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discovery of compounds able to inhibit those biomass was harvested and suspended in a 0.85%
mechanisms that confer resistance to antibiotics sodium chloride solution. The cell density was
by the bacteria. With this in mind, in our research adjusted by spectrophotometry (Merck SQ118,
a library of extracts from marine macroalgae Darmstadt, Germany) at λ 585 nm until 1.0 AU
from Mexico were screened as reversers of the (equivalent to 4.5 × 107 cells mL-1). The suspen-
resistance to antibiotics of three pathogenic bac- sions of each strain were used to inoculate the
teria to select unexplored sources of resistance- surface of Mueller-Hinton agar plates with an
reversing compounds. This choice is justified be- added sublethal concentration of an antibiotic.
cause marine algae are important sources of Escherichia coli and Staphylococcus aureus were
metabolites, such as acetogenins, polyphenolics, cultured on Mueller-Hinton agar with added
aromatic compounds, terpenes, and other related ampicillin (12 mg L-1, equivalent to 75% of the
structures9. To the best of our knowledge this is minimum inhibitory concentration (MIC)). The
the first time in which marine macroalgae col- medium for Streptococcus pyogenes was added
lected from the coasts of Baja California Sur, erythromycin (1.5 mg L-1, equivalent to 25% of
Mexico have been assessed as reversers of the re- the MIC). The drug susceptibility pattern of
sistance to antibiotics. Our finding has allowed each target strain was previously determined.
us to select a number of algae for further investi- The culturing conditions of strains under sub-
gation in search for the active compounds. lethal concentrations of antibiotic were exten-
sively tested and optimized. The assays were
done by putting, on the inoculated surface of the
agar plate, extract-impregnated paper discs (6.5
Materials and Methods diameter, Whathman No. 4). Each paper disc
was loaded with 100 µL of a stock solution of
The algae studied were gathered from several each algal extract (20 mg mL-1). The impregna-
locations along the coast of Baja California Sur tion of the disc was done under aseptic condi-
(Figure 1) between June 2004 and November tions. Solvent evaporation from the discs left
2007. The collection was done by free diving at 2.0 mg disc-1. Plates were incubated at 37°C.
1- to 2-m depth. Algal samples were cleaned of The inhibition zone (when observed) around
epiphytes, rinsed with tap water to remove any discs was measured after 24 h, and expressed in
associated debris, and the cleaned fresh materials mm. Every algal extract was tested in a medium
were dried in the sun, ground, and stored at added with antibiotic (as mentioned before) and
–20°C until extraction. Voucher specimens were its effect was contrasted by testing the same ex-
preserved for taxonomic identification and future tract, at the same time, in Mueller-Hinton agar
reference. without an antibiotic. Those extracts active
The extracts were obtained by maceration. against bacteria cultured on a medium with
Briefly, 100 g of each algal sample was soaked added antibiotic, but inactive in an antibiotic-
with 250 mL of distilled ethanol at 25-35°C. Af- free medium were considered as reversers of the
ter 48 h, the mixture was filtered through paper. resistance. Measures of the inhibition zones are
The residual algal tissue was extracted once presented as the mean values of two measure-
again under same conditions. After filtration, ments and its SD. Ethanol was always used as a
both extracts were pooled and concentrated to negative control.
dryness under vacuum at 40°C (Yamato RE500,
San Francisco, CA, USA). The crude extracts Statistical Analysis
were kept in dark and dry conditions at –20°C We have found a great difficult to perform a
until biological testing. statistical analysis. However, we have taken into
A modification of the classical agar disc-diffu- mind that:
sion method10 was used to test the effect of ex-
tracts on the reversion of the resistance of Es- • The substances produced by each alga do not
cherichia coli (ATCC BAA-196, expressing ex- necessarily correspond to the same chemical
tended spectrum β-lactamases), Staphylococcus group as well as we cannot assume that they
aureus (ATCC BAA-42, β-lactamases), and are in the same concentration, therefore a
Streptococcus pyogenes (ATCC BAA-946, efflux comparison between them we could give an
pump). Each strain was first cultured on Mueller- erroneous interpretation of the activity of each
Hinton agar plates at 37°C. After 18 h, the cell of the species.
740
Reversion of the antibiotic resistance
Figure 1. Map of Baja California peninsula (Mexico) showing the different collection locations.
• The effect of each alga on the three test bacte- in some cases there is a tendency to increase
ria cannot be compared because the mecha- the diameter of the halos when the algae ex-
nisms used by bacteria to resist antibiotics are tract is placed in the presence of the antibiotic,
different (β-lactamases and efflux pump as it cannot be directly interpreted as an effect of
mentioned in the introduction). Thus although inhibition of antibiotic resistance, because the
741
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
742
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
Family
Sample ID, species name, and authority (collection place)
Bangiaceae
06-020, Porphyra perforata Agardh, 1883 (2) 0±0 0±0 0±0 0±0 0±0 0±0
Chnoosporaceae
04-018, Chnoospora implexa Agardh, 1848 (8) 10.0 ± 0 7.0 ± 0 19.5 ± 07 12.0 ± 0 0±0 0±0
Cladophoraceae
06-001, Cladophora sp. (7) 10.0 ± 1.4 7.0 ± 0 16.0 ± 1.4 22.0 ± 1.4 0±0 0±0
Codiaceae
04-004, Codium amplivesiculatum Setchell & Gardner, 1924 (8) 8.0 ± 0 0±0 13.0 ± 0 12.0 ± 0 0±0 0±0(
06-012, Codium amplivesiculatum Setchell & Gardner, 1924 (6) 0±0 0±0 11.5 ± 0.7 10.0 ± 0±0 0±0
04-024, Codium cuneatum Setchell & Gardner, 1924 (8) 10.0 ± 0 9.5 ± 0.7 14.5 ± 0.7 11.0 ± 0 0±0 0±0
06-011, Codium cuneatum Setchell & Gardner, 1924 (6) 9.5 ± 2.1 0±0 15.0 ± 0 12.5 ± 0.7 0±0 0±0
06-010, Codium simulans Setchell & Gardner, 1924 (6) 9.5 ± 0.7 7.0 ± 0 11.5 ± 0.7 8.0 ± 0 0±0 0±0
Corallinaceae
07-001, Amphiroa valonioides Yendo, 1902 (7) 0±0 0±0 11.0 ± 0 17.5 ± 2.1 0±0 0±0
06-025, Corallina vancouveriensis Yendo, 1902 (4) 9±0 11.0 ±0 9.5 ± 0.7 13.5 ± 0.7 0±0 0±0
06-027, Corallina sp. (2) 15.5 ± 0.7 9.5 ± 0.7 15.5 ± 0.7 10.5 ± 0.7 0±0 0±0
Reversion of the antibiotic resistance
Cystocloniaceae
06-035, Hypnea johnstonii Setchell & Gardner, 1924 (2) 0±0 7.0 ± 0 10.0 ± 0 0±0 0±0 0±0
04-011, Hypnea valentiae (Turner) Montagne, 1841 (8) 8.0 ± 0 0±0 12.5 ± 0.7 10.5 ± 0.7 0±0 0±0
Dictyotaceae
06-016, Dictyota flabellata Setchell & Gardner, 1924 (6) 10.0 ± 0 7.5 ± 0.7 14.0 ± 0 18.5 ± 0.7 0±0 0±0
06-028, Dictyopteris undulata Holmes, 1896 (2) 18.0 ± 0 14.0 ± 1.4 21.5 ± 0.7 18.5 ± 0.7 0±0 0±0
06-029, Dictyopteris delicatula Lamouroux, 1809 (2) 14.5 ± 0.7 11.5 ± 1.4 13.0 ± 1.4 11.0 ± 0 0±0 0±0
04-002, Padina mexicana Thivy, 1945 (8) 9.5 ± 0.7 0±0 11.0 ± 0 10.0 ± 0 0±0 0±0
04-021, Padina mexicana Thivy, 1945 (8) 14.5 ± 0.7 7.0 ± 0 16.5 ± 0.7 10.0 ± 0 0±0 0±0
06-018, Padina concrescens Thivy, 1945 (2) 10.5 ± 0.7 7.0 ± 0 12.5 ± 0.7 11.0 ± 0.7 0±0 0±0
743
(Continued)
744
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
Gelidiaceae
04-008, Gelidium robustum Hollenberg & Abbott, 1965 (3) 11.0 ± 1.4 11.5 ± 0.7 22.5 ± 0.7 19.0 ± 0 0±0 0±0
06-024, Gelidium robustum Hollenberg & Abbott, 1965 (4) 14.0 ± 1.4 8.5 ± 0.7 14.0 ± 1.4 7.0 ± 0 0±0 0±0
06-033, Gelidium robustum Hollenberg & Abbott, 1965 (2) 12.5 ± 2.1 10.5 ± 0.7 14.5 ± 0.7 11.0 ± 0.7 0±0 0±0
Gigartinaceae
06-026, Chondracanthus canaliculatus Harvey, 1993 (2) 9.5 ± 0.7 7.5 ± 0.7 10.0 ± 1.4 0±0 0±0 0±0
07-010, Chondracanthus canaliculatus Harvey, 1993 (1) 9.0 ± 0 0±0 0±0 0±0 0±0 0±0
Gracilariaceae
07-007, Gracilaria marcialana Setchell & Gardner, 1924 (8) 0±0 0±0 11.0 ± 0 0±0 0±0 0±0
04-007, Gracilaria veleroae Dawson, 1944 (8) 0±0 0±0 0±0 0±0 0±0 0±0
04-010, Gracilaria vermiculophylla (Ohmi) Papenfuss, 1967 (5) 0±0 0±0 11.0 ± 0 11.0 ± 0 0±0 0±0
04-023, Gracilaria subsecundata Setchell & Gardner, 1924 (8) 13.5 ± 0.7 11.0 ± 1.4 14.0 ± 0 9.0 ± 1.4 0±0 0±0
Laminariaceae
06-019, Macrocystis pyrifera Agardh, 1820 (2) 9.0 ± 0 10.0 ± 0 11.0 ± 0 18.5 ± 0.7 0±0 0±0
Lessoniaceae
06-022, Eisenia arborea Areschoug, 1876 (2) 0±0 0±0 0±0 0±0 0±0 0±0
Liagoraceae
04-022, Ganonema farinosum Fan & Wang, 1974 (8) 12.5 ± 2.1 9.0 ± 0 17.0 ± 2.1 17.0 ± 0 0±0 0±0
06-008, Liagora californica Zeh, 1912 (8) 9.0 ± 0 0±0 13.5 ± 1.4 10.5 ± 0.7 0±0 0±0
06-013, Liagora californica Zeh, 1912 (6) 0±0 0±0 0±0 0±0 0±0 0±0
Rhodymeniaceae
06-003, Rhodymenia californica Kylin, 1931 (7) 7.0 ± 0 7.0 ± 0 14.0 ± 0 13.0 ± 0 0±0 0±0
06-032, Rhodymenia californica Kylin, 1931 (2) 0±0 0±0 0±0 0±0 0±0 0±0
(Continued)
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
Table I. Diameter of inhibition zone around the discs individually impregnated with sixty algal extracts collected from Baja California Sur (Mexico). Bacteria were cultured on
Mueller Hinton agar plates add with a sublethal concentration of antibiotic.
Rhodomelaceae
07-004, Acanthophora spicifera (M. Vahl) Børgesen, 1910 (8) 0±0 0±0 12.0 ± 0 10.5 ± 0.7 0±0 0±0
07-002, Laurencia gardneri Hollenberg, 1943 (7) 0±0 0±0 10.5 ± 0.7 8.5 ± 0.7 0±0 0±0
04-005, Laurencia johnstonii Setchell & Gardner, 1924 (8) 30.0 ± 0 30.5 ± 0.7 38.0 ± 0.7 26.5 ± 1.4 0±0 0±0
07-009, Laurencia johnstonii Setchell & Gardner, 1924 (8) 12.0 ± 0 11.5 ± 0.7 9.0 ± 1.4 10.5 ± 0.7 0±0 0±0
06-007, Laurencia pacifica Setchell & Gardner, 1924 (8) 19.5 ± 0.7 18.0 ± 0.7 31.5 ± 2.1 25.0 ± 0.7 0±0 0±0
06-015, Laurencia pacifica Setchell & Gardner, 1924 (6) 21.0 ± 0 12.0 ± 0.7 21.5 ± 2.1 19.5 ± 0.7 0±0 0±0
07-005, Laurencia sp. (8) 19.0 ± 0.7 14.5 ± 0.7 30.0 ± 0 30.5 ± 0.7 0±0 0±0
06-031, Neorhodomela larix (Turner) Masuda, 1982 (2) 12.0 ± 1.4 12.0 ± 0 11.0 ± 0 13.0 ± 0 0±0 0±0
07-003, Pterosiphonia bipinnata Falkenberg, 1901 (7) 0±0 0±0 10.5 ± 0.7 0±0 0±0 0±0
Sargassaceae
06-023, Cystoseira osmundacea (Turner) Agardh, 1820 (4) 0±0 0±0 11.0 ± 0.7 0±0 0±0 0±0
04-003, Sargassum horridum Setchell & Gardner, 1924 (8) 9.0 ± 0 0±0 12.0 ± 0 0±0 0±0 0±0
06-005, Sargassum horridum Setchell & Gardner, 1924 (8) 9.0 ± 0.7 7.0 ± 0 13.5 ± 0.7 14.0 ± 1.4 0±0 0±0
06-009, Sargassum horridum Setchell & Gardner, 1924 (8) 9.5 ± 0.7 7.0 ± 0 15.5 ± 0.7 14.5 ± 0.7 0±0 0±0
Scytosiphonaceae
04-017, Hydroclathrus clathratus (Agardh) Howe, 1920 (8) 10.5 ± 0.7 7.0 ± 0 13.0 ± 0 12.0 ± 0 0±0 0±0
06-006, Hydroclathrus clathratus (Agardh) Howe, 1920 (8) 9.5 ± 0.7 7.0 ± 0 12.5 ± 0.7 15.0 ± 0 0±0 0±0
04-015, Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 11.5 ± 0.7 0±0 15.0 ± 0 10.0 ± 0 0±0 0±0
04-020, Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 7.5 ± 0.7 7.0 ± 0 13.0 ± 0.7 13.0 ± 0 0±0 0±0
Reversion of the antibiotic resistance
06-004, Rosenvingea intricata (Agardh) Børgesen, 1914 (8) 9.0 ± 0 7.0 ± 0 14.0 ± 0 11.5 ± 0.7 0±0 0±0
06-017, Colpomenia tuberculata Saunders, 1898 (6) 10.5 ± 2.1 8.0 ± 0 20.5 ± 13.4 9.5 ± 0.7 0±0 0±0
06-030, Colpomenia tuberculata Saunders, 1898 (2) 13 ± 1.4 13 ± 0 13 ± 0 10 ± 0 0±0 0±0
07-008, Colpomenia sinuosa Derbés & Solier, 1851 (8) 11.0 ± 0 9.0 ± 0 10.5 ± 0.7 0±0 0±0 0±0
Spyridiaceae
04-025, Spyridia filamentosa (Wulfen) Harvey, 1833 (5) 10.5 ± 0.7 0±0 15.0 ± 0 7.0 ± 0 0±0 0±0
07-006, Spyridia filamentosa (Wulfen) Harvey, 1833 (5) 11.0 ± 0 9.0 ± 0 11.5 ± 0.7 0±0 0±0 0±0
Ulvaceae
04-001, Ulva rigida Agardh, 1823 (8) 0±0 0±0 15 ± 0 7.0 ± 0 0±0 0±0
06-002, Ulva dactylifera Setchell & Gardner, 1920 (7) 11.0 ± 0.7 7.0 ± 0 14.5 ± 0.7 11.0 ± 0 0±0 0±0
a
Mean values (n = 2) ± standard deviation. MAWA = cultured on medium added with 75% of the MIC of ampicillin; CWA = cultured without antibiotic; MAWE = cultured on
745
medium added with 25% of the MIC of erythromycin.
M. Muñoz-Ochoa, J.I. Murillo-Álvarez, L.A. Zermeño-Cervantes, S. Martínez-Diaz, R. Rodríguez-Riosmena
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––––––––––––––––––––
Acknowledgements
This study was done in partial fulfillment of the require-
ments of a Doctor in Sciences degree for MMO, with
grants from IPN (ref. SIP 20070020, SIP 20080216),
and CONACyT (ref. SEP-47942/A-1). Stephanie Castro
is acknowledged for technical assistance during the ex-
tractions. Authors thank COFAA-IPN and EDI-IPN for
the incentives granted. Thanks also to Dr. Ellis Glazier
for editing this English-language text.
747