidobacteria were streaked onto LSM al., 1999; Luna et al., 2000]. Purified
supplemented with 0.3 g L-1 cysteine, and amplicons were sequenced by cycle
incubated at 37C in an anaerobic chamber extension in an ABI 370 DNA sequencer
(Mac500, Down Whitley Scientific, West (Applied Biosystems, Foster City, Ca., USA).
Yorkshire, UK) (atmosphere 10% H2, 10% The microarray assay was performed
CO2, 80% N2) for 48 h. with an array containing 300 oligonucleotides
(50 to 60 base pairs long) specific for 250
Minimum inhibitory concentrations antibiotic resistance genes of the following
determined by the E-test. Individual classes: aminoglycosides, extended-
colonies from the agar plates were spectrum -lactamases, chloramphenicol,
suspended in 2-5 mL of sterile saline (Oxoid) macrolide-lincosamides- streptogramin,
until a density corresponding to McFarland sulphonamides, tetracyclines, trimethoprim,
standard 1 or its spectrophotometric and vancomycin. The spotting of the
equivalent (3x108 cfu mL-1) was obtained. oligonucleotides, hybridization conditions and
Bacterial suspensions were plated onto the analysis of the microarrays were as
the surface of MH (or LSM) agar with a sterile previously described [van Hoek et al., 2005].
cotton swab, and the plates allowed to dry for
approximately 15 min before applying the E- Computer analyses. DNA and protein
test strips (AB Biodisk, Solna, Sweden). MICs sequences were analyzed using the Clone
were recorded following the manufacturers Manager 5 computer program (Scientific and
recommendations after 48 h of incubation at Educational Software, Durham, N.C., USA).
30 or 37C. The susceptibilities of the LAB&B Homology searches were carried out using
strains were established using the MIC the BLAST server of the NCBI
breakpoints defined by the CLSI [CLSI, [http://www.ncbi.nlm.nih.gov/] (Bethesda,
2004], and compared to those of the Md., USA) and the software available on the
FEEDAP Panel [European Commission, the Ple-Bioinformatique Lyonnais
2005]. [http://pbil.univ-lyon1.fr/] (Lyon, France) web
page. Homology trees were constructed with
DNA techniques. Genomic DNA was the TreeTop-Phylogenetic Tree Prediction
isolated using a commercial kit (GenElute program [http://www.genebee.msu.su/]
bacterial genomic DNA kit; Sigma-Altdrich (Moscow, Russia).
Co., St. Louis, Mo., USA). Plasmid DNA
extraction was performed as previously Conclusion
described [OSullivan and Klaenhammer,
1993]. EcoRI- and HindIII-digested DNA was Antibiotic resistance is not a common trait
transferred to nylon membranes and of dairy and intestinal LAB&B species.
hybridized with probes obtained by PCR However, several strains were considered to
using oligonucleotide primers internal to be resistant to some antibiotics, and were
specific genes. Hybridization and detection thought to harbour specific resistance genes.
were all performed using the digoxigenin Moreover, genes identical to those described
DNA labelling and detection kit (Roche in other bacteria from the same environments
Molecular Biochemicals, Lewels, UK) as [tet(M), tet(O/W), tet(W), erm(B)] were
recommended by the manufacturer. identified in dairy and intestinal LAB&B
The presence of tetracycline resistance strains. When present, all these resistance
genes encoding ribosomal protection proteins determinants were located on the bacterial

83
chromosome, except for tet(M) which was Delgado S, Flrez AB, Mayo B: Antibiotic susceptibility of
Lactobacillus and Bifidobacterium species from the human
identified on plasmids in Lc. lactis. Non- gastrointestinal tract. Curr Microbiol 2005; 50:202-207.
specific mechanisms, such as those involving European-Commission: Opinion of the Scientific Panel on
Additives and Products in Substances used in Animal Feed
the membrane MDR transporters studied in on the updating of the criteria used in the assessment of
this work, might contribute to broaden the bacteria for resistance to antibiotics of human and
veterinary importance. The EFSA J. 2005; 223:1-12
antibiotic MIC ranges. The determination of Flrez AB, Delgado S, Mayo B: Antimicrobial susceptibility of
the molecular mechanisms underlying the lactic acid bacteria isolated from a cheese environment.
transfer of resistance in LAB and BB species Can J Microbiol 2005; 51:51-58.
Fons M, Hege T, Ladire M, Raibaud P, Ducluzeau R, Maguin
would be essential for the control of their E: Isolation and characterization of a plasmid from
spread via the food chain. Lactobacillus fermentum conferring erythromycin
resistance. Plasmid 1997; 37:199-203.
Gevers D, Masco L, Baert L, Huys G, Debevere J, Swings J:
Acknowledgments Prevalence and diversity of tetracycline resistant lactic acid
bacteria and their tet genes along the process line of
fermented dry sausages. Syst Appl Microbiol 2003; 26:277-
Work on antibiotic resistance in LAB&B 283.
Hagenbuch B, Dawson P: The sodium bile salt cotransport
strains at the authors laboratories was family SLC10. Pflugers Arch 2004; 447:566-570.
supported by an EU project within the sixth Hamilton-Miller JM, Shah S: Vancomycin susceptibility as an
aid to the identification of lactobacilli. Lett Appl Microbiol
Framework Programme (ref. 506214). M. S. 1998; 26:153-15
Ammor was awarded a postdoctoral Herzog-Velikonja B., Podlesek Z, Grabnar M: Conjugal
transfer of transposon Tn916 from Enterococcus faecalis to
fellowship from the Secretara de Estado de Bacillus licheniformis. Plasmid 1994 31:201-206.
Universidades e Investigacin of the Katla AK, Kruse H, Johnsen G, Herikstad H: Antimicrobial
Spanish Ministry of Education and Science susceptibility of starter culture bacteria used in Norwegian
dairy products. Int J Food Microbiol 2001; 67:147-152.
(ref. SB2004-0165). Jos Antonio Moreno, Kim KS, Morrison JO, Bayer AS:Deficient autholytic enzyme
IPLA-CSIC, Villaviciosa, Spain, and Douwe activity in antibiotic-tolerant lactobacilli. Infec Immun 1982;
36:582-585.
van Sinderen, Department of Microbiology, Klare I, Konstabel C, Muller-Bertling S, Reissbrodt R, Huys
University College Cork, Cork, Ireland, are G, Vancanneyt M, Swings J, Goossens H, Witte W:
Evaluation of new broth media for microdilution antibiotic
acknowledged for sharing unpublished susceptibility testing of lactobacilli, pediococci, lactococci,
sequence data. and bifidobacteria. Appl Environ Microbiol 2005; 71:8982-
8986.
Klein G, Hallmann C, Casas IA, Abad J, Louwers J, Reuter G:
References Exclusion of vanA, vanB and vanC type glycopeptide
resistance in strains of Lactobacillus reuteri and
Lactobacillus rhamnosus used as probiotics by polymerase
Ahn C, Collins-Thompson D, Duncan C, Stiles ME: chain reaction and hybridization methods. J Appl Microbiol
Mobilization and location of the genetic determinant of 2000; 89:815-824.
chloramphenicol resistance from Lactobacillus plantarum Lee EW, Huda MN, Kuroda T, Mizushima T, Tsuchiya T:
caTC2R. Plasmid 1992; 27:169-176. EfrAB, an ABC multidrug efflux pump in Enterococcus
Altenberg GA: Structure of multidrug-resistance proteins of the faecalis. Antimicrob Agents Chemother 2003; 47:3733-
ATP-binding cassette (ABC) superfamily. Curr Med Chem 3738.
Anticancer Agents 2004; 4:53-62. Lubelski J, Mazurkiewicz P, van Merkerk R, Konings WN,
Barbosa TM, Scott KP, Flint HJ: Evidence for recent Driessen AJ: ydaG and ydbA of Lactococcus lactis encode
intergeneric transfer of a new tetracycline resistance gene, a heterodimeric ATP-binding cassette-type multidrug
tet(W), isolated from Butyrivibrio fibrisolvens, and the transporter. J Biol Chem 2004; 279:34449-34455.
occurrence of tet(O) in ruminal bacteria. Environ Microbiol Luna VA, Cousin S, Jr., Whittington WL, Roberts MC:
1999; 1:53-64. Identification of the conjugative mef gene in clinical
Bates J, Jordens JZ, Griffiths DT: Farm animals as a putative Acinetobacter junii and Neisseria gonorrhoeae isolates.
reservoir for vancomycin-resistant enterococcal infection in Antimicrob Agents Chemother 2000; 44:2503-2506.
man. J Antimicrob Chemother 1994; 34:507-514. Margolles A, Moreno JA, van Sinderen D, de Los Reyes-
Blzquez J, Oliver A, Gmez-Gomez JM: Mutation and Gaviln CG: Macrolide resistance mediated by a
evolution of antibiotic resistance: antibiotics as promoters Bifidobacterium breve membrane protein. Antimicrob
of antibiotic resistance? Curr Drug Targets 2002; 3:345- Agents Chemother 2005; 49:4379-4381.
349. Moubareck C, Gavini F, Vaugien L, Butel MJ, Doucet-
Chung WO, Werckenthin C, Schwarz S, Roberts MC: Host Populaire F: Antimicrobial susceptibility of bifidobacteria. J
range of the ermF rRNA methylase gene in bacteria of Antimicrob Chemother 2005; 55:38-44.
human and animal origin. J Antimicrob Chemother 1999; Murray PR, Baron EJ, Jorgensen JH, Pfaller MA, Yolken RH:
43:5-14. Manual of Clinical Microbiology. Wahington DC, USA,
Clermont D, Chesneau O, De Cespedes G, Horaud T: New American Society for Microbiology, 2003.
tetracycline resistance determinants coding for ribosomal Nikaido H, Zgurskaya HI: AcrAB and related multidrug efflux
protection in streptococci and nucleotide sequence of tet(T) pumps of Escherichia coli. J Mol Microbiol Biotechnol 2001;
isolated from Streptococcus pyogenes A498. Antimicrob 3:215-218.
Agents Chemother 1997; 41:112-116. Nikolich MP, Hong G, Shoemaker NB, Salyers AA: Evidence
CLSI (Clinical and Laboratory Standards Institute): Methods for natural horizontal transfer of tetQ between bacteria that
for Antimicrobial Susceptibility Testing of Anaerobic normally colonize humans and bacteria that normally
Bacteria: Approved Standard-six edition. M11-A6, Vol. 24, colonize livestock. Appl Environ Microbiol 1994; 60:3255-
n 2. Wayne, Pennsylvania, USA, 2004. 3260.
Cui L, Ma X, Sato K, Okuma K, Tenover FC, Mamizuka EM, Normark BH, Normark S: Evolution and spread of antibiotic
Gemmell CG, Kim MN, Poy MC, El-Solh N, Ferraz V, resistance. J Intern Med 2002; 252:91-106.
Hiramatsu K: Cell wall thickening is a common feature of OSullivan DJ, Klaenhammer TR: Rapid mini-prep isolation of
vancomycin resistance in Staphylococcus aureus. J Clin high-quality plasmid DNA from Lactococcus and
Microbiol 2003; 41:5-14. Lactobacillus spp. Appl Environ Microbiol 1993; 59:2730-
Danielsen M: Characterization of the tetracycline resistance 2733.
plasmid pMD5057 from Lactobacillus plantarum 5057 Ouwehand AC, Salminen S, Isolauri E: Probiotics: an overview
reveals a composite structure. Plasmid 2002; 48:98-103. of beneficial effects. Antonie Van Leeuwenhoek 2002;
Danielsen M, Wind A: Susceptibility of Lactobacillus spp. to 82:279-289.
antimicrobial agents. Int J Food Microbiol 2003; 82:1-11.

84
Perreten V, Schwarz F, Cresta L, Boeglin M, Dasen G, Teuber resistance determinant (ermGT) from Lactobacillus reuteri
M: Antibiotic resistance spread in food. Nature 1997; 100-63. Plasmid 1994; 31:60-71.
389:801. Temmerman R, Pot B, Huys G, Swings J: Identification and
Price CE, Reid SJ, Driessen AJ, Abratt VR: The antibiotic susceptibility of bacterial isolates from probiotic
Bifidobacterium longum NCIMB 702259T ctr gene codes products. Int J Food Microbiol 2003; 81:1-10.
for a novel cholate transporter. Appl Environ Microbiol Teuber M, Meile L, Schwarz F: Acquired antibiotic resistance in
2006; 72:923-926. lactic acid bacteria from food. Antonie van Leeuwenhoek
Putman M, van Veen HW, Konings WN: Molecular properties of 1999; 76:115-137.
bacterial multidrug transporters. Microbiol Mol Biol Rev van Hoek AHAM, Scholtens IMJ, Cloeckaert A, Aarts HJM:
2000; 64:672-693. Detection of antibiotic resistance genes in different
Ravaud S, Do Cao MA, Jidenko M, Ebel C, Le Maire M, Jault Salmonella serovars by oligonucleotide microarray
JM, Di Pietro A, Haser R, Aghajari N: The ABC analysis. J Microbiol Methods 2005; 62:13-23.
transporter BmrA from Bacillus subtilis is a functional dimer van Veen HW, Margolles A, Muller M, Higgins CF, Konings
when in a detergent-solubilized state. Biochem J 2006; WN: The homodimeric ATP-binding cassette transporter
395:345-353. LmrA mediates multidrug transport by an alternating two-
Scott KP, Melville CM, Barbosa TM, Flint HJ: Occurrence of site (two-cylinder engine) mechanism. EMBO J 2000;
the new tetracycline resistance gene tet(W) in bacteria from 19:2503-2514.
the human gut. Antimicrob Agents Chemother 2000; Witte W: Selective pressure by antibiotic use in livestock. Int J
44:775-777. Antimicrob Agents 2000; 16:S19-S24.
Stroman P, Muller CC, Sorensen KI: Heat shock treatment Zarazaga M, Senz Y, Portillo A, Tenorio C, Ruiz-Larrea F,
increases the frequency of loss of an erythromycin Del Campo R, Baquero F, Torres C: In vitro activities of
resistance-encoding transposable element from the ketolide HMR3647, macrolides, and other antibiotics
chromosome of Lactobacillus crispatus CHCC3692. Appl against Lactobacillus, Leuconostoc, and Pediococcus
Environ Microbiol 2003; 69:7173-7180. isolates. Antimicrob Agents Chemother 1999; 43:3039-41.
Tannock GW, Luchansky JB, Miller L, Connell H, Thode- Zhou JS, Pillidge CJ, Gopal PK, Gill HS: Antibiotic
Andersen S, Mercer AA, Klaenhammer TR: Molecular susceptibility profiles of new probiotic Lactobacillus and
characterization of a plasmid-borne (pGT633) erythromycin Bifidobacterium strains. Int J Food Microbiol 2005; 98:211-
217.

85
Artculo VII.- Molecular characterization of tet(M) from two Lactococcus lactis strains and
conjugal transfer of tetracycline resistance to L. lactis and Enterococcus faecalis.

Objetivo: El objetivo de este trabajo fue la caracterizacin del determinante gentico


tet(M), responsable de la resistencia a tetraciclina en dos cepas de Lactococcus lactis
aisladas del queso de Cabrales. Una vez caracterizado el gen de resistencia, se estudi
su capacidad de transferencia mediante conjugacin a especies de lactococos,
lactobacilos y enterococos.

Resultados: La presencia de genes de resistencia en dos cepas de Lc. lactis resistentes a


tetraciclina (CIM 48 y 96 g/ml) se analiz mediante PCR. Mediante esta tcnica,
ambas cepas resultaron positivas para el gen tet(M). La secuenciacin del amplicn
mostr una gran homologa del gen con el presente en otras especies, lo que permiti la
utilizacin de cebadores para la amplificacin total del gen y las secuencias adyacentes,
y su posterior secuenciacin. La secuencia nucleotdica del locus result ser muy similar
a la descrita en diferentes cepas de Enterococcus y Lactobacillus. De hecho, la secuencia
de tet(M) result idntica a la del gen tet(M) del transposn Tn916 de Enterococcus
faecalis DS16. Mediante hibridacin se localiz el gen tet(M) en un plsmido de
aproximadamente 45-50kpb, en el que el transposn podra encontrarse completo. La
resistencia a tetraciclina pudo transferirse desde ambas cepas a otras de Lc. lactis y E.
faecalis, pero no a Lb. plantarum.

Conclusin: En este trabajo se ha caracterizado el gen tet(M) que confiere resistencia a


dos cepas de Lc. lactis. Este gen est localizado en el transposn Tn916, que parece
haberse insertado en plsmidos residentes. La transferencia de la resistencia a
tetraciclina mediante conjugacin demostr la funcionalidad del transposn.

87
Molecular characterization of tet(M) from two Lactococcus lactis strains
and conjugal transfer of tetracycline resistance to L. lactis and
Enterococcus faecalis.

Abstract

Specific PCR and sequencing showed tet(M) to be responsible for tetracycline resistance
in two Lactococcus lactis (Flrez, A. B., Delgado, S., and Mayo, B. 2005. Can. J. Microbiol.
51, 51-58). Sequencing of the tetracycline resistance loci, including the upstream and
downstream regions of the genes, showed them to be identical to one other and to the
tet(M) encoded by the conjugative transposon Tn916 first discovered in Enterococcus
faecalis. Using primers based on sequences from the right and left arms of the
transposon, segments of Tn916 were amplified from total DNA of the resistant strains,
suggesting the transposon is complete in L. lactis. Hybridisation experiments associated
tet(M) with a plasmid of around 30 kbp which showed a reduction in size comparable to
that expected after Tn916 curing. The functionality of the transposon in these two L.
lactis strains was demonstrated by conjugal transfer of tetracycline resistance to other
lactococcus and enterococcus strains.

Introduction al., 2005). Pioneering antibiotic surveys have


showed lactococcus strains to be susceptible
The indiscriminate use of antibiotics in the to most antimicrobial agents (Cogan, 1972;
treatment of infections, as prophylactic Reinbold and Reddy, 1974; Orberg and
agents, and as growth promoters, has Sandine, 1985; Elliot and Facklam, 1996)
facilitated the emergence and spread of with the notable exception of rifampicin.
acquired resistance. This is a public health Intrinsic resistance has been proposed to
problem of increasing importance (Davies, explain this (Orberg and Sandine, 1985;
1994; Normak and Normak, 2002). Of great Teuber et al., 1999). This contrasts with the
concern is the possibility that food-borne vast array of acquired resistances found
commensal bacterial populations become among the enterococci (Franz et al., 1999;
reservoirs for antibiotic resistance genes Kayser, 2003). More recently, however,
(Teuber et al., 1999; Witte, 2000), from which isolates of L. lactis resistant to
they could be horizontally transferred to chloramphenicol, erythromycin, streptomycin
opportunist and pathogenic bacteria. and tetracycline have been reported
Microorganisms intended for use in food properties they are thought to have
systems should therefore be carefully developed via the acquisition of new genes
examined to avoid the spread of acquired (Perreten et al., 1997; Delgado and Mayo,
antibiotic resistance determinants via the 2004; Raha et al., 2002). Noteworthy,
food chain (Teuber et al., 1999; European plasmid pK214 has been found to harbour
Commission, 2005). four resistance-associated genes, tet(S), cat,
Lactococcus lactis is a lactic acid str, and mdt(A), which code for a non-specific
bacterium used worldwide as a starter efflux system (Perreten et al., 1997; Perreten
organism in the dairy industry for the et al., 2001).
manufacture and ripening of cheese and This paper reports the molecular
other fermented products (Fox et al., 2000). characterization of tet(M), and shows it to be
The development of L. lactis in milk provides responsible for tetracycline resistance in two
optimal conditions for curd formation, L. lactis strains isolated from artisanal starter-
prevents the outgrowth of pathogenic and free cheeses (Flrez et al., 2005). To our
spoilage bacteria, and creates biochemical knowledge this is the first description of
conditions indispensable for ripening. Further, tet(M) in L. lactis. The gene was localized on
L. lactis participates in the development of copies of the conjugative transposon Tn916,
cheese texture and flavour via its proteolytic which was first characterized in Enterococcus
and amino acid catabolic systems (Smit et faecalis DS16 (Flannagan et al., 1994). The

89
transposon was found inserted into plasmids presence of tetracycline resistance genes
of about the same size in both strains, and was checked by PCR using both universal
was lost at varying frequencies after culturing (for genes encoding ribosomal protection
the cells without antibiotic. The tetracycline proteins) and specific primers. The universal
resistance phenotype was shown to be primers used were DI-DII (Clermont et al.,
transferred by conjugation to other lactococci 1997) and Tet1 and Tet2 (Barbosa et al.,
and enterococci. 1999). The specific primers used were those
for tet(W) (Scott et al., 2000), and tet(M),
Methods and Material tet(S), tet(O), tet(K) and tet(L) (Gevers et al.,
2003). The PCR conditions used in each
Bacterial strains, media and culture case were those described by the
conditions. Lactococcus lactis subsp. lactis corresponding authors. Amplicons were
IPLA 31008 and L. lactis subsp. lactis IPLA purified and sequenced by cycle extension in
31009 were originally isolated from a raw- an ABI 370 DNA sequencer (Applied
milk, starter-free, Cabrales cheese (Flrez et Biosystems, Foster City, Ca., USA).
al., 2006). Both strains were suspected of Sequences were then compared to others in
being tetracycline resistant during a recent public databases using the BLAST program
antibiotic resistance survey (Flrez et al., (Altschul et al., 1997).
2005). The strains were routinely cultivated in Given the degree of similarity of the
M17 broth (Oxoid, Hampshire, UK) and tet(M) sequences of the present isolates to
stored at 80C in the same medium with that of the E. faecalis Tn916 transposon
15% (w/v) glycerol (Merck; VWR (Flannagan et al., 1994; GenBank Accession
International, Darmstadt, Germany). No. NC_006372), the primers tetM-F1 and
Incubations were performed at 28C in tetM-R1 and the PCR conditions described
aerobic conditions for 48 h. by Stapleton et al. (2004) were used to
For susceptibility testing, cultures of the amplify the entire tet(M) genes, including the
cryopreserved L. lactis strains were promoter and terminator regions. The
recovered on Mueller-Hinton agar (Oxoid). amplicons were purified, sequenced and their
Isolated colonies were then streaked onto sequences compared as above.
Mueller-Hinton plates and incubated for 16-
18 h at 28C. Amplification and sequencing of Tn916-
derived sequences.To amplify Tn916-
Susceptibility testing: the Etest. Individual derived sequences, two pairs of primers
colonies from the Mueller-Hinton plates were based on the above mentioned sequence
suspended in a sterile glass or plastic tube were designed, matching positions at both
containing 2-5 ml of sterile saline (Oxoid) until extremities of the transposon: Tn916-F1 (5
a density corresponding to McFarland TTGCTGGCGAGGATAAAGTC 3) (positions
standard 1 (corresponding to around 3x108 391-410) and Tn916-R1 (5
cfu/ml) was obtained. A sterile cotton swab GTGGTTGAGCTTTGAATGAAC 3)
was dipped into the suspension and used to (positions 1000-1020); Tn916-F2 (5
inoculate fresh plates of the same medium. CTGTTAGCCTTTGCAAAAGC 3) (positions
The agar surface was then allowed to dry for 17235-17254) and Tn916-R2 (5
approximately 15 min before applying the CAAGACGCTCCTGTTGCTTCT 3)
tetracycline Etest strips (AB Biodisk, Solna, (positions 17830-17850). The amplification
Sweden). Readings were recorded after program included one denaturing cycle at
incubating at 28C for 48 h, following the 94C for 5 min, 35 amplification cycles at
manufacturers recommendations. 94C for 30 seconds, 55C for 1 min, 72C for
1 min, and a final extension step at 72C for
Isolation of total and plasmid DNA. Total 10 min.
genomic DNA from the strains was purified
from 1 ml of overnight cultures in M17 broth DNA hybridisation. Total and plasmid DNA
supplemented with 40 mM of DL-threonine from resistant strains was digested with
(Merck) using the Wizard Genomic EcoRI and HindIII restriction enzymes (Roche
Purification Kit (Promega Corporation, Applied Science, Basel, Switzerland) and
Southampton, UK). Plasmid DNA was blotted onto Hybond-N nylon membranes
extracted and purified following the procedure (GE Heath Care Bio-Sciences, Little Chalfont,
of OSullivan and Klaenhammer (1993). UK) using a standard protocol (Sambrook et
al., 1989). A segment of tet(M) obtained by
PCR detection and sequencing of PCR using the specific primers was used as
tetracycline resistance genes. The a probe after labelling with the Digoxigenin

90
Labelling and Detection Kit (Roche, GenBank database under Accession Nos.
Biochemicals, Lewels, UK). Labelling, DQ060147 and DQ060148 respectively.
hybridisation (using high-stringency
conditions), and detection were all performed Results
following the manufacturers
recommendations. The MICs of tetracycline for L. lactis IPLA
31008 and IPLA 31009 have previously been
Stability of the tetracycline resistance recorded as 16 g ml-1 in a microbroth assay
phenotype.To check the stability of the (Florez et al., 2005). These two strains were
tetracycline resistance phenotype, cells were isolated from the same sample of Cabrales
grown for around 100 generations in M17 cheese, but are different at the phenotypic
with 0.5% glucose (this allowed the strains to (IPLA 31008 grows well in milk and is lactose
grow even if the ability to metabolise lactose positive, while and IPLA 31009 does not grow
was lost). Fresh medium was inoculated daily and is lactose negative) and genetic (different
with overnight cultures at (1% v/v) and RAPD profiles) levels (data not shown). In the
incubated for 24 h. The cells were then present Etest, MICs of 48 and 96 g ml-1
serially diluted in a saline solution (0.9%, w/v) were obtained for IPLA 31008 and IPLA
and spread onto GM17 agar plates. One 31009 respectively. By way of comparison,
hundred colonies were then picked at random the MICs of tetracycline for 50 susceptible
from these plates and replated on media with strains ranged from 0.064 to 0.38 g ml-1
and without tetracycline (15 g ml-1). (results not shown). Thus, the two strains
Tetracycline-susceptible colonies were were considered to be truly resistant to this
examined for plasmids as above. antibiotic.
The two isolates were examined by PCR
Mating procedure. Both IPLA 31008 and to search for tetracycline resistance genes(s).
IPLA 31009 were used as donors in mating Universal primers were used to amplify
experiments. The recipients were L. lactis internal segments of the ribosome protection
subsp. cremoris BU-2-60 and L. lactis subsp. genes, as well as specific primers for tet(K),
cremoris MG1614, both resistant to tet(L), tet(M), tet(O) and tet(S), as indicated
streptomycin (500 g ml-1) and rifampicin above. Single PCR products of around 1100
(100 g ml-1), E. faecalis OG1RF, resistant and 1300 bp were obtained with the primer
to rifampicin (100 g ml-1) and fusidic acid pairs DI-DII and tet1-tet2 respectively.
(25 g ml-1), and L. plantarum NC8, resistant Positive amplifications were also obtained
to streptomycin (500 g ml-1). Donor and with specific primers for tet(M). Analysis of
recipient strains were grown separately at the nucleotide sequences of the amplicons
30C on GM17 for 16-18 h. After incubation, from the two strains showed them to be
they were mixed at a donor:recipient ratio of identical. Comparison of the sequences
10:1. Aliquots of the mating mixtures were against those in databases also showed them
filtered through 0.45 m nitrocellulose filters, to be identical to the internal segments of
which were incubated over the surface of tet(M) of several Enterococcus spp. strains,
GM17 agar plates. Matings were allowed to including the tet(M) sequence of transposon
proceed for 24 h at 30C, after which the Tn916 of E. faecalis DS16 (Su et al., 1992;
filters were suspended in fresh GM17 broth. Flannagan et al., 1994; NC_006372).
Serial dilutions of this medium were prepared Based on this similarity, primers were
and GM17 agar plates with appropriate designed to amplify the gene and its
antibiotics inoculated for separate counting of surrounding upstream and downstream
donor, recipient, and transconjugant cells. regions. Positive amplification of the whole
Tetracycline was used at a final concentration locus was obtained when DNA from both
of 10 g ml-1 for both the counting and resistant strains was used as a template.
selecting plates. Transconjugants were finally Compared to the tet(M) locus sequence in
subjected to plasmid analysis and typed with Tn916, analysis of a region of 2630 bp
primer RAP3 (5-TGC TCT GCC C-3) using showed a single nucleotide modification 196
the RAPD procedure of Delgado and Mayo positions upstream of the start codon in the
(2004). sequence of strain IPLA 31008. Complete
nucleotide similarity was seen for the 2630 bp
Nucleotide sequence accession numbers. sequence from IPLA 31009 and that of
The nucleotide sequences of the tet(M) Tn916. An open reading frame (ORF)
genes from IPLA 31008 and L. lactis subsp. encoding a protein of 639 amino acids
lactis IPLA 31009 were deposited in the corresponding to Tet(M) was found, preceded
by a typical ribosome binding site (RBS)

91
sequence (GGAGG) nine nucleotides location of the genes, a 1.2 kbp internal
upstream. A second small ORF encoding a segment of tet(M) was digoxigenin-labeled
putative leader peptide of 28 amino acids and hybridised against total and plasmid
was observed immediately upstream of the DNA, undigested and digested with the
tet(M) start codon. restriction enzymes EcoRI and HindIII.
Amplification was also obtained when Hybridisation signals were obtained at similar
primers based on the far left and right arms of positions in digested total and undigested
the transposon were used in PCR. Analysis and digested plasmid DNA samples from the
and comparison of the sequences of the two strains (Fig. 1; EcoRI-digested total DNA
resulting amplicons showed them to be from IPLA31008, in lane 2, was thought to be
identical to those of Tn916 from E. faecalis partially degraded). The presence of
(Flannagan et al., 1994; NC_006372). This hybridisation signals at the same level in
strongly suggests that both L. lactis strains undigested plasmid DNA, and identical
harbour complete copies of the transposon. hybridisation signals in total and plasmid
digested DNA, suggests the tetracycline
Plasmid analysis revealed three plasmids
resistance gene to be associated with the
in strain IPLA 31008 (2.5, 35 and 45 kbp
biggest plasmid in each strain. Signals
long), and four in strain IPLA 31009 (2.5, 12,
around the chromosome fragments were due
35 and 50 kbp log) (Fig. 1).To identify the
to broken segments of the plasmid.

A B
1 2 3 4 M 5 6 7 8 M 9 10 11 12 1 2 3 4 M 5 6 7 8 M 9 10 11 12

21.22

5.1/5.0
4.26
3.53

2.03
1.90
1.58
1.37

Figure 1. Gel electrophoresis (A) and Southern blot analysis (B) of total (digested with either HindIII or EcoRI, respectively) and plasmid (undigested or digested
with HindIII, respectively) DNA from the tetracycline resistant Lactococcus lactis IPLA 31008 (lines 1 through 4) and L. lactis IPLA 31009 (lines 5 through 8), and
a tetracycline-susceptible plasmid-cured derivative of L. lactis IPLA 31009 (lines 9 through 12). As a probe, a segment of tet(M) obtained by specific PCR and
digoxigenin-labelled was used. M, molecular weight marker (digoxigenin-labelled, EcoRI and HindIII-digested, lambda DNA).

Given the plasmid location of the kbp - a size that agrees well with the 18 kbp
tetracycline locus, the stability of resistance in of Tn916 (Flannagan et al., 1994). Moreover,
the two strains was examined. Expressed as when the DNA of susceptible strains was
the number of tetracycline-resistant colonies used as a template, no amplification of tet(M)
after growth for around 100 generations in or Tn916-related sequences was ever
the absence of antibiotic, the stability was obtained (not shown).
rather good in strain IPLA 31008 (92.3%),
Tn916 has been reported to be a
and quite poor in strain IPLA 31009 (<2.5%).
conjugative transposon that can be
In fact, after overnight culture of this last
transferred between and among Gram
strain in the absence of selection, only
positive and Gram negative organisms
26.92% retained their tetracycline resistance.
(Bertram et al., 1991). Because L. lactis is
Analysis of the tetracycline-susceptible
neither pathogenic for animals nor humans,
colonies showed the large plasmids to have
the greatest concern regarding antibiotic
disappeared, and shorter bands of around
resistant lactococci is their capacity to
30-35 kbp to have appeared (data not
transfer resistance determinants to harmful
shown). The lost whole plasmids were never
bacteria (Teuber et al., 1999; European
observed among the segregating
Commission, 2005). To examine the
tetracycline-susceptible strains. Hybridisation
transference of tet(M) from these two Tn916-
experiments with chromosomal and plasmid
bearing L. lactis strains, filter matings were
DNA from susceptible strains gave no signal
performed using plasmid-free strains of L.
(Fig. 1, lanes 10-12). This further showed that
lactis, E. faecalis, and L. plantarum as
the tet(M) gene is located on a plasmid, the
receptors. Strains growing in the
transposon occupies occupying some 18-20

92
transconjugant-selecting plates were Frequency
examined by RAPD to exclude mutation of Donor Recipient
(per donor cell)
the donor and recipient strains (Fig. 2).
Lactococcus lactis subsp. cremoris MG 1614 1.78 x 10-7
M 1 2 3 4 5 6 7 Lactococcus lactis subsp. cremoris Bu2-60 1.80 x 10-6
IPLA 31008
Enterococcus faecalis OG1RF 2.54 x 10-7
Lactobacillus plantarum NC8 0

Lactococcus lactis subsp. cremoris MG 1614 0


Lactococcus lactis subsp. cremoris Bu2-60 1.0 x 10-8
IPLA 31009
Enterococcus faecalis OG1RF 3.9 x 10-7
Lactobacillus plantarum NC8 0

Table 1.- Conjugal transfer of tetracycline resistance


from Lactococcus lactis subsp. lactis IPLA 31008 and L.
lactis subsp. lactis IPLA 31009.

Lactococcus strains are susceptible to


most antibiotics; only intrinsic resistance to
rifampicin has been consistently reported
(Cogan et al., 1972; Reinbold and Reddy,
1974; Orberg and Sandine, 1985; Elliot and
Facklam, 1996; Katla et al., 2001). However,
a few resistant strains have been found
Figure 2.- Random amplification of polymorphic DNA (Teuber et al., 1999; Delgado and Mayo,
(RAPD) analysis of the tetracycline resistant
2002; Raha et al., 2002; Flrez et al., 2005),
Lactococcus lactis IPLA 31008 used a donor, recipients
(L. lactis M1G 1614 or L. lactis Bu2-60) and and some acquired determinants have been
transconjugant strains obtained in the conjugation described, including cat, str, tet(S) (Perreten
experiments. Line 1, L. lactis MG 1614; lines 2 and 3, et al., 1997) and mdt(A) (Perreten et al.,
transconjugants of the mating between IPLA 31008 and
Mg 1614; line 4, L. lactis IPLA 31008 (donor strain); lines
2001). In the present work, tetracycline MICs
5 and 6, transconjugants in the mating between IPLA clearly deviating from the normal range for
31008 and Bu2-60; line 6, L. lactis Bu2-6. M, molecular susceptible populations were found in two L.
weight marker. lactis strains isolated from a Spanish
traditional starter-free cheese (48 and 96 g
-1
ml respectively). In both these strains, a
They were also all examined for their plasmid tet(M) determinant was identified as
content and checked by PCR for the responsible for the resistance phenotype.
presence of tet(M) (see Table 1). This is the first detailed molecular
Transfer of tetracycline resistance was characterization of a tet(M) gene in L. lactis
scored in the two L. lactis strains. In general, strains. The presence of tet(M) gene has
the transfer frequency was rather low (around previously been documented in some lactic
1.0x10-7 transformants per recipient in most acid bacteria, such as E. faecalis (Flannagan
matings). No L. plantarum NC8 tetracycline- et al., 1994) and Lactobacillus species
resistant transconjugants were seen. New (Gevers et al., 2003). It has also been
plasmids were never seen in transconjugants mentioned in Lactococcus in a recent review
(data not shown), suggesting that only the (Roberts, 2005), although the determinant
transposon is mobilized. was not referenced, and no sequences of
tet(M) associated with Lactococcus were
Discussion found in either the GenBank or EMBL
databases. tet(M) has been associated with
The emergence and spread of resistance Tn916 (first isolated from E. faecalis
to antibiotics in bacteria poses a serious [Flannagan et al., 1994]), a highly infective
threat to human and animal health; it could transposon that has contributed to the spread
also cause major economic problems of tetracycline resistance among Gram
(European Commission, 2005). At present positive and Gram negative bacteria (Rice,
there is concern that microbial cultures used 1998; Bertram et al., 1991). Indeed,
in food production or naturally occurring in transposon-related sequences were
foods could become vehicles for the encountered in the two resistant strains
transmission of acquired antibiotic resistance examined in this work. The transposon was
genes (Teuber et al., 1999; European found to be inserted into plasmids of similar
Commission, 2005). size in the two L. lactis strains. Further, the

93
stability of the transposon was completely Elliot, J. A., and Facklam, R. R. 1996. Antimicrobial
susceptibilities of Lactococcus lactis and Lactococcus
different in each strain after subculturing in garviae and a proposed method to discriminate between
antibiotic-free media, although whether this them. J. Clin. Microbiol. 34: 1296-1298.
European-Commission. 2005. Opinion of the Scientific Panel
was a consequence of different plasmid or on Additives and Products in Substances used in Animal
chromosomal backgrounds remains to be Feed on the updating of the criteria used in the assessment
of bacteria for resistance to antibiotics of human and
seen. These findings suggest that veterinary importance. The EFSA J. 223: 1-12.
independent transfer events of Tn916 Flannagan, S. E., Zitzow, L. A., Su, Y. A., and Clewell, D. B.
probably occurred from an unknown 1994. Nucleotide sequence of the 18-kb conjugative
transposon Tn916 from Enterococcus faecalis. Plasmid 32:
intermediate host. The functionality of the 350-354.
transposon in the strains of this study was Flrez, A. B., Delgado, S., and Mayo,B. 2005. Antimicrobial
susceptibility of lactic acid bacteria isolated from a cheese
demonstrated by conjugation. Only the environment. Canadian J. Microbiol. 51: 51-58.
transposon was transferred in matings with Flrez, A. B., lvarez-Martn, P., Lpez-Daz, T. M., and Mayo.
B. 2006. Microbiological characterisation of the traditional
other species, suggesting that the plasmids Spanish blue-veined Cabrales cheese: identification of
harbouring Tn916 are not conjugative and dominant lactic acid bacteria. Eur. Food Res. Technol. 223:
that the transposon cannot mobilize the 503-508.
Fox, P. F., Guinee, T. P., Cogan, T. M., and McSweeney, P. L.
plasmids. H. 2000. Fundamentals of Cheese Science. AN Aspen
Publishers Inc., Gaithersburg, Ma., USA.
Franz, C. M., Holzapfel, W. H., and Stiles, M. E. 1999.
In conclusion, the present study reports Enterococci at the crossroad of food safety? Int. J. Food
two L. lactis strains from an artisanal cheese Microbiol. 47: 1-24.
to harbour an acquired tetracycline resistance Gevers, D., Danielsen, M., Huys, G., and Swings, J. 2003.
Molecular characterization of tet(M) genes in Lactobacillus
phenotype encoded by tet(M). The gene is isolates from different types of fermented dry sausage.
located on a functional Tn916 transposon, Appl. Environ. Microbiol. 69: 1270-1275.
Kayser, F. H. 2003. Safety aspects of enterococci from the
inserted into resident plasmids of the parental medical point of view. Int. J. Food Microbiol. 88: 255-262.
tetracycline-susceptible strains from an Katla, A. K., Kruse, H., Johnsen, G., and Herikstad, H. 2001.
Antimicrobial susceptibility of starter culture bacteria used
unknown host. The presence of acquired in Norwegian dairy products. Int. J. Food Microbiol. 67:
antibiotic resistance genes in food- 147-152.
Normark, B. H., Normark, S. 2002. Evolution and spread of
transforming bacteria justifies the antibiotic antibiotic resistance. J. Intern. Med. 252: 91-106.
surveying of candidate starter culture strains. Orberg, P. K., Sandine, W. E. 1985 Survey of antimicrobial
resistance in lactic streptococci. Appl. Environ. Microbiol.
49: 538-542.
Acknowledgements OSullivan, D. J. and Klaenhammer, T. R. 1993. Rapid mini-
prep isolation of high-quality plasmid DNA from
Lactococcus and Lactobacillus spp. Appl. Environ.
This work was supported by an EU Microbiol. 59: 2730-2733.
project within the sixth Framework Perreten, V., Schwarz, F., Cresta, L., Boeglin, M., Dasen, G.,
and Teuber, M. 1997. Antibiotic resistance spread in food.
Programme (ACE-ART, Ref. 506214). M. S. Nature 389: 801-802.
Ammor was awarded a postdoctoral Perreten, V., Schwarz, F. V., Teuber, M., and Levy, S. B. 2001.
Mdt(A), a new efflux protein conferring multiple antibiotic
fellowship from the Secretara de Estado de resistance in Lactococcus lactis and Escherichia coli.
Universidades e Investigacin of the Antimicrob. Agents Chemother. 45:1109-14.
Raha, A. R., Ross, E., Yusoff, K., Manap, M. Y., and Ideris, A.
Spanish Ministry of Education and Science 2002. Characterisation and molecular cloning of an
(Ref. SB2004-0165). erythromycin resistance plasmid of Lactococcus lactis
isolated from chicken cecum. J. Biochem. Mol. Biol.
Biophys. 6: 7-11.
References Reinbold, G. W., and Reddy, M. S. 1974. Sensitivity or
resistance of dairy starter and associated microorganisms
to selected antibiotics. J. Milk Food Technol. 37: 517-521.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Rice, L. B. 1998. Tn916 family conjugative transposons and
Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped dissemination of antimicrobial resistance determinants.
BLAST and PSI-BLAST: a new generation of protein Antimicrob. Agents Chemother. 42: 1871-1877.
database search programs. Nucleic Acids Res. 25: 3389- Roberts, M. C. 2005. Update on acquired tetracycline resistance
3402. genes. FEMS Microbiol. Lett. 245: 195-203.
Barbosa, T. M., Scott, K. P., and Flint, H. J. 1999. Evidence for Sambrook, J., Fritsch, E. F., and Maniatis, T. 1989. Molecular
recent intergeneric transfer of a new tetracycline resistance Cloning: A Laboratory Manual. 2nd ed. Cold Spring Harbor
gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the Laboratory, Cold Spring Harbor, New York, USA.
occurrence of tet(O) in ruminal bacteria. Environ. Microbiol. Scott, K. P., Melville, C. M., Barbosa, T. M., and Flint, H. J.
1: 53-64. 2000. Occurrence of the new tetracycline resistance gene
Bertram, J., Stratz, M., Durre, P. 1991. Natural transfer of tet(W) in bacteria from the human gut. Antimicrob. Agents
conjugative transposon Tn916 between gram-positive and Chemother. 44: 775-777.
gram-negative bacteria. J. Bacteriol. 173: 443-448. Smit, G., Smit, B. A., and Engels, W. J. 2005. Flavour
Clermont, D., Chesneau, O., de Cespedes, G., and Horaud, T. formation by lactic acid bacteria and biochemical flavour
1997. New tetracycline resistance determinants coding for profiling of cheese products. FEMS Microbiol. Rev. 29:
ribosomal protection in streptococci and nucleotide 591-610.
sequence of tet(T) isolated from Streptococcus pyogenes Stapleton, P., Adams, V., Pike, R., Lucas, V., Roberts, G.,
A498. Antimicrob. Agents Chemother. 41: 112-116. Mullany, P., Rowbury, R., Wilson, M., and Richards, H.
Cogan, T. M. 1972. Susceptibility of cheese and yogurt starter 2004. Characterisation of viridans group streptococci with
bacteria to antibiotics. Appl. Microbiol. 23: 960-965. different levels of Tet(M)-mediated tetracycline resistance.
Davies, J. 1994. Inactivation of antibiotics and the dissemination Int. J. Antimicrob. Agents 24: 439-43.
of resistance genes. Science 264: 375-382. Su, Y. A., He, P., and Clewell, D. B. 1992. Characterization of
Delgado, S., and Mayo, B. 2004. Phenotypic and genetic the tet(M) determinant of Tn916: evidence for regulation by
diversity of Lactococcus lactis and Enterococcus spp. transcription attenuation. Antimicrob. Agents Chemother.
strains isolated from Northern Spain starter-free farmhouse 36: 769-778.
cheeses. Int. J. Food Microbiol. 90: 309-319.

94
Teuber, M., Meile, L., and Schwarz, F. 1999. Acquire antibiotic Witte, W. 2000. Selective pressure by antibiotic use in livestock.
resistance in lactic acid bacteria from food. Antonie van Int. J. Antimicrob. Agents 16: S19-S24.
Leeuwenhock 76: 115-137.

95
Artculo VIII.- Molecular anlisis of tetracycline resistance in dominant intestinal
Bifidobacterium species from healthy humans mediated by tet(W) genes.
Applied and Environmental Microbiology. 72:7377-7379 (2006).

Objetivos: El objetivo del trabajo fue la caracterizacin molecular de los determinantes


genticos que confieren resistencia a tetraciclina en un grupo cepas de bifidobacterias
de distintas especies.

Resultados: Unos 17 aislados de bifidobacterias presentaban CIMs a tetraciclina entre 4


y 32 g/ml, por lo que se consideraron fenotpicamente resistentes. Tras su tipificacin
mediante RAPD se consider que los aislados correspondan a siete cepas de las
especies Bifidobacterium longum (5), Bifidobacterium bifidum (1) y Bifidobacterium animalis
(1). Para la deteccin de genes de resistencia a tetraciclina se utilizaron
oligonucletidos universales y especficos. Los amplicones obtenidos se secuenciaron y
el anlisis de la secuencia mostr una homologa del 98-99% con el gen tet(W) descrito
en bacterias intestinales y del rumen. La secuenciacin completa del gen tet(W) y de la
regin anterior y posterior al mismo se realiz en la cepa H66 de B. longum. La
secuencia obtenida fue idntica a la del gen tet(W) descrito en Butyrivibrio fibrisolvens, a
excepcin de un nico nucletido en la regin codificadora. Sin embargo, en la cepa
H66 no se detect el transposn TnB1230 presente en B. fibrisolvens. El gen se encuentra
localizado en el cromosoma bacteriano en todas las cepas, sin embargo la organizacin
gentica vara ampliamente entre especies y cepas.

Conclusiones: Se han encontrado diversas especies de bifidobacterias resistentes a


tetraciclina formando parte de las poblaciones mayoritarias de individuos sanos sin
una historia reciente de tratamiento con antibiticos. El determinante responsable de la
resistencia fue, en todos los casos, tet(W), gen que se encuentra ampliamente
distribuido entre bacterias intestinales de distinto origen.

97
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2006, p. 73777379 Vol. 72, No. 11
0099-2240/06/$08.000 doi:10.1128/AEM.00486-06
Copyright 2006, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of tet(W) Gene-Mediated Tetracycline Resistance in


Dominant Intestinal Bifidobacterium Species from Healthy Humans
Ana Belen Florez, Mohammed Salim Ammor, Pablo Alvarez-Martn,
Abelardo Margolles, and Baltasar Mayo*
Instituto de Productos Lacteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Asturias, Spain
Received 28 February 2006/Accepted 19 August 2006

tet(W) was found responsible for tetracycline resistance (MICs, 4 to >32 g ml1) in dominant bifidobac-
terial species from the gastrointestinal tracts of healthy humans. The gene from Bifidobacterium longum H66
proved to be identical over a 2.6-kbp region to the recently described tet(W) determinant of Butyrivibrio
fibrisolvens.

The commensal intestinal microbiota of humans and an- recent history of antibiotic treatment (4). The object of this
imals may act as a reservoir of antibiotic resistance genes study was to investigate the nature and molecular organization
that could ultimately be transferred to pathogens (15, 18); in of the tetracycline determinant encoding this resistance.
fact, gene transfer between bacterial species in the gastro- MICs of tetracycline for bifidobacteria. The MICs of tetra-
intestinal tracts (GIT) of mammals is known to occur (11). cycline for a series of susceptible (14 isolates) and resistant (16
Bifidobacteria are among the dominant populations of the isolates) bifidobacterial species which had been assayed by
human GIT microbiota, where they are thought to play a microdilution (4) were precisely determined by Etest (AB Bio-
pivotal role in maintaining the microbial balance necessary disk, Solna, Sweden). Assays were performed in LSM test agar
for intestinal health (19). Bifidobacterial strains are there- (6) (90% IsoSensitest, 10% MRS, 1.5% agar; all from Oxoid,
fore frequently used as probiotics in the prophylaxis and New Hampshire, United Kingdom) supplemented with 0.3 g of
therapy of GIT disorders (12). This practice, however, re- cysteine liter1. Plates were incubated at 37C for 48 h in an
quires that they be screened for acquired antibiotic resis- anaerobic chamber (atmosphere, 10% H210% CO280% N2).
tance determinants if the latter are not to be propagated The MICs for susceptible isolates ranged from 0.25 to 1 g
through the food chain (14, 15, 18). ml1, whereas those for isolates thought to be resistant ranged
Tetracyclines inhibit protein synthesis by preventing the at- from 4 to 32 g ml1. MICs obtained for the same strains in
tachment of aminoacyl-tRNA to the bacterial ribosome (2). duplicate experiments never exceeded 1 order of dilution.
The broad-spectrum antimicrobial properties of these agents, To exclude the analysis of replicates, resistant and suscepti-
the absence of major adverse side effects, and their low price ble isolates were typed by rapid amplification of polymorphic
have led to their intensive use not only in the treatment of DNA (RAPD) analysis by PCR. Amplifications were per-
human and animal infections but also as prophylactic agents formed using primer OPA18 and PCR conditions described
and growth promoters in livestock raising and aquaculture (2). elsewhere (8). Seven different RAPD-PCR profiles identifying
This extensive use has promoted the appearance of resistance distinct strains were obtained among the resistant isolates, as
and its spread by horizontal acquisition (2, 13). Resistance is follows: Bifidobacterium longum (11 isolates, 5 strains), Bifido-
mediated through efflux proteins, ribosomal protection pro- bacterium bifidum (5 isolates, 1 strain), and Bifidobacterium
teins, or antibiotic-inactivating enzymes (for a review, see animalis subsp. animalis (1 isolate, 1 strain).
reference 13). Amplification and analysis of tetracycline resistance genes.
The tet(W) gene, which encodes a ribosomal protection pro- The presence of genes encoding ribosomal protection proteins
tein, has recently been described for a wide range of gram-positive was checked among resistant and susceptible strains by PCR
and gram-negative bacteria (1, 2), including bifidobacteria (7, 10, with two pairs of degenerate primers: DIDII (3) and Tet1
17). This gene has been associated with a conjugative transposon Tet2 (1). Resistant strains all yielded a single PCR product of
(TnB1230) in Butyrivibrio fibrisolvens (9) that has been shown to the same size when either of the primer pairs was used. Sur-
transfer at high frequencies (up to 5.1 103 transconjugants per prisingly, positive amplification was also obtained with DNA
recipient) (16). from the susceptible strain B. longum M21. Additional PCR
In a recent survey, a microbroth assay showed atypical (16 assays were then performed with primers TetWF and Tet2,
g ml1) MICs of tetracycline for human bifidobacterial specific for tet(W) (17), and primers DI and TetMR, specific
strains isolated from fecal samples of healthy adults without a for tet(M) (5). No DNA was amplified from either resistant or
susceptible isolates with primers for tet(M). In contrast, a prod-
uct of about 1,250 bp was obtained for all 16 resistant isolates
* Corresponding author. Mailing address: Instituto de Productos when primers for tet(W) were used. Purified amplicons were
Lacteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villavi-
ciosa, Asturias, Spain. Phone: 34 985 89 21 31. Fax: 34 985 89 22 33.
sequenced and the resulting sequences analyzed and compared
E-mail: baltasar.mayo@ipla.csic.es. to others held in public databases.

Published ahead of print on 25 August 2006. Analysis of the nucleotide sequences showed them to share

7377
7378 FLOREZ ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 1. Phylogenetic tree showing the evolutionary relationships of complete Tet(W) and other ribosome protection proteins from tetracycline-
resistant bacteria. GenBank accession numbers for the nucleotide sequences used to derive amino acid sequences are given in parentheses. The
rooted neighbor-joining tree was generated using DNAman software (Lynnon Corporation, Vaudreuil-Dorion, Quebec, Canada). Numbers beside
nodes indicate bootstrap values when these are greater than 95% (based on 500 trials). Bar, 0.05% difference in amino acid sequence.

99.8% identity. Comparison of the sequences showed a high protein sequences in unrelated bacterial and bifidobacterial
degree of similarity at the nucleotide level to several tetracy- species suggests that recent horizontal transfer has occurred
cline resistance genes. In particular, complete identity was seen (1, 16). Intriguing is the fact that different MICs are repeatedly
with an internal segment of the tet(W) gene from B. fibrisolvens reported for identical genes (7, 10, 17). Multiple loci, different
(1). Based on this homology, primers TetWSacF (5-CCCTG expression levels of tet(W), and/or the influence of the genetic
GAGCTCATGCTCATGTAC-3) and TetWSacR (5-CCAT background of the strains may account for the phenotypic
CGGAGCTCCATAACTTCTG-3) were designed to amplify differences.
the whole gene and its surrounding promoter and terminator Location of the tet(W) gene. The genetic location of tet(W) in
regions. Positive amplification was obtained only when DNA the distinct species was assessed by hybridization using as a
from the resistant strains was used as a template, suggesting probe a 1.2-kbp internal segment of the gene obtained by PCR
that the susceptible strain may harbor a shortened (nonfunc- and labeled with digoxigenin (Roche Applied Science, Basel,
tional) version of the gene. Switzerland). Total and plasmid DNAs digested with the re-
The amplicon was cloned into pUC19 and introduced into striction enzymes EcoRI and HindIII (Roche) were hybridized
Escherichia coli, which became resistant to tetracycline (MIC, using high-stringency standard conditions (hybridization at
64 g ml1). The insert was then double-strand sequenced. At 68C and two final washing steps in 0.5 SSC [1 SSC is 0.15
the nucleotide level, analysis of a 2,589-bp segment showed M NaCl plus 0.015 M sodium citrate]0.1% SDS at 68C for 15
that the sequence was identical to that of B. fibrisolvens 1.230 min). Figure 2 shows the results obtained with EcoRI-digested
(1, 9), except for a single oligonucleotide change in the coding total DNA. A positive hybridization signal was obtained with
region leading to a conservative amino acid replacement in its all tetracycline-resistant strains and, as expected, with the sus-
deduced Tet(W) protein. Further, the B. longum H66 sequence ceptible strain M21. The hybridization signal appeared at a
was found to share around 98% identity to recently reported different position in each of the strains (estimated sizes, 2.5 to
tet(W) genes from bifidobacterial species (GenBank accession 16 kbp), except for the related strains B. longum B93 and B94
no. AF202986) (7, 17). A phylogenetic tree showing the rela- (displaying distinct RAPD-PCR patterns but isolated from the
tionships of the deduced Tet(W) proteins and those of other same individual). This suggests that the genetic organization of
ribosome protection proteins is depicted in Fig. 1. The pres- the loci might be different in each strain. The tet(W) locus is
ence of resistance genes with nearly identical nucleotide and thought to be located on the bacterial chromosome, since only

100
VOL. 72, 2006 tet(W) GENE FROM HUMAN BIFIDOBACTERIUM SPECIES 7379

longum H66 has been deposited in GenBank (accession no.


DQ060146).

This work was supported by an EU project within the VI Framework


Programme (ACE-ART, ref. CT-2003-506214). M.S.A. was awarded a
postdoctoral fellowship from the Secretara de Estado de Univer-
sidades e Investigacion of the Spanish Ministry of Education and
Science (SB2004-0165).

REFERENCES
1. Barbosa, T. M., K. P. Scott, and H. J. Flint. 1999. Evidence for recent
intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated
from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacte-
ria. Environ. Microbiol. 1:5364.
FIG. 2. Hybridization of EcoRI-digested chromosomal DNA from 2. Chopra, I., and M. Roberts. 2001. Tetracycline antibiotics: mode of action,
different Bifidobacterium strains using a 1.2-kbp PCR amplicon ob- applications, molecular biology, and epidemiology of bacterial resistance.
Microbiol. Mol. Biol. Rev. 65:232260.
tained with the specific primers for tet(W) as a probe. The code num-
3. Clermont, D., O. Chesneau, G. De Cespedes, and T. Horaud. 1997. New
bers of the strains are given above the lane numbers. Tetracycline- tetracycline resistance determinants coding for ribosomal protection in
susceptible strains are asterisked. Lanes 1, 2, 3, 4, 5, 6, and 7, streptococci and nucleotide sequence of tet(T) isolated from Streptococcus
Bifidobacterium longum strains; lane 8, Bifidobacterium animalis E43; pyogenes A498. Antimicrob. Agents Chemother. 41:112116.
lanes 9 and 10, Bifidobacterium bifidum strains. Lanes M, molecular 4. Delgado, S., A. B. Florez, and B. Mayo. 2005. Antibiotic susceptibility of
weight marker (digoxigenin-labeled, EcoRI- and HindIII-digested Lactobacillus and Bifidobacterium species from the human gastrointestinal
lambda DNA). tract. Curr. Microbiol. 50:202207.
5. Donoghue, D. J. 2003. Antibiotic residues in poultry tissues and eggs: human
health concerns? Poult. Sci. 82:618621.
6. Klare, I., C. Konstabel, S. Muller-Bertling, R. Reissbrodt, G. Huys, M.
the resistant strain B. bifidum L71 was shown to harbor a Vancanneyt, J. Swings, H. Goossens, and W. Witte. 2005. Evaluation of new
broth media for microdilution antibiotic susceptibility testing of lactobacilli,
plasmid (of around 15 kbp), and its hybridization signals did pediococci, lactococci, and bifidobacteria. Appl. Environ. Microbiol.
not match the position of EcoRI and HindIII plasmid frag- 71:89828986.
ments (data not shown). 7. Masco, L., K. Van Hoorde, E. De Brandt, J. Swings, and G. Huys. 2006.
Antimicrobial susceptibility of Bifidobacterium strains from humans, animals
Since the tet(W) gene is encoded on a conjugative transpo- and probiotic products. J. Antimicrob. Chemother. 58:8594.
son in B. fibrisolvens 1.230 (TnB1230) (9, 17), primers based on 8. Matto, J., E. Malinen, M.-L. Suihko, M. Alander, A. Palva, and M. Saarela.
transposon sequences were also synthesized and used in PCR 2004. Genetic heterogeneity and functional properties of intestinal bifido-
bacteria. J. Appl. Microbiol. 97:459470.
experiments. However, no amplification products were ob- 9. Melville, C. M., R. Brunel, H. J. Flint, and K. P. Scott. 2004. The Butyrivibrio
tained with any strain, suggesting that there are no sequences fibrisolvens tet(W) gene is carried on the novel conjugative transposon
TnB1230, which contains duplicated nitroreductase coding sequences. J.
related to this mobile unit in these Bifidobacterium species. Bacteriol. 186:36563659.
Further, preliminary inverse-PCR experiments indicate that 10. Moubareck, C., F. Gavini, L. Vaugien, M. J. Butel, and F. Doucet-Populaire.
tet(W) is inserted into a putative open reading frame encoding 2005. Antimicrobial susceptibility of bifidobacteria. J. Antimicrob. Che-
mother. 55:3844.
a permease (GenBank accession no. AE014295) of an ABC 11. Netherwood, T., R. Bowden, P. Harrosin, A. G. ODonnel, D. S. Parker, and
transporter system in B. longum NCC2705 (data not shown). H. J. Gilbert. 1999. Gene transfer in the gastrointestinal tract. Appl. Environ.
In conclusion, these and other results indicate that dominant Microbiol. 65:51395141.
12. Ouwehand, A. C., S. Salminen, and E. Isolauri. 2002. Probiotics: an overview
Bifidobacterium species from the human GIT frequently har- of beneficial effects. Antonie Leeuwenhoek 82:279289.
bor acquired tetracycline resistance encoded by a tet(W) gene. 13. Roberts, M. C. 2005. Update on acquired tetracycline resistance genes.
FEMS Microbiol. Lett. 245:195203.
The fact that genes from Bifidobacterium showed more nucle- 14. Saarela, M., G. Mogensen, R. Fonden, J. Matto, and T. Mattila-Sandholm.
otide changes than those from species of different genera 2000. Probiotic bacteria: safety, functional and technological properties.
strongly suggests independent transfer events. The spread of J. Biotechnol. 84:197215.
15. Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal bacteria as
this gene among cattle rumen and human GIT organisms may reservoirs for antibiotic resistance genes. Trends Microbiol. 12:412416.
be occurring very rapidly by unknown mechanisms that merit 16. Scott, K. P., T. M. Barbosa, K. J. Forbes, and H. J. Flint. 1997. High-
further research. However, preliminary conjugation experi- frequency transfer of a naturally occurring chromosomal tetracycline resis-
tance element in the ruminal anaerobe Butyrivibrio fibrisolvens. Appl. Envi-
ments showed that tetracycline resistance from B. longum H66 ron. Microbiol. 63:34053411.
and L42 did not transfer to susceptible strains (data not 17. Scott, K. P., C. M. Melville, T. M. Barbosa, and H. J. Flint. 2000. Occurrence
of the new tetracycline resistance gene tet(W) in bacteria from the human
shown). Once acquired, genes could either be stably main- gut. Antimicrob. Agents Chemother. 44:775777.
tained in the absence of antibiotic selection or selected by 18. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in
continued exposure to antibiotics through dietary intake (5). lactic acid bacteria from food. Antonie Leeuwenhoek 76:115137.
19. Ventura, M., D. van Sinderen, G. F. Fitzgerald, and R. Zink. 2004. Insights
Nucleotide sequence accession number. The nucleotide se- into the taxonomy, genetics and physiology of bifidobacteria. Antonie Leeu-
quence of the tet(W) gene and its surrounding regions from B. wenhoek 86:205233.

101
Artculo IX.- Molecular anlisis of a chromosomally-encoded erm(B) gene and its flanking
insertion points in Lactobacillus johnsonii G41.
Antimicrobial Agents Chemotherapy. 50:4189-4190 (2006).

Objetivos: El objetivo de este trabajo fue la caracterizacin del determinante gentico


responsable de la resistencia a eritromicina y clindamicina en la cepa G41 de
Lactobacillus johnsonii. Se determin la secuencia completa del gen erm(B) y de las zonas
adyacentes, con el fin de determinar, si fuera posible, el mecanismo de transferencia.

Resultados: Teniendo en cuenta los puntos de corte descritos para los lactobacilos, la
cepa G41 fue considerada resistente a eritromicina y clindamicina ya que sus CIMs
respectivas fueron superiores a 256 g/ml. Por PCR se detect el gen erm(B), el ms
frecuente dentro de las bacterias Gram-positivas. Mediante tcnicas de hibridacin el
gen se localiz en el cromosoma bacteriano de Lb. johnsonii. La tcnica de PCR inversa
permiti el estudio de las secuencias circundantes, resultando idnticas a un fragmento
del plsmido pRE25 descrito en Enterococcus faecalis, incluyendo la zona codificadora
del gen erm(B) y las zonas adyacentes. Se especula que una secuencia de 9 pb presente
en una copia en el genoma de Lb. johnsonii y 19 veces repetida en el plsmido pRE25
podra estar involucrada en la integracin. En una posterior resolucin podran haberse
generado secuencias nucleotdicas nuevas alrededor de las zonas de insercin.

Conclusiones: Se ha secuenciado el gen erm(B) presente en Lb. johnsonii G41,


resultando idntico al gen erm(B) identificado en cepas de Streptococcus, Enterococcus y
Lactobacillus. Las secuencias anteriores y posteriores al gen sugieren la insercin de un
fragmento del plsmido pRE25 de E. faecalis en la cepa G41 o en una cepa intermedia,
seguida de una resolucin subsiguiente.

103
ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2006, p. 41894190 Vol. 50, No. 12
0066-4804/06/$08.000 doi:10.1128/AAC.00657-06
Copyright 2006, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of a Chromosome-Carried erm(B) Gene and Its


Flanking Insertion Points in Lactobacillus johnsonii G41
Ana Belen Florez, Mohammed Salim Ammor, Susana Delgado, and Baltasar Mayo*
Instituto de Productos Lacteos de Asturias (CSIC), Carretera de Infiesto s/n, 33300-Villaviciosa, Asturias, Spain
Received 30 May 2006/Returned for modification 28 July 2006/Accepted 12 September 2006

An erm(B) gene carried on the Lactobacillus johnsonii G41 chromosome and the upstream and downstream
regions were fully sequenced. Apparently, a 1,495-bp segment of pRE25 from Enterococcus faecalis carrying the
erm(B) gene became inserted, by an unknown mechanism, into the L. johnsonii chromosome.

The possession of no acquired resistance to antibiotics is a An amplicon of around 5.5 kbp was obtained with the EcoRI-
key safety criterion that candidate probiotic microorganisms digested ligation, from which the complete sequence of erm(B)
should fulfill besides functional and technological properties and the upstream and downstream regions were obtained
(6). A collection of intestinal lactic acid bacteria and bifidobac- (GenBank accession no. DQ518904).
teria of human origin was investigated for antibiotic resistance Two partial open reading frames (ORFs) with high homology
during a survey for new probiotic strains (1). Among them, one to two contiguous genes on the L. johnsonii NCC 533 chromo-
Lactobacillus johnsonii strain (G41) was suspected of being some (GenBank accession no. NC_005362) were observed at the
resistant to erythromycin. Characterization of antibiotic resis- extremes of the segment (Fig. 2). The first (5-orf1 in Fig. 2)
tance genes and the elements involved in their transfer might encoded the C-terminal part of a phosphoenolpyruvate phospho-
help to reduce the spread of antibiotic resistance by the food transferase component (protein identification no. ABF70486.1).
chain (2, 8). Besides mutations in the 23S rRNA molecule, A second ORF split into two parts by the erm(B) region (5-orf2
more than 40 genes encoding efflux proteins, methylases, and and 3-orf2 in Fig. 2) may code for a hypothetical protein (pro-
inactivating enzymes have been described previously (4). tein identification no. AAS09591.1).
The MIC of erythromycin for strain G41 was found to be The inserted sequence comprised 1,495 bp identical to those
256 g ml1 by Etest (AB Biodisk, Solna, Sweden). A MIC corresponding to the erm(B) locus in Enterococcus faecalis
of 4 g ml1 has been recently proposed as the microbio- plasmid pRE25 (7) (positions 12,577 to 14,022 of the sequence
logical breakpoint to separate susceptible from resistant with GenBank accession no. X92945). This segment included
strains (2). In addition, the MIC of clindamycin for this three complete ORFs related to the MLS phenotype [orf3,
strain was also 256 g ml1. encoding the putative MLS leader peptide; the erm(B) gene
Identification and location of the erm(B) determinant. The (orf4); and orf5, a second small ORF present at most erm(B)
genes ermA, erm(B), erm(C), erm(F), and mef(A) are widely loci (3, 5)] and some adjacent pRE25 sequences.
distributed among gram-positive and gram-negative organisms The insertion is flanked by a duplicated 9-bp sequence (AA
(3, 5). Using as a template total DNA from L. johnsonii G41, AGAAAAA) (Fig. 2), which is originally present once at the
positive amplification was only obtained for erm(B); the prim-
ers and PCR conditions used were reported elsewhere (5). The
sequence of this amplicon proved to be identical to erm(B)
sequences from Streptococcus, Enterococcus, and Lactobacillus
sp. strains.
As no plasmid DNA was detectable in L. johnsonii G41,
hybridization experiments, using as a probe a digoxigenin-la-
beled erm(B) internal segment and high-stringency conditions,
located the erm(B) determinant on the bacterial chromosome
(Fig. 1).
Sequence and analysis of the erm(B) gene and the surround-
ing regions. Total DNA from L. johnsonii was digested with
EcoRI and HindIII, self-ligated, and used as a template for
inverse PCR with primers erm(B)1f (5-CATCAAGCAATG
AAACACG-3) and emrB2r (5-GTCTGTTTCAAAACAGT
AGATG-3), which are based on erm(B) internal sequences. FIG. 1. Southern blot analysis of total genomic DNA from L.
johnsonii G41 left undigested or digested with EcoRI, BlgII, PstI, or
ClaI (lanes 1 through 5, respectively) and hybridized with an internal
* Corresponding author. Mailing address: Baltasar Mayo, Instituto segment of the erm(B) gene obtained by PCR and labeled with digoxi-
de Productos Lacteos de Asturias (CSIC), Carretera de Infiesto s/n, genin (Roche [Hoffmann-La Roche Ltd., Basel, Switzerland]). Lanes
33300-Villaviciosa, Spain. Phone: 34 985 89 21 31. Fax: 34 985 89 22 33. M, molecular size marker (digoxigenin-labeled EcoRI-HindIII-di-
E-mail: baltasar.mayo@ipla.csic.es. gested lambda DNA [Roche]). The values on the left are molecular

Published ahead of print on 25 September 2006. sizes in kilobase pairs.

4189
4190 NOTES ANTIMICROB. AGENTS CHEMOTHER.

FIG. 2. Diagram of the erm(B) gene of L. johnsonii G41 and the sequences and structures at its chromosomal integration position. Gray and
black bars indicate the segment homologous to the L. johnsonii NC 533 chromosome (GenBank accession no. NC_005362) and that identical to
the erm(B) locus in E. faecalis plasmid pRE25 (positions 12,527 to 14,022 of the sequence with GenBank accession no. X92945), respectively. ORFs
and sequences are as indicated in the text. This diagram is not drawn to scale.

start of corresponding orf2 in the L. johnsonii NCC 533 ge- was awarded a postdoctoral fellowship from the Secretara de Estado
nome and twice and in the same place and orientation in the de Universidades e Investigacion of the Spanish Ministry of Education
and Science (reference no. SB2004-0165).
pRE25 sequence. Chromosomal and plasmid sequences might
provide homology for the integration of pRE25 in a manner REFERENCES
similar to phage integration by attB and attP sites. However, 1. Delgado, S., A. B. Florez, and B. Mayo. 2005. Antibiotic susceptibility of
two sequences with no homology to other DNA sequences Lactobacillus and Bifidobacterium species from the human gastrointestinal
appeared around the junction points (Fig. 2): one at the 3 end tract. Curr. Microbiol. 50:202207.
2. European Commission. 2005. Opinion of the Scientific Panel on Additives
(AACGTATTTCTCGCAGCT), immediately downstream of and Products in Substances used in Animal Feed on the updating of the
the 9-bp sequence, and a second one at the 5 end (GCCAG criteria used in the assessment of bacteria for resistance to antibiotics of
CTTTAA), 75 bp upstream of the 9-bp sequence. These se- human and veterinary importance. EFSA J. 223:112.
3. Jensen, L. B., N. Frimodt-Moller, and F. M. Aarestrup. 1999. Presence of erm
quences suggest further DNA rearrangement during or after gene classes in gram-positive bacteria of animal and human origin in Den-
integration. mark. FEMS Microbiol. Lett. 170:151158.
4. Leclercq, R. 2002. Mechanisms of resistance to macrolides and lincosamides:
In conclusion, this work reports on the molecular character- nature of the resistance elements and their clinical implications. Clin. Infect.
ization of an erm(B) gene encoding erythromycin and clinda- Dis. 34:482492.
mycin resistance in L. johnsonii G41. The gene was found to be 5. Roberts, M. C., W. O. Chung, D. Roe, M. Xia, C. Marquez, G. Borthagaray,
W. L. Whittington, and K. K. Holmes. 1999. Erythromycin-resistant Neisseria
identical to that present in many gram-positive bacteria. Anal- gonorrhoeae and oral commensal Neisseria spp. carry known rRNA methylase
ysis of the surrounding regions suggested that a segment of the genes. Antimicrob. Agents Chemother. 43:13671372.
erm(B) locus from enterococcal plasmid pRE25 was integrated 6. Saarela, M., G. Mogensen, R. Fonden, J. Matto, and T. Mattila-Sandholm.
2000. Probiotic bacteria: safety, functional and technological properties.
and rearranged into the L. johnsonii genome via unknown J. Biotechnol. 84:197215.
mechanisms. 7. Schwarz, F. V., V. Perreten, and M. Teuber. 2001. Sequence of the 50-kb
conjugative multiresistance plasmid pRE25 from Enterococcus faecalis RE25.
Plasmid 46:170187.
This work was supported by an EU project within the sixth Frame- 8. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic resistance in
work Programme (ACE-ART, reference no. 506214). M. S. Ammor lactic acid bacteria from food. Antonie Leeuwenhoek 76:115137.

106
Artculo X.- Acquired macrolide resistance in Lactobacillus rhamnosus E41 associated with
a transition mutation in the 23S rRNA.

Objetivo: El objetivo de este trabajo fue determinar la causa de la resistencia a


macrlidos de la cepa E41 de Lb. rhamnosus. Estudios previos por PCR e hibridacin de
microarrays haban resultado negativos para un amplio nmero de genes de
resistencia. Por esta razn se examin mediante oligonucletidos universales la
presencia de mutaciones en el gen que codifica el ARNr 23S.

Resultados: En numerosos aislados clnicos se han descrito mutaciones puntuales en


genes o protenas ribosomales que afectan al sitio de unin de la eritromicina. Por
tanto, mediante la amplificacin de un fragmento del ARNr 23S y su posterior
restriccin se pudo observar una mutacin puntual sustituyendo una adenina por una
guanina en la posicin 2058 (siguiendo la numeracin de Escherichia coli). La mutacin
se comprob tambin mediante la secuenciacin de los amplicones. Ambas tcnicas
mostraron heterocigosis de secuencia en distintos operones, indicando la presencia de
genes originales y mutados en la misma cepa.

Conclusiones: La presencia de una mutacin en el gen que codifica el ARNr 23S de la


cepa Lb. rhamnosus E41 confiriendo resistencia a macrlidos es la primera que se
describe en una bacteria lctica. Dado que este tipo de resistencia no es transmisible, la
cepa podra explotarse por su potencial probitico, incluyendo su administracin
durante tratamiento con esta clase de antibiticos.

107
Acquired macrolide resistance in Lactobacillus rhamnosus E41
associated with a transition mutation in the 23S rRNA gene.

Abstract

Restriction fragment length polymorphism and DNA sequencing of PCR products


showed that a Lactobacillus rhamnosus strain of human origin, resistant to macrolides,
lincosamides and streptogramins (MLS phenotype) and in which no erythromycin
resistance determinants have been found by specific PCR or microarray screening,
carried an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of its
23S rRNA gene. The proved to be heterozygous, harbouring both wild-type and mutant
copies of the 23S rRNA gene. The low risk of horizontal dissemination of this type of
acquired resistance renders this strain acceptable for use in technological and probiotic
applications.

The resistance gene reservoir hypothesis intolerance, the boosting of the immune
suggests that beneficial and commensal response, and the lowering of cholesterol
bacterial populations in food and the levels. Benefit is also derived from their
gastrointestinal tract (GIT) of animals and possible anti-carcinogenic and anti-
humans may play a role in the transfer of mutagenic activities, etc. (Ouwehand et al.,
antibiotic resistance (Teuber et al., 1999; 2002; Sanders, 2003).
Salyers et al., 2004). To reduce the spread of Erythromycin and other macrolides are the
such resistance, the appropriate use of best alternatives for the treatment of
antibiotics is important (MARAN, 2002; penicillin-allergic patients. Bacterial
DANMAP, 2003), as is the screening for resistance to macrolides, lincosamides and
antibiotic resistance in bacteria intended to streptogramins (MLS phenotype) is often due
be used in food systems (Teuber et al., 1999; to efflux systems, methylases, and
European Commission, 2005). Distinguishing inactivating enzymes, for which more than 40
between intrinsic and acquired resistance is genes have been reported (Roberts et al.,
essential, and with respect to the latter it is 1999a). However, it can also be due to
important to determine whether it is caused mutations in ribosomal proteins L4 and L22
by genomic mutation or added genes; the last or in the 23S rRNA molecule (Leclercq,
of these poses the greatest risk of horizontal 2002). In fact, chromosomal mutations
transmission (Maiden, 1998; Normak and altering the erythromycin binding site in the V
Normak, 2002). domain of the 23S rRNA gene (at cognate
Lactobacillus species are members of the position A2058 [Escherichia coli numbering])
lactic acid bacteria (LAB) group, and are has been shown in a number of clinical
capable of colonising habitats as diverse as isolates, including Mycoplasma spp. (Meier et
fresh and fermented plant materials, meat al., 1994; Lucier et al., 1995), Helicobacter
products, fish, dairy, sourdoughs, fermented pylori (Versalovic et al., 1996),
beverages, and the human and animal GIT Propionibacterium spp. (Ross et al., 1997),
(Kandler and Weiss, 1986). The use of and Bordetella pertussis (Bartkus et al.,
selected species of lactobacilli as starter 2003). Here we report on the identification of
organisms in industrial food and feed a 23S rRNA mutation in a Lactobacillus
fermentations has a long tradition (Cogan, rhamnosus strain conferring resistance to
1996; Bernardau et al., 2006). Moreover, the erythromycin, clindamycin and some other
lactobacilli form one of the subdominant macrolides, in which PCR and microarray
bacterial populations of the human and screening techniques have failed to identify
animal GIT (Vaughan et al., 2002), where any macrolide resistance genes. To our
they are thought to exert an array of knowledge, this is the first report of such a
beneficial effects, including the inhibition of mutation in an LAB species.
pathogens, the alleviation of lactose

109
Minimum inhibitory concentration (MIC) of amplification was obtained with any of the
macrolides for L. rhamnosus E41. During a specific primer couples. Analysis of two
recent survey, two L. rhamnosus isolates original L. rhamnosus isolates for resistance
were identified as resistant to both genes using DNA microarrays with more than
erythromycin (MIC 1024 g ml-1) and 300 oligonucleotide probes, of which 42
clindamycin (MIC 256 g ml-1) in a corresponded to genes involved in MLS
microbroth assay (Delgado et al., 2005). phenotype, also returned negative results
Resistance to these antibiotics is confirmed in (Ammor et al., 2006).
the present work by the Etest method (AB
Biodisk, Solna, Sweden). In contrast, the PCR amplification and analysis of 23S
MICs of erythromycin and clindamycin in rRNA gene sequences. After ruling out the
susceptible L. rhamnosus isolates (E51, G92, presence of added genes as responsible for
G94, E52, E57, F46) and the industrial the MLS phenotype in strain E41, a search
probiotic strain L. rhamnosus LMG 18243 for mutations in its ribosomal components
(strain GG) were 1 and 0.5 g ml-1 was performed. The L. rhamnosus
respectively. Typing of the isolates by sequences for the genes encoding the L4
phenotypic (API50 CHL, bioMrieux, and L22 proteins, and that of 23S RNA, are
Montalieu-Vercieu, France) and genetic not available in public databases. Therefore
(random amplification of polymorphic DNA) to amplify a segment of the 23S RNA gene,
methods identified them as the same strain. including the critical 2058 residue (E. coli 23S
rRNA numbering) involved in macrolide
Identification of erythromycin resistance resistance, we made use of the recently
determinants in L. rhamnosus E41. Genes described universal primers 1104f and 2241r
ermA, erm(B), erm(C), erm(F) and mef(A), (Hunt et al., 2006). For comparison, the same
which confer macrolide resistance, are widely segment of the 23S rRNA from seven
distributed among Gram positive and Gram erythromycin- and clindamycin-susceptible
negative organisms (Jensen et al., 1999). strains, including L. rhamnosus GG, was
Some have already been characterized in amplified under identical conditions. An
plasmids (Tannock et al., 1994; Fons et al., amplicon of around 1200 bp was obtained
1997; Gfeller et al., 2003) and on the from all strains. This was purified using the
chromosome (Flrez et al., 2006) of Gen Elute PCR Clean Up kit (Sigma
lactobacillus species. In the present study, Chemical Co., St. Louis, Mo., USA) and
the presence of these five genes was subjected to restriction fragment length
therefore analysed by specific PCR using polymorphism (RFLP) analysis and
purified total DNA from E41 as a template. sequencing.
The primers and PCR conditions used were
those reported by Roberts et al. (1999b). No .

M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 7 8 M

2.0kbp
1.5kbp
1.0kbp
0.5kbp

BbsI HindIII
HindIII
Figure 1. PCR restriction fragment length polymorphism (RFLP) analysis of 23S gene amplicons from the macrolide resistant strain
Lactobacillus rhamnosus E41 (line 1) and a series of L. rhamnosus susceptible strains [E51, G92, G94, E52, E57, F46 and LMG 18243
(strain GG); lines 2 through 8, respectively] digested with the restriction enzymes BbsI (left) and HindIII (right). M, molecular weight
marker.

The transition mutation from A to G at amplicons were all digested with this enzyme
position 2058 in the erythromycin-resistant - which anticipates the mutation - before
23S rRNA sequence introduces a recognition analysis of the sequences. Fragments of the
site for the restriction enzyme BbsI. Thus, expected size were only observed in the

110
amplicon obtained from the erythromycin- The chromatograms showed a
resistant strain (Fig. 1, line 1). However, part fluorescence signal for an adenine residue in
of the amplicon appeared to be undigested, E41 as well as in the wild-type susceptible
suggesting either partial digestion with BbsI strains, indicating its heterozygous status.
had occurred, or the simultaneous presence Although no information is available on the
of wild-type and mutant copies of the 23S number of rRNA operons in L. rhamnosus, it
rRNA gene. As a control, amplicons were is expected to range from four to seven, as in
also digested with HindIII, the cleavage site other Lactobacillus species (Klaenhammer et
of which is in the middle of the amplified al., 2002). The sequences obtained in this
region in the 23S rRNA sequences of many work showed the highest homology (97%) to
lactobacillus species. With this enzyme, all a partial sequence of the L. casei 23S rRNA
the amplicons were digested, giving rise to gene (accession no. AF098107), and
identical fragments. This supports the idea of significant homology (93%) to the 23S rRNA
heterozygosity for the 23S rRNA genes in sequences from other lactobacilli and
the L. rhamnosus E41 studied. enterococci. Comparing the wild-type and
To support these results, all amplicons mutant sequencing signals in the resistant
were sequenced in an ABI PRISM 370 strain with the signal of the restriction
sequencer (Applied Biosystems, Foster City, fragments digested with BbsI, it is tempting
Ca., USA), and the sequences obtained to speculate that more than one copy of the
were compared to one another and to those mutated sequence is present in the L.
in databases. In the sequence corresponding rhamnosus E41 23S gene.
to the resistant strain, a transition mutation
(A to G) was observed at the corresponding In conclusion, this paper reports a
position of 2058; this was not seen in the chromosomal mutation of the 23S rRNA
susceptible strains (Fig. 2). gene as the most plausible cause of
macrolide resistance in L. rhamnosus E41.
2058 Analysis of other ribosomal components
thought to be involved in macrolide
resistance should exclude other possibilities.
Lactobacillus rhamnosus E41 shows
promising probiotic properties (Delgado and
Mayo, unpublished); it would be especially
useful to people undergoing long-term
macrolide treatment.

Nucleotide sequence accession numbers.


A A A A A The wild-type and mutant 23S rRNA gene
sequences of L. rhamnosus were assigned
L. rhamnosus LMG 18243 (strain GG) GenBank accession nos. EF030190 and
Susceptible to macrolides
EF030191, respectively.
2058
This work was supported by an EU project
within the VI Frame Program (ACE-ART, ref.
CT-2003-506214). M. S. Ammor was
awarded a postdoctoral fellowship from the
Secretara de Estado de Universidades e
Investigacin of the Spanish Ministry of
Education and Science (ref. SB2004-0165).
BCCMTM, University of Gent, Belgium, is
acknowledged for providing the Lactobacillus
A A A A rhamnosus LMG 18243 control strain.
A
REFERENCES
L. rhamnosus E41
Resistant to macrolides Ammor, M. S., A. B. Flrez, A. H. A. M. Van Hoek, C. G. de los
Reyes-Gaviln, H. J. M. Aarts, A. Margolles, and B.
Figure 2. Representative sequence chromatograms of 23S rRNA Mayo. 2006. Molecular characterization of intrinsic and
genes for the wild type macrolide-susceptible Lactobacillus acquired antibiotic resistance in lactic acid bacteria and
rhamnosus LMG 18243 (strain GG) (left) and that of the bifidobacteria. J. Mol. Microbiol. Biotechnol. (In Press).
macrolide-resistance L. rhamnosus E41 (right). Arrows indicate Bartkus, J. M., B. A. Juni, K. Ehresmann, C. A. Miller, G. N.
the nucleotide at position 2058 (of the Escherichia coli Sanden, P. K. Cassiday, M. Saubolle, B. Lee, J. Long,
numbering). Note the heterozygous nature (G/A) of strain E41 at A. R. Harrison, Jr., and J. M. Besser. 2003. Identification
this position. of a mutation associated with erythromycin resistance in

111
Bordetella pertussis: Implications for surveillance of Maiden, M. C. 1998. Horizontal genetic exchange, evolution, and
antimicrobial resistance. J. Clin. Microbiol. 41:1167-1172. spread of antibiotic resistance in bacteria. Clin. Infect. Dis.
Bernardeau, M., M. Gueguen, and P. Vernoux. 2006. 1:S12-20.
Beneficial lactobacilli in food and feed: long-term use, MARAN. 2002. Monitoring of antimicrobial resistance and
biodiversity and proposals for specific and realistic safety antibiotic usage in animals in The Netherlands in 2002,
assessments. FEMS Microbiol. Rev. 30:487-513. (http://www.vwa.nl).
Cogan, T. M. 1996. History and taxonomy of starter cultures, pp. 19. Meier, A., P. Kirschner, B. Springer, V. A. Seteingrube, B.
1-20. In T. M. Cogan and J.-P. Accolas (ed.), Dairy starter A. Brown, R. J. Wallace, Jr., and E. C. Botter. 1994.
cultures. VCH Publishers, Inc., New York. Identification of mutations in the 23S rRNA gene of
DANMAP. 2003. Use of antimicrobial agents and occurrence of clarithromycin-resistant Mycobacterium intracellulare.
resistance in bacteria from food animals, food and humans. Antimicrob. Agents Chemother. 38:381-384.
ISSN 1600-2003, (http://www.dfvf.dk). Normark, B. H., Normark, S. 2002. Evolution and spread of
Delgado,S., A. B. Florez, and B. Mayo. 2005. Antibiotic antibiotic resistance. J. Intern. Med. 252:91-106.
susceptibility of Lactobacillus and Bifidobacterium species Ouwehand, A. C., Salminen, S., and Isolauri, E. 2002.
from the human gastrointestinal tract. Curr. Microbiol. Probiotics: an overview of beneficial effects. Antonie van
50:202-207. Leeuwenhoek 82:279-289.
European Commission. 2005. Opinion of the FEEDAP Panel Roberts, M. C., J. Sutcliffe, P. Courvalin, L. B. Jensen, J.
on the updating of the criteria used in the assessment of Rood, and H. Seppala. 1999a. Nomenclature for
bacteria for resistance to antibiotics of human or veterinary macrolide and macrolide-lincosamide-streptogramin B
importance. EFSA J. 223:1-12. resistance determinants. Antimicrob. Agents. Chemother.
Flrez, A. B., M. S. Ammor, S. Delgado, and B. Mayo. 2006. 43:2823-2830.
Molecular analysis of a chromosomally-encoded erm(B) Roberts, M. C., W. O. Chung, D. Roe, M. Xia, C. Marquez, G.
gene and its flanking insertion points in Lactobacillus Borthagaray, W. L. Whittington, and K. K. Holmes.
johnsonii G41. Antimicrob. Agents Chemother. (In Press). 1999b. Erythromycin-resistant Neisseria gonorrhoeae and
Fons, M., T. Hege, M. Ladire, P. Raibaud, R. Ducluzeau, and oral commensal Neisseria spp. carry known rRNA
E. Maguin. 1997. Isolation and characterization of a methylase genes. Antimicrob. Agents Chemother. 43:1367-
plasmid from Lactobacillus fermentum conferring 1372.
erythromycin resistance. Plasmid 37:199-203. Ross, J. I., E. A. Eady, J. H. Cove, C. E. Jones, A. H. Ratyal,
Gfeller, K. Y., M. Roth, L. Meile, and M. Teuber. 2003. Y. W. Miller, S. Vyakrnam, and W. J. Cunliffe. 1997.
Sequence and genetic organization of the 19.3-kb Clinical resistance to erythromycin and clindamycin in
erythromycin- and dalfopristin-resistance plasmid pLME300 cutaneous propionibacteria isolated from acne patients is
from Lactobacillus fermentum ROT1. Plasmid. 50:190-201. associated with mutations in 23S rRNA. Antimicrob. Agents
Hunt, D. E., V. Mepac-Ceraj, S. G. Acinas, C. Gautier, S. Chemother. 41:1162-1165.
Bertilsson, and M. F. Polz. 2006. Evaluation of 23S rRNA Salyers, A. A., A. Gupta, and Y. Wang. 2004. Human intestinal
PCR primers for use in phylogenetic studies of bacterial bacteria as reservoirs for antibiotics resistance genes.
diversity. Appl. Environ. Microbiol. 72:2221-2225. Trends Microbiol. 12:412-416.
Jensen, L. B., N. Frimodt-Moller, and F. M. Aarestrup. 1999. Sander, P., T. Prammananan, A. Meier, K. Frischkorn, and E.
Presence of erm gene classes in Gram-positive bacteria of C. Bottger. 1997. The role of ribosomal RNAs in macrolide
animal and human origin in Denmark. FEMS Microbiol. resistance. Mol. Microbiol. 26:469-480.
Lett. 170:151-158. Sanders, M. E. 2003. Probiotics: considerations for human
13. Klander, O., and N. Weiss N. 1986. Genus Lactobacillus health. Nutr. Rev. 61:91-99.
Beijerinck 1901, 212AL, pp. 1208-1234. In P. H. A. Sneath, Tannock, G. W., J. B. Luchansky, L. Miller, H. Connell, S.
N. S. Mair, M. E. Sharpe and J. G Holt (ed.), Bergeys Thodeandersen, A. A. Mercer, and T. R. Kalenhammer.
Manual of Systematic Bacteriology, Vol. 2. Williams and 1994. Molecular characterization of a plasmid-borne
Wilkins, Baltimore. (pGT633) erythromycin resistance determinant (ermGT)
Klaenhammer, T., E. Alterman, F. Arigoni, A. Bolotin, F. from Lactobacillus reuteri 100-63. Plasmid 31:60-71.
Breidt, J. Broadbent, R. Cano, S. Chaillou, J. Teuber, M., L. Meile, and F. Schwarz. 1999. Acquired antibiotic
Deutscher, M. Gasson, M. van de Guchte, J. Guzzo, A. resistance in lactic acid bacteria from food. Antonie van
Haarte, T. Hawkins, P. Hols, R. Hutkins, M. Leeuwenhoek 76:115-137.
Klerebeezem, J. Kok, O. Kuipers, M. Lubbers, M. Vaughan, E. E., M.-C. de Vries, E. G. Zoetendal, K. Ben-Amor,
Maguin, L. McKay, D. Mills, A. Nauta, R. Overbeek, H. A. D. L. Akkermans, and W. M. de Vos. 2002. The
Pel, D. Pridmore, M. Saier, D. van Sinderen, A. Sorokin, intestinal LABs. Antonie van Leeuwenhoek 82:341-352.
J. Steele, D. OSullivan, W. de Vos, M. Zagorec, and R. Versalovic, J., D. Shortridge, K. Kibler, M. V. Griffy, J. Beyer,
Siezen. 2002. Discovering lactic acid bacteria by R. K. Flamm,, S. K. Tanaka, D. Y. Graham, and M. F. Fo.
genomics. Antonie van Leeuwenhoek 82:29-58. 1996. Mutations in the 23S rRNA are associated with
Leclercq, R. 2002. Mechanisms of resistance to macrolides and claritrhomycin resistance in Helicobacter pilory. Antimicrob.
lincosamides: nature of the resistance elements and their Agents Chemother. 40:477-480.
clinical implications. Clin. Infect. Dis. 34:482-492. Vester, B., and S. Douthwaite. 2001. Macrolide resistance
Lucier, T. S., K. Heitzman, S. K. Liu, and P. C. Hu. 1995. conferred by base substitutions in 23S rRNA. Antimicrob.
Transition mutations in the 23S rRNA of erythromycin- Agents Chemother. 45:1-12.
resistant isolates of Mycoplasma pneumoniae. Antimicrob.
Agents Chemother. 39:2770-2773.

112
Captulo IV.- Caracterizacin molecular de la resistencia intrnseca en BAL y
bifidobacterias mediada por transportadores inespecficos (MDR).
Artculo XI.- Two membrane proteins from Bifidobacterium breve UCC2003 constitute an
ABC-type multidrug transporter.
Microbiology. 152:3497-3505 (2006).
Artculo XII.- Ubiquity and diversisty of multidrug resistance genes in Lactococcus lactis
strains isolates between 1936 and 1995.
FEMS Microbiology Letters. 263:21-25 (2006).

Objetivo: Determinar el posible papel que pueden jugar en la resistencia a antibiticos


los sistemas inespecficos de transporte codificados por transportadores de tipo MDR
(Multidrug Resistance).

Resultados: Mediante la clonacin de dos ORFs del genoma de B. breve, altamente


homlogas con protenas MDR, en Lc. lactis se estudi el papel de estos transportadores
inespecficos en la resistencia a antibiticos. El estudio de la susceptibilidad de las
diferentes construcciones frente a 15 antibiticos, mostr diferencias significativas para
el antibitico polimixina B cuando ambas protenas se expresaban conjuntamente.
Resultados similares se obtuvieron para la nisina.
A fin de estudiar la abundancia y plasticidad de los transportadores MDR
previamente caracterizados en Lactococcus se seleccionaron 13 cepas de Lc. lactis subsp.
lactis, 4 Lc. lactis subsp. cremoris, 2 Lc. plantarum y 2 Lc. raffinolactis aisladas durante el
periodo 1936-1995. Los genes que codifican para LmrA, LmrP y LmrCD se detectaron en
todas las cepas de Lc. lactis, pero no en las especies Lc. plantarum y Lc. raffinolactis. El
anlisis de la secuencia de aminocidos mostr variaciones dependientes de la
subespecie. Adems, la pauta de lectura de los genes LmrA y/o LmrP estaba
interrumpida en 5 cepas, mientras que la pauta completa del gen LmrD estaba presente
en todas las cepas.

Conclusiones: Las protenas AbcA y AbcB de B. breve actan como un heterodmero


(transportador ABC) confiriendo resistencia a polimisina B y nisina, y siendo capaz de
transportar compuestos citotxicos. La existencia de transportadores MDR en Lc. lactis
antes del comienzo de la era de los antibiticos sugiere un papel principalmente
fisiolgico relacionado probablemente con su medio ambiente, en el que estn
presentes gran variedad de compuestos perjudiciales para las clulas.

115
Microbiology (2006), 152, 34973505 DOI 10.1099/mic.0.29097-0

Two membrane proteins from Bifidobacterium


breve UCC2003 constitute an ABC-type multidrug
transporter
Abelardo Margolles,1 Ana Belen Florez,1 Jose Antonio Moreno,1,2
Douwe van Sinderen2 and Clara G. de los Reyes-Gavilan1
1
Correspondence Instituto de Productos Lacteos de Asturias, Consejo Superior de Investigaciones Cientficas
Abelardo Margolles (CSIC), Ctra Infiesto s/n, 33300, Villaviciosa, Asturias, Spain
amargolles@ipla.csic.es 2
Department of Microbiology and Alimentary Pharmabiotic Centre, University College Cork,
Western Road, Cork, Ireland

Intrinsic resistance to drugs is one of the main determining factors in bacterial survival in the
intestinal ecosystem. This is mediated by, among others, multidrug resistance (MDR) transporters,
membrane proteins which extrude noxious compounds with very different chemical structures and
cellular targets. Two genes from Bifidobacterium breve encoding hypothetical membrane
proteins with a high homology with members of the ATP-binding cassette (ABC) family of multidrug
efflux transporters, were expressed separately and jointly in Lactococcus lactis. Cells co-expressing
both proteins exhibited enhanced resistance levels to the antimicrobials nisin and polymyxin B.
Furthermore, the drug extrusion activity in membrane vesicles was increased when both proteins
were co-expressed, compared to membranes in which the proteins were produced independently.
Both proteins were co-purified from the membrane as a stable complex in a 1 : 1 ratio. This is
Received 26 April 2006 believed to be the first study of a functional ABC-type multidrug transporter in Bifidobacterium and
Revised 24 August 2006 contributes to our understanding of the molecular mechanisms underlying the capacity of intestinal
Accepted 30 August 2006 bacteria to tolerate cytotoxic compounds.

INTRODUCTION representative species of its genus, is among the predomi-


nant bacteria present in the gastrointestinal tract of infants
Bifidobacteria are important members of the human gut
(Matsuki et al., 1999).
microbiota, in which they can be present at concentrations
as high as 1011 cells per g faeces, representing up to 91 % of Enteric bacteria have evolved to tolerate inhibitory factors in
the total microbial population in breast-fed infants the intestinal niche. Their survival depends on tolerance to
(Harmsen et al., 2000). Their presence has been associated host-produced substances, such as bile (Begley et al., 2005;
with beneficial effects. On the basis of their role in Yokota et al., 2000), and antimicrobial peptides (Ganzle et
promoting human wellbeing, some species and strains of al., 1999; Mahida et al., 1997), but they are also conditioned
the genus Bifidobacterium are considered to be probiotic and by exposure to exogenous cytotoxic agents, including
are used as active ingredients in functional foods, mainly antibiotics (Vedantam & Hecht, 2003). Nowadays, drug
dairy-based products (Ouwehand et al., 2002). Well- efflux, mediated by MultiDrug Resistance (MDR) trans-
documented clinically established applications of some porters, is considered as one of the main mechanisms
strains of this genus are the treatment of diarrhoea and the responsible for these resistances (Grkovic et al., 2002). These
balancing of the intestinal microbiota (Salminen & proteins can be subdivided into two groups according to
Gueimonde, 2004), while other health-promoting actions, structural and bioenergetic criteria. ATP-binding cassette
such as anticarcinogenic activity, immunomodulation and (ABC) transporters power the transport via the hydrolysis of
reduction of serum cholesterol levels, have been suggested ATP, whereas the activity of secondary transporters is
(Isolauri et al., 2004). Bifidobacterium breve, one of the dependent on the transmembrane electrochemical gradient,
typically the proton-motive force (Kim et al., 2004;
Abbreviations: ABC, ATP-binding cassette; AMP-PNP, 59-(b,c-imido) Mazurkiewicz et al., 2005). Most of the bacterial multidrug
triphosphate; Bbm, Bifidobacterium breve multidrug transporter; DDM, efflux systems characterized up to now belong to the second
n-dodecyl b-D-maltoside; MDR, multidrug resistance. class of transporters (Putman et al., 2000), and just a few of
The GenBank/EMBL/DDBJ accession number for the sequence of the them, such as LmrA and LmrCD from Lactococcus lactis
B. breve gene cluster is DQ486860. (Lubelski et al., 2004; van Veen et al., 1996), HorA from
117
0002-9097 G 2006 SGM Printed in Great Britain 3497
A. Margolles and others

Lactobacillus brevis (Sakamoto et al., 2001), MsbA from (AMP-PNP), chloramphenicol, erythromycin, nisin, polymyxin B,
Escherichia coli (Karow & Georgopoulus, 1993), BmrA imidazole and n-dodecyl b-D-maltoside (DDM) were purchased
from Sigma. Takara supplied all restriction enzymes, excluding
from Bacillus subtilis (Orelle et al., 2003) and EfrAB from
BspLU11I (Roche Applied Science). Platinum-Pfx DNA polymerase
Enterococcus faecalis (Lee et al., 2003), belong to the ABC- and Hoechst-33342 [29-(4-ethoxyphenyl)-5-(4-methyl-1-piperazi-
type family. nyl)-2,59-bi-1H-benzimidazole] were obtained from Invitrogen, and
Ni2+ nitriloacetic acid (Ni-NTA) agarose was supplied by Qiagen.
Recent evidence indicated that B. breve is more resistant to E-test strips were from AB Biodisk and ATP was from Amersham
antibiotics than other Bifidobacterium species (Moubareck Biosciences. All chemicals were reagent grade and all solutions were
et al., 2005), suggesting that this species could have a made with molecular biology reagent water (Sigma).
stronger intrinsic resistance. In a previous study, we
characterized BbmR, a membrane protein from B. breve Bacterial strains, plasmids, and culture conditions. Bacteria
which was able to confer resistance to macrolides and and plasmids used in this study are shown in Table 1. Bifi-
exhibited characteristics reminiscent of MDR proteins dobacterium breve UCC2003 was grown at 37 uC in MRS medium
(Margolles et al., 2005). Its homologue in Bifidobacterium (Merck) supplemented with 0?05 % (w/v) L-cysteine in an anaerobic
chamber (Mac500, Don Whitley Scientific). Lactococcus lactis subsp.
longum, named Ctr, was also found to confer resistance to
lactis NZ9000 and NZ9700 (Kuipers et al., 1993, 1998) were culti-
several antibiotics and to transport radioactive cholate vated at 30 uC in M17 broth (Oxoid) with 0?7 % (w/v) glucose
(Price et al., 2006). The current study presents work relating (GM17) and 5 mg chloramphenicol or 5 mg erythromycin ml21
to B. breve genes that play a role in its intrinsic resistance to when they contained pNZ8048 and its derivatives or pNZE8048 and
cytotoxic compounds. We describe the gene cloning and its derivatives respectively. Cells cotransformed with pNAbcA (or
functional characterization of a novel bifidobacterial ABC- pNHAbcA) and pNAbcB were grown under the same conditions,
type multidrug transporter in L. lactis, which shares both but in a medium that contained 3 mg chloramphenicol plus 3 mg
erythromycin ml21.
structural and functional properties with prokaryotic and
eukaryotic MDR proteins, being able to confer resistance to
several antimicrobials and to transport cytotoxic drugs. DNA and RNA manipulation, cloning of the genes, and
sequence analysis. DNA manipulations were carried out as
described by Sambrook et al. (1989). Total DNA was obtained from
METHODS B. breve as previously described (Margolles & de los Reyes-Gavilan,
2003). Vector pNZE8048 was constructed by amplifying a DNA
Chemicals. DNase A, creatine phosphokinase, phosphocreatine, fragment from the vector pNG8048 (Zuniga et al., 2002), with the
potassium EDTA, DTT, HEPES, adenosine 59-(b,c-imido) triphosphate primers emr-f and emr-r (Table 1). The resulting PCR product was

Table 1. Bacterial strains, plasmids and primers

Strain or plasmid Relevant phenotype or genotype Source or reference

B. breve UCC2003 Infant isolate MacConaill et al. (2003)


L. lactis NZ9000 L. lactis MG1363 pepN : : nisRK Kuipers et al. (1998)
L. lactis NZ9700 Nisin-producing strain Kuipers et al. (1993)
pNZ8048 Gene expression vector, PnisA, Cmr de Ruyter et al. (1996)
pNZE8048 Gene expression vector, PnisA, Emr This work
pNAbcA pNZE8048 derivative; abcA This work
pNAbcB pNZ8048 derivative; abcB This work
pNHAbcA pNZE8048 derivative; His-tag abcA This work
Primer Sequence*
emr-f 59-AAGGCCAGGAAGATCTCCGATTCACAAAAAATAGGCACACG-39 This work
emr-r 59-CGATATCGGATCCGTCGACCCGTGC-39 This work
abcA-f 59-TGCGATCACATGTACGTCGATCCGGGAATCAATACCAGCGC-39 This work
abcA-r 59-ATACGGAGAATTCTTATTAAAACCCCTGTGCGGCGCC-39 This work
abcB-f 59-TGCGACCACCATGGCACAACGCAATACATTTCGCG-39 This work
abcB-r 59-GCCGACTCTAGATTACTACTCAGTAGCCCCGGTGG-39 This work
abcAh-f 59-GCTGTATCAAGATTCGCAAGCTGC-39 This work
abcAh-r 59-TGCGATCAGAATTCTTATTAGTGATGGTGATGGTGATGAAAC- This work
CCCTGTGCGGCG CCGGACTGG-39
abcA-fq 59-ACGACTGGCAGCGATTCAAC-39 This work
abcA-rq 59-GACCTGCTCGACGATTGTGA-39 This work
abcB-fq 59-TTCTCCGACATCTTCACATTCTTC-39 This work
abcB-rq 59-CAGTACGAGGCTCCACAATGC-39 This work

*Restriction sites are underlined and the codons for the histidine tag are in bold italic.
118
3498 Microbiology 152
Bifidobacterium breve ABC-type MDR transporter

digested with SalI and BglII, and ligated with SalI/BglII-digested Prediction Software (http://www.fruitfly.org/seq_tools/) was used for
pNZ8048, yielding pNZE8048, in which the chloramphenicol resis- searching putative promoter regions. Homology trees were constructed
tance marker is replaced by an erythromycin resistance cassette. with the TreeTop-Phylogenetic Prediction program (http://www.
genebee.msu.su/).
The structural genes abcA and abcB were amplified from the genome of
B. breve by PCR using primers abcA-f and abcA-r (for gene abcA), and Preparation of inside-out membrane vesicles. For the isolation
abcB-f and abcB-r (for gene abcB) (Table 1), respectively. Given that of inside-out membrane vesicles of L. lactis NZ9000, cells were grown
the abcA gene contains an internal NcoI site, the enzyme BspLU11I, at 30 uC to an OD600 of about 0?4. At this point, 0?1 % (v/v) of the
which yields compatible ends with NcoI, was used in the cloning supernatant of the nisin-producing L. lactis strain NZ9700 was added
procedure. The abcA gene was amplified, digested with BspLU11I and to the culture to trigger transcription of the abcA or abcB gene, or
EcoRI, and ligated into pNZE8048, previously treated with NcoI and jointly the abcA/abcB genes from the nisA promoter. Subsequently,
EcoRI, yielding pNAbcA. For abcB, the amplicon was digested with the cells were incubated for 1 h at 30 uC, harvested at an OD600 of
NcoI and XbaI, and ligated with NcoI/XbaI-digested pNZ8048, approximately 0?8 by centrifugation, and membrane vesicles were
resulting in pNAbcB. Furthermore, in order to introduce a he- obtained as previously described (Margolles et al., 1999). The mem-
xahistidine tag at the C-terminus of AbcA, a fragment of abcA was brane vesicles were stored in small aliquots in liquid nitrogen.
amplified using the primers abcAh-f and abcAh-r (Table 1), digested
Antimicrobial susceptibility testing. Cells were grown at 30 uC
with ClaI and EcoRI, and ligated into pNAbcA, previously cut with the
same enzymes. This construction yielded plasmid pNHAbcA. The to an OD600 of about 0?4 in GM17 broth containing chlorampheni-
col or erythromycin, or both. To induce gene expression at this
vectors pNAbcA and pNAbcB were transformed into electrocompetent
point, 0?1 % (v/v) of the culture supernatant of L. lactis NZ9700 was
L. lactis NZ9000 cells using previously described methods (de Ruyter
added to the GM17 broth. Subsequently, the cells were incubated
et al., 1996), and transformants were screened by restriction analysis of
for 1 h. For MIC determinations, 1 ml of the culture was added to
the recovered plasmids. For co-expression of AbcA and AbcB, pNAbcA
30 ml soft (0?7 % agar) GM17 at 40 uC, containing 0?1 % of the L.
(or pNHAbcA) was introduced into electrocompetent NZ9000 cells
lactis NZ9700 culture supernatant. Then, the mixture was layered on
containing pNAbcB. To confirm that no PCR-borne mutations were
the top of 15 cm Petri dishes containing 50 ml GM17 (2 % agar), to
introduced, the fidelity of the inserts was verified by DNA sequencing
which supernatant of the L. lactis nisin-producing strain had been
of both strands with an ABI Prism 377 sequencer (Applied Biosystems).
added. E-test strips of 15 different antibiotics were applied with an
Real-time PCR was used to assess the expression levels of abcA and applicator, and MICs were determined after 24 h incubation. For
abcB. Primers abcA-fq, abcA-rq, abcB-fq and abcB-rq were chosen to resistance assays on agar media, cultures were centrifuged, resus-
amplify internal fragments of 128 and 84 bp of abcA and abcB, pended in GM17 broth and the OD600 was adjusted to 1. Then,
respectively (Table 1). Four independent cultures of L. lactis cells were serial 10-fold dilutions were performed, and 5 ml of each dilution
disrupted with glass beads (0?5 mm) in a FastPrep FP120 Instrument was spotted on the GM17 agar medium with or without the inhibi-
(Thermo Savant). Total RNA was extracted using Tri-Reagent solution tory agent (nisin or polymyxin B). The plates were incubated for
24 h and all the experiments were done at least in triplicate.
according to the manufacturers instructions (Sigma). Two micro-
grams of total RNA was treated with 2 units of DNase (Fermentas) for Hoechst-33342 transport in membrane vesicles. Inside-out
1 h at 37 uC. Then, cDNA was synthesized using the iScript cDNA membrane vesicles (1 mg of total membrane protein) were diluted
synthesis kit (Bio-Rad). Absence of chromosomal DNA contamination in 2 ml ATP regenerating buffer (50 mM potassium HEPES buffer,
was checked by real-time PCR. Real-time PCR reactions were carried pH 7?1, containing 5 mM MgSO4, 8?5 mM NaCl, 0?1 mg creatine
out using an ABI PRISM 7500 with a SYBR green PCR master mix kinase ml21 and 5 mM phosphocreatine) in a 3 ml quartz cuvette.
(Applied Biosystems). The efficiency was calculated based on the slope After 1 min incubation at 30 uC, Hoechst-33342 was added to a final
of a standard curve. In all cases, the 16S rRNA level was used as an concentration of 0?2 mM. Once the signal was stable, Mg2+-ATP or
internal control. Mg2+-AMP-PNP was added to a final concentration of 2 mM, and
the fluorescence intensity (excitation 355 nm, emission 457 nm) was
The stability of pNAbcA and pNAbcB co-existing in the same cell was followed with an Eclipse fluorescence spectrophotometer (Varian)
checked by growing the cells for more than 70 generations in GM17 provided with a magnetically stirred holder at 30 uC.
broth containing chloramphenicol and erythromycin (five consecutive
cultures inoculated with 0?0037 % (v/v) of a culture grown to an OD600 Affinity purification and identification of the purified pro-
of about 1?2). In parallel, cells were plated on GM17 agar with and teins. Inside-out membrane vesicles from L. lactis NZ9000 contain-
without antibiotics, to confirm that all cells contained the two ing pNHAbcA/pNAbcB (12 mg total membrane protein ml21) were
antibiotic markers. Subsequently, 24 independent colonies (12 colonies solubilized in 50 mM potassium phosphate buffer, pH 8?0, contain-
from plates inoculated with cultures grown for 42 generations, and 12 ing 10 % (v/v) glycerol, 100 mM NaCl and 1 % (w/v) DDM. The
colonies from plates inoculated with cultures grown for 71 generations) suspension was mixed and, after 30 min incubation at 4 uC, the
were analysed, and plasmids were extracted with the GenElute DNA kit insoluble material was removed by centrifugation (250 000 g,
(Sigma) and digested with BglII to verify that no DNA recombination 20 min, 4 uC). For purification of histidine-tagged AbcA, 1 ml solu-
had occurred. Furthermore, the genes abcA and abcB, and the upstream bilized membrane proteins was mixed and incubated for 1 h with
region corresponding to the nisin promoter in pNAbcA and pNAbcB, 200 ml Ni-NTA agarose, which was preequilibrated in buffer A
were sequenced after the five consecutive cultures to confirm that no [50 mM potassium phosphate, pH 8?0, 100 mM NaCl, 10 % (v/v)
mutations were produced during growth under selective pressure when glycerol, 0?05 % (w/v) DDM] plus 10 mM imidazole. After incuba-
both plasmids are present in the same cell. tion, the resin was transferred to a Bio-spin column (Bio-Rad) and
washed first with 25 column volumes of buffer A containing 10 mM
DNA and protein sequences were analysed using the computer imidazole, and subsequently with 12 column volumes of buffer A
program Clone Manager 5 (Scientific and Educational Software). (pH 7?0) containing 30 mM imidazole. The protein was eluted with
Homology searches and multiple sequence alignments (clustalw) were buffer A, pH 7?0, supplemented with 250 mM imidazole. All steps
carried out using the BLAST server of the National Center for were carried out at 4 uC.
Biotechnology Information (http://www.ncbi.nlm.nih.gov) and the
software available on the Web page of the Pole-Bioinformatique Proteins from the membrane and eluted fractions were checked by
Lyonnais (http://pbil.univ-lyon1.fr/). The Neural Network Promoter SDS-PAGE by using a Mini-Protean II system (Bio-Rad). SDS-PAGE
119
http://mic.sgmjournals.org 3499
A. Margolles and others

gels were stained with Coomassie BioSafe (Bio-Rad), and densito- role in multidrug resistance and their ability to transport
metric scanning was carried out by using the Gel Doc 2000 system with drugs.
the Quantity One software (Bio-Rad). For protein identification, bands
were excised from gels and submitted to tryptic digestion, and mass
spectrometry analyses were performed at the Servicio de Proteomica of Production of the ABC transporters and
the Centro Nacional de Investigaciones Cardiovasculares. All protein antimicrobial activity profiles
concentrations were determined by the Lowry method.
In recent years, molecular techniques for disrupting genes
and controlling gene expression have been extensively used
to functionally study MDR transporters (Doerrler & Raetz,
RESULTS AND DISCUSSION 2002; Hirata et al., 2004; Lubelski et al., 2004; Ravaud et al.,
2006). However, the lack of efficient transformation systems
Identification and sequence analysis of abcA and the paucity of effective molecular tools (e.g. cloning and
and abcB expression vectors and gene inactivation systems) have so
A 7930 bp DNA fragment was selected from the preliminary far severely limited functional studies in Bifidobacterium
genome sequence of B. breve UCC2003 (S. Leahy, J. A. (Ventura et al., 2004). Since previous studies from our
Moreno, M. OConnell-Motherway, H. G. Higgins, G. F. group have shown that the nisin-inducible system from
Fitzgerald & D. Van Sinderen, unpublished data). Its genetic the Gram-positive bacterium L. lactis can generate large
analysis revealed the presence of two adjacent ORFs quantities of bifidobacterial membrane and cytosolic
displaying significant homology to several hypothetical proteins (Margolles & de los Reyes-Gavilan, 2003;
MDR transporters, named abcA and abcB, putatively Margolles et al., 2005), we selected this bacterium as the
transcribed in the same direction and separated by host to produce AbcA and AbcB. We cloned (independently
202 bp. A putative promoter sequence was found 182 bp or together) abcA and abcB by constructing plasmids
upstream of the potential abcA start codon, but not pNAbcA and pNAbcB, which were then introduced into L.
upstream of the abcB gene. The first gene, abcA, possesses lactis cells. Both plasmids could be maintained in the same
a putative ribosome-binding site 8 bp upstream of its start host cell after more than 70 generations. Total cell counts
codon (GGTGAT), while the second gene, abcB, is followed indicated that all cells contained the two antibiotic markers,
by a transcription terminator-like (inverted repeat) and no recombinations or mutations were observed after
sequence (Fig. 1a). The abcA and abcB genes are predicted sequencing and restriction enzyme analysis of the plasmids
to encode 636 and 601 aa proteins, respectively, identified in any of the tested colonies. This proves that pNAbcA and
by a database enquiry (BLASTP) as putative ABC transpor- pNAbcB could co-exist and are stable in the same cell, and
ters. Hydropathy profile analysis using the Expasy shows that they did not segregate and were not maintained
Proteomic Server predicted that both proteins possess a in different cell subpopulations.
transmembrane domain, composed of six putative trans- Gene expression and protein synthesis were investigated by
membrane helices, followed by a hydrophilic portion with a real-time PCR and SDS-PAGE. Only the expression of AbcB
putative ATP-binding domain, containing the Walker A and was apparent on the protein electrophoresis profiles,
Walker B motifs, and the ABC signature sequence probably due to the coincidence of the molecular mass of
(Schneider & Hunke, 1998; Walker et al., 1982) (Fig. 1b). AbcA with one of the major membrane proteins of L. lactis
Since the ABC domain is highly conserved, in order to (Fig. 2). However, real-time PCR showed that both abcA
determine evolutionary relationships with other transpor- and abcB were transcribed, either when the genes were
ters the predicted permease domains of AbcA (339 N- cloned separately or together. Interestingly, in cells carrying
terminal amino acids) and AbcB (347 N-terminal amino both pNAbcA and pNAbcB, the abcB transcript appears to
acids) were subjected to BLASTP analysis and compared with be about two times more abundant than the abcA transcript
homologous sequences. Analysis of a multiple alignment of (2DDCT=21?040?059), whereas when abcA and abcB were
the primary sequence of these domains showed that they are expressed alone, their transcripts were 22?431?17 times and
closely related to a number of ABC transporters from 21?491?52 times more abundant, respectively, than the
bacteria (Fig. 1c, d). Both protein A and B domains corresponding transcripts of the cells containing both
displayed the highest homology scores with proteins that plasmids.
were located in tandem on the genomes. Interestingly, the
AbcA permease domain matched with proteins encoded by We investigated if AbcA and AbcB could be involved in
the upstream genes, whereas the AbcB permease domain conferring antimicrobial resistance. We determined the
matched with proteins encoded by the downstream genes of MIC of 15 antibiotics using E-test strips, a quantitative
the tandem-like structures. These results indicated that method that has emerged in the last few years as an accurate
AbcA/AbcB homologues are widely distributed in bacteria, alternative to the traditional methods, such as microdilution
and most likely they are orthologous systems performing and disk diffusion (Turnbull et al., 2004). The antibiotics
similar physiological functions. This prompted us to study tested were ampicillin, benzylpenicillin, clindamycin, cipro-
the functionality of both proteins (independently or floxacin, doxycycline, kanamycin, meropenem, minocy-
together) by investigating phenotypic changes of a cell cline, polymyxin, quinupristin-dalfopristin, rifampicin (low
that expresses these proteins, as well as determining their range, 0?002 to 32 mg ml21), streptomycin (high range,
120
3500 Microbiology 152
Bifidobacterium breve ABC-type MDR transporter

Fig. 1. (a) Organization of the B. breve UCC2003 genomic region containing the abcA and abcB genes. The white arrows
indicate the relative positions and direction of transcription of the ORFs. The pin-like symbol indicates a terminator-like
sequence. Relevant restriction sites and their locations in the sequence are also indicated. A putative promoter region is
indicated with a black arrow upstream of abcA. (b) Modular structure of AbcA and AbcB. The amino acid sequences in
parentheses are the consensus sequences of the ABC transporter family. TMD, transmembrane domain; X, any amino acid; h,
hydrophobic residue. (c, d) Phylogenetic relationship analysis of the permease domains of AbcA (c) and AbcB (d). Database
accession numbers are given in parentheses. Trees were constructed with the matrix of pair distances between sequences
using the cluster algorithm, and bootstrap values (100 replicates) are given at the branch points.

0?064 to 1,024 mg ml21), tetracycline, trimethoprim-sulfa- containing pNAbcB or pNAbcA/pNAbcB were 6?00 and
methoxazole, and vancomycin. Chloramphenicol and 6?71?2 mg ml21, respectively. Interestingly, a recent
macrolides were not tested since they are the selection report demonstrated that the increased expression of two
markers in the vectors. A small increase in resistance was multidrug ABC transporter-like genes is associated with
observed for ciprofloxacin. The MICs for the control strain, ethidium bromide and ciprofloxacin resistance in
harbouring the empty plasmid, and the strain harbouring Mycoplasma hominis (Raherison et al., 2005). In our case,
pNAbcA were 3?30?6 mg ml21, whereas those for cells the most significant changes were found for polymyxin B,
121
http://mic.sgmjournals.org 3501
A. Margolles and others

peptide nisin. Therefore, susceptibility differences against


nisin and polymyxin B between L. lactis cells harbouring
pNAbcA, pNAbcB or pNAbcA/pNAbcB, and bacterial cells
harbouring the control plasmid, were determined in GM17
agar. The joint expression of AbcA and AbcB resulted in an
increased resistance to nisin and polymyxin with respect to
the control, which became apparent by colony formation at
the highest dilution used. However, considerable nisin
resistance was also found under similar conditions for cells
expressing only AbcB (Fig. 3). This indicated that the
expression of AbcB alone could also reduce the cell
susceptibility to nisin, although to a lesser extent than
when both proteins are co-expressed. Consistent with the
above finding, it has previously been shown that certain
ABC transporters can act on bacteriocin-like compounds.
Fig. 2. Expression of AbcA and AbcB and co-purification of For example, the ABC-transporter LmrB from L. lactis
the AbcA/AbcB complex. Coomassie BioSafe-stained SDS- confers resistance to LsbA and LsbB, two class II bacteriocins
PAGE gel of inside-out membrane vesicles (30 mg protein/lane) (Gajic et al., 2003). It does so, most likely, by removing LsbA
from cells harbouring the empty vector (pNZ8048, lane 1) or and LsbB from the cytoplasmic membrane, which is the
cells expressing AbcA (pNAbcA, lane 2), AbcB (pNAbcB, lane target of these antimicrobial peptides. Other studies have
3), or jointly AbcA and AbcB (pNAbcA/pNAbcB, lane 4). The suggested that ABC transporters could play a key role in
purified histidine-tagged AbcA from a Ni-NTA column with generating resistance to nisin and other antimicrobial
250 mM imidazole and the co-eluted protein AbcB are repre- peptides in Gram-positive bacteria (Kok et al., 2005).
sented in lane 5. MM, protein markers (molecular masses in
kDa are indicated on the right). The arrows indicate the posi- Transport of Hoechst-33342 in membrane
tions of AbcA and AbcB.
vesicles
Hoechst-33342 is a cytotoxic drug extensively employed to
the resistance increasing more than fourfold for cells detect the activity of MDR transporters (Lubelski et al.,
expressing AbcA (MIC 42?79?2 mg ml21, compared to 2004; Margolles et al., 1999; Sakamoto et al., 2001; van Veen
9?32?3 for the control strain), and more than twelve-fold et al., 2000; Woebking et al., 2005). This probe has the
for cells expressing AbcB (128?00 mg ml21) or both property of being fluorescent when present in the lipid
proteins (149?340 mg ml21). No significant differences environment of biomembranes, while being essentially non-
between the control strain and the membrane-protein- fluorescent in the aqueous phase (Shapiro & Ling, 1995).
expressing cells were found for any other antibiotic using the Since MDR transporters are thought to pump out
E-test assay. hydrophobic drugs from (or close to) the cytoplasmic
membrane (Putman et al., 2000), this compound was used
Since polymyxin B is a polycationic antimicrobial peptide to study the extrusion of drugs directly from the membrane
that acts at the cell surface level, dissipating proton-motive to the aqueous phase, via the decrease of fluorescence
force by making pores in the cell membrane (Hancock & observed when extrusion activity is present (Margolles et al.,
Chapple, 1999), we decided to investigate the resistance- 1999; Shapiro & Ling, 1995; Woebking et al., 2005).
conferring capability of AbcA and AbcB to the antimicrobial Furthermore, the advantage of carrying out functional

Fig. 3. Effect of AbcA, AbcB, and AbcA/


AbcB expression on the susceptibility of L.
lactis to nisin and polymyxin. Results shown
are from cells harbouring pNZ8048 (1),
pNAbcA (2), pNAbcB (3), or pNAbcA and
pNAbcB (4) and spotted on GM17 plates
containing 78 ng nisin ml1 or 62 mg poly-
myxin B ml1.
122
3502 Microbiology 152
Bifidobacterium breve ABC-type MDR transporter

studies in isolated membranes is the avoidance of possible purification procedure (Fig. 2). When His-tagged AbcA was
interference caused by other cellular components that may purified, two protein bands were eluted, with molecular
modify the transport activity. Experiments performed in masses that correspond to AbcA and AbcB. The identity of
inside-out membrane vesicles of L. lactis NZ9000 cells the proteins was confirmed by mass spectrometry, the upper
showed the highest transport rate of Hoechst-33342 in band being identified as AbcA, and the lower as AbcB.
membranes harbouring AbcA and AbcB, although signifi- Purified proteins were analysed by densitometry of
cant transport was also detected in AbcB-containing Coomassie BioSafe-stained SDS-PAGE gels, showing that
membrane vesicles. AbcA-containing membranes displayed both proteins are present in approximately equal amounts,
an extremely low transport activity as compared to the other indicating the existence of a stable membrane-associated
two membrane systems (Fig. 4). In addition, the ATP- complex with a stoichiometry of 1 : 1 (Fig. 2).
dependency of the transport process was demonstrated,
since no activity was detected in the presence of the non- It has been experimentally proved that several prokaryotic
hydrolysable ATP analogue AMP-PNP. ATP-dependent ABC transporters act as dimers. Homodimerization has
transport of Hoechst-33342 was not observed in control been demonstrated for LmrA of L. lactis (van Veen et al.,
membrane vesicles. 2000), MsbA of E. coli (Chang & Roth, 2001) and BmrA of B.
subtilis (Ravaud et al., 2006), while a heterodimeric complex
was found to be the functional unit of the MDR transporter
AbcA and AbcB form a stable complex in the LmrCD from L. lactis (Lubelski et al., 2004). However, in
membrane our study we have found that AbcB can retain some activity
The results of the experiments described above point to by itself, conferring nisin resistance and extruding Hoechst-
cooperation between AbcA and AbcB that results in an 33342 from the membrane, although to a lesser extent than
increased resistance to nisin and polymyxin, and an when both proteins are present. A similar effect has also been
enhanced ability to extrude Hoechst-33342 from the reported for other ABC transporters. In eukaryotic cells, the
membrane of L. lactis. As suggested for other ABC mammalian proteins ADLP (adrenoleukodystrophy pro-
transporters (Abele & Tampe, 1999; Lee et al., 2003; tein), ALDRP (adrenoleukodystrophy-related protein) and
Lubelski et al., 2004), this is likely to happen through a direct PMP70 (70 kDa peroxisomal protein) were found to act as
interaction of AbcA and AbcB in the membrane, where these homo- as well as heterodimers, and it was proposed that the
two proteins are assumed to form a complex that represents different dimer combinations vary in activity and substrate
the functional unit of the transporter. To prove this specificity (Liu et al., 1999). Genetic evidence suggests that
assumption for the AbcAB proteins, a histidine tag was the substrate specificity of the traffic ATPase transporters
attached to the C-terminal part of AbcA to facilitate involved in the uptake of eye pigment precursors in
purification of the complex by affinity chromatography. For Drosophila melanogaster (the white, scarlet and brown
this purpose, membranes were isolated from cells harbour- gene products) depends on the dimer formed: white and
ing pNHAbcA/pNAbcB, solubilized with a DDM-contain- scarlet together form a tryptophan transporter, while the
ing buffer and subjected to a single-step Ni-NTA affinity white and brown gene products form a guanine transporter

100
Fluorescence (%)

80

60

(a) (b) (c)

200 400 600 800 200 400 600 800 200 400 600 800
Time (s)

Fig. 4. AbcA-, AbcB- and AbcA/AbcB-mediated extrusion of Hoechst-33342 from inside-out membrane vesicles.
Membranes were prepared from NZ9000 cells harbouring pNAbcA (a); pNAbcB (b), or pNAbcA/pNAbcB (c). The arrow
indicates the addition of 2 mM ATP (black lines) or AMP-PNP (grey lines). Hoechst-33342 transport in L. lactis NZ9000/
pNZ8048 control membranes was not observed under the same assay conditions (data not shown). The graphics shown are
representative of three independent experiments with three different batches of vesicles for each strain.
123
http://mic.sgmjournals.org 3503
A. Margolles and others

(Mackenzie et al., 1999). In a similar way, we have observed Escherichia coli and Listeria in a dynamic model of the stomach and
that when AbcA and AbcB are co-expressed in L. lactis cells, the small intestine. Int J Food Microbiol 48, 2135.
AbcB is produced about two times more than AbcA. This Grkovic, S., Brown, M. H. & Skurray, R. A. (2002). Regulation of
implies that in pNAbcA/pNAbcB-containing cells there bacterial drug export systems. Microbiol Mol Biol Rev 66, 671701.
would be a population of free AbcB, not bound to AbcA. Hancock, R. E. & Chapple, D. S. (1999). Peptide antibiotics.
Since we have shown that AbcB alone is active under certain Antimicrob Agents Chemother 43, 13171323.
conditions, this could partially mask the activity of the Harmsen, H. J., Wildeboer-Veloo, A. C., Raangs, G. C., Wagendorp,
heterodimer. Future reconstitution studies could address A. A., Klijn, L., Bindels, J. G. & Welling, G. W. (2000). Analysis of
these questions. intestinal flora development in breast-fed and formula-fed infants by
using molecular identification and detection methods. J Pediatr
In short, this study has provided evidence that AbcA and Gastroenterol Nutr 30, 6167.
AbcB act as a heterodimeric ABC-type multidrug transpor- Hirata, T., Saito, A., Nishino, K., Tamura, N. & Yamaguchi, A. (2004).
ter, conferring resistance to nisin and polymyxin and Effects of efflux transporter genes on susceptibility of Escherichia coli to
tigecycline (GAR-936). Antimicrob Agents Chemother 48, 21792184.
extruding cytotoxic compounds. Therefore, we propose to
rename AbcA and AbcB as BbmA (Bifidobacterium breve Isolauri, E., Salminen, S. & Ouwehand, A. C. (2004). Microbial-gut
interactions in health and disease. Probiotics. Best Pract Res Clin
multidrug transporter) and BbmB, respectively. These
Gastroenterol 18, 299313.
findings provide the basis for further biochemical studies
Karow, M. & Georgopoulos, C. (1993). The essential Escherichia coli
of BbmAB and the half-size transporters BbmA and BbmB,
msbA gene, a multicopy suppressor of null mutations in the htrB
and open some questions about the physiological role of this gene, is related to the universally conserved family of ATP-dependent
B. breve transporter in its natural environment, the intestinal translocators. Mol Microbiol 7, 6979.
niche. Kim, S. H., Chang, A. B. & Saier, M. H., Jr (2004). Sequence similarity
between multidrug resistance efflux pumps of the ABC and RND
superfamilies. Microbiology 150, 24932495.
ACKNOWLEDGEMENTS Kok, J., Buist, G., Zomer, A. L., van Hijum, S. A. & Kuipers, O. P.
(2005). Comparative and functional genomics of lactococci. FEMS
This work was financed by the European Union STREP project ACE-
Microbiol Rev 29, 411433.
ART (FP6-506214), European Union FEDER funds, and the Spanish
Plan Nacional de I+D (project AGL2004-06727-C02). J. A. Moreno Kuipers, O. P., Beerthuyzen, M. M., Siezen, R. J. & de Vos, W. M.
was the recipient of a post-doctoral contract from CSIC (I3P (1993). Characterization of the nisin gene cluster nisABTCIPR of
programme), Spain. The work was also financially supported by the Lactococcus lactis. Requirement of expression of the nisA and nisI
Department of Agriculture and Food FIRM programme (01/R&D/C/ genes for development of immunity. Eur J Biochem 216, 281291.
159), by the Higher Education Authority Programme for Research in Kuipers, O. P., de Ruyter, P. G., Kleerebezem, M. & de Vos, W. M.
Third Level Institutions, and by the SFI-funded Alimentary (1998). Quorum sensing-controlled gene expression in lactic acid
Pharmabiotic Centre. We thank S. Leahy, M. OConnell-Motherway, bacteria. J Biotechnol 64, 1521.
G. F. Fitzgerald and H. G. Higgins for sharing unpublished data with
Lee, E. W., Huda, M. N., Kuroda, T., Mizushima, T. & Tsuchiya, T.
us. We acknowledge Oscar Kuipers (Genetics Department, University
(2003). EfrAB, an ABC multidrug efflux pump in Enterococcus
of Groningen, The Netherlands) for providing us with the plasmid
faecalis. Antimicrob Agents Chemother 47, 37333738.
pNZ8048 and Daniel Linares (IPLA-CSIC) for excellent assistance in
carrying out the quantitative PCR. Liu, L. X., Janvier, K., Berteaux-Lecellier, V., Cartier, N., Benarous, R.
& Aubourg, P. (1999). Homo- and heterodimerization of perox-
isomal ATP-binding cassette half-transporters. J Biol Chem 274,
3273832743.
REFERENCES
Lubelski, J., Mazurkiewicz, P., van Merkerk, R., Konings, W. N. &
Abele, R. & Tampe, R. (1999). Function of the transport complex Driessen, A. J. (2004). ydaG and ydbA of Lactococcus lactis encode a
TAP in cellular immune recognition. Biochim Biophys Acta 1461, heterodimeric ATP-binding cassette-type multidrug transporter.
405419. J Biol Chem 279, 3444934455.
Begley, M., Gahan, C. G. & Hill, C. (2005). The interaction between MacConaill, L. E., Butler, D., OConnell-Motherway, M., Fitzgerald,
bacteria and bile. FEMS Microbiol Rev 29, 625651. G. F. & van Sinderen, D. (2003). Identification of two-component
Chang, G. & Roth, C. B. (2001). Structure of MsbA from E. coli: a regulatory systems in Bifidobacterium infantis by functional com-
homolog of the multidrug resistance ATP binding cassette (ABC) plementation and degenerate PCR approaches. Appl Environ
transporters. Science 293, 17931800. Microbiol 69, 42194226.
de Ruyter, P. G., Kuipers, O. P. & de Vos, W. M. (1996). Controlled Mackenzie, S. M., Brooker, M. R., Gill, T. R., Cox, G. B., Howells, A. J.
gene expression systems for Lactococcus lactis with the food-grade & Ewart, G. D. (1999). Mutations in the white gene of Drosophila
inducer nisin. Appl Environ Microbiol 62, 36623667. melanogaster affecting ABC transporters that determine eye coloura-
Doerrler, W. T. & Raetz, C. R. (2002). ATPase activity of the MsbA tion. Biochim Biophys Acta 1419, 173185.
lipid flippase of Escherichia coli. J Biol Chem 277, 3669736705. Mahida, Y. R., Rose, F. & Chan, W. C. (1997). Antimicrobial peptides
Gajic, O., Buist, G., Kojic, M., Topisirovic, L., Kuipers, O. P. & Kok, J. in the gastrointestinal tract. Gut 40, 161163.
(2003). Novel mechanism of bacteriocin secretion and immunity Margolles, A. & de los Reyes-Gavilan, C. G. (2003). Purification and
carried out by lactococcal multidrug resistance proteins. J Biol Chem functional characterization of a novel a-L-arabinofuranosidase from
278, 3429134298. Bifidobacterium longum NB667. Appl Environ Microbiol 69, 50965103.
Ganzle, M. G., Hertel, C., van der Vossen, J. M. & Hammes, W. P. Margolles, A., Putman, M., van Veen, H. W. & Konings, W. N. (1999).
(1999). Effect of bacteriocin-producing lactobacilli on the survival of The purified and functionally reconstituted multidrug transporter
124
3504 Microbiology 152
Bifidobacterium breve ABC-type MDR transporter

LmrA of Lactococcus lactis mediates the transbilayer movement of Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
specific fluorescent phospholipids. Biochemistry 38, 1629816306. a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold
Margolles, A., Moreno, J. A., van Sinderen, D. & de los Reyes-Gavilan, Spring Harbor Laboratory.
C. G. (2005). Macrolide resistance mediated by a Bifidobacterium breve Schneider, E. & Hunke, S. (1998). ATP-binding-cassette (ABC)
membrane protein. Antimicrob Agents Chemother 49, 43794381. transport systems: functional and structural aspects of the ATP-
Matsuki, T., Watanabe, K., Tanaka, R., Fukuda, M. & Oyaizu, H. hydrolyzing subunits/domains. FEMS Microbiol Rev 22, 120.
(1999). Distribution of bifidobacterial species in human intestinal Shapiro, A. B. & Ling, V. (1995). Reconstitution of drug transport by
microflora examined with 16S rRNA gene-targeted species-specific purified P-glycoprotein. J Biol Chem 270, 1616716175.
primers. Appl Environ Microbiol 65, 45064512. Turnbull, P. C., Sirianni, N. M., LeBron, C. I., Samaan, M. N., Sutton,
Mazurkiewicz, P., Driessen, A. J. & Konings, W. N. (2005). What do F. N., Reyes, A. E. & Peruski, L. F. (2004). MICs of selected
proton motive force driven multidrug resistance transporters have in antibiotics for Bacillus anthracis, Bacillus cereus, Bacillus thurin-
common? Curr Issues Mol Biol 7, 721. giensis, and Bacillus mycoides from a range of clinical and
Moubareck, C., Gavini, F., Vaugien, L., Butel, M. J. & Doucet- environmental sources as determined by the Etest. J Clin Microbiol
Populaie, F. (2005). Antimicrobial susceptibility of bifidobacteria. 42, 36263634.
J Antimicrob Chemother 55, 3844. van Veen, H. W., Venema, K., Bolhuis, H., Oussenko, I., Kok, J.,
Orelle, C., Dalmas, O., Gros, P., di Pietro, A. & Jault, J. M. (2003). Poolman, B., Driessen, A. J. & Konings, W. N. (1996). Multidrug
The conserved glutamate residue adjacent to the Walker-B motif is resistance mediated by a bacterial homolog of the human multidrug
the catalytic base for ATP hydrolysis in the ATP-binding cassette transporter MDR1. Proc Natl Acad Sci U S A 93, 1066810672.
transporter BmrA. J Biol Chem 278, 4700247008. van Veen, H. W., Margolles, A., Muller, M., Higgins, C. F. & Konings,
Ouwehand, A. C., Salminen, S. & Isolauri, E. (2002). Probiotics: an W. N. (2000). The homodimeric ATP-binding cassette transporter
overview of beneficial effects. Antonie Van Leeuwenhoek 82, 279289. LmrA mediates multidrug transport by an alternating two-site (two-
Price, C. E., Reid, S. J., Driessen, A. J. & Abratt, V. R. (2006). The
cylinder engine) mechanism. EMBO J 19, 25032514.
Bifidobacterium longum NCIMB 702259T ctr gene codes for a novel Vedantam, G. & Hecht, D. W. (2003). Antibiotics and anaerobes of
cholate transporter. Appl Environ Microbiol 72, 923926. gut origin. Curr Opin Microbiol 6, 457461.
Putman, M., van Veen, H. W. & Konings, W. N. (2000). Molecular Ventura, M., van Sinderen, D., Fitzgerald, G. F. & Zink, R. (2004).
properties of bacterial multidrug transporters. Microbiol Mol Biol Rev Insights into the taxonomy, genetics and physiology of bifidobac-
64, 672693. teria. Antonie van Leeuwenhoek 86, 205223.
Raherison, S., Gonzalez, P., Renaudin, H., Charron, A., Bebear, C. & Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982).
Bebear, C. M. (2005). Increased expression of two multidrug Distantly related sequences in the a- and b-subunits of ATP
transporter-like genes is associated with ethidium bromide and synthase, myosin, kinases and other ATP-requiring enzymes and a
ciprofloxacin resistance in Mycoplasma hominis. Antimicrob Agents common nucleotide binding fold. EMBO J 1, 945951.
Chemother 49, 421424. Woebking, B., Reuter, G., Shilling, R. A., Velamakanni, S., Shahi, S.,
Ravaud, S., do Cao, M. A., Jidenko, M., Ebel, C., le Maire, M., Jault, Venter, H., Balakrishnan, L. & van Veen, H. W. (2005). Drug-lipid A
M., di Pietro, A., Haser, R. & Aghajari, N. (2006). The ABC interactions on the Escherichia coli ABC transporter MsbA. J Bacteriol
transporter BmrA from Bacillus subtilis is a functional dimer in the 187, 63636369.
detergent-solubilized state. Biochem J 395, 345353. Yokota, A., Veenstra, M., Kurdi, P., van Veen, H. W. & Konings, W. N.
Sakamoto, K., Margolles, A., van Veen, H. W. & Konings, W. N. (2000). Cholate resistance in Lactococcus lactis is mediated by an
(2001). Hop resistance in the beer spoilage bacterium Lactobacillus ATP-dependent multispecific organic anion transporter. J Bacteriol
brevis is mediated by the ATP-binding cassette multidrug transporter 182, 51965201.
HorA. J Bacteriol 183, 53715375. Zuniga, M., Franke-Fayard, B., Venema, G., Kok, J. & Nauta, A.
Salminen, S. & Gueimonde, M. (2004). Human studies on (2002). Characterization of the putative replisome organizer of the
probiotics: what is scientifically proven? J Food Sci 69, 137140. lactococcal bacteriophage r1t. J Virol 76, 1023410244.

125
http://mic.sgmjournals.org 3505
Ubiquity and diversity of multidrug resistance genes in
Lactococcus lactis strains isolated between1936 and1995
Ana Belen Florez1, Clara G. de los Reyes-Gavilan1, Anette Wind2, Baltasar Mayo1 & Abelardo Margolles1
1
Instituto de Productos Lacteos de Asturias, Consejo Superior de Investigaciones Cientficas (CSIC), Villaviciosa, Asturias, Spain; and 2Molecular Strain
Characterisation, chr. Hansen A/S, Bge Alle 10-12, 2970, Hrsholm, Denmark

Correspondence: Abelardo Margolles, Abstract


Instituto de Productos Lacteos de Asturias,
Consejo Superior de Investigaciones
The presence and the nucleotide sequence of four multidrug resistance genes,
Cientficas (CSIC). Ctra. Infiesto s/n, 33300, lmrA, lmrP, lmrC, and lmrD, were investigated in 13 strains of Lactococcus lactis ssp.
Villaviciosa, Asturias, Spain. Tel.: 134 985 89 lactis, four strains of Lactococcus lactis ssp. cremoris, two strains of Lactococcus
21 31; fax: 134 985 89 22 33; e-mail: plantarum, and two strains of Lactococcus raffinolactis. Multidrug resistance genes
amargolles@ipla.csic.es were present in all L. lactis isolates tested. However, none of them could be detected
in the strains belonging to the species L. raffinolactis and L. plantarum, suggesting a
Received 26 April 2006; revised 24 May 2006;
different set of multidrug resistance genes in these species. The analysis of the four
accepted 21 June 2006.
First published online 21 August 2006. deduced amino acid sequences established two different variants depending on the
subspecies of L. lactis. Either lmrA, or lmrP, or both were found naturally disrupted
DOI:10.1111/j.1574-6968.2006.00371.x in five strains, while full-length lmrD was present in all strains.

Editor: Anthony George

Keywords
Lactococcus lactis ; multidrug resistance; MDR-
transporters.

Introduction to the ABC transporter family, whereas LmrP is a proton-


dependent secondary transporter (Bolhuis et al., 1995),
Bacterial resistance to drugs is one of the major causes of
and Mdt(A) shares sequence homology with both ABC and
antibiotic treatment failure against pathogenic infections.
secondary transporters (Perreten et al., 2001). LmrB
Microorganisms have developed various strategies to resist
and Mdt(A) are plasmid-encoded proteins, whereas LmrA,
the effect of cytotoxic substances. In some cases, this
LmrP, and LmrCD are encoded in the chromosome. Ex-
resistance can be mediated by multidrug resistance (MDR)
tensive biochemical and functional studies have been carried
transporters, membrane proteins that actively extrude nox-
out for LmrA, LmrP, and LmrCD, leading to a solid knowl-
ious compounds with very different chemical structures and
edge of their functional properties and substrate specifici-
cellular targets from the cell (Putman et al., 2000b). Multi-
ties. However, the plasticity of the genes coding for these
drug transporters can be amplified in drug-resistant micro-
proteins and its abundance in the genus Lactococcus has
organisms, and can shift their drug profiles, making them a
never been investigated. Thus, this study examines the
notorious menace to drug treatment (van Veen et al., 1999).
presence of these four MDR genes in several Lactococcus
These proteins can be classified into two groups. In ATP-
strains isolated in 60 years.
binding cassette (ABC) transporters, the transport is driven
via the hydrolysis of ATP, whereas the activity of secondary
transporters is dependent on the transmembrane electro-
Material and methods
chemical gradient, typically the proton motive force
Strains and growth conditions
(Putman et al., 2000b).
During the last decade, Lactococcus lactis has become one A total of 21 Lactococcus strains from dairy and vegetable
of the most important species to study MDR transporters in origins, including two strains of Lactococcus plantarum
gram-positive bacteria. Currently, five MDR transporters (LMG 8517T and LMG 11686), two strains of Lactococcus
have been described in this microorganism. LmrA (van Veen raffinolactis (LMG 13095T and LMG 13098), nine strains of
et al., 1996), LmrB (Gajic et al., 2003), and the hetero- Lactococcus lactis ssp. cremoris, and eight strains of Lactococ-
dimeric transporter LmrCD (Lubelski et al., 2004) belong cus lactis ssp. lactis (Table 1), isolated in different places

FEMS Microbiol Lett 263 (2006) 2125 


c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
127
22 A.B. Florez et al.

Table 1. Lactococcus lactis strains used in this study


% Protein identity
Isolation year Previous 16S RNA genetic
Strain Biological origin (or first reported) identification identification LmrAw LmrPw LmrCz LmrDz
NCDO 712 Cheese starter 1955 cremoris cremoris 100 100 95.8 97.1
IL 594 Cheese starter 1984 lactis lactis 96.6 96.0 100 100
NCIMB 700495 1948 cremoris cremoris (97.5) (96.8) 95.8 97.3
NCIMB 700608 1937 cremoris cremoris (97.5) (96.9) 95.8 97.3
NCIMB 10493 1936 lactis cremoris 100 99.5 95.8 97.3
NCIMB 6681T Plant material 1940 lactis lactis 96.8 97.0 99.7 99.8
CHCC 1416 Raw milk 1959 lactis lactis 96.6 (91.3) 99.7 100
CHCC 1418 Raw milk 1959 cremoris lactis 96.4 96.8 99.7 100
CHCC 7524 Dairy 1957 cremoris lactis 96.4 96.5 99.8 99.8
CHCC 7526 Dairy, raw milk 1958 cremoris lactis (88.5) 96.8 99.5 99.8
CHCC 7532 Raw milk 1959 cremoris lactis 96.6 (91.5) 99.7 100
CHCC 7533 Dairy 1957 cremoris lactis 96.6 96.5 99.8 99.8
LMG 8526 Chinese radish 1977 lactis lactis 96.4 97.0 99.0 100
LMG 8527 Frozen peas 1978 lactis lactis 96.4 97.3 ND 100
LMG 9446 Frozen peas 1966 lactis lactis 96.8 97.3 99.7 100
IPLA 2A14 Dairy products 1995 lactis lactis 96.6 97.0 99.7 99.8
IPLA 2A34 Dairy products 1995 cremoris lactis 96.6 96.8 99.7 100
For those genes whose ORF was interrupted, percentage of DNA identity (in brackets) is represented instead of protein identity.
w
Percentage of identity respect to LmrA and LmrP from L. lactis ssp. cremoris NCDO 712 (accession numbers U63741 and X89779, respectively).
Lactococcus lactis ssp. cremoris NCDO 712 was used as a control for the sequencing of lmrA and lmrP, as this is the original strain in which these two
genes have been described.
z
Percentage of identity respect to LmrC and LmrD from L. lactis ssp. lactis IL 594 (accession number AE006268). Lactococcus lactis ssp. lactis IL 594 was
used as a control for the sequencing of lmrC and lmrD, as this is the original strain in which these two genes have been described.

Based on accession number AAK04809.1 (for LmrA) and AAK06263.1 (for LmrP).
NCDO, National Collection of Dairy Organisms, Reading, UK; NCIMB, National Collections of Industrial and Marine Bacteria Ltd., Aberdeen, Scotland;
CHCC, Christian Hansen Culture Collection, Hrsholm, Denmark; LMG, Universiteit Gent/Belgium Co-ordinated Collection of Microorganisms, Gent,
Belgium; IPLA, Instituto de Productos Lacteos Collection, VIllaviciosa, Asturias, Spain; ND, not detected; , not known.

around the world, were used to analyze systematically the primers LmrCD-f (GTTAAAAATCCAAATGTCTT) and
presence of MDR genes and their nucleotide sequences. LmrCD-r (AGCATGTTGATGTGTAAAGAAG).
Cells were cultivated at 30 1C in M17 broth (Oxoid) with For species and subspecies determination, PCR primers
0.7% glucose. Y1 (TGG CTC AGG ACG AAC GCT GGC GGC) and Y2
(CCT ACT GCT GCC TCC CGT AGG AGT) were used to
amplify a 348 bp internal fragment of the 16S rRNA gene of
DNA purification and PCR analysis
all Lactococcus strains, as described previously (Ward et al.,
Total DNA was extracted from overnight cultures of Lacto- 1998).
coccus using a GenElute Bacterial Genomic DNA kit (Sigma)
following the manufacturers instructions. Primers LmrA-f
Sequencing and bioinformatic analysis
(GAAAGAGGTCCACAAATGGCCAATCG) and LmrA-r
(CAACAGTCAATTGTTCTGAAACATATTTAGC), LmrP-f Both DNA strands of the polymerase chain reaction pro-
(GTATGAAAGAGTTTTGGAATTTAGAC) and LmrP-r ducts were directly sequenced, carried out by the Servicio
(GTTTTCTGATGTCTATTTACAGCAACAAG), LmrC-f de Secuenciacion de DNA (Centro de Investigaciones
(GAAGCATAAATGGGTTGCCTTATTCTCAATCG) and Biologicas, Consejo Superior de Investigaciones Cientficas)
LmrC-r (GTCCTCCTGTGCTTTCTGTGTGTCGTAG), and with an ABI Prism 377 sequencer (Applied Biosystems).
LmrD-f (GAAAATACCAAATCAACAAGAAAAATGTCT DNA and the deduced protein sequences were analyzed
GACAC) and LmrD-r (CAAAAACAAATTGATTGTGA using the computer program Vector NTI (InvitrogenTM Life
TAAAGTTCAGAGTAG) were used to amplify the genes Science Software). Homology trees were constructed with
lmrA, lmrP, lmrC, and lmrD, respectively. At least 99.2% of the DNAMAN software (Lynnon Corporation). Homology
the full-length nucleotide sequence of the four genes was searches were carried out using the BLAST server of the
amplified with this set of primers. The intergenic DNA National Center for Biotechnology Information at the
region between LmrC and LmrD was amplified with the National Institutes of Health (Bethesda, MD) (http://


c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 263 (2006) 2125
Published by Blackwell Publishing Ltd. All rights reserved
128
Lactococcus lactis MDR transporters 23

LmrA LmrP NCDO 712


NCDO 712
100% NCIMB 10493
100% NCIMB 10493
LMG 8526 LMG 8526
97%
LMG 8527 IPLA 2A14
99% IPLA 2A14 LMG 9446
LMG 9446 100%
LMG 8527
99%
NCIMB 6681 NCIMB 6681
100% IL 594
CHCC 7524 CHCC 7526
CHCC 1418 IL 594
CHCC 7533 CHCC 7533
CHCC 7532 CHCC 7524
100% IPLA 2A34
IPLA 2A34
CHCC 1416 CHCC 1418

95% 100% 95% 100%

LmrC LmrD
NCIMB 700608 NCIMB 700608
NCIMB 700495
100% NCIMB 700495
NCIMB 10493 100% NCIMB 10493
NCDO 712
NCDO 712
96% 97% CHCC 7533
LMG 8526
CHCC 7526
IL 594 100%
99% IPLA 2A14
CHCC 7526
100% CHCC 7524
CHCC 7533 NCIMB 6681
CHCC 7524 IL 594
LMG 9446 LMG 9446
CHCC 7532 LMG 8527
NCIMB 6681 LMG 8526
CHCC 1418 CHCC 7532
CHCC 1416 CHCC 1416
IPLA 2A34 IPLA 2A34
IPLA 2A14 CHCC 1418

95% 100% 95% 100%

Fig. 1. Homology trees of deduced protein sequences of LmrA, LmrP, LmrC, and LmrD. The distances among the different strains are percentages of
different residues. Proteins for which the coding DNA sequence was interrupted are not represented in the graphic. Lactococcus lactis ssp. cremoris
strains are represented in bold letters.

www.ncbi.nlm.nih.gov) and the software available on the is different from those of L. lactis. The partial sequence of
Web page of the Pole-Bioinformatique Lyonnais (Lyon, the 16S rRNA gene from L. lactis strains allowed us to
France) (http://pbil.univ-lyon1.fr/). All the sequences of the reclassify the 17 L. lactis strains, 13 belonging to the sub-
MDR genes were deposited in the GenBank database species lactis and the other four belonging to the subspecies
(accession numbers from DQ516910 to DQ516970). cremoris (Table 1). Moreover, the analysis of the four
deduced amino acid sequences allowed us to nicely establish
two different groups according to the subspecies they
Results and discussion belong: lactis or cremoris (Fig. 1).
For the L. lactis strains, amplicons with the expected size The ABC transporter LmrA was found to confer resis-
were obtained for all genes, excluding lmrC from L. lactis tance to more than 20 different antibiotics (Putman et al.,
ssp. lactis LMG 8527. However, no PCR products could be 2000a) and is able to transport several cytotoxic drugs
obtained for any of the strains belonging to the species (Margolles et al., 1999; van Veen et al., 2000). LmrP confers
L. raffinolactis and L. plantarum. Taking into account the resistance to lincosamides, macrolides, streptogramins, and
considerable phylogenetic distance existing between these tetracyclines (Putman et al., 2001). Genes coding for both
two species and L. lactis (Pu et al., 2002), this likely indicates proteins were present in all the strains. However, for the
that the set of MDR genes in L. raffinolactis and L. plantarum L. lactis strains analyzed in the present work, the ORF of

FEMS Microbiol Lett 263 (2006) 2125 


c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
129
24 A.B. Florez et al.

lmrA was disrupted in the strain CHCC 7526 and the same hydrophobic molecules encountered in the natural environ-
was found for lmrP in the strains CHCC 7532 and CHCC ment of many organisms or the transport of specific
1416. Remarkably, the sequences of both genes were dis- metabolites) and the efflux of relevant antimicrobial com-
rupted in the strains NCIMB 700495 and NCIMB 700608 pounds can be achieved merely opportunistically (Grkovic
(Table 1). In relation to this, it is known that the deletion of et al., 2002). Toxic compounds have always been part of the
lmrA (Lubelski et al., 2004) or lmrP (Bolhuis et al., 1995) natural habitat of microorganisms, and the presence of
does not significantly alter the ability to secrete some drugs MDR among pathogenic and nonpathogenic microorgan-
from the cell, suggesting that these transporters are not isms suggests that MDR mechanisms did not arise recently
essential for some cell functions and probably other MDR in response to antimicrobial chemotherapy. This work
proteins can take over their roles in L. lactis. It is noteworthy indeed supports this hypothesis. In fact, LmrA was shown
that in several of our L. lactis strains, mutations changing to transport fluorescent phosphatidylethanolamine, a phos-
amino acid residues that could be involved in the activity of pholipid very abundant in biological membranes (Margolles
LmrA (V304A in the transmembrane segment VI in all L. et al., 1999), and to substitute functionally for the lipid A
lactis ssp. lactis strains) and LmrP (D128A in the cytoplas- transporter MsbA in Escherichia coli (Reuter et al., 2003).
mic loop between transmembrane segment IV and V in L. Other prokaryotic and eukaryotic MDR transporters are
lactis NCIMB 6681) were found (Mazurkiewicz et al., 2002; also involved in lipid transport (Pohl et al., 2004). In
Poelarends & Konings, 2002), which may affect the substrate addition, among the recent findings regarding LmrA phy-
specificity and activity of the transporters. With respect to siological role, it has been shown that this transporter
LmrCD, it is composed of two polypeptides, LmrC and mediates the cotransport of drugs and protons, and its
LmrD, that bind in the membrane to constitute the func- activity significantly affects the intracellular pH of L. lactis
tional unit of this transporter, a heterodimer (Lubelski et al., (Venter et al., 2003; Shilling et al., 2005). We have shown
2004). Full-length lmrC and lmrD were found in 16 of the 17 that the MDR transporters of L. lactis have been almost
L. lactis strains used in this work, whereas lmrD, but not unchanged since more than 70 years ago, even before the
lmrC, was found in the remaining strain. The intergenic beginning of the antibiotic era and before some of the more
region was constituted of 3 bp (GAC) for the 16 L. lactis widely used antibiotics or cytotoxic drugs that are substrates
strains in which both genes were identified. No interrup- of these transporters, such as the semi-synthetic dye ethi-
tions in the ORFs of both genes were found for any of the dium bromide, were synthesized (Swartz, 2000). This sug-
strains. Recently, it was shown that mutation of the putative gests that the MDR transporters of L. lactis have a main
catalytic residue in the ATP-hydrolyzing domain of LmrC physiological function, probably related to their environ-
did not strongly interfere in the transport activity of the ment, such as vegetables, in which alkaloids, other MDR
LmrCD complex, whereas when the same mutation was substrates, are very abundant (Lewis, 2001; Krulwich et al.,
introduced in LmrD drug transport of the LmrCD hetero- 2005).
dimer was abolished (Lubelski et al., 2006). Relating to this, In short, these findings open some questions about the
it was found that some ABC transporters can act as homo- physiological role of these MDR transporters in the natural
as well as heterodimers (Liu et al., 1999), although this fact environment of L. lactis, vegetables, and dairy-based pro-
remains to be studied in detail for LmrCD. Remarkably, it ducts.
has been previously found that deletion of lmrC and lmrD
deeply modified the susceptibility of L. lactis to drugs (van
den Berg van Saparoea et al., 2005), suggesting that this Acknowledgements
MDR transporter is indeed necessary to maintain some cell This work was financed by the European Union STREP
functions and it could be a housekeeping protein. project ACE-ART (FP6-506214). We appreciate the gift of
MDR genes are thought to be present in all living cells. the strain IL 594 from Marie-Christine Chopin. We thank
Currently, the genome sequence of more than 250 bacteria is the Belgian Co-ordinated Collections of Microorganisms for
publicly available, and in all of them MDR homologs have providing us with several strains used in this study.
been found. The function of these transporters has always
been very controversial. In L. lactis, LmrA, LmrP, and
LmrCD constitute, probably together with other MDR References
proteins whose function remains to be investigated, the first Bolhuis H, Poelarends G, van Veen HW, Poolman B, Driessen AJ
physical barrier of defense against cytotoxic compounds. & Konings WN (1995) The lactococcal lmrP gene encodes a
While some authors have suggested that the primary role of proton motive force-dependent drug transporter. J Biol Chem
these pumps is to confer resistance to antibiotics and drugs; 270: 2609226098.
others support the hypothesis that they have a pre-existing Gajic O, Buist G, Kojic M, Topisirovic L, Kuipers OP & Kok J
physiological function (such as protection against toxic (2003) Novel mechanism of bacteriocin secretion and


c 2006 Federation of European Microbiological Societies FEMS Microbiol Lett 263 (2006) 2125
Published by Blackwell Publishing Ltd. All rights reserved
130
Lactococcus lactis MDR transporters 25

immunity carried out by lactococcal multidrug resistance Putman M, van Veen HW, Degener JE & Konings WN (2000a)
proteins. J Biol Chem 278: 3429134298. Antibiotic resistance: era of the multidrug pump. Mol
Grkovic S, Brown MH & Skurray RA (2002) Regulation of Microbiol 36: 772773.
bacterial drug export systems. Microbiol Mol Biol Rev 66: Putman M, van Veen HW & Konings WN (2000b) Molecular
671701. properties of bacterial multidrug transporters. Microbiol Mol
Krulwich TA, Lewinson O, Padan E & Bibi E (2005) Do Biol Rev 64: 672693.
physiological roles foster persistence of drug/multidrug-efflux Putman M, van Veen HW, Degener JE & Konings WN (2001) The
transporters? A case study. Nat Rev Microbiol 3: 566572. lactococcal secondary multidrug transporter LmrP confers
Lewis KR (2001) In search of natural substrates and inhibitors of resistance to lincosamides, macrolides, streptogramins and
MDR pumps. J Mol Microbiol Biotechnol 3: 247254. tetracyclines. Microbiology 147: 28732880.
Liu LX, Janvier K, Berteaux-Lecellier V, Cartier N, Benarous R & Reuter G, Janvilisri T, Venter H, Shahi S, Balakrishnan L & van
Aubourg P (1999) Homo- and heterodimerization of Veen HW (2003) The ATP binding cassette multidrug
peroxisomal ATP-binding cassette half-transporters. J Biol transporter LmrA and lipid transporter MsbA have
Chem 274: 3273832743. overlapping substrate specificities. J Biol Chem 278:
Lubelski J, Mazurkiewicz P, van Merkerk R, Konings WN & 3519335198.
Driessen AJ (2004) ydaG and ydbA of Lactococcus lactis encode Shilling R, Federici L, Walas F, Venter H, Velamakanni S,
a heterodimeric ATP-binding cassette-type multidrug Woebking B, Balakrishnan L, Luisi B & van Veen HW (2005) A
transporter. J Biol Chem 279: 3444934455. critical role of a carboxylate in proton conduction by the ATP-
Lubelski J, van Merkerk R, Konings WN & Driessen AJ (2006) binding cassette multidrug transporter LmrA. FASEB J 19:
Nucleotide-binding sites of the heterodimeric LmrCD ABC- 16981700.
multidrug transporter of Lactococcus lactis are asymmetric. Swartz MN (2000) Impact of antimicrobial agents and
Biochemistry 45: 648656. chemotherapy from 1972 to 1998. Antimicrob Agents
Margolles A, Putman M, van Veen HW & Konings WN (1999) Chemother 44: 20092016.
The purified and functionally reconstituted multidrug van den Berg van Saparoea HB, Lubelski J, van Merkerk R,
transporter LmrA of Lactococcus lactis mediates the Mazurkiewicz P & Driessen AJ (2005) Proton motive force-
transbilayer movement of specific fluorescent phospholipids. dependent Hoechst 33342 transport by the ABC transporter
Biochemistry 38: 1629816306. LmrA of Lactococcus lactis. Biochemistry 44: 1693116938.
Mazurkiewicz P, Konings WN & Poelarends GJ (2002) Acidic van Veen HW, Venema K, Bolhuis H, Oussenko I, Kok J, Poolman
residues in the lactococcal multidrug efflux pump LmrP play B, Driessen AJ & Konings WN (1996) Multidrug resistance
critical roles in transport of lipophilic cationic compounds. mediated by a bacterial homolog of the human multidrug
J Biol Chem 277: 2608126088. transporter MDR1. Proc Natl Acad Sci USA 93: 1066810672.
Perreten V, Schwarz FV, Teuber M & Levy SB (2001) Mdt(A), a van Veen HW, Putman M, Margolles A, Sakamoto K & Konings
new efflux protein conferring multiple antibiotic resistance in WN (1999) Structure-function analysis of multidrug
Lactococcus lactis and Escherichia coli. Antimicrob Agents transporters in Lactococcus lactis. Biochem Biophys Acta 1461:
Chemother 45: 11091114. 201206.
Poelarends GJ & Konings WN (2002) The transmembrane van Veen HW, Margolles A, Muller M, Higgins CF & Konings WN
domains of the ABC multidrug transporter LmrA form a (2000) The homodimeric ATP-binding cassette transporter
cytoplasmic exposed, aqueous chamber within the membrane. LmrA mediates multidrug transport by an alternating two-site
J Biol Chem 277: 4289142898. (two-cylinder engine) mechanism. EMBO J 19: 25032514.
Pohl A, Devaux PF & Herrmann A (2004) Function of Venter H, Shilling RA, Velamakanni S, Balakrishnan L & van Veen
prokaryotic and eukaryotic ABC proteins in lipid transport. HW (2003) An ABC transporter with a secondary-active
Biochim Biophys Acta 1733: 2952. multidrug translocator domain. Nature 426: 866870.
Pu ZY, Dobos M, Limsowtin GK & Powell IB (2002) Integrated Ward L, Brown J & Graham D (1998) Two methods for the
polymerase chain reaction-based procedures for the detection genetic differentiation of Lactococcus lactis and cremoris based
and identification of species and subspecies of the Gram-positive on differences in the 16S rRNA gene sequence. FEMS Microbiol
bacterial genus Lactococcus. J Appl Microbiol 93: 353361. Lett 166: 1521.

FEMS Microbiol Lett 263 (2006) 2125 


c2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
131
DISCUSIN GENERAL
__________________________________________
_____________________________________
__________________________________________
Discusin

IV.-DISCUSIN

ANTIBITICOS Y RESISTENCIA

La utilizacin de agentes antimicrobianos ha revolucionado la medicina


humana y animal. As, mientras antes de 1940 ocho de las diez causas de muerte ms
probables tenan etiologa infecciosa, hoy solo las afecciones respiratorias estn en este
grupo (Madigan et al., 2000). Sin embargo, su utilizacin masiva en la prctica clnica y
como agentes profilcticos y promotores del crecimiento en ganadera, acuicultura y
agricultura (Levy, 1997; Chopra y Roberts, 2001; Wegener, 2003; McManus et al., 2002)
favorece la aparicin de resistencias y su posterior diseminacin. La utilizacin
continuada de antibiticos elimina a las bacterias sensibles, promueve la aparicin de
resistencias y selecciona las cepas resistentes, de tal forma que stas aumentan
rpidamente. La resistencia a antimicrobianos es un problema de salud pblica
creciente a escala mundial que complica y encarece el tratamiento de las enfermedades
(Stuart y Marshall, 2004). De hecho, el fenmeno se ha definido de forma reciente
como una epidemia en la sombra (http://www.apua.org). El coste econmico y
humano es difcil de estimar, pero parece claro que las infecciones por
microorganismos resistentes duplican la estancia media en el hospital y doblan la
mortalidad y la morbilidad de las enfermedades (Cosgrove et al., 2003).
La resistencia puede ser inherente a una especie o gnero bacteriano
(resistencia intrnseca) o puede ser adquirida, mediante una o varias mutaciones
secuenciales o por medio de la incorporacin de nuevo material gentico (European
Commission, 2005). Este ltimo tipo es, sin duda, el que presenta mayor riesgo de
transferencia horizontal. Las primeras bacterias resistentes se detectaron en los aos 40
en el ambiente hospitalario; muy pronto tras la introduccin de los antimicrobianos
(Levy y Marshall, 2004). Entre los aos 50 y 60 se aislaron las primeras bacterias con
resistencia mltiple en cepas entricas de Salmonella, Shigella y Escherichia coli. Sin
embargo, la preocupacin por la resistencia no tom cuerpo hasta mediados de los
aos 70 cuando se identificaron cepas patgenas de Haemophilus influenzae y Neisseria
gonorrhoeae resistentes a ampicilina (De Graaf et al., 1976; Elwell et al., 1977).
La resistencia a antibiticos est ntimamente ligada a genuinos procesos
evolutivos, siendo por tanto esperable y predecible. As, la aparicin de resistencias
depende del volumen de antimicrobiano utilizado, de la frecuencia de mutacin de los

133
__________________________________________
Discusin _____________________________________
__________________________________________

microorganismos y del coste biolgico de las resistencias (Barbosa y Levy, 2000;


Baquero et al., 2002). En la mayora de los casos, el coste biolgico de una mutacin no
parece ser grande y puede compensarse con mutaciones en otros genes (Andersson y
Levin, 1999). De hecho, la rpida aparicin de resistencias contrasta con la lenta
desaparicin de los microorganismos resistentes en ausencia del agente selectivo
(Andersson, 2003). Las resistencias podran mantenerse tambin por una exposicin
continuada a residuos de antibiticos en el ambiente (Donoghue, 2003). Adems, con
frecuencia, los genes de resistencia a antibiticos se encuentran ligados entre s o con
determinantes de resistencia a otros compuestos txicos, de manera que las
resistencias pueden mantenerse tambin por co-seleccin (Borgen et al., 2002).
Finalmente, desde un punto de vista ecolgico, los microorganismos resistentes
seleccionados por el tratamiento de un solo individuo se diluiran en un ambiente
colonizado por los microorganismos susceptibles; sin embargo, el tratamiento de
poblaciones enteras con el mismo agente aumenta la presin selectiva por
microorganismos resistentes y reduce la recolonizacin por los sensibles (Levy, 1997).
Los genes de resistencia se disponen de forma frecuente en elementos genticos
mviles (como plsmidos o transposones) con gran capacidad de transferencia y
movilizacin (Normak y Normak, 2002). Algunos transposones contienen integrones:
transposones complejos que disponen de un sitio dedicado a la insercin conjunta de
genes de resistencia a antibiticos, o de otro tipo, que se expresan desde un promotor
nico (Hall et al., 1999; Nandi et al., 2004). Unos y otros pasan de unas clulas a otras,
saltando las barreras de especie y gnero, por los tres mecanismos de transferencia
gnica mencionados. El resultado final es que, en la actualidad, algunas cepas de las
especies Acinetobacter bumanii, Enterobacter cloacae, Enterococcus faecium, Klebsiella
pneumoniae, Mycobacterium tuberculosis, Pseudomonas aeruginosa, S. aureus y St.
pneumoniae son resistentes a los antimicrobianos disponibles para el tratamiento de las
infecciones que causan (Wright, 2003; Levy y Marshall, 2004); motivo por el que llegan
a ser fatales. Cabe destacar, sin embargo, el carcter clonal de los microorganismos
infecciosos (Levy y Marshall, 2004), lo que sugiere que puede haber otros factores,
adems de la resistencia a antibiticos, que contribuyan al xito de las cepas
patgenas. En todo caso, la resistencia se conjuga en la actualidad, tal y como se ha
comentado en la Introduccin, con el aparente abandono de la bsqueda de nuevos
antibiticos por las compaas farmacuticas. La industria achaca este abandono a una

134
__________________________________________
_____________________________________
__________________________________________
Discusin

legislacin rigurosa, el gran coste de los ensayos clnicos y la corta proteccin de los
descubrimientos (Projan, 2003; Shales et al., 2004).
En este escenario, por tanto, sern esenciales todas las estrategias dirigidas a
prevenir la aparicin de resistencias y a reducir, en lo posible, su diseminacin. Los
programas de seguimiento resultan, pues, esenciales para indicar el estado de las
resistencias en determinados grupos o especies microbianas y en diferentes
localizaciones. Ser conveniente tambin el aislamiento de los enfermos colonizados
por microorganismos resistentes, para evitar su diseminacin as como el ensayo de
nuevas formas de tratamiento (identificacin ms rpida y fiable de los agentes
infecciosos, menor duracin de los tratamientos, desarrollo de agentes teraputicos
contra nuevas dianas moleculares, desarrollo de vacunas, inhibicin especfica de la
infeccin, etc.) (Levy y Marshall, 2004).

MICROORGANISMOS COMENSALES Y RESISTENCIA A ANTIBITICOS

Se conoce desde hace tiempo que diversas poblaciones de organismos


comensales constituyen reservorios de elementos de resistencia (plsmidos,
transposones) capaces de transferirse a los microorganismos patgenos (DeFlaun y
Levy, 1989). As, las cepas comensales de Haemophilus parainfluenzae suministran
plsmidos que codifican -lactamasas a las cepas de H. influenzae (Levy, 1985). De
forma similar, las cepas de Streptococcus epidermidis sirven como reservorio de genes y
plsmidos de resistencia para las cepas de S. aureus (Cohen et al., 1982). Sin estar
codificada en un elemento mvil, el origen de la resistencia a penicilina del patgeno
St. pneumoniae se encuentra en la especie comensal resistente natural al antibitico
Streptococcus viridans (Balsalobre et al., 2003). La preocupacin, por tanto, de que los
microorganismos comensales se conviertan en reservorios de resistencias desde donde
stas puedan transferirse a bacterias patgenas es una preocupacin bien
fundamentada cientficamente (Levy y Marshall, 2004; Salyers et al., 2004;
http://www.roarproject.org).
Pero adems, algunas bacterias como E. coli y especies de los gneros
Clostridium, Bacteroides y Enterococcus son patgenos oportunistas y en determinadas
condiciones pueden causar infeccin. Aunque de forma an ms rara, tambin los
representantes genuinos de las bacterias del cido lctico, considerados seguros o
beneficiosos desde el punto de vista de la salud, pueden estar involucrados en

135
__________________________________________
Discusin _____________________________________
__________________________________________

infecciones (Gasser, 1994; Cannon, 2005). La resistencia a antibiticos en estos tipos


bacterianos, como los patgenos, complica el tratamiento y la evolucin de las
enfermedades; como ocurre en la infeccin de individuos inmunocomprometidos por
E. faecium (Goosens, 1999).

RESISTENCIA A ANTIBITICOS EN LAS BACTERIAS LCTICAS

Las bacterias del cido lctico (BAL), o bacterias lcticas, son un grupo
heterogneo de microorganismos cuya caracterstica comn es la produccin de cido
lctico a partir de la fermentacin de los carbohidratos. En la actualidad, las BAL ms
tpicas se agrupan en los gneros Bifidobacterium, Lactobacillus, Lactococcus, Leuconostoc,
Pediococcus y Propionibacterium (Carr et al., 2002). Especies de estos gneros llevan a
cabo importantes fermentaciones alimentarias (leche, carne, vegetales), modificando la
materia prima hasta obtener un producto final con propiedades organolpticas y de
conservacin mejoradas (cambios en la textura, desarrollo de compuestos de aroma y
sabor, inhibicin de microorganismos patgenos y alterantes, etc.). A travs de los
productos fermentados, las BAL forman parte de la dieta del hombre desde tiempo
inmemorial. Su ingesta segura, y la constatacin de que algunos tipos constituyen las
poblaciones mayoritarias en el intestino de los lactantes (Reuter, 2001), las ha
encumbrado como beneficiosas en el mantenimiento del equilibrio microbiano
intestinal necesario para la salud (Guarner y Malagelada, 2003); razones por las que se
utilizan de forma extendida como probiticos (Ouewehand et al., 2002; Ljungh y
Wadstrom, 2006).
Dado que las materias primas no pueden esterilizarse porque se alteran de
forma irreversible sus caractersticas organolpticas, las BAL entran en contacto con
bacterias patgenas y oportunistas durante las fermentaciones alimentarias y, por
supuesto, durante el trnsito gastrointestinal de los productos fermentados. En este
contexto, cobra todo su sentido la hiptesis del reservorio, y ms an cuando varios
autores sostienen que la cadena alimentaria es una de las principales vas de
transmisin de las resistencias a antibiticos (Teuber et al., 1999; Wegener, 2003;
Salyers et al., 2004; European Commission, 2005). Por tanto, las cepas que se vayan a
utilizar en sistemas alimentarios (fermentaciones, probiticos) habrn de estar libres
de marcas de resistencia, para lo que ser esencial disponer de puntos de corte fiables
para diferenciar a los microorganismos resistentes de los sensibles. El estudio de

136
__________________________________________
_____________________________________
__________________________________________
Discusin

resistencia a antibiticos en las BAL es tambin importante para conocer las clases y
tipos de resistencias ya diseminadas entre los distintos componentes del grupo.
Finalmente, estas bacterias podran constituir un buen modelo para esclarecer las
causas y factores que propician la diseminacin de resistencias entre microorganismos,
determinar los ecosistemas en los que tienen lugar las transferencias y precisar los
flujos de los determinantes.

METODOLOGA

Es importante que los mtodos de anlisis que se utilizan para determinar los
niveles de resistencia/susceptibilidad estn estandarizados y sean reconocidos
internacionalmente, de modo que los resultados de distintos ensayos y en distintos
laboratorios sean comparables. Para los microorganismos de inters clnico se dispone
de mtodos muy contrastados y puntos de corte bien definidos (CLSI, 2004), lo que no
sucede para las BAL y las bifidobacterias. En la actualidad, se utilizan diversos
mtodos como la difusin en disco, la dilucin en placa, el Etest y los sistemas de
microdilucin (Herrero et al., 1996; Charteris et al., 1998; Felten et al., 1999; Katla et al.,
2001; Danielsen y Wind 2003). A la diversidad de mtodos se une una multiplicidad de
medios de cultivo (M17, MRS, Mueller-Hinton, IsoSensitest, etc.), resultando en una
nueva fuente de confusin. Los distintos medios tienen pHs distintos, y
concentraciones diferentes de compuestos crticos (timina, cido flico, etc.) que, sin
duda, influyen en los niveles de susceptibilidad a determinados antibiticos (Huys et
al., 2002; Klare et al., 2004). El requerimiento de condiciones anaerbicas de algunos
tipos supone un grado de dificultad mayor para la realizacin de los ensayos (Roe et
al., 2002; CLSI, 2004). Otros factores que influyen en la variabilidad de los resultados
son la densidad del inculo, la temperatura y el tiempo de incubacin (Egervarn et al.,
2006). El efecto de la concentracin de inculo en la determinacin de la CIM a
tetraciclina de varias cepas de Lb. plantarum ha sido patente tambin en este trabajo;
cuando utilizbamos un inculo de 108 ufc/ml (lo que corresponde aproximadamente
a la dilucin 1 de McFarland) varias cepas se clasificaban como resistentes (CIM entre
96 y 256 g/ml). Sin embargo, la repeticin del ensayo con inculos menores (105
ufc/ml) resultaba en una CIM de 16 g/ml, considerndose las cepas sensibles.
Durante el transcurso de esta Tesis hemos participado en un estudio de
armonizacin interlaboratorios para escoger los mejores medios de cultivo y establecer

137
__________________________________________
Discusin _____________________________________
__________________________________________

procedimientos estandarizados de anlisis para el sistema Etest y el sistema de


microdilucin en placa microttulo. Basndose en estudios preliminares, se ha
precisado la concentracin de inculo, el medio de cultivo y las condiciones de
crecimiento para distintas especies. Los protocolos armonizados (Standardized
Operating Procedures) se relacionan como material complementario a la Tesis en los
Anexos 2 y 3.
Aunque se relaciona en ltimo lugar, no cabe duda que es indispensable una
correcta identificacin de los aislados que se someten a los ensayos, puesto que los
niveles de susceptibilidad/resistencia de las distintas especies es diferente y podran
cometerse errores graves en la interpretacin de los resultados. En este sentido, se ha
publicado varias veces la deteccin de cepas de Lc. lactis resistentes a vancomicina
(Green et al., 1990; Elliot y Facklam, 1996), que han resultado ser, tras una correcta
identificacin, especies de Enterococcus. Este gnero comparte muchas caractersticas
bioqumicas con el gnero Lactococcus, por lo que es comn la equivocacin, sobre todo
cuando los microorganismos se identifican por mtodos fenotpicos tradicionales
(Klein, 2003).

RESISTENCIAS INTRNSECAS

Como se ha venido repitiendo a lo largo de toda la Tesis, la resistencia a un


antibitico puede ser intrnseca o adquirida. La resistencia intrnseca es inherente a un
gnero o a una especie y no entraa riesgo de transferencia horizontal (European
Commission, 2005). El conocimiento de las resistencias intrnsecas de las especies es
importante para distinguirlas de las adquiridas; asociadas estas ltimas de forma
frecuente a la presencia de determinantes especficos con gran capacidad de
transferencia.
Uno de los ejemplos ms claros es la resistencia a vancomicina, antibitico al
que son resistentes de forma natural los leuconostocs, pediococos y muchas especies
de lactobacilos (Elisha y Courvalin, 1995). Esta propiedad puede ser til para su
aislamiento selectivo o para distinguirlos de otras bacterias Gram-positivas (Simpson
et al. 1988; Hamilton-Miller and Shah 1998). Tambin es comn un elevado nivel de
resistencia de las BAL a los aminoglicsidos (Lim et al., 1993; Charteris et al., 1998;
Katla et al., 2001; Danielsen y Wind, 2003), que se debe a un ineficiente transporte de
los antibiticos por carecer las bacterias de sistemas de transporte asociados a la

138
__________________________________________
_____________________________________
__________________________________________
Discusin

cadena de citocromos (Bryan y Kwan, 1981b). Los lactococos, por su parte, presentan
una resistencia intrnseca y diferenciada a la rifampicina que est bien documentada
(Orberg y Sandine, 1985; Elliot y Facklam, 1996; Teuber et al., 1999; Katla et al., 2001).
La rifampicina inhibe de manera especfica la subunidad de la ARN polimerasa
bacteriana que codifica el gen rpoB (Wehrli, 1983). Sin embargo, el enzima de
Lactococcus y Enterococcus no parece tener afinidad por este antibitico. Por ltimo, la
mayora de las BAL presentan resistencia intrnseca al metronidazol. Esta resistencia
podra deberse a la ausencia de actividad hidrogenasa (Church et al. 1996), ya que este
enzima es el encargado de reducir el antibitico dentro de la clula generando el
compuesto activo.
Es destacable tambin, el papel que pueden ejercer en las resistencias los
sistemas generales de exportacin, como los que codifican los transportadores MDR.
Estos sistemas estn siendo ampliamente estudiados en distintos tipos de BAL
(Bolhuis et al., 1995; van Veen et al., 1996; Perreten et al., 2001; Sakamoto et al., 2001;
Gajic et al., 2003; Lubelski et al., 2004) incluidas las bifidobacterias (Margolles et al.,
2005; Price et al., 2006). Y aunque segn este trabajo y el de otros autores, no parecen
aportar resistencias elevadas a ninguna clase de antibiticos, en determinadas
circunstancias o en determinadas cepas podran sobreexpresarse y llegar a conferir
niveles de resistencia propios de los tipos adquiridos. De hecho, pensamos que la
variabilidad en los niveles de susceptibilidad/resistencia que se da entre cepas puede
deberse, al menos en parte, a esta causa.

RESISTENCIAS ADQUIRIDAS

La resistencia adquirida es producto de la mutacin del material gentico de un


microorganismo o de la incorporacin de genes capaces de conferir la resistencia. En
todo caso, las cepas resistentes de una especie determinada se distinguen sobre un
fondo mayoritario de cepas sensibles. De esta forma, el estudio de la distribucin de
las resistencias a un antibitico en las poblaciones es de gran ayuda para identificar las
cepas resistentes. Sin embargo, existen distintos trminos para distinguir cepas
resistentes de sensibles dependiendo de la utilidad. As, tenemos el punto de corte
clnico, que hace referencia a los valores fisiolgicos que alcanzan los antibiticos en
el tratamiento; el punto de corte microbiolgico, que se introdujo para tratar de
identificar las resistencias codificadas por determinantes transferibles (Olsson-

139
__________________________________________
Discusin _____________________________________
__________________________________________

Liljequist et al., 1997); y el valor de distincin susceptible/resistente, ligado a la


estimacin del riesgo que supone la presencia de resistencias. Es esencial, sin embargo,
que los ensayos fenotpicos se complementen con mtodos moleculares para
identificar y caracterizar los determinantes de resistencia.
Trabajos previos de susceptibilidad en BAL indican que no es comn la
presencia de resistencias adquiridas en cepas de estas especies. Sin embargo, el
aislamiento de cepas resistentes parece estar aumentando (Perreten et al., 1997a; Raha
et al., 2002; Gevers et al., 2003). El aumento pudiera deberse tambin a la atencin que
se le presta en los ltimos aos. En todo caso, la posibilidad de encontrar cepas
resistentes en los alimentos justifica los ensayos de susceptibilidad/resistencia como
criterio de seleccin negativo para la caracterizacin de cepas que lleguen a formar
parte de cultivos iniciadores o de probiticos. A lo largo de la Tesis se han detectado
numerosas cepas resistentes a tetraciclina, eritromicina y/o clindamicina. En estas
cepas, primero se supuso la intervencin de determinantes de resistencia adquirida
por transferencia horizontal y, en algunos casos, despus se demostr. La resistencia
mltiple no es corriente en las BAL; como atestigua el hecho de que slo se ha descrito
en una cepa de Lc. lactis, resistente a tres antibiticos: tetraciclina, cloranfenicol y
estreptomicina (Perreten et al., 1997a). De las cepas analizadas en esta Tesis, tan solo
(Lb. johnsonii G41) present resistencias adquiridas a dos antibiticos de distinto tipo
(tetraciclina y eritromicina). Ambas resistencias resultaron estar codificadas por
determinantes adquiridos. Tampoco es frecuente la descripcin de mutaciones como
base gentica de las resistencias. En este trabajo detectamos la presencia de una cepa
de Lb. rhamnosus resistente a eritromicina y otros macrlidos, lincosamidas y
estreptogramina B, con una mutacin puntual en el gen que codifica el ARNr 23S; de
forma especfica, en la posicin 2058 (segn la numeracin de Escherichia coli). El
concurso de esta mutacin se ha descrito recientemente en varios tipos bacterianos,
como Mycoplasma spp. (Lucier et al., 1995), Mycobacterium spp. (Meier et al., 1994),
Helicobacter pylori (Versalovic et al., 1996), Propionibacterium spp. (Ross et al., 1997) o
Bordetella pertussis (Bartkus et al., 2003), entre otros. Esta es la primera vez, sin
embargo, que la mutacin se describe en un representante de las BAL. Dado que la
resistencia no tiene riesgo de transferencia (European Commission, 2005), y que la
cepa presenta caractersticas adecuadas de probiosis, pudiera utilizarse en este sentido
en tratamientos prolongados con antibiticos de estos grupos.

140
__________________________________________
_____________________________________
__________________________________________
Discusin

Los aislados intestinales de bifidobacterias y lactobacilos parecen presentar una


mayor incidencia y diversidad de resistencias atpicas (adquiridas) que los aislados de
BAL de los quesos tradicionales. En general estos resultados concuerdan con los que
refieren otros autores para cepas de estos dos ambientes (Orberg y Sandine, 1985; Lim
et al., 1993; Charteris et al., 1998 y b; Teuber et al., 1999; Katla et al., 2001; Danielsen y
Wind, 2003; Moubareck et al., 2005; Masco et al., 2006). Esto sugiere que existe una
presin selectiva diferencial en los ecosistemas. La presin antibitica en el tracto
gastrointestinal humano podra ser mayor como consecuencia de la administracin de
antibiticos por va oral. Los determinantes de resistencia ms estudiados son los de
tetraciclina, y solo de forma ocasional han aparecido tambin cepas resistentes a
eritromicina y a cloranfenicol. Estos antibiticos no son los ms utilizados en clnica
por lo que su supuesta presin selectiva podra tener otras causas.
Es destacable, finalmente, la extraa distribucin de las CIMs a estreptomicina
(y se puede ver de una forma ms clara en los captulos dedicados al ensayo de las
cepas de Lc. lactis y Lb. plantarum). Ya hemos apuntado que, en general, las BAL no son
sensibles a los aminoglicsidos debido a la ausencia de transporte, pero no
encontramos explicacin para la enorme variabilidad de CIM entre cepas. En algunas
especies (Lb. acidophilus, Lb. salivarius y P. acidilactici) se han detectado genes de
resistencia (Tenorio et al., 2001). En nuestro caso, no hemos identificado genes de esta
naturaleza ni siquiera en las cepas que se examinaron mediante hibridacin con
microarrays. Aunque pertenece a otro grupo de antibiticos, la distribucin de los
perfiles de resistencia a clindamicina en las cepas de Lb. plantarum se encuentra en una
situacin parecida: gran variabilidad entre cepas y ausencia de una clara distribucin
unimodal.

GENES DE RESISTENCIA

Los genes de resistencia a antibiticos se diseminan rpidamente en las


comunidades microbianas de manera vertical (ya se ha comentado que las cepas
patgenas con resistencias tienen un carcter clonal) y horizontal, mediante los
procesos de intercambio gnico. La transmisin se ve potenciada por la localizacin de
los determinantes en plsmidos conjugativos, transposones, elementos de insercin,
etc. Las BAL entran en contacto con nmeros elevados de microorganismos en la
matriz de los alimentos y en el tracto gastrointestinal, lo que posibilita el paso de

141
__________________________________________
Discusin _____________________________________
__________________________________________

informacin de unos tipos microbianos a otros. Los microorganismos con resistencias


pueden transferir los genes a las BAL, convirtindose en portadoras para transmitirlos
a continuacin a microorganismos patgenos o patgenos oportunistas (Teuber et al.,
1999; Salyer et al., 2004). Numerosos determinantes genticos que codifican resistencia
para uno o varios antibiticos han sido previamente caracterizados en especies de BAL
y bifidobacterias (Tannock et al., 1994; Perreten et al., 1997a; Schwarz et al., 2001;
Danielsen, 2002; Gfeller et al., 2003; Gevers et al., 2003; Scott et al., 2000; Masco et al.,
2006; Anexo 4).
En esta Tesis se han detectado dos cepas de Lc. lactis resistentes a tetraciclina
procedentes de una elaboracin artesanal de queso de Cabrales (Captulo I. Artculo I;
Flrez et al., 2005). El determinante gentico, que se describe por vez primera en esta
especie, fue en ambos casos el gen tet(M). El gen es completamente idntico al que se
encuentra asociado al transposn Tn916 de la cepa E. faecalis DS16 (Su et al., 1992;
Flannagan et al., 1994). Por lo que pudimos comprobar, el transposn parece estar
completo en estas cepas de Lc. lactis; de forma que el transposn se encuentra
totalmente activo. Adems, son capaces de transferir la resistencia a otras cepas de Lc.
lactis y E. faecalis. La presencia del transposn en cepas distintas y en plsmidos
diferentes sugiere procesos de transferencia independientes desde otro hospedador.
En las bifidobacterias, y en algunos lactobacilos, el gen responsable de la
resistencia a tetraciclina es tet(W) que codifica una protena de proteccin ribosomal.
Este gen parece abundantemente diseminado en especies Gram-positivas y Gram-
netativas del rumen y del intestino (Scott et al., 1997; Barbosa et al., 1999; Scott et al.,
2000). La gran homologa a nivel de nucletidos entre el gen que presentan distintas
especies de bifidobacterias (B. longum, B. bifidum y B. animalis) y la secuencia del
Butyrivibrio fibrisolvens (Barbosa et al., 1999), superando incluso la homologa con el
gen de otras bifidobacterias [B. longum F8 (Scott et al. 2000) y Bifidobacterium sp.
ISO3519 (AF202986)], sugiere (como en el caso de tet(M) en los lactococos), procesos de
transferencia independientes antes que un origen clonal. Sin embargo, puesto que no
se detecta la presencia del transposn que alberga tet(W) en B. fibrisolvens, poco
podemos decir del origen de la resistencia y del flujo gnico. Las bifidobacterias
podran ser, pues, receptoras o donadoras de esta resistencia. La caracterizacin
preliminar de las zonas adyacentes del gen en las distintas especies y cepas sugiere
una casustica diversa, aunque, en algunos casos, parece estar involucrado un
transposn que pudiera ser especfico de las bifidobacterias.

142
__________________________________________
_____________________________________
__________________________________________
Discusin

El gen de resistencia a eritromicina detectado en la cepa Lb. johnsonii G41


muestra homologa con el gen erm(B) presente en varias especies de Streptococcus,
Enterococcus y Lactobacillus, siendo ste el gen de resistencia a eritromicina ms
extendido entre las bacterias Gram-positivas (Weisblum, 1995; Jensen et al., 1999;
Roberts et al., 1999). La secuencia del gen erm(B) y las secuencias anteriores y
posteriores presentan una identidad total con la secuencia nucleotdica del plsmido
pRE25 de E. faecalis (Schwarz et al., 2001), dando la impresin de que un segmento de
aproximadamente 1,5 kpb que incluye el gen erm(B) se ha insertado en el cromosoma
del lactobacilo. Parece claro, que tanto en este caso como en el de tet(M), el origen del
gen se situara en los enterococos, desde donde ambos habran sido transferidos
horizontalmente a los lactococos y a esta cepa de Lb. johnsonii, respectivamente. La
participacin de enterococcus en estas transferencias no es casual puesto que estos
microorganismos estn presentes en nmeros elevados en muchas fermentaciones
alimentarias y son resisdentes habituales en el tracto gastrointestinal del hombre y los
animales (Franz et al., 1999; Giraffa, 2002). Ya se ha comentado, adems que las
especies del gnero Enterococcus estn cargadas de resistencias (Eaton y Gasson, 2001;
Foulqui-Moreno et al., 2006; Mathur y Singh, 2005). Estos resultados avalan la
hiptesis del reservorio, de tal manera que las cepas con resistencias de un hbitat
determinado actuarn de transmisores de genes de resistencia hacia las especies libres
de marcas que ocupan el mismo nicho ecolgico. Entre las resistencias transferidas por
los enterococcus destaca por sus implicaciones en clnica la resistencia a vancomicina
(DeLisle y Perl, 2003) que ha sido transferida recientemente desde los enterococos a la
especie patgena S. aureus (Pfeltz y Wilkinson, 2004).

Aunque el cuadro an no est completo, si parece claro que en los ltimos aos
se ha incrementado el nmero de cepas de BAL y bifidobacterias resistentes a
antibiticos. Parte de este aumento puede deberse a un mayor inters por el estudio de
las resistencias en este grupo de microorganismos comensales, pero el hecho de que
los determinantes genticos responsables de las resistencias sean iguales a los que se
han descrito con anterioridad en otras bacterias sugiere que las BAL estn recibiendo
genes de resistencia de otros tipos microbianos. La evaluacin continuada del nivel y
los tipos de resistencias en este grupo, utilizando tcnicas y procedimientos
estandarizados, podra ser de gran ayuda para desentraar los mecanismos por los
que los genes de resistencia a antibiticos se extienden en las comunidades

143
__________________________________________
Discusin _____________________________________
__________________________________________

microbianas y la forma en que los biotipos resistentes se seleccionan o se fijan en los


distintos ecosistemas. Todo ello, como ya hemos indicado, sera de gran ayuda para
una utilizacin ms racional de los antibiticos y el desarrollo de estrategias que
minimicen, en lo posible, la diseminacin de los microorganismos resistentes y los
determinantes de resistencia, de forma que los antibiticos mantengan la eficacia y los
beneficios que han a la salud humana en los ltimos 65 aos.

144
CONCLUSIONES
___________________________________________
____________________________________
_______________________________________
Conclusiones

V.- CONCLUSIONES

1. El ensayo por medio del sistema de microdilucin comercial Sensititre Anaero3


mostr que las BAL procedentes de quesos tradicionales presentaron pocas
resistencias a los antibiticos ensayados; de stas, algunas se suponen resistencias
intrnsecas (como la resistencia a cefoxitina, o la resistencia a vancomicina de
lactobacilos y leuconostoc). Ahora bien, dos cepas de Lc. lactis subsp. lactis resultaron
sospechosas de portar resistencia adquirida a tetraciclina; tal y como demostramos
posteriormente.

2. Con el mismo mtodo, la gran mayora de los aislados intestinales de


lactobacilos y bifidobacterias se mostraron sensibles a los antibiticos analizados. El
rango de CIM para los antibiticos cefoxitina, metronidazol y moxifloxacino sugiere
una resistencia inespecfica que, sin embargo, vara notablemente entre especies y
cepas. Por el contrario, las CIMs a tetraciclina, eritromicina y clindamicina en unas
pocas cepas sugirieron una resistencia adquirida. Algunos de los determinantes
responsables se caracterizaron con posterioridad en este trabajo.

3. La presencia de cepas de BAL y bifidobacterias multirresistentes no parece ser


habitual, y tan slo una cepa de Lb. johnsonii se mostr resistente a tres antibiticos
(tetraciclina, eritromicina y clindamicina). Tal y como analizamos a continuacin, los
responsables de estas resistencias fueron el gen erm(B) y un gen mosaico
[tet(W/O/32)].

4. Mediante el ensayo de 93 cepas de Lc. lactis y 121 de Lb. plantarum por el


mtodo Etest se proponen los siguientes nuevos puntos de susceptibilidad/resistencia
a algunos antibiticos. Tetraciclina (2 g/ml), estreptomicina (64 g/ml), cloranfenicol
(12 g/ml) y vancomicina (6 g/ml), para la especie Lc. lactis, y clindamicina (16
g/ml ) y tetraciclina (64 g/ml ) para la especie Lb. plantarum. Estos puntos de corte
harn ms sencilla la separacin de las cepas sensibles de las resistentes y la
identificacin, en un siguiente paso, de los determinantes genticos responsables.

5. Dos cepas de Lc. lactis aisladas de una elaboracin artesanal de queso de


Cabrales portan el gen tet(M) que confiere resistencia a tetraciclina. Aparentemente, el
gen se encuentra localizado en una copia del transposn Tn916 inserto, a su vez, en
plsmidos de las cepas resistentes. La resistencia que presentan estas cepas pudo ser

145
___________________________________________
Conclusiones ____________________________________
_______________________________________

transferida mediante conjugacin a otras cepas de Lc. lactis y E. faecalis, demostrando


su capacidad de transferencia. Sin embargo, no se detect transferencia a una cepa de
Lb. plantarum.

6. Cepas dominantes de varias especies de bifidobacterias del tracto


gastrointestinal humano presentaron el gen tet(W) como responsable de la resistencia a
tetraciclina. El gen mostr una identidad nucleotdica absoluta entre los distintos
aislados, sin embargo se encontraba ubicado en diferentes posiciones del cromosoma
bacteriano en las distintas especies y cepas, lo que sugiere una diseminacin reciente
desde otro hospedador. Los genes tet(W) analizados en este trabajo son ms parecidos
a los genes de otros microorganismos intestinales que los presentes en otras especies
de bifidobacterias de otras comunidades humanas, lo que parece indicar que las
resistencias no son de origen clonal y sugiere procesos de transferencia
independientes.

7. Las resistencias a eritromicina y clindamicina descritas en la cepa G41 de Lb.


johnsonii estn codificadas por el determinante gentico erm(B). Este gen es idntico al
gen erm(B) caracterizado en distintas cepas de Streptococcus, Enterococcus y
Lactobacillus. El hecho de que las regiones adyacentes al gen tengan secuencias
nucleotdicas idnticas a las del plsmido pRE25 de E. faecium indica la insercin total
o parcial del plsmido en el cromosoma de Lb. johnsonii y una resolucin posterior, por
algn mecanismo desconocido. En los procesos de insercin o resolucin parecen
haberse generado secuencias nuevas que no estn presentes en pRE25 y si en el
cromosoma de Lb. jonhsonii.

8. La resistencia a macrlidos presente en la cepa E41 de Lb. rhamnosus se debe a


una mutacin cromosmica en la posicin 2058 del ARN ribosmico 23S (siguiendo la
numeracin de E. coli). La sustitucin de una adenina por una guanina (transicin)
parece ser responsable de la resistencia a elevadas concentraciones de eritromicina,
clindamicina y otros macrlidos, dado que la cepa no posee otros genes de resistencias
conocidos. Las potencialidades probiticas de la cepa, incluyendo su utilizacin para
mantener el equilibrio microbiano intestinal durante el tratamiento con este tipo de
antibiticos, podran ser aprovechadas puesto que el riesgo de transferencia de la
mutacin es muy reducido.

146
___________________________________________
____________________________________
_______________________________________
Conclusiones

9. El transportador MDR de B. breve estudiado en este trabajo no parece conferir


niveles elevados de resistencia a los grupos de antibiticos ensayados. Sin embargo, se
postula que una actividad diferencial del transportador en distintas cepas podra
inducir un distinto nivel de susceptibilidad/resistencia a los antibiticos.

10. Los genes que codifican para los transportadores MDR de Lc. lactis LmrA, LmrP
y LmrCD son muy ubicuos en esta especie. LmrCD parece tener una funcin fisiolgica
ms importante que LmrA y LmrP puesto que el gen se conserva intacto en todas las
cepas. Aunque no se ha examinado en este trabajo, el papel de estos transportadores
en la resistencia a antibiticos pudiera ser similar al de otros MDR analizados.

147
BIBLIOGRAFA
_______________________________________
___________________________________
_________________________________
Bibliografa

VI.- BIBLIOGRAFA

Aarestrup FM, Seyfarth AM, Emborg HD, Baquero F. 1997. Gram-positive resistance:
Pedersen K, Hendriksen RS, y Bager F. challenge for the development of new
2001. Effect of abolishment of the use of antibiotics. J. Antimicrob. Chemoth. 39
antimicrobial agents for growth Suppl. A: 16.
promotion on occurrence of Barbosa TM, y Levy SB. 2000. The impact of
antimicrobial resistance in fecal antibiotic use on resistance development
enterococci from food animals in and persistence. Drug Resist. Update. 3:
Denmark. Antimicrob. Agents Ch. 45: 303-311.
2054-2059. Barbosa TM, Scott KP, y Flint HJ. 1999.
Ahn C, Collins-Thompson D, Duncan C, y Evidence for recent intergeneric transfer
Stiles ME. 1992. Mobilization and of a new tetracycline resistance gene,
location of the genetic determinant of tet(W), isolated from Butyrivibrio
chloramphenicol resistance from fibrisolvens, and the occurrence of tet(O)
Lactobacillus plantarum caTC2R. Plasmid. in ruminal bacteria. Environ. Microbiol. 1:
27:169-76. 53-64.
Alanis AJ. 2005. Resistance to antibiotics: Are Bartkus JM, Juni BA, Ehresmann K, Miller
we in the post-antibiotic era?. Arch. Med. CA, Sanden GN, Cassiday PK, Saubolle
Res. 36: 697-705. M, Lee B, Long J, Harrison AR, y Besser
Andersson DI. 2003. Persistence of antibiotic JM. 2003. Identification of a mutation
resistant bacteria. Curr. Opin. Microbiol. associated with erythromycin resistance
6: 452-456. in Bordetella pertussis: Implications for
Andersson DA, y Levin BR. 1999. The surveillance of antimicrobial resistance. J.
biological cost of antibiotic resistance. Clin. Microbiol. 41: 1167-1172.
Curr. Opin. Microbiol. 2: 487-491. Bernardeau M, Guguen M, y Vernoux JP.
Asahara T, Takahashi M, Nomoto K, 2006. Beneficial lactobacilli in food and
Takayama H, Onoue M, Morotomi M, feed: long-term use, biodiversity and
Tanaka R, Yokokura T, y Yamashita N. proposals for specific and realistic safety
2003. Assessment of safety of assessments. FEMS Microbiol. Rev. 30:
Lactobacillus strains based on resistance 487-513.
to host innate defense mechanisms. Clin. Berruti G, Tosi L, y Morelli L. 2005.
Diagn. Lab. Immunol. 10:169-173. Phenotypic and molecular
Aslim B, y Beyatli Y. 2004. Antibiotic characterization of erythromycine-
resistance and plasmid DNA contents of resistant strains of Streptococcus
Streptococcus thermophilus strains isolated thermophilus. 8th Symposium on Lactic
from Turkish yogurts. Turk. J. Vet. Anim. Acid Bacteria: Genetics, Metabolism, and
Sci. 28: 257-263. Applications. Egmond aan Zee, The
Axelsson LT, Ahrne SE, Andersson MC, y Netherlands.
Stahl SR. 1988. Identification and Bolhuis H, Poelarends G, van Veen HW,
cloning of a plasmid-encoded Poolman B, Driessen AJ, y Konings WN.
erythromycin resistance determinant 1995. The lactococcal lmrP gene encodes a
from Lactobacillus reuteri. Plasmid. 20: proton motive force-dependent drug
171-174. transporter. J. Biol. Chem. 270: 26092-
Balsalobre L, Ferrandiz MJ, Linares J, 26098.
Tubau F, y de la Campa AG. 2003. Bolotin A, Wincker P, Mauger S, Jaillon O,
Viridans group streptococci are donors Malarme K, Weissenbach J, Ehrlich SD,
in horizontal transfer of topoisomerase y Sorokin A. 2001. The complete genome
IV genes to Streptococcus pneumoniae. sequence of the lactic acid bacterium
Antimicrob. Agents Ch. 47: 2072-2081. Lactococcus lactis ssp. lactis IL1403.
Baquero F, Baquero-Artigao G, Cantn R, y Genome Res. 11: 731-53.
Garca-Rey C. 2002. Antibiotic BolotinA, Quinquis B, Renault P, Sorokin A,
consumption and resistance selection in Ehrlich SD, Kulakauskas S, Lapidus A,
Streptococcus pneumoniae. J. Antimicrob. Goltsman E, Mazur M, Pusch GD,
Chemother. 50: 27-37. Fonstein M, Overbeek R, Kyprides N,
Purnelle B, Prozzi D, Ngui K, Mazuy D,

149
_______________________________________
Bibliografa ___________________________________
_________________________________

Hancy F, Burteau S, Boutry M, Delcour of potentially probiotic Lactobacillus


J, Goffeau A, y Hols P. 2004. Complete species. J. Food Protect. 61: 1636-43.
sequence and comparative genome Chopra I, y Roberts M. 2001. Tetracycline
analysis of the dairy bacterium antibiotics: mode of action, applications,
Streptococcus thermophilus. Nat. molecular biology, and epidemiology of
Biotechnol. 22: 1523-1524. bacterial resistance. Microbiol. Mol. Biol.
Borgen K, Wasteson Y, Kruse H, y Willems R. 65: 232-260.
RJL. 2002. Vancomycin-resistant Church DL, Bryant RD, Sim V, y Laishley
Enterococcus faecium (VREF) from EJ. 1996. Metronidazole susceptibility
Norwegian poultry cluster with VREF and the presence of hydrogenase in
from poultry from the United Kingdom pathogenic bacteria. Anaerobe. 2: 147-
and the Netherlands in an amplified 153.
fragment length polymorphism Clewell DB, Yagi Y, Dunny GM, y Schultz
genogroup. Appl. Environ. Microb. 68: SK. 1974. Characterization of three
3133-3137. plasmids deoxyribonucleic acid
Bronzwaer SL, Cars O, Buchholz U, molecules in a strain of Streptococcus
Molstad S, Goettsch W, Veldhuijzen IK, faecalis: identification of a plasmid
Kool JL, Sprenger MJ, y Degener JE. determining erythromycin resistance. J.
2002. A European study on the Bacteriol. 117: 283-289.
relationship between antimicrobial use CLSI. Clinical and Laboratory Standards
and antimicrobial resistance. Emerg. Institute. 2004. Methods for
Infect. Dis. 8: 278-82. antimicrobial susceptibility testing of
Bryan LE, y Kwan S. 1981a. Mechanisms of anaerobic bacteria: Approved Standard-
aminoglycoside resistance of anaerobic sixth edition. M11-A6, paikka, USA.
bacteria and facultative bacteria grown Cohen ML, Wong ES, y Falkow S. 1982.
anaerobically. J. Antimicrob. Chemother Common R-plasmids in Staphylococcus
8 Suppl NaN: 18 aureus and Staphylococcus epidermidis
Bryan LE, y Kwan S. 1981b. during a nosocomial Staphylococcus
Aminoglycoside-Resistant mutants of aureus outbreak. Antimicrob. Agents Ch
Pseudomonas aeruginosa deficient in 21: 210-215.
cytochrome-D, nitrite reductase, and Cohen ML. 2000. Changing patterns of
aerobic transport. Antimicrob. Agents Ch infectious disease. Nature. 406: 762-767.
19: 958-964. Condon S. 1983. Aerobic metabolism of lactic
Bussiere DE, Muchmore SW, Dealwis CG, acid bacteria. Irish J. Food Sci. Technol. 7:
Schluckebier G, Nienaber VL, Edalji 15-25.
RP, Walter KA, Ladror US, Holzman TF, Coppola R, Succi M, Tremonte P, Reale A,
y Abad-Zapatero C. 1998. Crystal Salzano G, y Sorrentino E. 2005.
structure of ermC, an rRNA Antibiotic susceptibility of Lactobacillus
methyltransferase which mediates rhamnosus strains isolated from
antibiotic resistance in bacteria. Parmigiano Reggiano cheese. Lait. 85:
Biochemistry-US. 37: 71037112. 193-204.
Cannon JP, Lee TA, Bolanos JT, y Danziger Cosgrove SE, y Carmeli Y. 2003. The impact
LH. 2005. Pathogenic relevance of of antimicrobial resistance on health and
Lactobacillus: a retrospective review of economic outcomes. Clin. Infect. Dis. 36:
over 200 cases. Eur. J. Clin. Microbiol. 14331437.
Dis. 24: 31-40. Danielsen M, y Wind A. 2003. Susceptibility
Carr FJ, Chill D, y Maida N. 2002. The lactic of Lactobacillus spp. to antimicrobial
acid bacteria: a literature survey. Crit. agents. Int. J. Food Microbiol. 82: 1-11.
Rev. Microbiol. 28: 281-370. Danielsen M. 2002. Characterization of the
Cataloluk O, y Gogebakan B. 2004. Presence tetracycline resistance plasmid pMD5057
of drug resistance in intestinal from Lactobacillus plantarum 5057 reveals
lactobacilli of dairy and human origin in a composite structure. Plasmid. 48: 98-
Turkey. FEMS Microbiol. Lett. 236: 7-12. 103.
Charteris WP, Kelly PM, Morelli L, y Danielsen M, Simpson PJ, O'Connor EB,
Collins JK. 1998. Antibiotic susceptibility Ross RP, y Stanton C. 2006.
Susceptibility of Pediococcus spp. to

150
_______________________________________
___________________________________
_________________________________
Bibliografa

antimicrobial agents. J. Appl. lactic acid bacteria. Antimicrob. Agents


Microbiol.(On line). Ch. (On line).
Davidson BE, Kordias N, Dobos M, y Elisha BG, y Courvalin P. 1995. Analysis of
Hillier AJ. 1996. Genomic organization genes encoding D-alanine:D-alanine
of lactic acid bacteria, p. 65-87. In G ligase related enzymes in Leuconostoc
Venema, Huis int Veld JHJ, Hugenholtz mesenteroides and Lactobacilli spp.
J (ed.), Lactic Acid Bacteria: Genetics, Gene. 152: 79-83.
Metabolism and Applications. Kluwer Elliot JA, y Facklam RR. 1996.
Academic Publishers, Dordrecht, The Antimicrobial susceptibilities of
Netherlands. Lactococcus lactis and Lactococcus garviae
Davies J. 1994. Inactivation of antibiotics and and a proposed method to discriminate
the dissemination of resistance genes. between them. J. Clin. Microbiol. 34:
Science. 264: 375-382. 1296-1298.
De Graaf J, Elwell LP, y Falkow S. 1976. Elwell L, Roberts M, Mayer L, y Falkow S.
Molecular nature of two -lactamases- 1977. Plasmid-mediated -lactamase
specifying plasmids isolated from production in Neisseria gonorrhoeae.
Haemophilus influenzae type b. J. Bacteriol. Antimicrob. Agents Ch 11: 528-533.
126: 439-446. European Commission. 2005. Opinion of
DeFlaun MF, y Levy SB. 1989. Genes and the scientific panel on additives and
their varied hosts. En, Gene Transfer in products in substances used in animal
the Environment. S. B. Levy y R. V. feed on the updating of the criteria used
Miller (eds.). pp. 1-32. McGraw-Hill, in the assessment of bacteria for
New York, USA. resistance to antibiotics of human and
DeLisle S, y Perl TM. 2003. Vancomycin- veterinary importance. The EFSA J. 223:
resistant enterococci: a road map on how 1-12.
to prevent the emergence and Evans RP, y Macrina FL. 1983.
transmission of antimicrobial resistance. Streptococcal R plasmid pIP501:
Chest. 123: 50418. endonuclease site map, resistance
Dellaglio F, de Roissart H, Torriani S, determinant location, and construction
Curk MC, y Janssens D. 1994. of novel derivatives. J. Bacteriol. 154:
Caractristiques gnrales des bactries 1347-1355.
lactiques, In Bactries Lactiques, vol. I, Falker S, Schmidt MA, y Heusipp G. 2005.
pp. 25116. Edited by H. de Roissart & DNA methylation in Yersinia
F. M. Luquet. Uriage, France: Lorica. enterocolitica: role of the DNA adenine
Donoghue DJ. 2003. Antibiotic residues in methyltransferase in mismatch repair
poultry tissues and eggs: human health and regulation of virulence factors.
concerns? Poultry Sci. 82: 618-621. Microbiology. 151: 2291-2299.
Dougherty BA, Hill C, Weidman JF, Felten A, Barreau C, Bizet C, Lagrange PH,
Richardson DR, Venter JC, y Ross RP. y Philippon A. 1999. Lactobacillus
1998. Sequence and analysis of the 60 kb species identification, H2O2
conjugative, bacteriocin-producing production, and antibiotic resistance
plasmid pMRC01 from Lactococcus lactis and correlation with human clinical
DPC3147. Mol. Microbiol. 29:1029-1038. status. J. Clin. Microbiol. 37: 729-733.
Eaton TJ, y Gasson MJ. 2001. Molecular Fitzgerald GF, y Gasson MJ. 1988. In vivo
screening of Enterococcus virulence gene transfer systems and transposons.
determinants and potential for genetic Biochimie. 70: 489-502.
exchange between food and medical Flannagan SE, Zitzow LA, Su YA, y
isolates. Appl. Environ. Microb. 67: Clewell DB. 1994. Nucleotide-Sequence
1628-1635. of the 18-Kb Conjugative Transposon
Edwards D. 1980. Antimicrobiol Drug Tn916 from Enterococcus faecalis.
Action, MacMillion Pvt. Co., London, Plasmid. 32: 350-354.
pp 193-216. Flrez AB, Delgado S, y Mayo B. 2005.
Egervarn M, Lindmark H, Roos S, Huys G, Antimicrobial susceptibility of lactic
y Lindgren S. 2006. Effects of inoculum acid bacteria isolated from a cheese
size and incubation time on broth environment. Can. J. Microbiol. 51: 51-8.
microdilution susceptibility testing of

151
_______________________________________
Bibliografa ___________________________________
_________________________________

Florez AB, Ammor MS, Delgado S, y Mayo Gevers D. 2002. Tetracycline resistance in
B. 2006a. Molecular analysis of a lactic acid bacteriaisolated from
chromosome-carried erm(B) gene and fermented dry sausages. Tesis Doctoral.
its flanking insertion points in Ghent University, Belgium.
Lactobacillus johnsonii G41. Antimicrob. Gevers D, Danielsen M, Huys G, y Swings
Agents Chemother. 50: 4189-4190. J. 2003. Molecular Characterization of
Flrez AB, Ammor MS, lvarez-Martn, tet(M) Genes in Lactobacillus Isolates
Margolles A, y Mayo B. 2006b. from Different Types of Fermented Dry
Molecular analysis of tet(W) gene- Sausage. Appl. Environ. Microb. 69:
mediated tetracycline resistance in 1270-1275.
dominant intestinal Bifidobacterium Gfeller KY, Roth M, Meile L, y Teuber M.
species from healthy humans. Appl. 2003 Sequence and genetic organization
Environ. Microb. 72: 7377-7379. of the 19.3-kb erythromycin- and
Fons M, Hege T, Ladire M, Raibaud P, dalfopristin-resistance plasmid
Ducluzeau R, y Maguin E. 1997. pLME300 from Lactobacillus fermentum
Isolation and characterization of a ROT1. Plasmid. 50: 190-201.
plasmid from Lactobacillus fermentum Giraffa G. 2002. Enterococci from foods.
conferring erythromycin resistance. FEMS Microbiol. Rev. 26:163-171.
Plasmid. 37: 199-203. Goosens H. 1999. The epidemiology of
Food and Drug Administration (FDA). vancomycin-resistant enterococci. Curr.
2000. FDA approves zyvox, the first Opin. Infect. Dis. 12: 537-541.
antimicrobial drug in a new class. Granizo JJ, Aguilar L, Casal J, Dal-Re R, y
http://www.fda.gov/bbs/topics/ANS Baquero F. 2000. Streptococcus pyogenes
WERS/ANS01010.html. resistance to erythromycin in relation to
Foulquie-Moreno MR, Sarantinopoulos P, macrolide consumption in Spain (1986-
Tsakalidou E, y De Vuyst L. 2006. The 1997). J. Antimicrob. Chemoth. 46: 959-
role and application of enterococci in 964.
food and health. Int. J. Food Microbiol. Green M, Wadowsky RM, y Barbadora K.
106: 1-24. 1990. Recovery of Vancomycin-
Franz CMAP, Holzappel WH, y Stiles ME. Resistant Gram-Positive Cocci from
1999. Enterococci at the crossroad of Children. J. Clin.Microbiol. 28: 484-488.
food safety. Int. J. Food Microbiol. 47: 1 Guarner F, y Malagelada JR. 2003. Gut
24. flora in health and disease. Lancet. 361:
Gajic O, Buist G, Kojic M, Topisirovic L, 512-519.
Kuipers OP, y Kok J. 2003. Novel Hall RM, Collis CM, Kim MJ, Partridge
mechanism of bacteriocin secretion and SR, Recchia GD, y Stokes HW. 1999.
immunity carried out by lactococcal Mobile gene cassettes and integrons in
multidrug resistance proteins. J. Biol. evolution. Ann. NY. Acad. Sci. 870: 68-
Chem. 278: 34291-34298. 80.
Garau J. 2002. Treatment of drug-resistant Hamilton-Miller JMT, y Shah S. 1998.
pneumococcal pneumonia. Lancet Vancomycin susceptibility as an aid to
Infect. Dis. 2: 404-15. the identification of lactobacilli. Lett.
Gasser F. 1994. Safety of lactic acid bacteria Appl.Microbiol. 26: 153-154.
and their occurrence in human clinical Hawkey PM. 2000. Mechanisms of
infections. Bull. Inst. Pasteur. 92: 45-67. resistance to antibiotics. Intens. Care
Gasson MJ, y Fitzgerald GF. 1994. Gene Med. 26 Suppl 1:S9-13.
transfer systems and transposition. In, Helmark S, Hansen ME, Jelle B, Sorensen
Genetics and Biotechnology of Lactic KI, y Jensen PR. 2004. Transformation
Acid Bacteria. M.J. Gasson and W.M. de of Leuconostoc carnosum 4010 and
Vos (eds.). pp. 22-46. Blackie Academic evidence for natural competence of the
& Professional, London. organism. Appl. Environ. Microb. 70:
Gasson MJ, Godon JJ, Pillidge CJ, Eaton 3695-3699.
TJ, Jury K, y Shearman CA. 1995. Herrero M, Mayo B, Gonzlez B, y Surez
Characterization and exploitation of JE. 1996. Evaluation of technologically
conjugation in Lactococcus lactis. Int. important traits in lactic acid bacteria
Dairy J. 5: 757. isolated from spontaneous

152
_______________________________________
___________________________________
_________________________________
Bibliografa

fermentations. J. Appl. Microbiol. 81: bifidobacteria. Int. J. Med. Microbiol.


565-570. 294: 215.
Herreros MA, Sandoval H, Gonzalez L, Klein G. 2003. Taxonomy, ecology and
Castro JM, Fresno JM, y Tornadijo ME. antibiotic resistance of enterococci from
2005. Antimicrobial activity and food and the gastro-intestinal tract. Int.
antibiotic resistance of lactic acid J. Food Microbiol. 88: 123-131.
bacteria isolated from Armada cheese (a Lacroix JM, y Walker CB. 1995. Detection
Spanish goats' milk cheese). Food and incidence of the tetracycline
Microbiol. 22: 455-459. resistance determinant tet(M) in the
Huys G, DHaene K, y Swings J. 2002. microflora associated with adult
Influence of the culture medium on periodontitis. J. Periodontol. 66: 102-
antibiotic susceptibility testing of food- 108.
associated lactic acid bacteria with the Langella P, y Chopin A. 1989. Conjugal
agar overlay disc diffusion method. transfer of plasmid pIP501 from
Lett. Appl. Microbiol. 34: 402-6. Lactococcus lactis to Lactobacillus
Huys G, DHaene K, Collard JM, y Swings delbruckii subsp. bulgaricus and
J. 2004 Prevalence and molecular Lactobacillus helveticus. FEMS Microbiol.
characterization of tetracycline Lett. 51:149-152.
resistance in Enterococcus isolates from Levy SB, y Marshall B. 2004. Antibacterial
food. Appl. Environ. Microb. 70: 1555- resistance world wide: causes,
1562. challenges and responses. Nat. Med. 10:
Jacoby GA, y Archer GL. 1991. New 122-129.
mechanisms of bacterial resistance to Levy SB. 1985. Ecology of plasmids and
antimicrobial agents. New Engl. J. Med. unique DNA sequences. En, Engineered
324: 601-612. Organisms in the Environment:
Jensen LB, Frimodt-Moller N, y Aarestrup Scientific Issues. H. O. Haalvorson, D.
FM. 1999. Presence of erm gene classes Pramer y M. Rogul (eds.). ASM Press,
in Gram-positive bacteria of animal and Washington DC.
human origin in Denmark. FEMS Levy SB. 1997. Antibiotic resistance: an
Microbiol. Lett. 170: 151-158. ecological imbalance. In: Antibiotic
Katla AK, Kruse H, Johnsen G, y Resistance: Origins, Evolution, Selection
Herikstad H. 2001. Antimicrobial and Spread. Ciba Foundation
susceptibility of starter culture bacteria Symposium. 207 Suppl. 1-14, Wiley,
used in Norwegian dairy products. Int. Chichester.
J. Food Microbiol. 67: 147-152. Lim KS, Huh CS, y Baek YJ. 1993.
Kastner S, Perreten V, Bleuler H, Antimicrobial susceptibility of
Hugenschmidt G, Lacroix C, y Meile L. bifidobacteria. J. Dairy Sci. 76: 2168-74.
2006. Antibiotic susceptibility patterns Lin CF, Fung CF, Wu CL, y Chung TC.
and resistance genes of starter cultures 1996. Molecular characterization of a
and probiotic bacteria used in food. plasmid-borne (pTC82)
Syst. Appl. Microbiol. 29: 145-155. chloramphenicol resistance determinant
Klander O, y Weiss N. 1986. Regular, (cat-Tc) from Lactobacillus reuteri G4.
Nonsporing Gram-Positive Rods In: Plasmid 36: 116-124.
Sneath, P.H.A.; Mair, N.S.; Sharpe, Lin CF, y Chung TC. 1999. Cloning of
M.E.; Holt, J.G. (eds). Bergey's Manual erythromycin-resistance determinants
of Systematic Bacteriology. Williams and replication origins from indigenous
and Wilkins, Baltimore. plasmids of Lactobacillus reuteri for
Klare I, Konstabel C, Reissbrodt R, potential use in construction of cloning
Muller-Bertling S, Huys G, vectors. Plasmid. 42: 31-41.
Vancanneyt M, Swings J, Goossens H, Ljungh A, y Wadstrom T. 2006. Lactic acid
y Witte W. 2004. Standardized bacteria as probiotics. Curr. Issues
microdilution test with the newly Intest. Microbiol. 7: 73-89.
developed LSM broth for antibiotic Lubelski J, Mazurkiewicz P, van Merkerk
susceptibility testing of lactobacilli, R, Konings WN, y Driessen AJ. 2004.
lactococci, pediococci, and ydaG and ydbA of Lactococcus lactis
encode a heterodimeric ATP-binding

153
_______________________________________
Bibliografa ___________________________________
_________________________________

cassette-type multidrug transporter. J. intracellulare. Antimicrob. Agents Ch.


Biol. Chem. 279: 34449-34455. 38: 381384.
Lucier TS, Heitzman K, Liu SK, y Hu PC. Mercenier A, Pouwels PH, y Chassy BM.
1995. Transition mutations in the 23S 1993. Genetic engineering of lactobacilli,
rRNA of erythromycin resistant isolates leuconostocs and Streptococcus
of Mycoplasma pneumoniae. Antimicrob. thermophilus. In MJ Gasson, de Vos WM
Agents Ch. 39: 27702773. (ed.), Genetics and Biotechnology of
Madigan MT, Martinko JM, y Parker J. Lactic Acid Bacteria. Blackie Academic
Brock. 2000. Biologa de los & Professional, Glasgow, United
microorganismos. 8 edicin. Prentice Kingdom.
Hall. Madrid. Miller JH. 1996. Spontaneous mutators in
Mandell, Douglas y Bennet. 1997. bacteria: insights into pathways of
"Enfermedades Infecciosas, principios y mutagenesis and repair. Annu. Rev.
prctica " 4 Edicin., Editorial Microbiol. 50: 625-43.
Panamericana. Moellering RC. 2003. Linezolid: The First
Margolles A, Moreno JA, van Sinceren D, Oxazolidinone Antimicrobial. Ann.
y Reyes-Gavilan CGD. 2005. Macrolide Intern. Med. 138: 135-142.
resistance mediated by a Bifidobacterium Moubareck C, Gavini F, Vaugien L, Butel
breve membrane protein. Antimicrob. MJ, y Doucet-Populaire F. 2005.
Agents Ch. 49: 4379-4381. Antimicrobial susceptibility of
Martel A, Meulenaere V, Devriese LA, bifidobacteria. J. Antimicrob.
Decostere A, y Haesebrouck F. 2003. Chemother. 55: 38-44.
Macrolide and lincosamide resistance in Nandi S, Maurer JJ, Hofacre C, y Summers
the gram-positive nasal and tonsillar AO. 2004. Gram-positive bacteria are a
flora of pigs. Microb. Drug Resist. 9: major reservoir of Class 1 antibiotic
293-297. resistance integrons in poultry litter. P.
Masco L, Van Hoorde K, De Brandt E, Natl Acad. Sci. USA. 101: 7118-22.
Swings J, y Huys G. 2006. Normark BH, y Normark S. 2002. Evolution
Antimicrobial susceptibility of and spread of antibiotic resistance. J.
Bifidobacterium strains from humans, Intern. Med. 252: 91-106.
animals and probiotic products. J. Olsson-Liljequist B, Larsson P, Walder M,
Antimicrob. Chemother. 58: 85-94. y Miorner H. 1997. Antimicrobial
Mathur S, y Singh R. 2005. Antibiotic susceptibility testing in Sweden. III.
resistance in food lactic acid bacteria a- Methodology for susceptibility testing.
review. Int. J. Food Microbiol. 105: 281- Scand. J. Infect. Dis. 105: 13-23.
95. Orberg PK, y Sandine WE. 1985. Survey of
Matteuzzi D, Crociani F, y Brigidi P. 1983. antimicrobial resistance in lactic
Antimicrobial susceptibility of streptococci. Appl. Environ. Microb. 49:
Bifidobacterium. Ann. Inst. Past. 538542.
Microbiol. 134A: 339-349. Ouwehand AC, Salminen S, y Isolauri E.
Mayo B, Hardisson C, y Braa AF. 1990. 2002. Probiotics: an overview of
Characterization of wild strains of beneficial effects. Anton. Leeuw. Int. J.
Lactococcus lactis isolated from Cabrales G. 82: 279-89.
cheese. J. Dairy Res. 57: 125-134. Perreten V, Schwarz F, Cresta L, Boeglin
Mazel D, y Davies J. 1999. Antibiotic M, Dasen G, y Teuber M. 1997a.
resistance in microbes. Cell. Mol. Life Antibiotic resistance spread in food.
Sci. 56: 742-754. Nature. 389: 801-802.
McManus PS, Stockwell VO, Sundin GW, Perreten V, Kolloffel B, y Teuber M. 1997b.
y Jones AL. 2002. Antibiotic use in plant Conjugal transfer of the Tn916-like
agriculture. Ann. Rev. Phytopath. 40: transposon TnFO1 from Enterococcus
443465. faecalis isolated from cheese to other
Meier A, Kirschner P, Springer B, Gram-positive bacteria. Syst. Appl.
Steingrube VA, Brown BA, Wallace Microbiol. 20: 27-38.
RJ, y Bttger EC. 1994. Identification of Perreten V, Schwarz FV, Teuber M, y Levy
mutations in 23S rRNA gene of SB. 2001. Mdt(A), a new efflux protein
clarithromycin-resistant Mycobacterium conferring multiple antibiotic resistance

154
_______________________________________
___________________________________
_________________________________
Bibliografa

in Lactococcus lactis and Escherichia coli. Rojo-Bezares B, Saenz Y, Poeta P, Zarazaga


Antimicrob. Agents Ch. 45: 1109-1114. M, Ruiz-Larrea F, y Torres C. 2006.
Pfeltz RF, y Wilkinson BJ. 2004. The Assessment of antibiotic susceptibility
escalating challenge of vancomycin within lactic acid bacteria strains
resistance in Staphylococcus aureus. Curr. isolated from wine. Int. J. Food
Drug Targets. 4: 273-294. Microbiol. 111: 234-240.
Piddock LJ. 2006. Multidrug-resistance Ross JI, Eady EA, Cove JH, Jones CE,
efflux pumps - not just for resistance. Ratyal AH, Miller YW, Vyakrnam S, y
Nat. Rev. Microbiol. 4: 629-636. Cunliffe WJ. 1997. Clinical resistance to
Price CE, Reid SJ, Driessen AJM, y Abratt erythromycin and clindamycin in
VR. 2006. The Bifidobacterium longum cutaneous propionibacteria isolated
NCIMB 702259(T) ctr gene codes for a from acne patients is associated with
novel cholate transporter. Appl. mutations in 23S rRNA. Antimicrob.
Environ. Microb. 72: 923-926. Agents Ch. 41: 1162-1165.
Projan SJ. 2003. Why is big Pharma getting Sakamoto K, Margolles A, van Veen HW,
out of antibacterial drug discovery? y Konings WN. 2001. Hop resistance in
Curr. Opin. Microbiol. 6: 427-430. the beer spoilage bacterium Lactobacillus
Putman M, van Veen HW, y Konings WN. brevis is mediated by the ATP-binding
2000a. Molecular properties of bacterial cassette multidrug transporter HorA. J.
multidrug transporters. Microbiol. Mol. Bacteriol. 183: 5371-5375.
Biol. R. 64: 672-693. Salyers AA, Gupta A, y Wang Y. 2004.
Putman M, van Veen HW, Degener JE, y Human intestinal bacteria as reservoirs
Konings WN. 2000b. Antibiotic for antibiotic resistance genes. TRENDS
resistance: era of the multidrug pump. Microbiol. 12: 412-416.
Mol. Microbiol. 36: 772-773. Sandine WE, Elliker PR, Allen LK, y WC
Raha AR, Ross E, Yusoff K, Manap MY, y Brown. 1962. Genetic exchange and
Ideris A. 2002. Characterisation and variability in lactic streptococcis starter
molecular cloning of an erythromycin organisms. J. Dairy Sci. 45: 1266-1271.
resistance plasmid of Lactococcus lactis Schleifer KH, y Ludwig W. 1995.
isolated from chicken cecum. J. Phylogenetic relationships of lactic acid
Biochem. Mol. Biol. Biophys. 6: 7-11. bacteria. p. 7-18, in: B.J.B. Wood & W.H.
Rauch PJ, Beerthuyzen MM, y de Vos WM. Holzapfel (eds) The Genera of Lactic
1994. Distribution and evolution of Acid Bacteria. Blackie Academic &
nisin-sucrose elements in Lactococcus Professional. London.
lactis. Appl. Environ. Microb. 60: 1798- Schwarz FV, Perreten V, y Teuber M. 2001.
1804. Sequence of the 50-kb conjugative
Reuter G. 2001. The Lactobacillus and multiresistance plasmid pRE25 from
Bifidobacterium microflora of the human Enterococcus faecalis RE25. Plasmid. 46:
intestine: composition and succession. 170-187.
Curr. Issues Intest. Microbiol. 2: 43-53. Scott KP, Barbosa TM, Forbes KJ, y Flint
Roberts MC, Sutcliffe J, Courvalin P, HJ. 1997. High-frequency transfer of a
Jensen LB, Rood J, y Seppala H. 1999. naturally occurring chromosomal
Nomenclature for macrolide and tetracycline resistance element in the
macrolide-lincosamide-streptogramin B ruminal anaerobe Butyrivibrio
resistance determinants. Antimicrob. fibrisolvens. Appl. Environ. Microb. 63:
Agents Ch. 43: 2823-2830. 3405-3411.
Roberts MC. 2005. Update on acquired Scott KP, Melville CM, Barbosa TM, y
tetracycline resistance genes. FEMS Flint HJ. 2000. Occurrence of the new
Microbiol. Lett. 245: 195-203. tetracycline resistance gene tet(W) in
Roe DE, Finegold SM, Citron DM, bacteria from the human gut.
Goldstein EJC, Wexler HM, Antimicrob. Agents Ch. 44: 775-777.
Rosemblatt JE, Cox ME, Jenkins SG y Sgorbati B, Scardovi V, y Leblanc DJ. 1982.
Hecht DW. 2002. Multilaboratory Plasmids in the genus Bifidobacterium. J.
comparison of anaerobe susceptibility Gen. Microbiol. 128: 2121-31.
results using 3 different agar media.
Clin. Infect. Dis. 35: 40-46.

155
_______________________________________
Bibliografa ___________________________________
_________________________________

Shales DM, Projan SJ, y Edwards JE. 2004. Tannock GW, Luchansky JB, Miller L,
Antibiotic discovery: state of the state. Connell H, Thode-Andersen S, Mercer
ASM News. 70: 275-281. AA, y Klaenhammer TR. 1994.
Shaw KJ, Rather PN, Hare RS, y Miller Molecular characterization of a
GH. 1993. Molecular genetics of plasmid-borne (pGT633) erythromycin
aminoglycoside resistance genes and resistance determinant (ermGT) from
familial relationships of the Lactobacillus reuteri 100-63. Plasmid. 31:
aminoglycoside-modifying enzymes. 60-71.
Microbiology. 57: 138163. Tannock GW. 2005. Probiotics and
Simpson WJ, Hammond JRM, y Miller RB. Prebiotics: Scientific Aspects Horizon
1988. Avoparcin and vancomycin- .Bioscience/Horizon Scientific Press
useful antibiotics for the isolation of Ltd.
brewery lactic acid bacteria. J. Appl. Temmerman R, Pot B, Huys G, y Swings J.
Microbiol. 64: 299-309. 2003. Identification and antibiotic
Sjlund M. 2004. Development and susceptibility of bacterial isolates from
stability of antibiotic resistance. probiotic products. Int. J. Food
Uppsala University, Sweden. Doctoral Microbiol. 81: 1-10.
Thesis. Tenorio C, Zarazaga M, Martinez C, and
Stiles ME. 1996. Biopreservation by lactic Torres C. 2001. Bifunctional enzyme 6 '-
acid bacteria. Anton. Leeuw. Int. J. G. N-aminoglycoside acetyltransferase-2 ''-
70: 331-45. O-aminoglycoside phosphotransferase
Strman P, Muller CC, y Sorensen KI. in Lactobacillus and Pediococcus isolates
2003. Heat shock treatment increases of animal origin. J. Clin. Microbiol. 39:
the frequency of loss of an 824-825.
erythromycin resistance-encoding Teuber M, Meile L, y Schwarz F. 1999.
transposable element from the Acquired antibiotic resistance in lactic
chromosome of Lactobacillus crispatus acid bacteria from food. Anton. Leeuw.
CHCC3692. Appl. Environ. Microbiol. Int. J. G. 76: 115-37.
69: 7173-7180. Thanassi DG, Suh GS, y Nikaido H. 1995.
Stuart BL, y Marshall B. 2004. Antibacterial Role of outer membrane barrier in
resistance worldwide: causes, efflux-mediated tetracycline resistance
challenges and responses. Nat. Med. 10: of Escherichia coli. J. Bacteriol. 177: 998-
122 - 129 1007.
Su YA, Ping H, y Clewell DB. 1992. van Veen HW, Venema K, Bolhuis H,
Characterization of the tet(M) Oussenko I, Kok J, Poolman B,
determinant of Tn916 - evidence for Driessen AJ, y Konings WN. 1996.
regulation by transcription attenuation. Multidrug resistance mediated by a
Antimicrob. Agents Ch. 36: 769-778. bacterial homolog of the human
Surez JE, y Mendoza MC. 1991. Plasmid- multidrug transporter MDR1. Proc.
encoded fosfomycin resistance. Natl. Acad. Sci. U.S.A. 93: 10668-10672.
Antimicrob. Agents Ch. 35: 791-795. Versalovic J, Shortridge D, Kibler K,
Swanson JK. 2003. Antibiotic resistance of Griffy MV, Beyer J, Flamm RK,
Propionibacterium acnes in acne vulgaris. Tanaka SK, Graham DY, y Go M.F.
Dermatol. Nurs. 15: 359-362. 1996. Mutations in 23S rRNA are
Swenson JM, Facklam RR, y Thornsberry associated with clarithromycin
C. 1990. Antimicrobial susceptibility of resistance in Helicobacter pylori.
vancomycin-resistant Leuconostoc, Antimicrob. Agents Ch. 40: 477-480.
Pediococcus, and Lactobacillus species. Vescovo M, Morelli L, Bottazzi V, y
Antimicrob. Agents Ch. 34: 543-9. Gasson MJ. 1983. Conjugal transfer of
Tankovic J, Leclercq R, y Duval J. 1993. broad-host-range plasmid pAM1 into
Antimicrobial susceptibility of enteric species of lactic acid bacteria.
Pediococcus spp. and genetic basis of Appl. Environ. Microb. 46: 753-755.
macrolide resistance in Pediococcus Villedieu A, Diaz-Torres ML, Hunt N,
acidilactici HM3020. Antimicrob. Agents McNab R, Spratt DA, Wilson M, y
Ch. 37: 789-792. Mullany P. 2003. Prevalence of
tetracycline resistance genes in oral

156
_______________________________________
___________________________________
_________________________________
Bibliografa

bacteria. Antimicrob. Agents Ch. 47: dissemination of antibiotic resistance


878-882. genes. Cell. Mol. Life Sci. 59: 2044-2054
Walsh CT. 2000. Molecular mechanisms Williams JD. 2001. Antibiotic resistance in
that confer antibacterial drug resistance. hospital pathogensacquisition or
Nature. 406: 775-781. spread?. Int. J. Antimic. Agents 18: 295
Wegener HC. 2003. Antibiotics in animal 298.
feed and their role in resistance Wright GD. 2003. Mechanisms of resistance
development. Curr. Opin. Microbiol. 6: to antibiotics. Curr. Opin. Chem. Biol. 7:
439-445. 563-569.
Wehrli W. 1983. Rifampin: mechanisms of Zarazaga M, Senz Y, Portillo A, Tenorio
action and resistance. Rev. Infect. Dis. 5 C, Ruiz-Larrea F, Del Campo R,
Suppl 3, S407-S411. Baquero F, y Torres C. 1999. In vitro
Weisblum B. 1995. Erythromycin resistance activities of ketolide HMR3647,
by ribosome modification. Antimicrob. macrolides, and other antibiotics
Agents Ch. 39: 577-585. against Lactobacillus, Leuconostoc, and
Whitehead TR, y Cotta MA. 2001. Sequence Pediococcus isolates. Antimicrob. Agents
analyses of a broad host-range plasmid Ch. 43: 3039-41.
containing ermT from a tylosin-resistant Zhou JS, Pillidge CJ, Gopal PK, y Gill HS.
Lactobacillus sp. Isolated from swine 2005. Antibiotic susceptibility profiles of
feces. Curr. Microbiol. 43: 17-20. new probiotic Lactobacillus and
Whittle G, Shoemaker NB, y Salyers AA. Bifidobacterium strains. Int. J. Food
2002. The role of Bacteroides Microbiol. 98: 211-217.
conjugative transposons in the

157
ANEXOS
Anexo 1.- Direciones tiles de internet dedicadas a la resistencia de
antibiticos

Direcciones de organizaciones y proyectos dedicados a la investigacin, control y


difusin de la resistencia a antibiticos:
www.antibioticos.msc.es, sitio del Ministerio de Sanidad Espaol.
- www.who.int, sitio de la Organizacin Mundial de la Salud (OMS WHO).
- www.apua.org, sitio de la Alliance for the Prudent Use of Antibiotics
(APUA).
- www.roarproject.org; sitio del proyecto The Reservoirs of Antibiotic
Resistance (ROAR) de la APUA.
- www.react-group.org, sito de la asociacin Action on Antibiotic Resistance.
- www.antibioticresistance.org.uk, sitio de la National electronic Library of
Infection (NeLI) sobre resistencia a antibiticos.
- www.cdc.gov/drugresistance/, sitio dedicado a la resistencia a antibiticos del
Centers for Disease Control and Prevention (antigua CDC).
- www.rivm.nl/earss/, sitio del European Antimicrobial Resistance
Surveillance System (EARSS).
- www.enare.org, sitio de la European Network for Antimicrobial Resistance
and Epidemiology (ENARE).
- www.esbic.de/esbic/ind_esar.htm, sitio de la European Surveillance of
Antibiotic Resistance (ESAR).

Direcciones de inters didctico general:

- www.antibiotics.net, historia, clases y modo de accin de los antibiticos.


- www.familydoctor.org, informacin sobre antibiticos de la American
Academy of Family Physicians (AAFP).
- www.keepantibioticsworking.com, campaa para una utilizacin racional de
los antibiticos.
- www.antibiotics-info.org, pgina web del National Information Program on
Antibiotics de diversas organizaciones de salud de Canad.

159
Anexo 2.- Protocolo estandarizado de anlisis mediante el sistema Etest.

UNIVERSITEIT
GENT
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)

SOP
Standard Operating Procedure

Author: Acronym: Date last modified:


Geert Huys ACE-ART-ETEST 21-10-2005

Title:

ANTIBIOTIC SUSCEPTIBILITY TESTING OF


LACTIC ACID BACTERIA
WITH THE ETEST METHOD

References:
See section C
Reviewed & approved by: all ACE-ART partners

__________________________________________________________________________________________________
_Author: Geert Huys 161
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

ANTIBIOTIC SUSCEPTIBILITY TESTING OF LACTIC ACID BACTERIA


WITH THE ETEST METHOD

A. PURPOSE & PRINCIPLE


The purpose of this SOP is to describe a standardized procedure for determining the Minimal
Inhibitory Concentration (MIC) of a series of antibiotics applicable for non-enterococcal lactic acid
bacteria (LAB). In contrast to the disk diffusion method, which is a semi-quantitative method,
determination of MIC values provides a quantitative measure for the level of resistance expressed by
the test organism. Next to the agar dilution method, the Etest method provides an alternative to
determine MICs for organisms grown on agar media. The method is based on the inoculation of a
standardized inoculum of the test strain on the surface of the agar plate followed by the placement of an
Etest strip. According to the preformed antibiotic gradient of the strip, the MIC can be read at the
intersection between clear visual growth and inhibition of growth.

B. METHOD DESCRIPTION

I. Bacterial cultivation and material preparations (DAY 1)

Prior to the actual susceptibility assay, the LAB strains to be tested should be precultured overnight
(16-24 h, except for bifidobacteria which usually require 40-48 h) on the agar version of the test
medium under the recommended incubation conditions (Table 1). Strains stored as cryopreserved
cultures (e.g. in glycerol broth or on beads) can first be recovered in MRS broth prior to overnight
preculturing on the recommended test medium. The basis of each test medium is Isosensitest
medium (IST) supplemented with MRS medium (LSM) for lactobacilli, with MRS medium and
cysteine.HCl (LSM + cysteine) for bifidobacteria or with lactose (IST + lactose) for S.
thermophilus.
Depending on whether the tests are performed at 28C or at 37C, Lactobacillus plantarum ATCC
14917T (= LMG 6907T) or Enterococcus faecalis ATCC 29212 (= LMG 8222) needs to be included
as control strain during each susceptibility assay, respectively.
Prepare the necessary number of agar plates of the test medium and culture tubes containing 2-5 ml
sterile saline.
For testing bifidobacteria, it is recommended to pre-reduce the culture media in an anaerobic
chamber or jar one day prior to testing.

__________________________________________________________________________________________________
Author: Geert Huys
162
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

Table 1. Growth and test conditions for antimicrobial susceptibility testing of LAB

LAB species* Medium Temperature Atmosphere Time


Bifidobacterium spp. LSM+cysteine 37C Anaerobic** 48 h
Lb. acidophilus LSM 37C Anaerobic 24-48 h
Lb. amylovorus LSM 37C Anaerobic 24-48 h
Lb. brevis LSM 28C Anaerobic 24-48 h
Lb. casei/paracasei LSM 28C Anaerobic 24-48 h
Lb. delbreuckii LSM 37C Anaerobic 24-48 h
Lb. fermentum LSM 37C Anaerobic 24-48 h
Lb. gasseri LSM 37C Anaerobic 24-48 h
Lb. helveticus LSM 37C Anaerobic 24-48 h
Lb. johnsonii LSM 37C Anaerobic 24-48 h
Lb. plantarum/pentosus LSM 28C Anaerobic 24-48 h
Lb. reuteri LSM 37C Anaerobic 24-48 h
Lb. rhamnosus LSM 37C Anaerobic 24-48 h
Lb. sakei LSM 28C Anaerobic 24-48 h
Lc. lactis IST 32C Anaerobic 24 h
S. thermophilus IST+lactose 42C Anaerobic 24 h
*Strains of some LAB species such as Lb. amylovorus may display insufficient growth when using the agar formulation of
the recommended medium. Possibly, strains of these species may only be tested using the broth microdilution method
described in SOP-ACE-ART-MDIL.
**The anaerobic atmosphere should be generated preferably by Anoxomat (MART, The Netherlands) or AnaeroGen
(Oxoid) kits or in an anaerobic cabinet containing app. 10% CO2.

II. Preparation of the inoculum and application of Etest strips (DAY 2)

After overnight incubation, the agar culture is checked for purity. For inoculum preparation,
individual colonies are suspended in a sterile glass or plastic culture tube containing 2 - 5 ml sterile
saline until a density corresponding to McFarland (McF) standard 1 or an spectrophotometric
equivalent (3.10E8 CFU/ml) is obtained. Ideally, the culture tubes should fit into a portable
spectrophotometer for easy adjustment of the inoculum density until the OD590 or OD625 equivalent
of McF 1 is reached. According to the website of Quelab
(http://www.quelab.com/htmleng/2900a.html), a suspension density of McF 1 corresponds to an
OD625 equivalent of 0.16 0.2. The inocula of bifidobacteria should be prepared in pre-reduced
LSM+cysteine broth.

__________________________________________________________________________________________________
Author: Geert Huys
163
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

A sterile cotton swab is dipped in the standardized inoculum and used to inoculate an agar plate of
the recommended test medium (Table 1). The swab should be sufficiently moistened however
without forming droplets upon touching the agar plate. The agar plate is swabbed closely in three
directions or by moving it slowly from one end to another and back again if using a plate turntable.
Allow the agar surface to dry for approximately 15 minutes before applying the Etest.
Etests with preformed gradient of a specific antibiotic will be applied (Table 2). Using the Etest
applicator or forceps, 2-3 Etests can be placed in opposite directions on a small plate. Application of
more strips per plate may cause interference between inhibition zones when using conventional 9-10
cm diameter plates. Do not move the Etests as the antibiotic diffuses immediately upon contact with
the agar surface. Incubate the agar plates under the recommended conditions (Table 1).

Table 2. Test ranges of Etest strips

Antibiotic Class Concentration range AB Biodisk product no*


Tetracycline Tetracyclines 0.016 - 256 g/ml 5100 2258
Erythromycin Macrolides 0.016 - 256 g/ml 5100 1058
Streptomycin Aminoglycosides 0.016 - 256 g/ml 5100 2188
Gentamicin Aminoglycosides 0.016 - 256 g/ml 5100 1258
Clindamycin Lincosamides 0.016 - 256 g/ml 5100 0958
Ampicillin Penicillins 0.016 - 256 g/ml 5100 0158
Vancomycin** Glycopeptides 0.016 - 256 g/ml 5100 2558
*cfr. 2005 product list of ABBiodisk
**Only for bifidobacteria and members of the Lb. acidophilus group.

III. Reading of the MIC (DAY 3)

After 24-48 h incubation, a confluent layer of bacteria has grown on the plate except close to the
Etest strip where the concentration of antibiotic is inhibiting growth. The inhibition should be a
clear ellipse along the strip with no growth of bacteria. For the purpose of the harmonization
study, Etest assays should be read after 24 and 48h. For routine testing, however, 48h readings
are recommended to ensure sufficient growth of all LAB species. In the EU-ACE-ART
project, MIC values will serve to determine the microbiological breakpoint rather than the
clinical breakpoint of LAB species. Following the EUCAST definition (http://www.eucast.org),
a microbiological breakpoint serves to distinguish between wild type organisms (free of
acquired and mutational resistance mechanisms to the tested agent) and non-wild type
organisms (harbouring an acquired or mutational resistance mechanism to the tested agent).

__________________________________________________________________________________________________
Author: Geert Huys
164
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

In some cases, reading of Etests is straightforward whereas in other cases reading may require
assistance of a highly experienced technician. Even though the procedure is simple, there are several
pitfalls. The MIC is determined as the first value on the Etest strips where growth does not occur.
For bacteriostatic antibiotics (e.g. tetracyclines, macrolides and lincosamides), the MIC is read at
the point where growth is inhibited by 80% (i.e. the first point of significant inhibition as judged by
the naked eye). Accurate reading of clindamycin MICs can be especially difficult for
unexperienced workers as different shapes of inhibition ellipses can be observed. If the ellipse
has a bulb-like form, the MIC should be read where the "head of the bulb" ends, that is extrapolate
the ellipse edge towards the strip (Fig. 1). In this way microcolonies are excluded and the strip is
read at 80 % inhibition. However, if the ellipse is narrowing, MIC should be read at the actual
intersection (Fig. 1). Distinct individual colonies within the lower part of the narrowing ellipse
should be included if they are situated very close to the strip. Even if the ellipse is narrow,
clindamycin should be read at 80 % so that microcolonies are excluded. In cases of doubt, refer to
the manufacturer's instructions (an Etest Technical Manual is available on-line) or submit digital
images to techsupport@abbiodisk.se for interpretation.

Fig. 1. Narrowing and bulb-like ellipse shapes during clindamycin MIC determination with Etest
IV. Harmonization of method between collaborating laboratories

__________________________________________________________________________________________________
Author: Geert Huys
165
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

In order to correlate MIC data determined in different laboratories, it is necessary to harmonize the
inter-laboratory logistics at the start of each major survey. For this purpose, the results of MIC
determination for a set of control and reference strains should be compared between laboratories.
As mentioned in I, one of the two control strains (ATCC 29212 and LMG 6907) should be
included in each batch of MIC determinations depending on the recommended incubation
temperature. For the purpose of the harmonization study, Etest assays should be read after 24
and 48h.
It is proposed that the type strains of the 25 LAB species targetted in the ACE-ART project are
included in such a harmonization study. To be statistically relevant to some extent, each strain needs
to be tested in min. three independent laboratories. In each laboratory, five independent replicates
(i.e. starting from new fresh cultures) are needed per strain.

C. REFERENCES

http://www.abbiodisk.se

D. MATERIALS

IST (for Lc. lactis)


- Isosensitest agar (ISA; #CM 473) (Oxoid, Basingstoke, UK)

Media should be prepared with distilled or freshly deionised water according to the manufacturers
instructions. The pH is set at pH 7.4 + 0.2. Thickness of the agar layer should be app. 4 mm ( i.e.
24-25 ml in 9 cm petri dishes or 58 ml in 15 cm petri dishes). Poured ISA plates may be stored for
up to 2 weeks in air-tight plastic bags at 2-8 C. Immediately prior to inoculation ISA plates should
be moist but free of droplets, which should not be present on either the agar surface nor on the petri
dish lids. If necessary plates may be dried by incubation at 30-37 C or in a laminar flow cabinet
for a maximum of 30 min. A representative sample of each batch of ISB tubes or ISA plates should
be examined for sterility by incubation at 28 C for 72 h.

IST+lactose (for S. thermophilus)


- Isosensitest agar (ISA; #CM 473) (Oxoid, Basingstoke, UK)
- Lactose (In Italy: Carlo Erba Cod 457557)

ISA plates containing 1% (w/v) lactose are prepared with distilled or freshly deionised water
according to the manufacturers instructions (see preparation of IST). A filter-sterilized 10% (w/v)
__________________________________________________________________________________________________
Author: Geert Huys
166
UNIVERSITEIT
GENT
SOP-ACE-ART-ETEST
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

lactose solution is added to the medium in a 1/10 ratio after autoclaving the medium to obtain a
final concentration of 1% (w/v) in the medium.

LSM (for lactobacilli)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid)
- MRS broth (#CM0359; Oxoid)
- Bacteriological agar no. 1 (BA1; #LP0011; Oxoid)

For composition of the agar formula of LSM, a mixture of 90% (v/v) ISB and 10% (v/v) MRS broth
is supplemented with 1.5% (w/v) BA1 (see preparation of IST). The pH is finally adjusted to 6.7
after autoclaving with sterile HCl solution. Poured LSM plates may be stored for up to 1 week in
air-tight plastic bags at 2-8 C.

LSM+cysteine (for bifidobacteria)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid)
- MRS broth (#CM0359; Oxoid)
- Bacteriological agar no. 1 (BA1; #LP0011; Oxoid)
- Cysteine.HCl (AppliChem (A3698) in Austria; Merck 1.02839)

For composition of the agar formula of LSM + cysteine, a mixture of 90% (v/v) ISB, 10% (v/v) MRS
broth and 0.3 g l-1 cysteine.HCl is prepared (see preparation of IST). The agar version of LSM is
supplemented with 1.5% (w/v) BA1. The pH is finally adjusted to 6.7 after autoclaving with sterile
HCl solution. Poured plates may be stored for up to 1 week in air-tight plastic bags at 2-8 C.

Sterile saline
- Suspension medium (2 or 5 ml) (BioMerieux, #20 150)
or
- Sterile tubes with 2-5 ml 0.85% NaCl solution in distilled or deionised water

Etest strips
- Etest applicator (ABBiodisk, SE, #5100 5987)
- Etest strips (ABBiodisk; see Table 2 for product numbers [cfr. 2005 product list])
The Etest strips must be stored in a freezer at -20C. Unused Etest strips from an opened package
must be stored in an airtight storage tube containing silica gel. Place the Etests at room
temperature for 45 minutes before use.

__________________________________________________________________________________________________
Author: Geert Huys
167
Anexo 3.- Protocolo estandarizado de anlisis mediante el sistema de microdilucin.

UNIVERSITEIT
GENT
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)

SOP
Standard Operating Procedure

Author: Acronym: Date last modified:


Geert Huys ACE-ART-MDIL 21-10-2005

Title:

ANTIBIOTIC SUSCEPTIBILITY TESTING OF


LACTIC ACID BACTERIA
WITH THE BROTH MICRODILUTION METHOD

References:

Reviewed & approved by: all ACE-ART partners

__________________________________________________________________________________________________
_Author: Geert Huys 169
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

ANTIBIOTIC SUSCEPTIBILITY TESTING OF LACTIC ACID BACTERIA


WITH THE BROTH MICRODILUTION METHOD

A. PURPOSE & PRINCIPLE


The purpose of this SOP is to describe a standardized procedure for determining the Minimal
Inhibitory Concentration (MIC) of a series of antibiotics applicable for non-enterococcal lactic acid
bacteria (LAB). In contrast to the disk diffusion method, which is a semi-quantitative method,
determination of MIC values provides a quantitative measure for the level of resistance expressed by
the test organism. Next to the agar dilution method and the Etest, the broth dilution method is one of
the most frequently used methods to determine MICs. The method is based on the inoculation of a
standardized broth inoculum of the test strain in a dilution series of the antibiotic for which the MIC is
determined. The first concentration in the dilution series at which no visual growth can be determined
is then considered as the MIC.

B. METHOD DESCRIPTION

I. Bacterial cultivation and material preparations (DAY 1)

Prior to the actual susceptibility assay, the LAB strains to be tested should be precultured overnight
(16-24 h, except for bifidobacteria which usually require 40-48 h) on the agar version of the test
medium under the recommended incubation conditions (Table 1). Strains stored as cryopreserved
cultures (e.g. in glycerol broth or on beads) can first be recovered in MRS broth prior to overnight
preculturing on the recommended test medium. The basis of each test medium is Isosensitest
medium (IST) supplemented with MRS medium (LSM) for lactobacilli, with MRS medium and
cysteine.HCl (LSM+cysteine) for bifidobacteria or with lactose (IST+lactose) for S. thermophilus.
Depending on whether the tests are performed at 28C or at 37C, Lactobacillus plantarum ATCC
14917T (= LMG 6907T) or Enterococcus faecalis ATCC 29212 (= LMG 8222) needs to be included
as control strain during each susceptibility assay, respectively.
Prepare the necessary number of culture tubes containing 2-5 ml sterile saline or containing broth
test medium (min. 5 mL per strain excluding the volume needed to calibrate during
spectrophotometric readings and as negative control).
For testing bifidobacteria, it is recommended to pre-reduce the culture media in an anaerobic
chamber or jar one day prior to testing.

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
170
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

Table 1. Growth and test conditions for antimicrobial susceptibility testing of LAB

LAB species* Medium Temperature Atmosphere Time


Bifidobacterium spp. LSM+cysteine 37C Anaerobic** 48 h
Lb. acidophilus LSM 37C Anaerobic 24-48 h
Lb. amylovorus LSM 37C Anaerobic 24-48 h
Lb. brevis LSM 28C Anaerobic 24-48 h
Lb. casei/paracasei LSM 28C Anaerobic 24-48 h
Lb. delbreuckii LSM 37C Anaerobic 24-48 h
Lb. fermentum LSM 37C Anaerobic 24-48 h
Lb. gasseri LSM 37C Anaerobic 24-48 h
Lb. helveticus LSM 37C Anaerobic 24-48 h
Lb. johnsonii LSM 37C Anaerobic 24-48 h
Lb. plantarum/pentosus LSM 28C Anaerobic 24-48 h
Lb. reuteri LSM 37C Anaerobic 24-48 h
Lb. rhamnosus LSM 37C Anaerobic 24-48 h
Lb. sakei LSM 28C Anaerobic 24-48 h
Lc. lactis IST 32C Anaerobic 24 h
S. thermophilus IST+lactose 42C Anaerobic 24 h
*Strains of some LAB species such as Lb. amylovorus may occassionally display insufficient growth when using the agar
formulation of the recommended medium. Possibly, strains of these species may only be tested using the broth
microdilution method described in SOP-ACE-ART-MDIL.
**The anaerobic atmosphere should be generated preferably by Anoxomat (MART, The Netherlands) or AnaeroGen
(Oxoid) kits or in an anaerobic cabinet containing app. 10% CO2.

II. Preparation of the inoculum and inoculation of the microdilution plates (DAY 2)

After overnight incubation, the agar culture is checked for purity. For inoculum preparation,
individual colonies are suspended in a sterile glass or plastic culture tube containing 2 - 5 ml sterile
saline until a density corresponding to McFarland (McF) standard 1 or an spectrophotometric
equivalent (3.10E8 CFU/ml) is obtained. Ideally, the culture tubes should fit into a portable
spectrophotometer for easy adjustment of the inoculum density until the OD590 or OD625 equivalent
of McF 1 is reached. According to the website of Quelab
(http://www.quelab.com/htmleng/2900a.html), a suspension density of McF 1 corresponds to an
OD625 equivalent of 0.16 0.2. The inoculated saline suspension is then diluted 1:1000 (for
inoculation of VetMICTM ACE-ART microdilution plates) or 1:500 (for inoculation of vancomycin
__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
171
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

microdilution plates) in the recommended test medium (Table 1) to obtain a final concentration of
3.10E5 CFU/ml or 6.10E5 CFU/ml, respectively.

Using the VetMICTM ACE-ART microdilution plates, two strains can be tested per plate for
determination of MICs of six antibiotics (Table 2; Figure 1). Testing of vancomycin resistance
(only for bifidobacteria and members of the Lactobacillus acidophilus group i.e. L. acidophilus, L.
amylovorus, L. johnsonii and L. gasseri) needs to be performed in a separate microdilution assay
(Table 3; Figure 2).
For VetMICTM ACE-ART plate assays, 100 l of the 3.10E5 CFU/ml inoculum is added to each
well (= 3.10E4 CFU/well) in columns 1-6 (strain 1) or 7-12 (strain 2) of the microdilution plate
within 30 min after the preparation of the standardized inoculum. According to the manufacturer, no
additional homogenization step is required because the antibiotic in each well dissolves easily in the
test medium and diffuses to equilibrium throughout the well. Plates are incubated under the species-
specific conditions listed in Table 1. When using anaerobic jars, plates should be piled with a lid
between every plate to generate a homogeneous environment in the jar. As a negative control, a tube
of non-inoculated test medium is incubated together with the plates.

Table 2. Test ranges of antibiotics included in VetMICTM ACE-ART microdilution plates

Antibiotic Class Concentration range


Tetracycline Tetracyclines 0.5 - 128 g/ml
Erythromycin Macrolides 0.12 - 16 g/ml
Streptomycin Aminoglycosides 2 - 256 g/ml
Gentamicin Aminoglycosides 0.5 - 32 g/ml
Clindamycin Lincosamides 0.12 - 8 g/ml
Ampicillin Penicillins 0.12 - 8 g/ml

Figure 1. Lay-out of the VetMICTM ACE-ART microdilution panel

Strain 1 Strain 2

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
172
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

1 2 3 4 5 6 7 8 9 10 11 12
A Tc Cl Sm Em Gm Am Tc Cl Sm Em Gm Am
64 8 256 16 32 8 64 8 256 16 32 8

B
32 4 128 8 16 4 32 4 128 8 16 4

C
16 2 64 4 8 2 16 2 64 4 8 2

D
8 1 32 2 4 1 8 1 32 2 4 1

E
4 0.50 16 1 2 0.50 4 0.50 16 1 2 0.50

F
2 0.25 8 0.50 1 0.25 2 0.25 8 0.50 1 0.25

G
1 0.12 4 0.25 0.50 0.12 1 0.12 4 0.25 0.50 0.12

H Tc dist tri-cit Tc dist tri-cit


0.50 128 2 0.12 contr contr 0.50 128 2 0.12 contr contr

Tc Oxytetracycline Em Erythromycin
Cl Clindamycin Gm Gentamicin
Sm Streptomycin Am Ampicillin

tri-cit, growth control in citric acid medium; dist. contr., positive control (no antibiotic in well)

For the vancomycin microdilution assays, a stock solution of vancomycin is prepared in water
from which 5-fold intermediate dilutions are prepared in LSM (for members of L. acidophilus
group) or LSM+cysteine (for bifidobacteria) broth as outlined in Table 3. Subsequently, 50 l of the
final solutions obtained in steps 2-12 are dispensed in the microdilution plates as illustrated in
Figure 2. The trays are sealed in plastic bags and immediately frozen at -20 C until needed.
Upon usage, plates are thawed under anaerobic conditions. Each well is inoculated with 50 l of the
bacterial inoculum (6.10E5 CFU/ml) in LSM (for members of L. acidophilus group) or in
LSM+cysteine (for bifidobacteria) broth. The last row in each plate is used as sterility control and is
inoculated with pure LSM or LSM+cysteine broth.
Table 3. Step-wise preparation of the vancomycin working dilutions

Step Concentratio Source Volume Distilled Intermediate Final concentration


no n water concentratio at 1:5 dilution in

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
173
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

(g/ml) n LSM (+cysteine)

1280 stock in - - 1280 256


water
1 1280 stock 2 2 640 128
2 640 step 1 2 2 320 64
3 640 step 1 1 3 160 32
4 640 step 1 1 7 80 16
5 80 step 4 2 2 40 8
6 80 step 4 1 3 20 4
7 80 step 4 1 7 10 2
8 10 step 7 2 2 5 1
9 10 step 7 1 3 2.5 0.5
10 10 step 7 1 7 1.25 0.25

Figure 2. Lay-out of the vancomycin microdilution panel

Strain Final vancomycin concentration


1 2 3 4 5 6 7 8 9 10 11 12
A
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
B
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
C
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
D
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
E
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
F
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
G
128 64 32 16 8 4 2 1 0.5 0.25 0.12 0
Sterility
control 128 64 32 16 8 4 2 1 0.5 0.25 0.12 0

III. Reading of the MIC (DAY 3)

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
174
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

Following incubation, the negative control tube is checked for presence of visual growth. If
contamination is noted, all data generated from the involved strain should be rejected. During the
harmonization study, plates are read after 24 h and 48 h incubation. For routine testing,
however, 48h readings are recommended to ensure sufficient growth of all LAB species. In the
EU-ACE-ART project, MIC values will serve to determine the microbiological breakpoint
rather than the clinical breakpoint of LAB species. Following the EUCAST definition
(http://www.eucast.org), a microbiological breakpoint serves to distinguish between wild type
organisms (free of acquired and mutational resistance mechanisms to the tested agent) and
non-wild type organisms (harbouring an acquired or mutational resistance mechanism to the
tested agent).
The absence of growth in the tri-cit control well implies that the tested strain is sensitive to the citric
acid included in the buffer used for Am. In such cases, reading of MIC for Am is not relevant.
If negative control and tri-cit control are checked and approved, growth is determined visually for
each antibiotic by comparing with the positive and negative control. The panel is preferably placed
on top of a viewing device, in form of a rack with an enlarging mirror. A bench lamp giving indirect
light facilitates reading. Bacterial growth is easily detected in the mirror as a pellet in the bottom of
the well. Any series of wells where discontinuity in growth is observed (e.g. growth at 16 and 64
g/ml but not at 32 g/ml) should be discarded. The end-point is defined as the lowest antibiotic
concentration for which there is no visual growth. This concentration should be reported as the MIC
of that antibiotic for that particular strain. If trailing end-points are observed, this should be
reported as a remark. Irrespective of the bacteriocidal or bacteriostatic mechanism of the tested
agent, a 80% reduction in growth should be reported as end-point.

IV. Harmonization of method between collaborating laboratories

In order to correlate MIC data determined in different laboratories, it is necessary to harmonize the
inter-laboratory logistics at the start of each major survey. For this purpose, the results of MIC
determination for a set of control and reference strains should be compared between laboratories.
As mentioned in I, one of the two control strains (ATCC 29212 and LMG 6907) should be
included in each batch of MIC determinations depending on the recommended incubation
temperature.
It is proposed that the type strains of the 25 LAB species targetted in the ACE-ART project are
included in such a harmonization study. To be statistically relevant to some extent, each strain needs
to be tested in min. three independent laboratories. In each laboratory, five independent replicates
(i.e. starting from new fresh cultures) are needed per strain.

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
175
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

C. REFERENCES

To be inserted

D. MATERIALS

IST (for Lc. lactis)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid, Basingstoke, UK)

Media should be prepared with distilled or freshly deionised water according to the manufacturers
instructions. The pH is set at pH 7.4 + 0.2. Poured ISA plates may be stored for up to 2 weeks in
air-tight plastic bags at 2-8 C. Immediately prior to inoculation ISA plates should be moist but free
of droplets, which should not be present on either the agar surface nor on the petri dish lids. If
necessary plates may be dried by incubation at 30-37 C or in a laminar flow cabinet for a
maximum of 30 min. A representative sample of each batch of ISB tubes or ISA plates should be
examined for sterility by incubation at 28 C for 72 h.

IST+lactose (for S. thermophilus)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid, Basingstoke, UK)
- Lactose (In Italy: Carlo Erba Cod 457557)

ISA and ISB solutions containing 1% (w/v) lactose are prepared with distilled or freshly deionised
water according to the manufacturers instructions (see preparation of IST). A filter-sterilized 10%
(w/v) lactose solution is added to the medium in a 1/10 ratio after autoclaving the medium to obtain
a final concentration of 1% (w/v) in the medium.

LSM (for lactobacilli)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid)
- MRS broth (#CM0359; Oxoid)
- Bacteriological agar no. 1 (BA1; #LP0011; Oxoid)

ISB and MRS broth solutions are prepared separately with distilled or freshly deionised water
according to the manufacturers instructions (see preparation of IST). For composition of the broth
formula of LSM, a mixture of 90% (v/v) ISB and 10% (v/v) MRS broth is prepared. The agar
version of LSM is supplemented with 1.5% (w/v)BA1. The pH is finally adjusted to 6.7 after
__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
176
UNIVERSITEIT
GENT
SOP-ACE-ART-MDIL
Laboratory of Microbiology
K.L. Ledeganckstr. 35
B-9000 Gent (BELGIUM)
___________________________________________________________________________________

autoclaving with sterile HCl solution. Poured LSM plates may be stored for up to 1 week in air-tight
plastic bags at 2-8 C.

LSM+cysteine (for bifidobacteria)


- Isosensitest agar (ISA; #CM 473) and broth (ISB; #CM471) medium (Oxoid)
- MRS broth (#CM0359; Oxoid)
- Bacteriological agar no. 1 (BA1; #LP0011; Oxoid)
- Cysteine.HCl (AppliChem (A3698) in Austria; Merck 1.02839)

ISB and MRS broth solutions are prepared separately with distilled or freshly deionised water
according to the manufacturers instructions (see preparation of IST). For composition of the broth
formula of LSM + cysteine, a mixture of 90% (v/v) ISB, 10% (v/v) MRS broth and 0.3 g l-1
cysteine.HCl is prepared. The agar version of LSM+cysteine is supplemented with 1.5% (w/v) BA1.
The pH is finally adjusted to 6.7 after autoclaving with sterile HCl solution. Poured plates may be
stored for up to 1 week in air-tight plastic bags at 2-8 C.

Sterile saline
- Suspension medium (2 or 5 ml) (BioMerieux, #20 150)
or
- Sterile tubes with 2-5 ml 0.85% NaCl solution in distilled or deionised water

VetMICTM ACE-ART microdilution plates (Fig. 1)


National Veterinary Institute (Uppsala, Sweden)
Store @ room temperature (can be kept for 2 years).

Vancomycin
- Sigma V2002
The step-wise procedure to prepare the working dilutions is outlined in Table 3.

__________________________________________________________________________________________________
Author: Geert Huys Date last modification: 21/10/2005
177
Anexo 4.- Genes de resistencia a antibiticos identificados en diferentes especies de bacterias del cido lctico (no enterococos) y de
bifidobacterias.

Especie/ Origen Gen(es) de resistencia N de acceso en GenBank Referencia


Nombre o nmero de cepas

Bifidobacterium longum
H66 Intestino humano tet(W) DQ060146 (Flrez et al., 2006b)
F5, F8, and F10 Flora fecal humana tet(W) - (Scott et al., 2000)
B36 Hombre, animals, probiticos tet(W) - (Moubareck et al., 2005)
23 aislados Placa sublingual (muestras clnicas) tet(M) - (Lacroix and Walker, 1995)
NCIMB 702259 Intestino humano ctr DQ017587 (Price et al, 2006)

Bifidobacterium breve
UCC2003 Aislado nio bbmR (Margolles et al, 2005)

Bifidobacterium spp.

179
Varias cepas de diferentes especies Intestino humano tet(W) - (Flrez et al., 2006b)
DSM 10140 Intestino humano tet(W) - (Kastner et al., 2006)
Varias cepas de diferentes especies Hombre, animals, probiticos tet(W) - (Masco et al., 2006)

Lactobacillus acidophilus
1 aislado Heces de cerdo o pjaros aaa(6)Ie-aph(2)Ia - (Tenorio et al., 2001)
8 aislados Humano y productos lcteos tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

Lactobacillus alimentarius
DG500, DG499, DG498 Embutido fermentado tet(M) AY149587, AY149586, AY149585 (Gevers et al., 2003)

Lactobacillus animalis
- Cavidad tonsilar y nasal de cerdo erm(B) - (Martel et al., 2003)

Lactobacillus casei
6 aislados Humano y productos lcteos tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)
Lactobacillus crispatus
CHCC3692 Esfago y faringe de cerdo erm(B) AY262353 (Strman et al., 2003)
5 aislados Humano tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

Lactobacillus curvatus
DG524, DG484 DG142, DG143, Embutido fermentado tet(M) AY149595, AY149580, AY149576, (Gevers et al., 2003)
G048 AY149577 AY149575

Lactobacillus fermentum
LEM89 Heces de cerdo erm(B) U48430 (Fons et al., 1997)
ROT1 Producto de leche cruda erm(LF), vat(E-1), tet(M) AJ488494 (Gfeller et al., 2003)
20 aislados Humano tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

Lactobacillus gasseri
49 aislados Hunano y productos lcteos tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

Lactobacillus johnsonii
- Cavidad tonsilar y nasal de cerdo erm(B) - (Martel et al., 2003)
Twenty six isolates Humano tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

180
G41 Humano erm(B) (Flrez et al., 2006a)

Lactobacillus plantarum
5057 Ensilado tet(M) AF440277 (Danielsen, 2002)
DG507 Embutido fermentado tet(M), erm(B) AY149588 (Gevers et al., 2003)
DG533, DG522, DG520, DG515, Embutido fermentado tet(M) AY149597, AY149594, AY149593, (Gevers et al., 2003)
DG512, DG509, DG013 AY149591, AY149590, AY149589,
AY149574
caTC2R Carne de cerdo cat-TC - (Ahn et al., 1992)
8 aislados Humano y productos lcteos tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)
CCUG 43738 Humano tet(S) AM039486-AM030490

C709 Vino aac(6)-aph(2) (Rojo-Bezares et al., 2006)


J62 Vino ant(6) (Rojo-Bezares et al., 2006)
J65 Vino aac(6)-aph(2) (Rojo-Bezares et al., 2006)
J75 Vino aac(6)-aph(2) (Rojo-Bezares et al., 2006)
J77 Vino aac(6)-aph(2), aph(3)- (Rojo-Bezares et al., 2006)
IIIa
E43 Vino tet(M) (Rojo-Bezares et al., 2006)

Lactobacillus reuteri
1044 Intestino de cerdo erm(B) - (Axelsson et al., 1988)
100-63 Carne erm(T) M64090 (Tannock et al., 1994)
G4 Carne cat-TC U75299 (Lin et al., 1996)
N16, L1 Intestino de pollo erm(B) AF080450, AF030064 (Lin and Chung, 1999)
- Cavidad tonsilar y nasal de cerdo erm(B) - (Martel et al., 2003)
SD 2112 Humano tet(W), lnu(A) - (Kastner et al., 2006)

Lactobacillus rhamnosus
43 aislados Humano tet(M), erm(B) - (Cataloluk and Gogebakan, 2004)

Lactobacillus sakei
DG516, DG489, DG488, DG485, Embutido fermentado tet(M) AY149592, AY149583, AY149582, (Gevers et al., 2003)
DG483, DG165 AY149581, AY149579, AY149578

Lactobacillus salivarius

181
5 aislados Heces de cerdo aac(6)-aph(2) - (Tenorio et al., 2001)
Cavidad tonsilar y nasal de cerdo erm(B) - (Martel et al., 2003)

Lactobacillus spp.
- - tet(O), tet(Q) - (Chopra and Roberts, 2001)
PC121B Heces de cerdo erm(T) AF310974 (Whitehead & Cotta, 2001)
T22-5 Cavidad oral humana tet(W) - (Villedieu et al., 2003)
9 aislados Tracto urogenital humano tet(O) - (Villedieu et al., 2003)
- - tet(K), tet(M), tet(S), - (Roberts, 2005)
tet(W), tet(36)

Lactococcus lactis subsp. lactis


AA21aTc, AA29 Queso azul (Cabrales) tet(M) DQ060148, DQ060147 (Flrez and Mayo, unpublished)
K214 Queso fresco de leche cruda mdt(A), tet(S), cat, str X92946 (Perreten et al., 2001)
C5 Heces de pollo erm(T) - (Raha et al., 2002)
MG1363 lmrA U63741 (Van veen et al, 1996)
MN5 lmrB AF056207 (Gajic et al, 2004)
MG1363 lmrP X89779 (Bolhuis et al, 1995)
IL1403 lmrCD AE006268 (Lubelski et al, 2004)

Leuconostoc citreum
- Lnea de procesado de embutido tet(S) - (Gevers, 2002)

Oenococcus oeni
IS18 Vino aac(6)-aph(2) (Rojo-Bezares et al., 2006)
Vino aac(6)-aph(2), aph(3)- (Rojo-Bezares et al., 2006)
IS83
IIIa

Pediococcus acidilactici
HM3020 Heces (muestras clnicas) erm(B) - (Tankovic et al., 1993)
AR-63 Heces de cerdo o de mascotas erm(B) - (Zarazaga et al., 1999)
1 aislado Heces de cerdo o de mascotas aaa(6)-aph(2) - (Tenorio et al., 2001)
6990 Queso tradicional erm(B) - (Danielsen et al., 2006)
J83 Vino erm(B) (Rojo-Bezares et al., 2006)

Pediococcus parvulus

182
J82 Vino ant(6), tet(L) (Rojo-Bezares et al., 2006)
J103 Vino aac(6)-aph(2), ant(6) (Rojo-Bezares et al., 2006)

Pediococcus pentosaceus
C711 Vino aac(6)-aph(2) (Rojo-Bezares et al., 2006)
Anexo 5.- Secuencia nucleotdica de los genes de resistencia caracterizados en esta
tesis.

LOCUS DQ060147 2630 bp DNA linear BCT 12-JUN-2005


DEFINITION Lactococcus lactis subsp. lactis plasmid pAA291 transposon Tn916
TetM leader peptide and TetM genes, complete cds.
ACCESSION DQ060147
VERSION DQ060147.1 GI:67005924
KEYWORDS .
SOURCE Lactococcus lactis subsp. lactis
ORGANISM Lactococcus lactis subsp. lactis
Bacteria; Firmicutes; Lactobacillales; Streptococcaceae;
Lactococcus.
REFERENCE 1 (bases 1 to 2630)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Tetracycline tetM resistance gene from Lactococcus lactis AA29
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 2630)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Direct Submission
JOURNAL Submitted (13-MAY-2005) Instituto de Productos Lacteos de Asturias
(CSIC), Carretera de Infiesto s/n, Villviciosa 33300, Spain
FEATURES Location/Qualifiers

source 1..2630
/organism="Lactococcus lactis subsp. lactis"
/mol_type="genomic DNA"
/strain="AA29"
/sub_species="lactis"
/db_xref="taxon:1360"
/plasmid="pAA291"
repeat_region <1..>2630
/transposon="Tn916"
CDS 427..513
/note="regulates the transcription of TetM by attenuation"
/codon_start=1
/transl_table=11
/product="TetM leader peptide"
/protein_id="AAY62598.1"
/db_xref="GI:67005925"
/translation="MLCMPMVMHKNPSDKSIYHWDFYALLGF"
repeat_region 451..497
/rpt_type=inverted
CDS 529..2448
/function="tetracycline resistance"
/note="ribosome protection protein"
/codon_start=1
/transl_table=11
/product="TetM"
/protein_id="AAY62599.1"
/db_xref="GI:67005926"

/translation="MKIINIGVLAHVDAGKTTLTESLLYNSGAITELGSVDKGTTRTDNTLLERQRGITIQTGITSFQWENT
KVNIIDTPGHMDFLAEVYRSLSVLDGAILLISAKDGVQAQTRILFHALRKMGIPTIFFINKIDQNGIDLSTVYQDIKEKLSA
EIVIKQKVELYPNVCVTNFTESEQWDTVIEGNDDLLEKYMSGKSLEALELEQEESIRFQNCSLFPLYHGSAKSNIGIDNLIE
VITNKFYSSTHRGPSELCGNVFKIEYTKKRQRLAYIRLYSGVLHLRDSVRVSEKEKIKVTEMYTSINGELCKIDRAYSGEIV
ILQNEFLKLNSVLGDTKLLPQRKKIENPHPLLQTTVEPSKPEQREMLLDALLEISDSDPLLRYYVDSTTHEIILSFLGKVQM
EVISALLQEKYHVEIELKEPTVIYMERPLKNAEYTIHIEVPPNPFWASIGLSVSPLPLGSGMQYESSVSLGYLNQSFQNAVM
EGIRYGCEQGLYGWNVTDCKICFKYGLYYSPVSTPADFRMLAPIVLEQVLKKAGTELLEPYLSFKIYAPQEYLSRAYNDAPK
YCANIVDTQLKNNEVILSGEIPARCIQEYRSDLTFFTNGRSVCLTELKGYHVTTGEPVCQPRRPNSRIDKVRYMFNKIT"

ORIGIN
1 aactggtaaa tcctattcac aatcgtaagg ataatcaagt cacggtatcg ctgacagtgg

183
61 agtatatcga ccagcagacc aaagcaacgc aggtatctca atttgatttg gtacttgaaa
121 agaacgggag taattggaag attatagaat aacaaatatt ggtacattat tacagctatt
181 ttgtaatcac gtactctctt tgataaaaaa ttggagattc ctttacaaat atgctcttac
241 gtgctattat ttaagtatct atttaaaagg agttaataaa tatgcggcaa ggtattatta
301 aataaactgt caatttgata gcgggaacaa attattggat gtcctttttt aggagggctt
361 agttttttgt acccagttta agaatacctt tatcatgtga ttctaaagta tccggagaat
421 atctgtatgc tttgtatgcc tatggttatg cataaaaatc ccagtgataa gagtatttat
481 cactgggatt tttatgccct tttgggcttt tgaatggagg aaaatcacat gaaaattatt
541 aatattggag ttttagctca tgttgatgca ggaaaaacta ccttaacaga aagcttatta
601 tataacagtg gagcgattac agaattagga agcgtggaca aaggtacaac gaggacggat
661 aatacgcttt tagaacgtca gagaggaatt acaattcaga caggaataac ctcttttcag
721 tgggaaaata cgaaggtgaa catcatagac acgccaggac atatggattt cttagcagaa
781 gtatatcgtt cattatcagt tttagatggg gcaattctac tgatttctgc aaaagatggc
841 gtacaagcac aaactcgtat attatttcat gcacttagga aaatggggat tcccacaatc
901 ttttttatca ataagattga ccaaaatgga attgatttat caacggttta tcaggatatt
961 aaagagaaac tttctgccga aattgtaatc aaacagaagg tagaactgta tcctaatgtg
1021 tgtgtgacga actttaccga atctgaacaa tgggatacgg taatagaggg aaacgatgac
1081 cttttagaga aatatatgtc cggtaaatca ttagaagcat tggaactcga acaagaggaa
1141 agcataagat ttcagaattg ttctctgttc cctctttatc atggaagtgc aaaaagtaat
1201 atagggattg ataaccttat agaagttatt actaataaat tttattcatc aacacatcga
1261 ggtccgtctg aactttgcgg aaatgttttc aaaattgaat atacaaaaaa aagacaacgt
1321 cttgcatata tacgccttta tagtggagta ctacatttac gagattcggt tagagtatca
1381 gaaaaagaaa aaataaaagt tacagaaatg tatacttcaa taaatggtga attatgtaag
1441 attgatagag cttattctgg agaaattgtt attttgcaaa atgagttttt gaagttaaat
1501 agtgttcttg gagatacaaa actattgcca cagagaaaaa agattgaaaa tccgcaccct
1561 ctactacaaa caactgttga accgagtaaa cctgaacaga gagaaatgtt gcttgatgcc
1621 cttttggaaa tctcagatag tgatccgctt ctacgatatt acgtggattc tacgacacat
1681 gaaattatac tttctttctt agggaaagta caaatggaag tgattagtgc actgttgcaa
1741 gaaaagtatc atgtggagat agaactaaaa gagcctacag tcatttatat ggagagaccg
1801 ttaaaaaatg cagaatatac cattcacatc gaagtgccgc caaatccttt ctgggcttcc
1861 attggtttat ctgtatcacc gcttccgttg ggaagtggaa tgcagtatga gagctcggtt
1921 tctcttggat acttaaatca atcatttcaa aatgcagtta tggaagggat acgctatggt
1981 tgtgaacaag gattgtatgg ttggaatgtg acggactgta aaatctgttt taagtatggc
2041 ttatactata gccctgttag taccccagca gattttcgga tgcttgctcc tattgtattg
2101 gaacaagtct taaaaaaagc tggaacagaa ttgttagagc catatcttag ttttaaaatt
2161 tatgcgccac aggaatatct ttcacgagca tacaacgatg ctcctaaata ttgtgcgaac
2221 atcgtagaca ctcaattgaa aaataatgag gtcattctta gtggagaaat ccctgctcgg
2281 tgtattcaag aatatcgtag tgatttaact ttctttacaa atggacgtag tgtttgttta
2341 acagagttaa aagggtacca tgttactacc ggtgaacctg tttgccagcc ccgtcgtcca
2401 aatagtcgga tagataaagt acgatatatg ttcaataaaa taacttagtg tattttatgt
2461 tgttatataa atatggtttc ttgttaaata agatgaaata ttttttaata aagatttgaa
2521 ttaaagtgta aaggaggaga tagttattat aaactacaag tggatattgt gtcctgtatg
2581 tggaaataaa acacgattaa agataaggga agatactgaa ttaaaaaaat
//

LOCUS DQ060148 2630 bp DNA linear BCT 12-JUN-2005


DEFINITION Lactococcus lactis subsp. lactis plasmid pAA211 transposon Tn916
TetM leader peptide and TetM genes, complete cds.
ACCESSION DQ060148
VERSION DQ060148.1 GI:67005927
KEYWORDS .
SOURCE Lactococcus lactis subsp. lactis
ORGANISM Lactococcus lactis subsp. lactis
Bacteria; Firmicutes; Lactobacillales; Streptococcaceae;
Lactococcus.
REFERENCE 1 (bases 1 to 2630)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Tetracycline resistant tetM gene from Lactococus lactis subsp.
lactis AA21aTc
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 2630)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Direct Submission
JOURNAL Submitted (13-MAY-2005) Instituto de Productos Lacteos de Asturias
(CSIC), Carretera de Infiesto s/n, Villviciosa 33300, Spain
FEATURES Location/Qualifiers

184
source 1..2630
/organism="Lactococcus lactis subsp. lactis"
/mol_type="genomic DNA"
/strain="AA21aTc"
/sub_species="lactis"
/db_xref="taxon:1360"
/plasmid="pAA211"
repeat_region <1..>2630
/transposon="Tn916"
CDS 427..513
/note="regulates the transcription of TetM by attenuation"
/codon_start=1
/transl_table=11
/product="TetM leader peptide"
/protein_id="AAY62600.1"
/db_xref="GI:67005928"
/translation="MLCMPMVMHKNPSDKSIYHWDFYALLGF"
repeat_region 451..497
/rpt_type=inverted
CDS 529..2448
/function="tetracycline resistance"
/note="ribosome protection protein"
/codon_start=1
/transl_table=11
/product="TetM"
/protein_id="AAY62601.1"
/db_xref="GI:67005929"

/translation="MKIINIGVLAHVDAGKTTLTESLLYNSGAITELGSVDKGTTRTDNTLLERQRGITIQTGITSFQWENTKV
NIIDTPGHMDFLAEVYRSLSVLDGAILLISAKDGVQAQTRILFHALRKMGIPTIFFINKIDQNGIDLSTVYQDIKEKLSAEIVI
KQKVELYPNVCVTNFTESEQWDTVIEGNDDLLEKYMSGKSLEALELEQEESIRFQNCSLFPLYHGSAKSNIGIDNLIEVITNKF
YSSTHRGPSELCGNVFKIEYTKKRQRLAYIRLYSGVLHLRDSVRVSEKEKIKVTEMYTSINGELCKIDRAYSGEIVILQNEFLK
LNSVLGDTKLLPQRKKIENPHPLLQTTVEPSKPEQREMLLDALLEISDSDPLLRYYVDSTTHEIILSFLGKVQMEVISALLQEK
YHVEIELKEPTVIYMERPLKNAEYTIHIEVPPNPFWASIGLSVSPLPLGSGMQYESSVSLGYLNQSFQNAVMEGIRYGCEQGLY
GWNVTDCKICFKYGLYYSPVSTPADFRMLAPIVLEQVLKKAGTELLEPYLSFKIYAPQEYLSRAYNDAPKYCANIVDTQLKNNE
VILSGEIPARCIQEYRSDLTFFTNGRSVCLTELKGYHVTTGEPVCQPRRPNSRIDKVRYMFNKIT"

ORIGIN
1 aactggtaaa tcctattcac aatcgtaagg ataatcaagt cacggtatcg ctgacagtgg
61 agtatatcga ccagcagacc aaagcaacgc aggtatctca atttgatttg gtacttgaaa
121 agaacgggag taattggaag attatagaat aacaaatatt ggtacattat tacagctatt
181 ttgtaatcac gtactctctt tgataaaaaa ttggagattc ctttacaaat atgctcttac
241 gtgctattat ttaagtatct atttaaaagg agttaataaa tatgcggcaa ggtattatta
301 aataaactgt caatttgata gcgggaacaa ataattggat gtcctttttt aggagggctt
361 agttttttgt acccagttta agaatacctt tatcatgtga ttctaaagta tccggagaat
421 atctgtatgc tttgtatgcc tatggttatg cataaaaatc ccagtgataa gagtatttat
481 cactgggatt tttatgccct tttgggcttt tgaatggagg aaaatcacat gaaaattatt
541 aatattggag ttttagctca tgttgatgca ggaaaaacta ccttaacaga aagcttatta
601 tataacagtg gagcgattac agaattagga agcgtggaca aaggtacaac gaggacggat
661 aatacgcttt tagaacgtca gagaggaatt acaattcaga caggaataac ctcttttcag
721 tgggaaaata cgaaggtgaa catcatagac acgccaggac atatggattt cttagcagaa
781 gtatatcgtt cattatcagt tttagatggg gcaattctac tgatttctgc aaaagatggc
841 gtacaagcac aaactcgtat attatttcat gcacttagga aaatggggat tcccacaatc
901 ttttttatca ataagattga ccaaaatgga attgatttat caacggttta tcaggatatt
961 aaagagaaac tttctgccga aattgtaatc aaacagaagg tagaactgta tcctaatgtg
1021 tgtgtgacga actttaccga atctgaacaa tgggatacgg taatagaggg aaacgatgac
1081 cttttagaga aatatatgtc cggtaaatca ttagaagcat tggaactcga acaagaggaa
1141 agcataagat ttcagaattg ttctctgttc cctctttatc atggaagtgc aaaaagtaat
1201 atagggattg ataaccttat agaagttatt actaataaat tttattcatc aacacatcga
1261 ggtccgtctg aactttgcgg aaatgttttc aaaattgaat atacaaaaaa aagacaacgt
1321 cttgcatata tacgccttta tagtggagta ctacatttac gagattcggt tagagtatca
1381 gaaaaagaaa aaataaaagt tacagaaatg tatacttcaa taaatggtga attatgtaag
1441 attgatagag cttattctgg agaaattgtt attttgcaaa atgagttttt gaagttaaat
1501 agtgttcttg gagatacaaa actattgcca cagagaaaaa agattgaaaa tccgcaccct
1561 ctactacaaa caactgttga accgagtaaa cctgaacaga gagaaatgtt gcttgatgcc
1621 cttttggaaa tctcagatag tgatccgctt ctacgatatt acgtggattc tacgacacat
1681 gaaattatac tttctttctt agggaaagta caaatggaag tgattagtgc actgttgcaa

185
1741 gaaaagtatc atgtggagat agaactaaaa gagcctacag tcatttatat ggagagaccg
1801 ttaaaaaatg cagaatatac cattcacatc gaagtgccgc caaatccttt ctgggcttcc
1861 attggtttat ctgtatcacc gcttccgttg ggaagtggaa tgcagtatga gagctcggtt
1921 tctcttggat acttaaatca atcatttcaa aatgcagtta tggaagggat acgctatggt
1981 tgtgaacaag gattgtatgg ttggaatgtg acggactgta aaatctgttt taagtatggc
2041 ttatactata gccctgttag taccccagca gattttcgga tgcttgctcc tattgtattg
2101 gaacaagtct taaaaaaagc tggaacagaa ttgttagagc catatcttag ttttaaaatt
2161 tatgcgccac aggaatatct ttcacgagca tacaacgatg ctcctaaata ttgtgcgaac
2221 atcgtagaca ctcaattgaa aaataatgag gtcattctta gtggagaaat ccctgctcgg
2281 tgtattcaag aatatcgtag tgatttaact ttctttacaa atggacgtag tgtttgttta
2341 acagagttaa aagggtacca tgttactacc ggtgaacctg tttgccagcc ccgtcgtcca
2401 aatagtcgga tagataaagt acgatatatg ttcaataaaa taacttagtg tattttatgt
2461 tgttatataa atatggtttc ttgttaaata agatgaaata ttttttaata aagatttgaa
2521 ttaaagtgta aaggaggaga tagttattat aaactacaag tggatattgt gtcctgtatg
2581 tggaaataaa acacgattaa agataaggga agatactgaa ttaaaaaaat
//

LOCUS DQ060146 4174 bp DNA linear BCT 06-NOV-2006


DEFINITION Bifidobacterium longum TetW gene, complete cds.
ACCESSION DQ060146
VERSION DQ060146.2 GI:109693571
KEYWORDS .
SOURCE Bifidobacterium longum
ORGANISM Bifidobacterium longum
Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales;
Bifidobacteriaceae; Bifidobacterium.
REFERENCE 1 (bases 1 to 4174)
AUTHORS Florez,A.B., Ammor,M.S., Alvarez-Martin,P., Margolles,A. and
Mayo,B.
TITLE Molecular Analysis of tet(W) Gene-Mediated Tetracycline Resistance
in Dominant Intestinal Bifidobacterium Species from Healthy Humans
JOURNAL (er) Appl. Environ. Microbiol. 72 (11), 7377-7379 (2006)
PUBMED 16936047
REFERENCE 2 (bases 1 to 4174)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Direct Submission
JOURNAL Submitted (13-MAY-2005) Instituto de Productos Lacteos de Asturias
(CSIC), Carretera de Infiesto s/n, Villviciosa 33300, Spain
REFERENCE 3 (bases 1 to 4174)
AUTHORS Mayo,B. and Florez,A.B.
TITLE Direct Submission
JOURNAL Submitted (28-JUN-2006) Instituto de Productos Lacteos de Asturias
(CSIC), Carretera de Infiesto s/n, Villviciosa 33300, Spain
REMARK Sequence update by submitter
COMMENT On Jun 28, 2006 this sequence version replaced gi:67005922.
FEATURES Location/Qualifiers
source 1..4174
/organism="Bifidobacterium longum"
/mol_type="genomic DNA"
/isolate="H66"
/db_xref="taxon:216816"
misc_feature 521..1585
/note="similar to N-terminal part of probable permease
protein of ABC transporter system from Bifidobacterium
longum NCC2705 deposited in GenBank Accession Numbers
AAN24033 and AE014295"
CDS 2244..4163
/function="tetracycline resistance"
/note="ribosome protection protein"
/codon_start=1
/transl_table=11
/product="TetW"
/protein_id="AAY62597.1"
/db_xref="GI:67005923"

186
/translation="MKIINIGILAHVDAGKTTLTESLLYASGAISEPGSVEKGTTRTDTMFLERQRGITIQAAVTSFQWHRCKV
NIVDTPGHMDFLAEVYRSLAVLDGAILVISAKDGVQAQTRILFHALRKMNIPTVIFINKIDQAGVDLQSVVQSVRDKLSADIII
KQTVSLSPEIVLEENTDIEAWDAVIENNDELLEKYIAGEPISREKLAREEQQRVQDASLFPVYHGSAKNGLGIQPLMDAVTGLF
QPIGEQGGAALCGSVFKVEYTDCGQRRVYLRLYSGTLRLRDTVALAGREKLKITEMRIPSKGEIVRTDTAYQGEIVILPSDSVR
LNDVLGDQTRLPRKRWREDPLPMLRTAIAPKTAAQRERLLDALTQLADTDPLLRCEVDSITHEIILSFLGRVQLEVVSALLSEK
YKLETVVKEPSVIYMERPLKAASHTIHIEVPPNPFWASIGLSVTPLSLGSGVQYESRVSLGYLNQSFQNAVRDGIRYGLEQGLF
GWNVTDCKICFEYGLYYSPVSTPADFRSLAPIVLEQALKESGTQLLEPYLSFILYAPQEYLSRAYHDAPKYCATIETAQVKKDE
VVFTGEIPARCIQAYRTDLAFYTNGRSVCLTELKGYQAAVGQPVIQPRRPNSRLDKVRHMFQKVM"

ORIGIN
1 ctgcaggacc gaattgatgg tccggtctgc tttagccaga tatgggtact cgtcgatgac
61 gaaggcgatg cgttgctttg cggccagcgc gaacaccgct tccagtgcgg attcaaagct
121 gcggtatacg ggcgctgtct cgaacgcctc tgagcccttt tcatacagag caatgctgcg
181 gcttaacgat tcgagattct tggaagtgct ggattcgatt gccgaaaaga aaatcgtggg
241 gcgatcttcg aggaagcggt tgatgagtgt ggtcttgccc acgcggcgac ggccgtagac
301 cacgacgcac tcgaactgct gtgagctcca gcggcgttcc agtgccgaaa gctccttatc
361 tcgcccgacg aacatggccc ttgtctcctt gctttgctgt ctgccctggc gcacacgcgc
421 aaattataga atcgtaattt acttaattat gattctataa tttggtcgac tggtatgcaa
481 caggtctcga gcgtgcattg ccgcatgcgc aacgtttcgc gcgacacatt ccggggaatg
541 ttgcgcttag agagccgcgt atcgttgcta atgcggggaa cacgcagagt tgcgggccgt
601 tgcgattaga cgcaggctgt ctagactagt acgcgtttgc gaaagtcgaa cgtgcttcga
661 ccgatgaagg agagcttcgg tcatgcggcc aatgacaggc gcagtgcgaa gcacggctga
721 tcgtgaatcg cggcggcagc gattgccgcg tgcggcgcag gagcatgccg caccatgcca
781 caagcaaaca atccccaaga aacggtgatc tcctcacatg tacgtcgatc cgggaatcaa
841 taccagcgca agcaatctca aatgggtgct gccatactgc aaaccagacc tgccccgcgt
901 ggtaggcgcg cttctactgt tcatcgcaaa taacacgatg gccctgacca ttccgattct
961 gtccggtatc atcgtggacc gcgtcatcgt cggcggacat gccgacgaac tgccacgttt
1021 gtgtggactg atgatcctca tgaccatcat ccgcgtgggc gcgcgatacg gctaccagat
1081 gtggatggag cgtttcggcc agaactcggt ctaccgcctc gtctccgacg aatacgagaa
1141 gctgcacgag ctcgacttca cgtatttcaa ccacacacgc accggcgaca tcatgagccg
1201 cctgacctcc gacaccgacg cgatccgcca ctgcctgagt tgggtgagct atcagattgc
1261 cgactgcgtg gtcatgttcg tgggcgccct gtgcgtgatg ttcgccatcg attggcgact
1321 cgcgctcgtc ctggcctgcg tcacgccgtt catttttgtg ctcacgcgcg gcctgtccac
1381 gcacgcccac ccgctgttct tcgccatccg caactcgctg gcctccctca actccatggt
1441 cgaagagaac atcgagggca accgtgtggt caaggcattc gtgcgcgaac cgtacgagac
1501 cgaaaagttc gacgagcaca acgacgatta catgcagtgc aacatggcct tggcctacaa
1561 ctcgcgcaag tacatgccgt ggctgaatgc tcatgtacgg taaggaagca agcaaccgaa
1621 aacaagagat agaccgccag cactacacta ttccgaacca aagaccaaaa gataagcatt
1681 gtgggaaaat ctaaactttt ggattttcct acaatgccaa ctacggcgga atccctccca
1741 ctccttatat ctttctgtat acattgaatt tgtatttagt aaaatgcaga caacaccacg
1801 gatcggcttt tggttggaca attccaacca aacaccacag cagacagcag aaaacattct
1861 gaacgctagg aagccggtat gattgttaca tataagggga agaaaaattt cttttaggta
1921 cttgctttcc taaaactgat gtgatacaat gatttaatcc agaaaaggag taaaaaatat
1981 gcggcaaggt tttcttaaat aaaactataa tcaaatagtg ggaacaaagg attatgatag
2041 ctccttttgt aggggcttag ttttttgtac ccaatttaag aatacttttg ccttatcaat
2101 tttgacatat ccccaaaaac agcaatcaca aacaggtgta tgctgtatat gtgtatgtcc
2161 gcaacttata atccccagtg gtaaaagtat tttactgctg gggattttta tgccctttgg
2221 ggctgtaaag ggaggacaat cacatgaaaa taatcaatat tggaattctt gcccatgtag
2281 acgctggaaa gacgaccttg acggagagcc tgctatatgc cagcggagcc atttcagaac
2341 cggggagcgt cgaaaaaggg acaacgagga cggacaccat gtttttggag cggcagcgtg
2401 ggattaccat tcaagcggca gtcacttcct tccagtggca cagatgtaaa gttaacattg
2461 tggatacgcc cggccacatg gattttttgg cggaggtgta ccgctctttg gctgttttag
2521 atggggccat cttggtgatc tccgctaaag atggcgtgca ggcccagacc cgtattctgt
2581 tccatgccct gcggaaaatg aacattccca ccgttatctt tatcaacaag atcgaccagg
2641 ctggcgttga tttgcagagc gtggttcagt ctgttcggga taagctctcc gccgatatta
2701 tcatcaagca gacggtgtcg ctgtccccgg aaatagtcct ggaggaaaat accgacatag
2761 aagcatggga tgcggtcatc gaaaataacg atgaattatt ggaaaagtat atcgcaggag
2821 aaccaatcag ccgggaaaaa cttgcgcggg aggaacagca gcgggttcaa gacgcctccc
2881 tgttcccagt ctatcatggc agcgccaaaa atggccttgg cattcaaccg ttgatggatg
2941 cggtgacagg gctgttccaa ccgattgggg aacagggggg cgccgcccta tgcggcagcg
3001 ttttcaaggt tgagtacacc gattgcggcc agcggcgtgt ctatctacgg ttatacagcg
3061 gaacgctgcg cctgcgggat acggtggccc tggccgggag agaaaagctg aaaatcacag
3121 agatgcgtat tccatccaaa ggggaaattg ttcggacaga caccgcttat cagggtgaaa
3181 ttgttatcct tcccagcgac agcgtgaggt taaacgatgt attaggggac caaacccggc
3241 tccctcgtaa aaggtggcgc gaggaccccc tccccatgct gcggacggcg attgcgccga
3301 aaacggcagc gcaaagagaa cggctgctgg acgctcttac gcaacttgcg gatactgacc
3361 cgcttttgcg ttgcgaagtg gattccatca cccatgagat cattctttct tttttgggcc

187
3421 gggtgcagtt ggaggttgtt tccgctttgc tgtcggaaaa atacaagctt gaaacagtgg
3481 taaaggaacc ctccgtcatt tatatggagc ggccgctcaa agcagccagc cacaccatcc
3541 atatcgaggt gccgcccaac ccgttttggg catccatagg actgtctgtt acaccactct
3601 cgcttggctc cggtgtacaa tacgagagcc gggtttcgct gggatacttg aaccagagtt
3661 ttcaaaacgc tgtcagggat ggtatccgtt acgggctgga gcagggcttg ttcggctgga
3721 acgtaacgga ctgtaagatt tgctttgaat acgggcttta ttacagtccg gtcagcacgc
3781 cggcggactt ccgctcattg gccccgattg tattggaaca ggcattgaag gaatcgggga
3841 cgcagctgct ggaaccttat ctctccttca tcctctatgc gccccaggaa tacctttcca
3901 gggcttatca tgatgcaccg aaatactgtg ccaccatcga aacggcccag gtaaaaaagg
3961 atgaagttgt ctttactggc gagattcccg cccgctgtat acaggcatac cgtactgatc
4021 tggcctttta caccaacggg cggagcgtat gccttacaga gctgaaagga tatcaggccg
4081 ctgtcggtca gccggtcatc cagccccgcc gtccaaacag ccgcctggac aaggtgcgcc
4141 atatgtttca gaaggtaatg taaagataca taat
//

LOCUS DQ518904 2151 bp DNA linear BCT 31-MAY-2006


DEFINITION Lactobacillus johnsonii strain G41 PEP-PTS component gene, partial
cds; and MLS leader peptide, erythromycin and macrolide resistance
protein (erm(B)), and hypothetical protein genes, complete cds.
ACCESSION DQ518904
VERSION DQ518904.1 GI:102231545
KEYWORDS .
SOURCE Lactobacillus johnsonii
ORGANISM Lactobacillus johnsonii
Bacteria; Firmicutes; Lactobacillales; Lactobacillaceae;
Lactobacillus.
REFERENCE 1 (bases 1 to 2151)
AUTHORS Florez,A.B., Ammor,M.S., Delgado,S. and Mayo,B.
TITLE Molecular analysis of the erythromycin resistance gene (ermB) and
its flanking insertion sequences in Lactobacillus johnsonii G41
JOURNAL Unpublished
REFERENCE 2 (bases 1 to 2151)
AUTHORS Florez,A.B., Ammor,M.S. and Mayo,B.
TITLE Direct Submission
JOURNAL Submitted (27-APR-2006) Biotechnology and Food Characterization
Dairy Products, IPLA-CSIC, Carretera de Infiesto s/n, Villaviciosa,
Asturias 33300, Spain
FEATURES Location/Qualifiers
source 1..2151
/organism="Lactobacillus johnsonii"
/mol_type="genomic DNA"
/strain="G41"
/db_xref="taxon:33959"
CDS <1..477
/note="phosphoenolpyruvate-dependent sugar
phosphotransferase system EIID; probable
N-acetylgalactosamine specific"
/codon_start=1
/transl_table=11
/product="PEP-PTS component"
/protein_id="ABF70486.1"
/db_xref="GI:102231546"

/translation="ETQFLGAIIPTICGSIGAYMGLRGNPFGAFLWIIVNLIVLFTRFGFLNLGYSQGAKLIDSASGKLNAITD
SAILLGVTVVGALIPTVVNVKVPFVYRAGKVTLKMQTILNQIMPSLVPVLLVALVYWLLGKKHVTSTKMIWFVLILGIVLSYFH
ILSA"
misc_feature 564..573
/note="may be involved in the integration process"
RBS 813..818
CDS 826..909
/codon_start=1
/transl_table=11
/product="MLS leader peptide"
/protein_id="ABF70487.1"
/db_xref="GI:102231547"

188
/translation="MLVFQMRNVDKTSTVLKQTKNSDYADK"
gene 1023..1771
/gene="erm(B)"
RBS 1023..1028
/gene="erm(B)"
CDS 1034..1771
/gene="erm(B)"
/note="MLS methylase; ErmB"
/codon_start=1
/transl_table=11
/product="erythromycin and macrolide resistance protein"
/protein_id="ABF70488.1"
/db_xref="GI:102231548"

/translation="MNKNIKYSQNFLTSEKVLNQIIKQLNLKETDTVYEIGTGKGHLTTKLAKISKQVTSIELDSHLFNLSSEK
LKLNTRVTLIHQDILQFQFPNKQRYKIVGNIPYHLSTQIIKKVVFESRASDIYLIVEEGFYKRTLDIHRTLGLLLHTQVSIQQL
LKLPAECFHPKPKVNSVLIKLTRHTTDVPDKYWKLYTYFVSKWVNREYRQLFTKNQFHQAMKHAKVNNLSTITYEQVLSIFNSY
LLFNGRK"
CDS 1776..1907
/codon_start=1
/transl_table=11
/product="hypothetical protein"
/protein_id="ABF70489.1"
/db_xref="GI:102231549"

/translation="MSRFFKFGKLHVTKGNGDKLLDILLTASKKLKRSLAPTGNLYR"
misc_feature 2047..2056
/note="may be involved in the integration process"

ORIGIN
1 gagacacaat ttttgggcgc tattattcct acaatctgtg gatcaattgg tgcatatatg
61 ggattacgag gaaatccttt tggagctttc ttgtggataa ttgttaattt gattgtttta
121 ttcacaagat ttggcttttt aaacctcggt tattcacagg gagcaaaatt aattgattct
181 gcttcaggta agttaaatgc aattaccgac tctgctattt tacttggggt taccgtcgtt
241 ggggctttga taccaacagt agtgaatgtt aaagtaccat ttgtatatcg agcaggcaaa
301 gtaacattaa agatgcaaac aattctaaat caaattatgc catctttagt acctgtttta
361 cttgtagctt tagtttactg gttactagga aagaaacatg tgacgtccac taagatgatc
421 tggtttgttt tgattttagg aattgtttta agttacttcc atatcttaag tgcctaaagc
481 cagctttaat atttttaatg ctgaaatgga ggctattgtt ctataattaa aagaaacaat
541 aagattgata aagggacttt tatgaaagaa aaacgaaatg atacaccaat cagtgcaaaa
601 aaagatataa tgggagataa gacggttcgt gttcgtgctg acttgcacca tatcataaaa
661 atcgaaacag caaagaatgg cggaaacgta aaagaagtta tggaaataag acttagaagc
721 aaacttaaga gtgtgttgat agtgcattat cttaaaattt tgtataatag gaattgaagt
781 taaattagat gctaaaaatt tgtaattaag aaggagggat tcgtcatgtt ggtattccaa
841 atgcgtaatg tagataaaac atctactgtt ttgaaacaga ctaaaaacag tgattacgca
901 gataaataaa tacgttagat taattcctac cagtgactaa tcttatgact ttttaaacag
961 ataactaaaa ttacaaacaa atcgtttaac ttctgtattt gtttatagat gtaatcactt
1021 caggagagat tacatgaaca aaaatataaa atattctcaa aactttttaa cgagtgaaaa
1081 agtactcaac caaataataa aacaattgaa tttaaaagaa accgataccg tttacgaaat
1141 tggaacaggt aaagggcatt taacgacgaa actggctaaa ataagtaaac aggtaacgtc
1201 tattgaatta gacagtcatc tattcaactt atcgtcagaa aaattaaaac tgaatactcg
1261 tgtcacttta attcaccaag atattctaca gtttcaattc cctaacaaac agaggtataa
1321 aattgttggg aatattcctt accatttaag cacacaaatt attaaaaaag tggtttttga
1381 aagccgtgcg tctgacatct atctgattgt tgaagaagga ttctacaagc gtaccttgga
1441 tattcaccga acactagggt tgctcttgca cactcaagtc tcgattcagc aattgcttaa
1501 gctgccagcg gaatgctttc atcctaaacc aaaagtaaac agtgtcttaa taaaacttac
1561 ccgccatacc acagatgttc cagataaata ttggaagcta tatacgtact ttgtttcaaa
1621 atgggtcaat cgagaatatc gtcaactgtt tactaaaaat cagtttcatc aagcaatgaa
1681 acacgccaaa gtaaacaatt taagtaccat tacttatgag caagtattgt ctatttttaa
1741 tagttatcta ttatttaacg ggaggaaata attctatgag tcgctttttt aaatttggaa
1801 agttacacgt tactaaaggg aatggagata aattattaga tatactactg acagcttcca
1861 agaagctaaa gaggtcccta gcgcctacgg ggaatttgta tcgataaggg gtacaaattc
1921 ccactaagcg ctcgggaccc cttgtaggaa aatgtcctaa gtgtggcaac aatattgtat
1981 taaaaaaatc gttttatggt tgttcaaatt atcctgaatg taagtttact ttagctgaac
2041 attttagaaa gaaaaaacta accaaaacaa cgtatttctc gcagctatct ggatatgtat
2101 gctggtattg gcaatatagt tttgtggaat agatataaga aaactaatta a
//

189
LOCUS DQ486860 4255 bp DNA linear BCT 17-DEC-2006
DEFINITION Bifidobacterium breve UCC2003 ABC transporter gene, complete cds.
ACCESSION DQ486860
VERSION DQ486860.1 GI:94983656
KEYWORDS .
SOURCE Bifidobacterium breve UCC2003
ORGANISM Bifidobacterium breve UCC2003
Bacteria; Actinobacteria; Actinobacteridae; Bifidobacteriales;
Bifidobacteriaceae; Bifidobacterium.
REFERENCE 1 (bases 1 to 4255)
AUTHORS Margolles,A., Florez,A.B., Moreno,J.A., van Sinderen,D. and de los
Reyes-Gavilan,C.G.
TITLE Two membrane proteins from Bifidobacterium breve UCC2003 constitute
an ABC-type multidrug transporter
JOURNAL Microbiology (Reading, Engl.) 152 (12), 3497-3505 (2006)
REFERENCE 2 (bases 1 to 4255)
AUTHORS Margolles,A., Florez,A.B., Moreno,J.A., van Sinderen,D. and de los
Reyes-Gavilan,C.G.
TITLE Direct Submission
JOURNAL Submitted (10-APR-2006) Biotechnology, IPLA - CSIC, Ctra. Infiesto
s/n, Villaviciosa, Asturias 33300, Spain
FEATURES Location/Qualifiers
source 1..4255
/organism="Bifidobacterium breve UCC2003"
/mol_type="genomic DNA"
/db_xref="taxon:326426"
CDS 251..2161
/codon_start=1
/transl_table=11
/product="ABC transporter"
/protein_id="ABF50545.1"
/db_xref="GI:94983657"

/translation="MYVDPGINTSASNLKWVLPYCKPDLPRVAGALLLFIANNTMALTIPILSGIIVDRVIVGGHTDELPRLCG
LMILMTIIRVGARYGYQMWMERFGQNSVYRLVSDEYEKLHELDFTYFNHTRTGDIMSRLTSDTDAIRHCLSWVSYQIADCVVMF
IGALCVMFAIDWRLALALACATPFIFVLTRGLSTHAHPLFFAIRNSLASLNSMVEENIEGNRVVKAFVREPYETEKFDEHNDDY
MQRNMALAYNSRKYMPWLDGLGFSLQLITLGLGGFLVIKGYMTLGNLVSFNSFLWMIDGPVRASGWLINDWQRFNASCIKIRKL
LTEQPRIVEKPDAAEAVKQAVTIVEQVKRDDEATGVTGAPGASGTSGAQQSAERKSCPLRIKGDIRFAHVSFAFPDDPETPILK
DLDFTVPAGSKLGILGETGAGKSTLVSLISRFYDPTVGHVLIDGIDARDWPLATLRSQVCIVAQDTFLFSDTIGGNIGFGASDD
SDDRYIQKMAQIAGADNFIRSMPQGYDTVVGERGVGLSGGQRQRLSLARALADDPSILIMDDTTSAVDMETEAEIQKHLKEMDG
GKTIVTIAHRISSVKDSDLILVLEHGRIVERGTHAELVAAHGRYWGIYHKQLGLQSGAAQGF"

ORIGIN
1 ggggaacacg cagggttgtg ggctgttgcg attggacgca agccgcctag actagtacgc
61 gtttacgaaa gtcgaacgtg cttcgaccgg tgaaggagag ctccggtcgt gcgaccaatc
121 gccaatgatt gtcgcagtac gaagcacggc tgatcgtgaa tcgtggcggc aacgattgcc
181 gcgtgcggcg caggagcatg ccgcaccata ccacaagcaa acaatcccca agaaacggtg
241 atcccctcac atgtacgtcg atccgggaat caataccagc gcaagcaacc tcaaatgggt
301 gctgccatac tgcaaaccag acctgccccg cgtggcaggc gcgctcctac tgttcatcgc
361 caataacacg atggccctga ccattccgat tctgtccggc atcatcgtgg accgtgtcat
421 cgtcggcggg cataccgacg aactgccacg tctgtgcgga ctgatgatcc tcatgaccat
481 catccgcgtg ggcgcgcgat acggctacca gatgtggatg gagcgtttcg gccagaactc
541 ggtctaccgc ctcgtctccg acgaatacga gaagctgcac gagctcgact tcacgtactt
601 caaccacaca cgcaccggcg acatcatgag ccgcctgacc tccgacaccg acgcgatccg
661 ccactgcctg agttgggtga gctaccagat cgccgactgc gtggtcatgt tcatcggtgc
721 cctgtgtgtg atgtttgcca tcgactggcg acttgcgctc gccctggcct gcgccacgcc
781 gttcattttt gtgctcacgc gcggcctgtc cacgcacgcc cacccgctgt tcttcgccat
841 ccgcaactcg ctggcctccc tcaactccat ggtcgaggag aacatcgagg gcaaccgtgt
901 ggtcaaggca ttcgtgcgcg aaccgtacga aaccgaaaag ttcgacgaac acaacgacga
961 ctacatgcag cgcaacatgg ccctggccta caactcgcgc aagtacatgc cctggctgga
1021 cggcctcggc ttttcgctgc agctcatcac gctcggcctc ggcggcttcc tggtcatcaa
1081 gggctacatg acccttggca acttagtgag cttcaactcc ttcctgtgga tgatcgacgg
1141 cccggtgcgt gcctccggct ggctcatcaa cgactggcag cgattcaacg ccagctgtat

190
1201 caagattcgc aagctgctca ccgaacagcc gcgcatcgtg gagaagcccg acgcggccga
1261 agccgtcaag caggcggtca caatcgtcga gcaggtcaag cgcgatgatg aggctaccgg
1321 tgttaccgga gcgcccgggg cttccggaac atccggtgcc cagcagtccg ccgaacgcaa
1381 gtcctgcccg ctgcgcatca aaggcgacat ccgctttgcc cacgtcagtt tcgccttccc
1441 ggacgacccg gaaaccccga tcctcaaaga cctcgacttc accgtgcctg ccggctccaa
1501 actcggcatc ctcggcgaga ccggtgccgg caagtccacg ctcgtcagcc tcatctcccg
1561 cttctacgac ccaaccgtgg gccatgtgct catcgacggc atcgatgcac gcgactggcc
1621 gctggccacc ttgcgctcgc aggtgtgcat cgtggcgcag gacaccttct tgttctccga
1681 caccatcggt ggcaatatcg gctttggtgc cagcgatgat agcgacgacc gatatatcca
1741 gaaaatggcc cagatcgccg gcgcggacaa tttcatcagg tcgatgccgc agggttatga
1801 caccgtcgtg ggcgaacgcg gcgtgggcct gtccggcggg cagagacagc gcctgtcgct
1861 ggcccgcgcg ctcgcggatg atccgtcgat cctgattatg gatgatacga cctcggccgt
1921 cgatatggag accgaggcgg aaatccagaa gcacctcaag gagatggacg gcggcaagac
1981 catcgtcacc atcgcccacc gcatctcgtc ggtcaaggat tccgacctga ttctggtgct
2041 cgaacacgga cgcatcgtcg aacgcggcac ccacgccgaa ctcgtggccg cacacggccg
2101 ctactggggc atctaccaca agcagctcgg actccagtcc ggcgccgcac aggggtttta
2161 accgtcgttc aacctccctc tgacgagggc tgagccgtga agaaaacagc cctgtgggct
2221 gtttttaggc gagggcgaac cgataggtga gcaataaata aaggaagcga tgacggaggg
2281 ggagattttg acgacatatc tttcccccag gcagcttcgc tgacagcccc ctcgccggag
2341 ggggcttgag aagaggactt atggcacaac gcaatacatt tcgcgaggat gaggagctgg
2401 aggagtccat caacctccac gacatcgctc gcgtcggcaa atatctcaaa ccgtacattt
2461 cgcgcattgt gcgcatcctg gcggtcgtgg tctcgatgag ctgcatcgtg gtcagcgtcc
2521 cgtacctgac caagattatg attgacgacg cgattccgaa caaggatctc ggcaaactgg
2581 ccatgctggc cggcgggctg ttctgcctga tcgtgatcta cgaactcgcg ctgcgctacc
2641 gcacggtggc cattactcgc gtgggccagc tcatgctcaa ggacatgcgc cgcgatctgt
2701 tcacacacat ccagaccctg ccgttcagct actttgactc gcgcccgcac ggcaagatcc
2761 tgatccgcgt ggtcaactat gtgaacaccc tttcggacgc gctctcctcg ggtctgatca
2821 acgtgttctc cgacatcttc acattcttcg tgacgctgat cgtcatgttc gccatcgact
2881 ggcagctggc attgtggagc ctcgtactgt tcccggtgct catcatctgg gtgcgtgtgc
2941 tgcagtactt ccagcgcaag gcttatcagg tgctgtccaa caagcagagc aatctgaacg
3001 ccttcatcca cgagtcaatc gccggcgtga agaccacgca gaccttcgcc cgcgagaagg
3061 agcagttcga caccttccag cagcagcaag acgacgtacg taccgcctgg atggccgcca
3121 agcacattga attcctgatg tggccgggcg tgcagaccat ctccgtgatg accatcgcgt
3181 tcatctactt cgtgggcatc accggcttcg gcggcgtgag cgtgaccacc ggtatgctta
3241 tcgcgttcgt gggctacgcg aacaacttct ggaacccggt gatcaacatc ggcaacttct
3301 acaaccagct ggtcacctgc tccgcctacc tcgaacgcat cttcgagacg ctggacgtca
3361 agcccgagat cgcgaacaag ccgggtgcca ccgagctgcc cgccattcaa ggcaaggtcg
3421 atttcaacga cgtcgtgttc cggtacgagg acgacggccg taacatcctg aacttggtcg
3481 acttccacgt caccccgggc aagaccatcg ccctggtcgg cccgaccggc gccggcaaga
3541 ccaccatcgt gagcctgctg tcccggttct acgacgtgtc ggaaggttcg gtgaccattg
3601 atggccacga cgtgcgcgac gtgacgcttg aatcgttgcg ccggcagatg ggcgtgatgc
3661 tgcaggacac gttcatcttc tccggcaacg tacgtgacaa catccgctac ggcaagctcg
3721 acgccaccga cgaggagatc gaggcggcgg cgaaggccgt gcacgcgcac gagttcatca
3781 tggagctgcc tgatggctac gacaccacgg tggaggagcg cggctcgacc ctgtccgccg
3841 ggcagcgcca gctcatcgcg ttcgcgcgtg tgctgctcgc cgacccgcgc atcctgattc
3901 tggacgaggc gacctcgaac atcgacaccc gcaccgagga ggcgttgcag gccggcttgc
3961 ggcatctgct caagggtcgc acgagcttca tcatcgccca ccggctgtcc accatcgaga
4021 acgcggacca gatcttctac atcgatcacg gccagatcgt ggagcacggc acgcacgccg
4081 aactgctgga ggccaagggc gcgtactacc gcctctacga gtcccaatac gcgatgattc
4141 gcgtggccac cggggctact gagtagctcg gccacccgta gctgccccat atccccaatc
4201 tcccgaattt gtactgctgg tcgttactga gtagtacgaa ttcgggagat gggga
//

191