Connectivity dynamics in Irish mudflats between microorganisms including Vibrio spp., common cockles Cerastoderma edule, and shorebirds.

Shellfish, including the key species the common cockle Cerastoderma edule, living and feeding in waters contaminated by infectious agents can accumulate them within their tissues. It is unknown if microbial pathogens and microparasites can subsequently be transmitted via concomitant predation to their consumers, including shorebirds. The objective of this study was to assess if pathogens associated with C. edule could be detected seasonally in the faeces of shorebirds that feed on C. edule and in the physical environment (sediment) in which C. edule reside, along the Irish and Celtic Seas. Two potentially pathogenic global groups, Vibrio and Haplosporidia, were detected in C. edule. Although Haplosporidia were not detected in the bird faeces nor in the sediment, identical strains of Vibrio splendidus were detected in C. edule and bird faecal samples at sites where the oystercatcher Haematopus ostralegus and other waders were observed to be feeding on cockles. Vibrio spp. prevalence was seasonal and increased in C. edule and bird faecal samples during the warmer months, possibly due to higher seawater temperatures that promote the replication of this bacteria. The sediment samples showed an overall higher prevalence of Vibrio spp. than the bird faecal and C. edule samples, and its detection remained consistently high through the sites and throughout the seasons, which further supports the role of the sediment as a Vibrio reservoir. Our findings shed light on the fact that not all pathogen groups are transmitted from prey to predator via feeding but bacteria such as V. splendidus can be. As most of the wading birds observed in this study are migratory, the results also indicate the potential for this bacterium to be dispersed over greater geographic distances, which will have consequences for areas where it may be introduced.

Vibrio aestuarianus subsp. cardii subsp. nov., pathogenic to the edible cockles Cerastoderma edule in France, and establishment of Vibrio aestuarianus subsp. aestuarianus subsp. nov. and Vibrio aestuarianus subsp. francensis subsp. nov.

Cockle mortality events have been reported in northern France since 2012. In the present study, we describe and investigate the implication of a potential bacterial causative agent in cockle mortality. Bacteria isolated from five different cockle mortality events were characterized and studied. Using phenotypic analysis combined with DNA-DNA hybridization (DDH) and whole genome sequencing, the isolates were shown to belong to Vibrio aestuarianus, a species regularly detected in France during oyster mortality events. Comparison of the strains from cockles with strains from French oysters and the type strain showed that the strains from cockles were genetically different to those from oysters and also different to the V. aestuarianus type strain. Moreover, the cockle and oyster strains were classified into two different, but close, groups both separated from the type strain by: (1) analyses of the ldh gene sequences; (2) DDH assays between 12/122 3T3T (LMG 31436T=DSM 109723T), a representative cockle strain, 02/041T (CIP 109791T=LMG 24517T) representative oyster strain and V. aestuarianus type strain LMG 7909T; (3) average nucleotide identity values calculated on the genomes; and (4) phenotypic traits. Finally, results of MALDI-TOF analyses also revealed specific peaks discriminating the three representative strains. The toxicity of representative strains of these cockle isolates was demonstrated by experimental infection of hatchery-produced cockles. The data therefore allow us to propose two novel subspecies of Vibrio aestuarianus: Vibrio aestuarianus subsp. cardii subsp. nov. for the cockle strains and Vibrio aestuarianus subsp. francensis subsp. nov. for the Pacific oyster strains, in addition to an emended description of the species Vibrio aestuarianus.

Prevalence of virulence and antimicrobial resistance genes in Aeromonas species isolated from marketed cockles (Tegillarca granosa) in Korea.

This study aimed to examine incidence, virulence and antimicrobial properties in Aeromonas spp. isolated from cockles (Tegillarca granosa) in Korea. Firstly, genomic DNA was extracted from 32 Aeromonas spp. isolates, and PCR screening for virulence, antimicrobial resistance genes was carried out. The disk diffusion assay was used to examine antimicrobial susceptibility. Aeromonas spp. isolates comprised, A. hydrophila (n = 8), A. veronii (n = 15), A. media (n = 2), A. salmonicida (n = 2), A. allosaccharophila (n = 1), A. bestiarum (n = 1), A. culicicola (n = 1), A. enteropelogenes (n = 1) and A. rivipollensis (n = 1). High prevalence of virulence-related genes reported as; act (69%), alt (47%), ast (41%), aerA (56%), lip (50%), ahyB (47%), ser (28%), fla (66%), gcat (44%), ascV (50%) and hlyA (72%). All isolates were multidrug resistant, while highest resistance level observed for ampicillin (100%), followed by imipenem (81%), rifampicin (78%), cephalothin (72%), piperacillin (47%) and Colistin sulfate (31%). The presence of blaSHV , blaCTX , tetE, aac(6')-Ib, strA-strB, qnrS, qnrB and IntI1 genes were reported in varying combinations. Nevertheless, blaTEM , blaIMP , tetA, tetB, qnrA, qnrB and aphAI-IAB genes and the class1 integron were not detected. The high occurrence of virulence and antimicrobial resistance genes in cockles reveals that it can be a potential health risk source for consumers.

Prevalence of virulence and extended-spectrum ß-lactamase (ESBL) genes harbouring Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea.

The present study aimed to investigate the incidence, virulence and antibiotic properties in Vibrio spp. isolated from cockles (Tegillarca granosa) marketed in Korea. A total of 32 Vibrio spp. isolates including V. parahaemolyticus (n = 4), V. alginolyticus (n = 11), V. diabolicus (n = 14) and V. harveyi (n = 3) were detected using gyrB sequencing. The phenotypic pathogenicity revealed that the DNase, amylase and phospholipase activities were 100%, while lipase, slime production, gelatinase and caseinase were detected in 72, 88, 88 and 81% of the isolates respectively. The PCR amplification for the detection of V. parahaemolyticus species-specific tdh, tlh, trh and toxR genes were positive in 4 (13%), 16 (50%), 0 (0%) and 4 (13%) isolates respectively. The V. alginolytuicus species-specific tdh, tlh, trh, toxR and vac genes were carried by 15 (47%), 29 (91%), 0 (0%), 15 (47%) and 25 (78%) of the isolates respectively. In addition, multidrug resistance was observed by 27 (84%) isolates, whereas higher resistant rates were observed against ampicillin, piperacillin, streptomycin and cephalothin. The occurrence of blaCTX (78%), blaTEM (40%), blaSHV (22%) and aac(6')-Ib (94%) were prevalent, while strAB, tetB, aphAI-IAB, intl1 and aadA1 gene cassettes were also detected. The results signify the potential health risks resulting from the consumption of raw cockles in Korea. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrios are well known to cause human infections following consumption of raw or undercooked seafood. This phenomenon has undoubtedly increased the number of health issues over the past few years in Korea. Among the identified Vibrio spp., we could detect V. diabolicus and V. harveyi for the first time in marketed cockles in Korea. The presence of species-specific genes (tdh-VA, tlh-VP, tlh-VA and toxR-VA) in V. diabolicus exhibits the close genetic affinity among V. parahaemolyticus and V. alginolyticus. Furthermore, the prevalence of extended-spectrum ß-lactamases (ESBLs) and other antibiotic resistance genes along with multidrug resistance signifies the potential threat for consumers.

Paracoccus tegillarcae sp. nov., isolated from the gastrointestinal tract of a blood cockle (Tegillarca granosa).

A novel bacterial isolate, designated as strain BM15T, was isolated from the gastrointestinal tract of a blood cockle, Tegillarca granosa, which was collected from the foreshore of Beolgyo-eup, Republic of Korea. Strain BM15T was Gram-stain-negative, non-motile, strictly aerobic and short-rod-shaped. Optimum growth of the isolate occurred at 20 °C, in the presence of 4 % (w/v) NaCl and at pH 6. The 16S rRNA gene sequence analysis showed that strain BM15T belonged to the genus Paracoccus and had more than 97 % 16S rRNA gene sequence similarity to 'Paracoccus zhejiangensis' J6 (97.40 % similarity) and Paracoccus lutimaris HDM-25T (97.04 %). The polar lipid profile of strain BM15T comprised phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, an unidentified glycolipid and two unidentified lipids. The predominant respiratory quinone was ubiquinone-10. The major cellular fatty acid (>20 %) was summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c). The complete genome sequence of strain BM15T comprised 3,759,866 bp with 62.2 mol% G+C content. The results of the phylogenetic, phenotypic and genotypic analyses indicated that strain BM15T represents a novel species in the genus Paracoccus, for which the name Paracoccus tegillarcae is proposed. The type strain is BM15T (=KCTC 72032T=JCM 33289T).

A novel monoclonal Perkinsus chesapeaki in vitro isolate from an Australian cockle, Anadara trapezia.

A monoclonal Perkinsus chesapeaki isolate was established from 1 of 10 infected Australian Anadara trapezia cockles. Morphological features were similar to those of described P. chesapeaki isolates, and also included a unique vermiform schizont cell-type. Perkinsus olseni-specific PCR primers amplified DNAs from all 10 cockles. Perkinsus chesapeaki-specific primers also amplified DNAs from 4/10 cockles, including DNA from the isolate source cockle. Three different sets of DNA sequences from the monoclonal isolate grouped with the homologous, previously deposited, P. chesapeaki sequences in phylogenetic analyses. In situ hybridization assays detected both P. chesapeaki and P. olseni cells in histological sections from the source cockle for monoclonal isolate ATCC PRA-425.

Parasites, pathological conditions and resistance to Marteilia cochillia in lagoon cockle Cerastoderma glaucum from Galicia (NW Spain).

A histopathological survey revealed parasites and pathological conditions affecting lagoon cockles Cerastoderma glaucum along the Galician coast; serious pathological threats were not detected because the potentially pathogenic conditions (infections with a Marteilia-like parasite and bucephalid sporocysts, disseminated neoplasia and a condition involving large foci of heavy haemocytic reaction) were rare, while more prevalent parasites had negligible or limited pathogeny. Considering that C. edule and C. glaucum are sympatric in some Galician rias, it is remarkable that C. glaucum was not seriously affected by Marteilia cochillia while C. edule suffered an intense outbreak of this parasite associated with massive mortality. Comparison of the digestive gland between cockle species showed co-occurrence of digestive tubules in different phases, with abundant disintegrated tubules, in the case of C. glaucum, while C. edule showed synchronicity and absence of fully disintegrated tubules; these differences could influence their susceptibility to M. cochillia because the main location of this parasite in common cockles is the epithelia of the digestive gland. Moreover, the observation of histological sections through the digestive gland easily allows differentiating the 2 cockle species.

Serogroup, virulence, and molecular traits of Vibrio parahaemolyticus isolated from clinical and cockle sources in northeastern Thailand.

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n=74) and cockles (Anadara granosa) (n=74) in Thailand were analyzed by serotyping, determination of virulence and related marker genes present, response to antimicrobial agents, and genetic relatedness. Serological analysis revealed 31 different serotypes, 10 of which occurred among both clinical and cockle samples. The clinical isolates commonly included the pandemic serogroup O3:K6, while a few of the cockle isolates exhibited likely pandemic serovariants such as O3:KUT and O4:KUT, but not O3:K6. The pandemic (orf8 gene-positive) strains were more frequently found among clinical isolates (78.4%) than cockle isolates (28.4%) (p<0.001). Likewise, the virulence and related marker genes were more commonly detected among clinical than cockle isolates; i.e., tdh gene (93.2% versus 29.7%), vcrD2 (97.3% versus 23.0%), vopB2 (89.2% versus 13.5%), vopT (98.6% versus 36.5%) (all p<0.001) and trh (10.8% versus 1.4%) (p<0.05). Pulsed-field gel electrophoresis of NotI-digested genomic DNA of 41 randomly selected V. parahaemolyticus isolates representing different serotypes produced 33 pulsotypes that formed 5 different clusters (clonal complexes) (A-E) in a dendrogram. Vibrio parahaemolyticus O3:K6 and likely related pandemic serotypes were especially common among the numerous clinical isolates in cluster C, suggesting a close clonal link among many of these isolates. Most clinical and cockle isolates were resistant to ampicillin. This study indicates that O3:K6 and its likely serovariants based on the PFGE clusters, are causative agents. Seafoods such as cockles potentially serve as a source of virulent V. parahaemolyticus, but further work is required to identify possible additional sources.

Molecular analysis of Vibrio vulnificus isolated from cockles and patients in Thailand.

Vibrio vulnificus can cause septicemia, wound infection and gastroenteritis. The most severe infections are related to consumption of raw or undercooked seafood. Virulence genes, biomarkers, antimicrobial resistance, and genetic relationships among V vulnificus isolated from clinical and environmental sources in Thailand have not hitherto been investigated. ViuB encoding vulnibactin siderophore was detected in 33% and 50% of clinical and environmental (cockle) V. vulnificus isolates, respectively, and capsular polysaccharide allele 1 in 67% and 75% of clinical and environmental isolates, respectively. Analysis of the 16 S rDNA gene revealed that type B was the most frequent in both clinical and environmental isolates (67%) whereas the non type-able (30%) was detected only in environmental isolates. The virulence-correlated gene (vcg) with both type C and E together was the most frequently found among the clinical (67%) and environmental (72%) isolates. Pulsed-field gel electrophoresis differentiated V vulnificus into 2 clusters; most cockle samples (83%) and all clinical isolates grouped into cluster II, indicating a possible clonal relationship between V. vulnificus isolated from patients and cockles. Only 20% of environmental isolates were resistant to ampicillin. These studies suggest that V vulnificus isolated from cockles has virulence genes similar to those in clinical isolates and thus may have the potential of causing disease.

Antibiotic resistance and molecular typing among cockle (Anadara granosa) strains of Vibrio parahaemolyticus by polymerase chain reaction (PCR)-based analysis.

Genomic DNA of Vibrio parahaemolyticus were characterized by antibiotic resistance, enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis. These isolates originated from 3 distantly locations of Selangor, Negeri Sembilan and Melaka (East coastal areas), Malaysia. A total of 44 (n = 44) of tentatively V. parahaemolyticus were also examined for the presence of toxR, tdh and trh gene. Of 44 isolates, 37 were positive towards toxR gene; while, none were positive to tdh and trh gene. Antibiotic resistance analysis showed the V. parahaemolyticus isolates were highly resistant to bacitracin (92%, 34/37) and penicillin (89%, 33/37) followed by resistance towards ampicillin (68%, 25/37), cefuroxime (38%, 14/37), amikacin (6%, 2/37) and ceftazidime (14%, 5/37). None of the V. parahaemolyticus isolates were resistant towards chloramphenicol, ciprofloxacin, ceftriaxone, enrofloxacin, norfloxacin, streptomycin and vancomycin. Antibiogram patterns exhibited, 9 patterns and phenotypically less heterogenous when compared to PCR-based techniques using ERIC- and RAPD-PCR. The results of the ERIC- and RAPD-PCR were analyzed using GelCompare software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the V. parahaemolyticus isolates into 6 clusters and 21 single isolates at a similarity level of 80%. While, RAPD-PCR with primer Gen8 discriminated the V. parahaemolyticus isolates into 11 clusters and 10 single isolates and Gen9 into 8 clusters and 16 single isolates at the same similarity level examined. Results in the presence study demonstrated combination of phenotypically and genotypically methods show a wide heterogeneity among cockle isolates of V. parahaemolyticus.

Application of tetraplex PCR for detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle.

A tetraplex PCR method was developed for simultaneous detection of Vibrio cholerae, V. parahaemolyticus, V. vulnificus and V. mimicus in cockle samples in comparison with conventional culture method. Specific primers targeting ompW of V. cholerae, tl of V. parahaemolyticus, hsp60 of V. vulnificus and sodB of V. mimicus were employed in the same PCR. Detection limit of the tetraplex PCR assay was 104 cfu/ml (400 cfu/PCR reaction) for pure cultures of all four species of Vibrio. In Vibrio spiked cockle samples, the limit of detection after 6 hours enrichment in alkaline peptone water was 1 cfu/10 g of cockle tissue for three Vibrio spp, except for V. mimicus that was 102 cfu/10 g of cockle tissue. When the tetraplex PCR and culture methods were applied to 100 cockle samples, V. parahaemolyticus, V. vulnificus, V. cholerae and V. mimicus were detected in 100, 98, 80 and 9% of the samples by tetraplex PCR and in 76, 42, 0 and 0% by the culture method, respectively. This developed tetraplex PCR method should be suitable for simultaneous and rapid detection of Vibrio species in food samples and for food safety assessment.

Duplex PCR for detection of Salmonella and Shigella spp in cockle samples.

Salmonella and Shigella spp are important causative agents of foodborne diseases. A sensitive, specific and rapid method is essential for detection of these pathogens. In this study, a duplex PCR method was developed for simultaneous detection of Salmonella and Shigella spp in cockle samples and compared with the traditional culture method. Enrichment broths for Salmonella spp recovery were also compared. Sensitivity of the duplex PCR for simultaneous detection of Salmonella and Shigella spp from pure culture was 10(3) CFU/ml (40 CFU/PCR reaction), and that of sterile cockle samples spiked with these two pathogens was 1 CFU/10 g of cockle tissue after 9 hours enrichment [3 hours in buffered peptone water (BPW), followed by 6 hours in Rappaport Vasiliadis (RV) broth or tetrathionate (TT) broth for Salmonella spp and 6 hours enrichment in Shigella broth (SB) for Shigella spp]. There was no significant difference in detection sensitivity between enrichment in RV and TT broths. Salmonella spp detected in cockles in Khon Kaen, Thailand by duplex PCR and culture method was 17% and 13%, respectively but Shigella spp was not detected. The duplex PCR technique developed for simultaneous detection of Salmonella and Shigella spp in cockle samples was highly sensitive, specific and rapid and could serve as a suitable method for food safety assessment.

Dynamics of bacterial communities in cockles (Cerastoderma edule) with respect to trematode parasite (Bucephalus minimus) infestation.

The bacterial communities associated with the cockle (Cerastoderma edule) were investigated at the individual level through a 10-month monitoring programme. Temporal changes and those changes associated with a common parasite of the cockle, Bucephalus minimus, were investigated by monthly sampling of individuals, selected based on their shell length (cohort monitoring). Cockle bacterial community abundance (CBCA) and diversity (CBCD) were estimated by epifluorescence microscopy counts and automated ribosomal intergenic spacer analysis, respectively. CBCA showed a temporal pattern peaking at 30 × 10(6) cells per gram of cockle flesh and intervalval liquid in October and a significant 1.8-fold increase linked with B. minimus occurrence. CBCD was characterized by 112 ± 26 intergenic transcribed spacer (ITS) per individual and showed a relative homology between individuals (52 ± 6%, Jaccard similarity) in spite of more than 30% of rare ITS. Consistent with an undisturbed evolution of the condition index of the studied cohort individuals as an estimate of their physiological state, neither temporal nor parasite-induced change in CBCA has been related to marked changes in CBCD.

Metabolites of saxitoxin analogues in bivalves contaminated by Gymnodinium catenatum.

Bivalve metabolites of saxitoxin analogues, not present in microalgae, were recently described as an important toxin fraction in mussels contaminated by Alexandrium tamarense. These possess very low fluorescence, and require mass spectrometry detection. HILIC-MS was implemented to look for these metabolites in bivalves contaminated during Gymnodinium catenatum blooms at the Portuguese coast. The presence of M1 was tentatively identified in several bivalves, ranging from estuarine (Mytilus galloprovinciallis, Cerastoderma edule and Ruditapes decussatus) to oceanic habitat (Donax trunculus and Ensis spp.). It was hypothesized that M1 could contribute to an important fraction of the profile of STX analogues. M1 was more abundant in estuarine bivalves that retain longer PSP toxins, in the following order: mussels>cockles>clams. These data highlight that the study by fluorimetry alone of the carbamoyl, N-sulfocarbamoyl, and decarbamoyl families is manifestly insufficient to fully understand toxin dynamics in bivalves feeding on G. catenatum without a proper study of hydroxybenzoate and hydroxylated M-toxins.

Profiles of fatty acids and 7-O-acyl okadaic acid esters in bivalves: can bacteria be involved in acyl esterification of okadaic acid?

The presence of 7-O-acyl okadaic acid (OA) esters was studied by LC-MS in the digestive glands of blue mussel (Mytilus galloprovincialis) and common cockle (Cerastoderma edule) from Albufeira lagoon, located 20km south of Lisbon. The profile of free and total fatty acids (FA) was analysed using a similar LC separation with a reversed phase C8 column and mass spectrometry detection. In mussel the free FA profile was reflected in the FA esterified to OA, being palmitic acid for instance the most abundant in both cases. In cockle, 7-O-acyl esters with palmitic acid were almost absent and esters with a C16:0 isomer were dominant, followed by esters with C15:1 and C15:0. The cockle free FA profile was similar to mussel, and in accordance with literature findings in bivalves. After hydrolysis, a major difference in the FA profile occurred in both species, presenting a high percentage of a C16:0 isomer. The isomer found in general lipids and bound to OA seemed to be related, presenting similar relative retention times (RRT) to C16:0, differing from expected RRT of monomethyl-branched isomers (iso- or anteiso-). A tentative identification was made with the multimethyl-branched isoprenoid, 4,8,12-trimethyltridecanoic acid (TMTD). TMTD is a product of phytol degradation. This was also suspected when the proportion of this compound in relation to palmitic acid was reduced in vivo in mussels fed a chlorophyll-free diet. Extensive esterification of OA by, among others, phytol-degrading bacteria is discussed as a plausible hypothesis in cockle, but not in mussel, due to the relatively high specific proportion of odd-numbered and branched FA.

Interactive effects of metal contamination and pathogenic organisms on the marine bivalve Cerastoderma edule.

The present study evaluated the interactive effects of cadmium contamination and pathogenic organisms (trematodes Himasthla elongata and bacteria Vibrio tapetis) singularly and in combination during 7 days on the bivalve Cerastoderma edule. Some defense-related activities were analyzed such as genetic expression, metallothionein and immune responses. Trematode metacercarial infection, similar whatever the treatment, induced the strongest responses of immune parameters. Particularly, the interaction between cadmium and parasite exposures induced unusual responses on gene expression and immune responses. No effect of bacterial challenge appeared on bivalve responses, nevertheless a strong mortality of V. tapetis infected cockles occurred between 7 and 14 days. Cadmium bioaccumulation was significantly modulated by both pathogenic organisms. Furthermore, an antagonistic effect of trematodes and bacteria was shown on metal bioaccumulation of co-infected cockles. These results highlighted the importance of considering the multiplicity of perturbation sources in coastal ecosystems to assess the health status of organisms.

Novel plasmid-encoded ceftazidime-hydrolyzing CTX-M-53 extended-spectrum beta-lactamase from Salmonella enterica serotypes Westhampton and Senftenberg.

We describe the characterization of a novel CTX-M beta-lactamase from Salmonella enterica. Four S. enterica isolates (three of serotype Westhampton and one of serotype Senftenberg) resistant to extended-spectrum cephalosporins (cefotaxime and ceftazidime) were recovered in 2004 from living cockles in three supermarkets located in distant geographic areas in France, which got their supplies from the same fishery. The isolates were found to produce a novel extended-spectrum beta-lactamase (ESBL) belonging to the CTX-M-1 phylogenetic group and named CTX-M-53. The CTX-M-53 beta-lactamase harbored the substitution Asp240Gly, like the CTX-M-15 enzyme, which is specifically implicated in a higher catalytic efficiency against ceftazidime. The bla(CTX-M-53) gene was located on a mobilizable 11-kb plasmid, pWES-1. The complete sequence of pWES-1 revealed the presence of a novel insertion sequence, ISSen2, and an IS26 element upstream and downstream of the bla(CTX-M-53) gene, respectively; however, transposition assays of the bla(CTX-M-53) gene were unsuccessful. IS26 elements may have contributed to the acquisition of the bla(CTX-M-53) gene. Interestingly, the mobilization module of the pWES-1 plasmid was similar to that of quinolone resistance plasmids (carrying the qnrS2 gene) from aquatic sources. Although belonging to two serotypes differentiated on the basis of the O-antigen structure (E1 or E4 groups), the isolates were found to be genetically indistinguishable by pulsed-field gel electrophoresis. Multilocus sequence typing showed that the isolates of serotype Westhampton had a sequence type, ST14, common among isolates of serotype Senftenberg. This is the first characterization of the CTX-M-53 ESBL, which represents an additional ceftazidime-hydrolyzing CTX-M enzyme.

First isolation of Shiga toxin 1d producing Escherichia coli variant strains in shellfish from coastal areas in France.

AIMS: This study was carried out to evaluate the presence of Shiga toxin-producing Escherichia coli (STEC) and E. coli O157:H7 in shellfish from French coastal environments. METHODS AND RESULTS: Shellfish were collected in six growing areas or natural beds (B category) and nonfarming areas (D category) from July 2002 to August 2004. PCR detection of stx genes was performed on homogenized whole shellfish and digestive gland tissues enrichments. STEC strains were detected by colony DNA hybridization using a stx-specific gene probe and E. coli O157 strains were additionally searched by immunomagnetic separation with O157-specific magnetic beads. Stx genes were detected in 40 of 144 (27.8%) sample enrichments from mussels, oysters or cockles, 32 of 130 enrichments (24.6%) were from B-category areas and eight of 14 (57.1%) from the D-category area. Five strains carrying stx(1) or stx(1d) genes and one stx negative, eae and ehxA positive E. coli O157:H7 were isolated from six of 40 stx-positive enrichments. No relation was found between the total E. coli counts in shellfish and the presence of STEC strains in the samples. CONCLUSIONS: The STEC strains of different serotypes and stx types are present in shellfish from French coastal environments. It is the first isolation of STEC stx1d strains in France. SIGNIFICANCE AND IMPACT OF THE STUDY: Shellfish collected in coastal environments can serve as a vehicle for STEC transmission.