Journal of Microbiological Methods 80 (2010) 281–286

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Journal of Microbiological Methods

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j m i c m e t h

Identification of five human and mammal associated Arcobacter species by a novel

multiplex-PCR assay
Laid Douidah a, Lieven De Zutter a, Peter Vandamme b, Kurt Houf a,⁎
a
Ghent University, Faculty of Veterinary Medicine, Department of Veterinary Public Health and Food Safety, Salisburylaan 133, 9820 Merelbeke, Belgium
b
Ghent University, Department of Biochemistry, Physiology and Microbiology, Faculty of Sciences, K. L. Ledeganckstraat 35, 9000 Ghent, Belgium

a r t i c l e i n f o a b s t r a c t

Article history: A multiplex-PCR assay with seven primers was developed for the identification of the five human and
Received 17 October 2009 mammal related species of the emerging foodborne pathogen Arcobacter. The assay was validated using 58
Received in revised form 8 January 2010 reference and 358 collection strains isolated from humans and mammals. The selected primers on the 23 S
Accepted 13 January 2010
RNA gene amplify a 2061 bp fragment from A. butzleri, a 1590 bp fragment from A. thereuis, a 1125 bp
Available online 21 January 2010
fragment from A. cibarius and an A. skirrowii specific fragment of 198 bp. For A. cryaerophilus, a primer set on
Keywords:
the gyrA gene amplified a specific fragment of 395 bp. No PCR product was generated for closely related
Arcobacter bacteria including Campylobacter and Helicobacter species. Furthermore, examination of the 23 S RNA gene
Multiplex PCR of A. cryaerophilus revealed, besides large heterogeneity, the presence of intervening sequences ranging from
Identification 87 to 196 bp.
Foodborne pathogen © 2010 Elsevier B.V. All rights reserved.

1. Introduction (Vandenberg et al., 2004). Although some potential virulence factors

The genus Arcobacter, created in 1991 to include the former
aerotolerant campylobacters (Vandamme et al., 1991), comprises at
present nine fully characterized species. Three species, Arcobacter
butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii were
immediately associated both with human and animal diseases,
however without clear-cut evidence for a pathogenic role. Isolation
of those species over the last decade from diverse foods of animal origin
resulted in the classification of arcobacters as emerging foodborne
pathogens by the International Commission on Microbiological Speci-
fications for Foods (ICMSF) (International Commission on Microbiolog-
ical Specifications for Foods, 2002). Two species, Arcobacter cibarius
(Houf et al., 2005) and Arcobacter thereius (Houf et al., 2009) were more
recently isolated from poultry and pigs, but not from human specimens
yet. Besides, the species Arcobacter nitrofigilis (Mcclung et al., 1983),
Arcobacter halophilus (Donachie et al., 2005), Arcobacter mytili (Collado
et al., 2009) and recently Arcobacter marinus (Kim et al., in press) have
been isolated from environmental matrices and shellfish, but not from
mammals. They are phylogenetically distinct from the other five species
and their metabolic requirements, especially those of A. nitrofigilis and
A. halophilus, suggest an unlikely importance in veterinary and human
medicine.
Arcobacter skirrowii has rarely been isolated from human patients
(Samie et al., 2007; Wybo et al., 2004), but predominantly A. butzleri and
A. cryaerophilus are associated with enteritis and bacteraemia in humans
⁎ Corresponding author. Tel.: + 32 09 264 73 41; fax: + 32 09 264 73 41.
E-mail address: Kurt.Houf@UGent.be (K. Houf).
have recently been identified, the mechanisms of their pathogenicity
are not elucidated yet (Houf and Stephan, 2007). Clinical symptoms of
Arcobacter infections are similar to those of campylobacteriosis, but a
higher frequency of persistent and watery diarrhea has been reported
(Vandenberg et al., 2004). Contaminated drinking water has been
identified as a major infection source in developing countries (Taylor
et al., 1991), though the manipulation, consumption and cross-
contamination of raw and undercooked meat products are more likely
infection routes in industrialized countries (Ho et al., 2006). Recently,
close contact with pets has been suggested as another potential
infection source (Fera et al., 2009; Houf et al., 2008; Petersen et al.,
2007). The common presence of arcobacters on poultry products has
frequently been reported, but its origin is still debated (Ho et al., 2008;
Van Driessche and Houf, 2007b). Arcobacters have rarely been isolated
from the intestinal content of poultry, and therefore, in contrast to pigs
and cattle (Van Driessche et al., 2003, 2004; Van Driessche and Houf,
2007a), a fecal origin of the carcass contamination is questionable.
Arcobacters are, like campylobacters, metabolically inert which
hampers correct identification (On, 1996). Therefore, molecular
methods have soon taken over identification based on biochemical
tests. The DNA-based assays developed for the identification of
arcobacters at genus and species levels, target mainly the small or
large subunits of the ribosomal RNA genes (Figueras et al., 2008;
Harmon and Wesley, 1997; Houf et al., 2000; Kabeya et al., 2003).
None of the current molecular identification assays allow to
differentiate between the five mammal associated Arcobacter species.
Moreover, the growing number of new Arcobacter species raises
questions about their specificity. The aim of the present study was to
therefore examine the 16S and 23S RNA genes, the rpoB and the gyrA

0167-7012/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.mimet.2010.01.009
282 L. Douidah et al. / Journal of Microbiological Methods 80 (2010) 281–286

genes to select species-specific primers and to develop and validate a
single run multiplex-PCR assay (m-PCR) to allow a rapid and
simultaneous identification of A. butzleri, A. cryaerophilus, A. skirrowii,
A. cibarius and A. thereuis.
2. Materials and methods
2.1. Reference bacterial strains and isolation of chromosomal DNA
Reference strains of Arcobacter species and closely related bacteria
were obtained from the Belgian Co-ordinated Collections of Micro-
organisms (BCCM), the Laboratory of Microbiology Ghent (LMG)
Ghent University (Ghent, Belgium) and from the Collection of the
Institute Pasteur (CIP, France) (Table 1). Arcobacter reference strains
were grown on Mueller Hinton agar (CM 0337, Oxoid, Basingstoke,
UK) supplemented with 5% defibrinated horse blood (E&O Laborato-
ries Ltd., Bonnybridge, Scotland) and incubated for 48 h at 30 °C under
microaerobic conditions by evacuating 80% of the normal atmosphere
and introducing a gas mixture of 8% CO2, 8% H2 and 84% N2 into the jar.
Cultivation of the other test organisms was performed according to
their specific needs. A cell suspension of each strain was prepared in
10 ml of sterile water with an optical density of 0.074 ± 0.002
(measured at 660 nm) which corresponded to approximately
107 CFU/ml. Template DNA was extracted from a 0.5 ml cell
suspension of each strain by the guanidinium thiocyanate method
described by Pitcher et al. (1989). Five µl of each DNA preparation was
size separated by electrophoresis to evaluate the integrity of the DNA
extract. The concentration of each DNA template was determined spec-
trophotometrically (Biophotometer, Eppendorf, Hamburg, Germany) at
260 nm and adjusted to 50 ng µl− 1. The DNA templates were stored at
−20 °C.
2.2. Primer selection
For the development of primers, partial 16S rRNA and 23S rRNA gene
sequences of A. butzleri LMG 10828T, A. cryaerophilus taxon 1A LMG
9865 and LMG 10210, A. cryaerophilus taxon 1B LMG 9867 and LMG
10209, A. skirrowii LMG 6621T, A. cibarius LMG 21996T, A. thereuis LMG
24486T, A. nitrofigilis LMG 7604T, A. mytili LMG 24559T and A. halophilus
CIP 108450T were obtained from previous studies (Houf et al., 2005,
2009) or determined in the present study as previously described using
a QIA quick spin column (Qiagen, Hilden, Germany) to purify the PCR
product and an ABI 3730xl sequencer (Applied Biosystems, Foster City,
USA) with the Prism BigDye terminator cycle sequencing kit (Applied
Biosystems) (Houf et al., 2005, 2009). The partial sequences of the 23S
rRNA genes of A. butzleri LMG 10828T, A. cryaerophilus taxon 1A LMG
9865 and LMG 10210, A. cryaerophilus taxon 1B LMG 9867, LMG 10209,
A. skirrowii LMG 6621T, A. cibarius LMG 21996T and A. mytili LMG 24559T
are deposited in the EMBL database as FN 600698, 600699, 600700,
600701, 600702, 600703, 600704 and 600705 respectively. Besides,
partial sequences of the rpoB gene, which code for the β-subunit of RNA
polymerase were retrieved from GenBank® (National Institutes of
Health) and examined for the presence of species-specific regions
(accession numbers: AB104478, AB104480 and AB104481, AB104482,
AB104483, AB104484, AF136503, AF136504, and AF136505). For the
gyrA gene, which encodes the A subunit of DNA gyrase, partial
sequences were additionally determined for the species A. thereuis
and A. mytili in this study using the primers forward: 5′-AGGTG-
GAAAGGWAAAGTTGC-3′ and reverse 5′-TCAACTCTAATCATTTTWCCA-
3′ based on the sequences of the different species deposited in
GenBank® (accession numbers DQ464331, DQ464332, DQ464333,
DQ464334, DQ464335, DQ464336, DQ46437, DQ464338, DQ464339,
EF067876, EF067877, FJ754218 and FJ754219). All sequences of each
gene were aligned using FastPCR (Kalendar, 2007) and BioEdit 7.0.9
(Hall, 1999) and examined for Arcobacter genus and species-specific
regions. The corresponding genes of Campylobacter and Helicobacter
species, Escherichia coli and Salmonella Enteritidis were used as outliers
to avoid targeting highly conserved or non-specific regions (Table 1).
2.3. PCR conditions and electrophoresis
PCR reactions were performed in a reaction mixture (50 µL final
volume) composed of water (W4502, Sigma-Aldrich, Irvine, Ayrshire,
UK), 5 µL 10× PCR buffer (Invitrogen, Carlsbad, California, USA), 1.5 U
Taq polymerase (Invitrogen) and a deoxynucleotide triphosphate
mixture at a final concentration of 0.2 mM each (Invitrogen). Several
concentrations of primers, MgCl2 (Invitrogen) and DNA template were
tested. Annealing was performed at different temperatures ranging
from 50 to 68 °C. Prior to cycling, samples were heated at 94 °C for 3 min.
Cycles were extended to 5 min during the final elongation step. For all
experiments, the Veriti™ 96-Well Thermal Cycler (Applied Biosystems,
Foster city, USA) was used. The PCR products were size separated in 2%
agarose gels (Invitrogen) in 0.5× Tris–borate–EDTA buffer (Sigma) at
120 V for 2 h. Gels were stained with ethidium bromide (E8751, Sigma)
1 µg/ml. An UV transilluminator and photograph system (MICROdoc,

Table 1
Reference strains included for specificity in this study.

Species Strain number Amplification products

Arcobacter butzleri LMG 6620, 9869, 9906, 9910, 9939, 10220, 10223, 10828T, 10900, 11120, 11632, 14714 and 15557 2061 bp
Arcobacter cryaerophilus subgroup 1 LMG 9065, 9863, 9865, 9904T and 10210 395 bp
Arcobacter cryaerophilus subgroup 2 LMG 9867, 9947, 9948, 10216, 10209, 10211, 10212, 10213, 10216, 10217, 10218, 10219, 10221, 395 bp
10222, 10224, 10225, 10226, 10228, 10230, 10231, 10232, 10233, 10237, 10241, 10242 and 10244
Arcobacter skirrowii LMG 6621T, 9801, 9911, 9912, 10234 and 14985 198 bp
Arcobacter cibarius LMG 21996T and 21997 1125 bp
Arcobacter thereius LMG 24486T and 24487 1590 bp
Arcobacter nitrofigilis LMG 7547 and 7604T –
Arcobacter mytili LMG 24559T –
Arcobacter halophilus CIP 108450T –
Campylobacter jejuni LMG 6444T and 7790 –
Campylobacter coli LMG 6440T and 8530 –
Campylobacter lari 8845 and 8846T –
Campylobacter upsaliensis 8850T and 8852 –
Helicobacter pullorum 16 317T and 16318 –
Escherichia coli 2092T –
Salmonella Enteritidis 7233T –
T
: type strain.
–: no amplicon generated.
LMG: LMG: Culture Collection of the Laboratory for Microbiology, Ghent University, Belgium.
CIP: Collection de l'Institut Pasteur, France.
 L. Douidah et al. / Journal of Microbiological Methods 80 (2010) 281–286 283

Table 2 A. thereuis strains from pigs, 34 A. skirrowii strains from pigs and cattle,
Sequence and origin of the sets of primers. 11 A. cibarius strains from broiler carcasses and 86 A. cryaerophilus
Gene Primers Primer sequence strains from humans, pigs, cattle, sheep, dogs and poultry products. All
strains were additionally identified by amplified fragment length
23S rRNA ButR 5′-TCCTGATACAAGATAATTGTACG-3′
23S rRNA TherR 5′-GCAACCTCTTTGGCTTACGAA-3′ polymorphism assay according to Debruyne et al. (2010) and
23S rRNA CibR 5′-CGAACAGGATTCTCACCTGT-3′ characterized below the species level using ERIC-PCR to avoid the
23S rRNA SkiR 5′-TCAGGATACCATTAAAGTTATTGATG-3′ inclusion of duplicate strains (Houf et al., 2002a). In addition, serial
23S rRNA ArcoF 5′-GCYAGAGGAAGAGAAATCAA-3′
dilutions of spectrophotometrically quantified DNA (ranging from
23S rRNA CryF 5′-CAGAGGAAGAGAAATCAAAT-3′
23S rRNA CryR 5′-CCCACTATTCCATCAGTGAG-3′ 400 ng to 5 ng per reaction) of each species were tested. The robustness
Gyrase A GyrasF 5′-AGAACATCACTAAATGAGTTCTCT-3′ was tested by using DNA lysates prepared by boiling bacterial
Gyrase A GyrasR 5′-CCAACAATATTTCCAGTYTTTGGT-3′ suspensions for 10 min as previously described (Houf et al., 2002a)
F: forward; R: reverse. instead of DNA extracts, and by performing the assay on two additional
An artificial nucleotide substitution (T→A) in the TherR primer is shown in bold and cyclers: the icycler Thermal cycler (Biorad) and Geneamp PCR system
underlined. 9700 (Applied Biosystems).

2.5. Heterogeneity of the A. cryaerophilus 23S RNA gene

Cleaver Scientific, Warwickshire, UK) with an analyst computer
program (Easy software, Kodak) was used for visualization.
2.4. Sensitivity and specificity assessment
The validation of the m-PCR assay was performed by testing 58
reference strains of Arcobacter species and their nearest phylogenetic
neighbours (Table 1). Furthermore, a collection of 358 Arcobacter strains
from humans and animals isolated during several studies (Houf et al.,
2002b, 2005, 2008; Van Driessche et al., 2004, 2005) was used to further
evaluate the sensitivity and specificity of the m-PCR assay. These
strains have all been isolated from different biological specimens by the
method of Houf et al. (2001a) which uses amphotericin, cefoperazone,
5-fluorouracil, novobiocin, and trimethoprim as selective supplements.
All strains have been stored on beads (Microbank, Pro-Lab Diagnostics,
Richmond Hill, On, Canada) at −80 °C and were cultivated for the
present study as described above. This collection included 113 A. butzleri
strains from humans, pigs, cattle, sheep, dogs and broiler carcasses, 114
The alignment of partial 23S rRNA gene fragments of A. cryaerophilus
strains revealed that the length of the 23S rRNA gene of A. cryaerophilus
strains was highly variable. To evaluate this heterogeneity further a
restriction fragment length polymorphism analysis study was per-
formed on all 31 A. cryaerophilus reference strains (Table 1) and on a
random selection of 46 A. cryaerophilus collection strains. First, a PCR
assay using the primer set CryF-CryR targeting the 23S rRNA
gene (Table 2) was performed under the same conditions described
below for the m-PCR assay. Restriction enzyme digestion was performed
in a mix of 10 µL of the PCR mix after amplification, 2 µL of 10× enzyme
buffer, 2 µL restriction enzyme and 6 µL of nuclease free water and
incubated for 3 h at a temperature according to specific enzyme needs.
Twenty µl of the enzyme digested mixture was separated in a 1.5%
agarose gel for 1.5 h. Gels were stained as described above. The
restriction enzymes tested were TaqI, SduI, Alw21I, and XapI, preselected
by the Fast-PCR program based on 23S RNA gene sequences of
A. cryaerophilus present in the GenBank® and those sequenced in the
present study.

Fig. 1. Agarose gel showing the relative sizes of the PCR products for the five Arcobacter species and the specificity of the m-PCR. Lane 1, 100-bp marker (100 to 1500 and 2072 bp);
2 and 3, A. butzleri (LMG 6620, 10828T); 4 and 5, A. thereuis (LMG 24486T, 24487); 6 and 7, A. cibarius (LMG 21996T, 21997); 8, A. cryaerophilus 1A (LMG 9904T); 9, A. cryaerophilus 1B
(LMG 9947); 10 and 11, A. skirrowii (LMG 6621T, 14985); 12 and 13, A. nitrofigilis (LMG 7547, 7604T); 14, A. mytili (LMG 24559T); 15, A. halophilus (CIP108450T); 16, C. jejuni (LMG
6444T); 17 H. pullorum (LMG 16317T); 18, E. coli (LMG 2092T); 19, S. enteritidis (LMG 10395T); 20, negative control. T: type strain. LMG: Culture Collection of the Laboratory for
Microbiology, Ghent University, Belgium.
284 L. Douidah et al. / Journal of Microbiological Methods 80 (2010) 281–286

Fig. 2. Agarose gel showing the five major patterns (I–V) and deviated patterns (I′, II′, VI and VII) obtained after RFLP-PCR analysis of the 23S RNA gene of A. cryaerophilus strains.
Lanes 1 and 19, 100-bp marker (100 to 1500 and 2072 bp); 2, R360; 3, R345; 4, LMG 10222; 5, LMG 10219; 6, LMG 9867; 7, LMG 10231; 8, LMG 10230; 9, R212; 10, R 8631; 11, R159;
12, LMG 10209; 13, LMG 9944; 14, LMG 10210; 15, LMG 6622; 16, LMG 9865; 17, LMG 9065; 18, LMG 9863. LMG: Culture Collection of the Laboratory for Microbiology, Ghent
University, Belgium. R = field isolate.

3. Results Both crude lysates as well as DNA extracts are equally useful and
there is no need for preceding DNA concentration adjustment (data
3.1. Primer design and PCR conditions not shown).

Examination of the alignments of the 16S rRNA genes revealed no 3.2. Heterogeneity of the A. cryaerophilus 23S RNA gene
regions useful for the development of Arcobacter species-specific
primers. In contrast, the 23S rRNA gene proved useful and an Arco- An expected fragment of 1292 bp using primers cryF and cryR
bacter genus specific forward primer (ArcoF) was combined with (Table 2) targeting the 23S RNA gene was generated for 87% (67 of 77)
species-specific reverse primers which resulted in the development of of the strains. The restriction enzyme TaqI was the most discrimina-
a m-PCR assay (Table 2). tory enzyme; its use resulted in five (I–V) different fragments profiles
The ArcoF-ButR primer pair amplified a 2061 bp fragment and was within A. cryaerophilus (Fig. 2, Table 3). The restriction profile of the
specific for all A. butzleri strains tested. With ArcoF-TherR primers, a strains did not correlate with their taxonomic status (belonging to
1590 bp fragment was generated for A. thereuis strains and an amplicon taxon 1A or 1B), or with an isolation source or geographical origin. Of
of 1125 bp was obtained for A. cibarius strains using the primer each of the five patterns, at least one strain was selected for
combination ArcoF-CibR. Combining the primers ArcoF with SkirR subsequent 23S rRNA sequence analysis. Alignment of the resulting
generated an A. skirrowii specific fragment of 198 bp. For A. cryaerophilus sequences using BioEdit 7.0.9 software (Hall, 1999) revealed that
the combination of a genus specific and species-specific primer set pattern I can be considered the ancestor pattern with patterns II to V
appeared not possible: testing multiple sets of primers specific for being the result of single or multiple base mutations creating
A. cryaerophilus, resulted in no amplicon or amplicons longer than additional restriction sites. For nine strains, PCR amplicons of 87 bp
predicted when applied to the A. cryaerophilus reference and collection (5 strains), 119 bp (1 strain) or 196 bp (3 strains) longer than 1292 bp
strains. Therefore, the partial sequences of the rpoB and gyrA genes of were obtained and restriction analysis resulted in patterns I′, VI and
Arcobacter and related species were examined for Arcobacter species- VII respectively (Table 3, Fig. 1). Sequencing the 23S rRNA gene
specific regions. Examination for primer combinations based on the fragments of those strains showed that the intervening sequence of
partial sequences of the rpoB gene revealed no species-specific regions 119 bp is composed of a 109 bp fragment with 10 extra bases inserted
(data not shown). For the gyrA gene, the primer set GyrasF and GyrasR in three positions. The 196 bp sequence comprises the 87 and the
amplified a stable A. cryaerophilus specific fragment of 395 bp (Table 1). 109 bp fragments which were located at different positions within the
All primers were first separately subjected and then combined in a gene (Fig S1). For the pattern II′ (strain R8631, Fig. 2, Fig S1), no
multiplex set-up to various compositions of PCR mixes and cycling intervening sequences were detected.
conditions. Finally, a concentration of 1.5 mmol of MgCl2 and 50 pmol
of each primer, ButR, SkiR, TherR, CibR, ArcoF, GyrasF and GyrasR
Table 3
showed to be the optimal PCR mix. The PCR assay involved 30 cycles
Restriction fragment length polymorphism of a partial 23S RNA gene sequence of 77
of denaturation (94 °C, 45 s), primer annealing (58 °C, 45 s) and chain A. cryaerophilus strains.
extension (72 °C, 2 min). After electrophoresis under the conditions
Amplicon size Pattern Main fragments (bp) > 150 bp Number of strains
provided above, a clear-cut species identification of the five species
was possible, as illustrated in Fig. 1. 1292 bp I 269, 914 41
With this set-up, all 58 reference and 358 collection strains were 1380 bp I′ 269, 1001 5
1292 bp II 269, 384, 530, 914 4
correctly identified and no fragments were generated for A. nitrofigilis, 1292 bp II′ 269, 384, 530, 914 1
A. mytili and A. halophilus, nor for the other non-arcobacters included 1292 bp III 269, 384, 530 9
in the test panel (Table 1). To achieve this result, a base substitution of 1292 bp IV 177, 196, 269, 384, 530 1
thymine into adenine in the original A. thereuis primer (TherR) was 1292 bp V 177, 196, 269, 530 12
1414 bp VI 269, 506, 528, 1034 1
necessary to prevent a weak amplification of A. halophilus in the final
1492 bp VII 269, 496, 618, 1114 3
conditions (Table 1).
 L. Douidah et al. / Journal of Microbiological Methods 80 (2010) 281–286
4. Discussion
Though arcobacters are commonly isolated from animals, food and
environmental samples, and cases of human infections are regularly
reported worldwide, several aspects actually hamper the risk
assessment for these bacteria. Besides the fact that human clinical
samples are not routinely tested for Arcobacter, most isolations are
currently performed by using methods designed for Campylobacter
species. The selectivity of those media is mostly achieved by the
incorporation of antimicrobial agents and although arcobacters have
sporadically been isolated in this way, studies on the susceptibility to
the antimicrobials included showed that none of the Campylobacter
selective supplements allowed the growth of all Arcobacter species or
strains and at the same time sufficiently suppressed the accompa-
nying flora (Houf et al., 2001b). Correct identification is a subsequent
major challenge as arcobacters are often misidentified, especially
when phenotypic methods are applied (On, 1996). Due to their
relatively metabolic inertness and antigenic heterogeneity, biochem-
ical or serological identification of arcobacters is not recommended.
Over the years, several DNA-based assays have been applied with
ribotyping (Kiehlbauch et al., 1994; Schroeder-Tucker et al., 1996),
restriction fragment length polymorphism (RFLP) analysis (Kiehl-
bauch et al., 1991) and PCR-RFLP assays (Cardarelli-Leite et al., 1996;
Gonzalez et al., 2000; Hurtado and Owen, 1997; Marshall et al., 1999)
as first attempts. Some of these assays allow differentiation between
A. butzleri and A. cryaerophilus, though mostly only genus level
differentiation between Campylobacter, Helicobacter, Arcobacter and
Wolinella is obtained. Recently, PCR-RFLP based identification of
arcobacters at the species level was reintroduced (Figueras et al.,
2008). The use of Arcobacter specific primers targeting the 16S rRNA
gene followed by MseI-restriction enzyme digestion and fragment
separation in a polyacrylamide gel, allowed to identify Arcobacter
species which were then established but also of the new species,
A. mytili isolated from mussels (Collado et al., 2009; Figueras et al.,
2008). Yet, in silico analysis revealed identical fragments of 548, 216
and 138 bp for A. thereuis and A. butzleri. This, but also the need for
polyacrylamide gel electrophoresis renders the technique not useful
for routine identification of mammal related Arcobacter species.
Based on the partial knowledge of the nucleic acid composition of
the 16S and 23S rRNA genes of Arcobacter species, genus- and species-
specific DNA-probes and primers have been developed (Al Rashid
et al., 2000; Bastyns et al., 1995; Gonzalez et al., 2000; Harmon and
Wesley, 1996; Houf et al., 2000; Kabeya et al., 2003; Neubauer and
Hess, 2006). The first described PCR-based species identification assay
included genus- and species-specific primers targeting the 23S
rRNA gene, but three separate amplications were needed (Bastyns
et al., 1995). Combination of those primers with primers based on
the Arcobacter and A. butzleri-specific DNA-probes (Wesley et al.,
1995) resulted in the first m-PCR (Harmon and Wesley, 1997). Two
m-PCR assays combined with agarose electrophoresis are commonly
applied for the simultaneous identification of the three human-
related Arcobacter species (Houf et al., 2000; Kabeya et al., 2003).
However, testing A. thereuis in the m-PCR of Houf et al. (2000) results
in amplicons of 401 bp and 257 bp corresponding respectively with
A. butzleri and A. cryaerophilus. Moreover, not all A. cryaerophilus
strains are correctly identified. With the m-PCR assay of Kabeya et al.
(2003), differentiation between A. cryaerophilus subgroup 1A and 1B
is reported. Besides the practical difficulty to differentiate fragments
of 728 bp (A. cryaerophilus subgroup 1A) and 692 bp (A. butzleri) by
means of agarose gel electrophoresis in a routine set-up, analysis of
the partial sequences of the 23S rRNA gene of A. cryaerophilus strains
included in the present study revealed that the primer for the specific
amplification of A. cryaerophilus 1B also amplified some A. cryaer-
ophilus 1A reference strains. Moreover, some A. cryaerophilus 1A
reference strains could not be detected using the primer specifically
developed for this subgroup (data not shown).
285
The new Arcobacter species described recently rendered rRNA
based identification of all Arcobacter species impossible. Especially the
large subunit of the A. cryaerophilus rRNA gene is heterogeneous and
contains different intervening sequences. This phenomenon was also
reported in Helicobacter species and other genera (Hurtado et al.,
1997). Also the rpoB gene showed no specific regions. In contrast,
primers targeting the gyrA gene were the most reliable to detect all
different A. cryaerophilus variants. In two studies, specific primers for
some Arcobacter species based on rpoB and 23S RNA genes (Brightwell
et al., 2007) and more recently on the 16S RNA and gyrA genes
(Pentimalli et al., 2009) for the use in real-time PCR have been
developed. They have been tested on a limited number of reference
strains and their use in routine analysis needs to be further assessed.
Other techniques as AFLP (On et al., 2003; Debruyne et al., 2010) and
partial sequencing (Lau et al., 2002; Woo et al., 2001) have been
applied but are yet not really accessible for routine analysis or, as in
the case of AFLP, needs a comparative database.
With the development of the present m-PCR, all five mammal
related species can be identified in a fast, cheap and routinely
applicable set-up. In this way, it is a useful tool towards a more
accurate identification of this emerging foodborne pathogen.
Acknowledgements
The skilled assistance provided by Evie Debrandt and Prof. Dr. Anne
Willems with the sequencing work is greatly appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.mimet.2010.01.009.
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