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Appl Environ Microbiol. 2009 December; 75(23): 7574–7577.
Published online 2009 October 2. doi:  10.1128/AEM.00772-09
PMCID: PMC2786417

Detection and Identification of tdh- and trh-Positive Vibrio parahaemolyticus Strains from Four Species of Cultured Bivalve Molluscs on the Spanish Mediterranean Coast[down-pointing small open triangle]

Abstract

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.

Gastroenteritis caused by Vibrio parahaemolyticus has been reported worldwide, though only sporadic cases have been reported in Europe (7, 14). The bacterium can be naturally present in seafood, but pathogenic isolates capable of inducing gastroenteritis in humans are rare in environmental samples (2 to 3%) (15) and are often not detected (10, 19, 20).

The virulence of V. parahaemolyticus is based on the presence of a thermostable direct hemolysin (tdh) and/or the thermostable direct hemolysin-related gene (trh) (1, 5). Both are associated with gastrointestinal illnesses (2, 9).

Spain is not only the second-largest producer in the world of live bivalve molluscs but also one of the largest consumers of bivalve molluscs, and Catalonia is the second-most important bivalve producer of the Spanish Autonomous Regions. Currently, the cultivation of bivalves in this area is concentrated in the delta region of the Ebro River. The risk of potentially pathogenic Vibrio spp. in products placed on the market is not assessed by existing legislative indices of food safety in the European Union, which emphasizes the need for a better knowledge of the prevalence of diarrheal vibrios in seafood products. The aim of this study was to investigate the distribution and pathogenic potential of V. parahaemolyticus in bivalve species exploited in the bays of the Ebro delta.

Thirty animals of each species of Mytilus galloprovincialis, Crassostrea gigas, Ruditapes decussatus, and Ruditapes philippinarum were collected. They were sampled from six sites of the culture area, three in each bay of the Ebro River delta, at the beginning (40°37′112"N, 0°37′092"E [Alfacs]; 40°46′723"N, 0°43′943"E [Fangar]), middle (40°37′125"N, 0°38′570"E [Alfacs]; 40°46′666"N, 0°45′855"E [Fangar]), and end (40°37′309"N, 0°39′934"E [Alfacs]; 40°46′338"N, 0°44′941"E [Fangar]) of the culture polygon. Clams were sampled from only one site per bay as follows: in the Alfacs Bay from a natural bed of R. decussatus (40°37′44"N, 0°38′0"E) and in the Fangar Bay from an aquaculture bed of R. philippinarum (40°47′3"N, 0°43′8"E). In total, 367 samples were analyzed in 2006 (180 oysters, 127 mussels, 30 carpet shell clams, and 30 Manila clams) and 417 samples were analyzed in 2008 (178 oysters, 179 mussels, 30 carpet shell clams, and 30 Manila clams).

All animals were individually processed and homogenized, and 1 ml of the homogenate was inoculated into 9 ml of alkaline peptone water (Scharlau, Spain). Following a 6-h incubation at 37°C, one loopful of the contents of each tube of alkaline peptone water was streaked onto CHROMagar vibrio plates (CHROMagar, France) and incubated for 18 h at 37°C. Mauve-purple colonies were purified, and each purified isolate was cryopreserved at −80°C (135 isolates in 2006 and 96 in 2008). From the initial homogenate portion, 100 μl was inoculated onto marine agar (Scharlau, Spain) and onto thiosulfate citrate-bile salts-sucrose agar (Scharlau, Spain) for total heterotrophic marine bacteria counts and total vibrio counts, respectively (Table (Table11).

TABLE 1.
Vibrio parahaemolyticus isolates, serotypes, and origins and total number of vibrios/heterotrophic bacteria contained in the bivalvea

Total DNA was extracted from each purified isolate using the Wizard genomic DNA purification kit (Promega), following the instructions of the manufacturer. A one-step PCR analysis was performed to identify/confirm which isolates were tl positive (species marker for V. parahaemolyticus). Further detection of the tdh or trh gene was carried out on all positive tl strains. All PCR analyses were carried out using the primers described by Bej et al. (2) with the following amplification conditions on the thermocycler (Eppendorf Mastercycler Personal): an initial denaturation at 95°C for 8 min, followed by 40 cycles of a 1-min denaturation at 94°C, annealing at 55°C for 1 min, elongation at 72° for 1 min, and a final extension of 10 min at 72°C. Positive and negative controls were included in all reaction mixtures: two positive controls, tl and tdh CAIM 1400 and trh CAIM 1772 (Collection of Aquatic Important Microorganisms [http://www.ciad.mx/caim/CAIM.html]), and negative control DNA-free molecular grade water (Sigma-Aldrich, Spain). Expected amplicons were visualized in 2% agarose gels stained with ethidium bromide.

Fifty-eight isolates contained the gene tl in 2006 and 96 in 2008, which confirmed their identity as V. parahaemolyticus. In 2006, the distribution of the 58 isolates was as follows: 7 from 127 mussels, 34 from 180 oysters, and 17 from 30 R. decussatus clams. No tl-positive isolates were found in R. philippinarum. PCR analysis of the tl-positive isolates for the presence of the tdh or trh gene indicated that eight isolates contained the tdh gene and four contained the trh gene. In 2008, the source of the confirmed V. parahaemolyticus isolates was as follows: 31 from 88 oysters, 44 from 89 mussels, 9 from 30 R. decussatus clams, and 12 from 30 R. philippinarum clams. Of these, 17 were found to contain the tdh gene and 7 contained the trh gene. Two isolates (I806 and I1042) contained both toxigenic genes, tdh and trh.

Putative tdh- and trh-positive PCR products were purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer's instructions and were sequenced bidirectionally by Macrogen Inc. Sequences were aligned using BioEdit (8) and analyzed using BLAST (National Center for Biotechnology Information). None of the toxigenic isolates was found positive by PCR analysis for the presence of open reading frame 8 of the phage 237 (16), a marker for the pandemic strain O3:K6.

The isolates were fingerprinted by repetitive extragenic palindromic PCR (rep-PCR) as described previously (3), and the resulting electrophoretic band patterns were analyzed with the GelCompar II software (v4.5; Applied Maths). The similarity matrix was calculated with the Jaccard coefficient with a band position tolerance of 0.8%, and the dendrogram was constructed with the Ward algorithm. A high level of genomic diversity was found among the 32 toxigenic isolates characterized by rep-PCR. Three clonal groups were identified (those having identical rep-PCR band patterns) (Fig. 1a to c).

FIG. 1.
rep-PCR dendrogram of toxigenic isolates of V. parahaemolyticus isolated in the Ebro delta. Letters denote clonal groups of isolates.

In vitro antibiotic susceptibility tests were performed using the diffusion disc test following a previously described protocol (18). The antibiotics used were gentamicin (10 μg), oxolinic acid (10 μg), amoxicillin (25 μg), polymyxin B (300 UI), vancomycin (30 μg), trimethoprim sulfamethoxazole (1.25/23.75 μg), nitrofurantoin (300 μg), doxycyclin (30 μg), ceftazidime (30 μg), streptomycin (10 μg), neomycin (30 UI), penicillin (6 μg), flumequine (30 μg), tetracycline (30 μg), ampicillin (10 μg), kanamycin (30 μg), ciprofloxacin (5 μg), and sulfonamide (300 μg). All tests were performed in duplicate. A Student t test for two samples with unequal variance was performed to compare the sensitivity of all 2006 isolates against the sensitivity of 2008 isolates for each antibiotic (Microsoft Office Excel 97-2003). Antibiogram results revealed a lower susceptibility in 2008 than in 2006, indicating a possible shift in overall susceptibility. Results from the t test indicated that significantly lower susceptibility in 2008 was detected (P ≤ 0.05; n = 36) for the following antibiotics: vancomycin, polymyxin B, ampicillin, amoxicillin, gentamicin, neomycin, trimethoprim sulfamethoxazole, nitrofurantoin, doxycyclin, ceftazidime, tetracycline, flumequine, and ciprofloxacin.

The serological types for 27 strains were determined by the agglutination method using commercially available V. parahaemolyticus antisera (Denka Seiken Ltd.; Cosmos Biomedical Ltd, United Kingdom) following the manufacturer's instructions. Potentially toxigenic V. parahaemolyticus isolates collected in 2006 were serologically heterogeneous (8 out of the 11 isolates) (Table (Table1).1). In isolates collected in 2008, results were more homogenous, with seven serotypes found among 19 isolates analyzed. The O3:K6 serotype was not detected in any of the strains analyzed, in agreement with the open reading frame 8 PCR results.

The present study is the first to report the detection of potentially diarrheal V. parahaemolyticus strains isolated from cultured bivalves on Spanish Mediterranean coasts, providing data on the presence of both tdh- and trh-positive isolates. V. parahaemolyticus has previously been detected in several European countries (4, 13, 21, 22). A recent study carried out in Spain detected tdh-positive V. parahaemolyticus strains from patients who had consumed fresh oysters in a market in Galicia on the Atlantic coast of Spain (12) and potentially pathogenic V. parahaemolyticus strains have also been reported in France (17). These studies indicate that the risk of infections caused by V. parahaemolyticus in Europe is low compared to that in America or Asia (15). However, this risk could have been underestimated, since V. parahaemolyticus is not included in the current European surveillance programs, such as the European Network for Epidemiological Surveillance and Control of Communicable Diseases.

Toxigenic V. parahaemolyticus strains detected in this study were genomically and serologically heterogeneous. The pandemic serotype O3:K6 was not detected, and although attempts to isolate O3:K6 from the environment and from seafood have not always been successful in previous studies reviewed by Nair and coauthors (15), this finding seems to be in agreement with the fact that no outbreak of diarrhea was observed in the area. Interestingly, isolates I806 and I1042 have been found positive for both tdh and trh in PCR tests. The coexistence of tdh and trh genes has already been reported in isolates from Japan, the United States, and Mexico (3, 6, 11, 19, 23). To our knowledge, no occurrence of an environmental isolate positive for both tdh and trh had previously been reported in Europe. All isolates tested were slightly different in their antibiotic resistance profiles. Typically, a high level of resistance could be determined. The detection of tdh- and/or trh-positive V. parahaemolyticus strains for the first time on the Mediterranean coast emphasizes the need to monitor for the presence of potentially diarrheal vibrios and bacterial gastroenteritis, and these data should be taken into consideration to revise the European legislation on the requirements for shellfish harvested for consumption in order to include the surveillance of these pathogens in Europe.

Nucleotide sequence accession numbers.

The following nucleotide sequence accession numbers were deposited in GenBank: EU908021 (tdh I628), EU908022 (tdh I678), EU908023 (tdh I775), EU908024 (tdh I793), EU908025 (tdh I805), EU908026 (tdh I806), EU908027 (tdh I809), EU908028 (tdh I745), EU982193 (trh I712), and EU982194 (trh I806).

Acknowledgments

The present study was financed by INIA (Spanish Ministry of Education and Science) project numbers RTA-2005-00079-00-00 and RTA-2008-00063-00-00, awarded to A.R. C.L.-J. has a doctoral scholarship provided by INIA. B.G.-G. was awarded two visiting grants, one by MEC, Spain (October 2007-February 2008) and one by CONACYT, Mexico (September 2008-August 2009).

We are grateful to Josep Maria Reverte and the staff from the USM at IRTA for the sample collection and to Margarita Fernandéz and Jorge Diogene for the environmental data.

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 October 2009.

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