Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds

ORIGINAL RESEARCH published: 01 December 2015 doi: 10.3389/fmicb.2015.01356 Edited by: Anton F. Post, University of Rhode Island, USA Reviewed by: Erin Katherine Lipp, University of Georgia, USA Dale Leavitt, Roger Williams University, USA *Correspondence: Michèle Gourmelon [email protected] Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Microbiology Received: 14 August 2015 Accepted: 16 November 2015 Published: 01 December 2015 Citation: Balière C, Rincé A, Blanco J, Dahbi G, Harel J, Vogeleer P, Giard J-C, Mariani-Kurkdjian P and Gourmelon M (2015) Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds. Front. Microbiol. 6:1356. doi: 10.3389/fmicb.2015.01356 Prevalence and Characterization of Shiga Toxin-Producing and Enteropathogenic Escherichia coli in Shellfish-Harvesting Areas and Their Watersheds Charlotte Balière1, Alain Rincé2, Jorge Blanco3, Ghizlane Dahbi3,JoséeHarel 4, Philippe Vogeleer4, Jean-Christophe Giard5, Patricia Mariani-Kurkdjian6,7,8 and Michèle Gourmelon1* 1Laboratoire Santé Environnement et Microbiologie, Unité Santé, Génétique et Microbiologie des Mollusques, Département Ressources Biologiques et Environnement, Ifremer, Plouzané, France, 2U2RM EA4655 Stress/Virulence, Normandie-Université, University of Caen Normandy, Caen, France, 3Departamento de Microbioloxía e Parasitoloxía, Facultade de Veterinaria, Universidade de Santiago de Compostela, Lugo, Spain, 4Groupe de Recherche sur les Maladies Infectieuses du Porc, Département de Pathologie et Microbiologie, Faculté de Médecine Vétérinaire, Centre de Recherche d’Infectiologie Porcine et Avicole, Université de Montréal, Saint-Hyacinthe, QC, Canada, 5U2RM EA4655 Antibio-Résistance, Normandie-Université, University of Caen Normandy, Caen, France, 6Service de Microbiologie, CNR Associé Escherichia coli, AP-HP, Hôpital Robert-Debré, Paris, France, 7Infection, Antimicrobials, Modelling, Evolution, UMR 1137, INSERM, Paris, France, 8Infection, Antimicrobials, Modelling, Evolution, UMR 1137, Université Paris Diderot – Sorbonne Paris Cité, Paris, France During a 2-years study, the presence of Shiga-toxin producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) was investigated in shellfish (n=238), seawater (n=12), and surface sediment (n=39) collected from three French coastal shellfishharvesting areas and freshwaters (n=216) in their watersheds. PCR detection of Shiga toxin- (stx1/stx2) and intimin- (eae) genes following enrichment from these samples revealed the presence of least one of the stx genes in 30.3% of shellfish batches, 85.9% of freshwater, 41.7% of seawater, and 28.2% of sediment samples, while the eae gene was observed in 74.8, 100, 100, and 43.6% of shellfish batches, freshwater, seawater, and sediment samples, respectively. Twenty-eight STEC and 89 EPEC strains were isolated and analyzed in order to determine their serotype, phylogroup, and genetic relatedness and to evaluate the presence of the saa and ehxA genes encoding the STEC autoagglutinating adhesin and the enterohemolysin A, respectively. Finally, the ability to form biofilms and antimicrobial susceptibility were investigated for a selection of strains. Eighteen serotypes were identified among the STEC isolates and 57 among the EPEC isolates. A high diversity was observed within these strains, as 79 different PFGE patterns and 48 distinguishable sequence types were identified. Strains were found to belong mainly to phylogroups B1 and B2 and virulence was observed to be low as more than 85% of the strains possessed only stx1,stx2, or eae genes. One STEC and several EPEC strains belonged to three of the five highly pathogenic serogroups (i.e., O26, O103, and O145). The subset of strains tested for their capacity to form biofilms was mainly strongly to moderately adherent and Frontiers in Microbiology | www.frontiersin.org 1December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment more strains formed a strong biofilm at 18 than at 30◦C. Finally, more than 85% of analyzed strains were found to be sensitive to the 16 tested antibiotics. These data suggest the low risk of human infection by STEC if shellfish from these shellfishharvesting areas were consumed. Keywords: STEC, EPEC, shellfish, water, MLST, PFGE, biofilms INTRODUCTION The microbiological quality of coastal environments can be impacted by urban and agricultural fecal wastes from watersheds. Moreover, shellfish can accumulate and concentrate pathogenic micro-organisms, such as Salmonella, pathogenic Escherichia coli (E. coli) and noroviruses present in surrounding waters by their filter-feeding activities (Potasman et al., 2002). This can lead to closures or downgrading of shellfish-harvesting areas and to outbreaks of food poisoning through consumption of contaminated shellfish (Iwamoto et al., 2010). Enumeration of E. coli, a fecal bacterial indicator, is the standard way to assess the level of fecal microorganisms in water and shellfish and indirectly, to estimate the associated potential risk to human health from all waterborne enteric pathogens (e.g., through classification of bathing areas and shellfish-harvesting areas; Anonymous, 2004). However, in addition to being a fecal indicator and a commensal bacterium, E. coli includes strains that can be pathogenic to humans. These can cause diarrhea and extra-intestinal diseases after acquiring virulence genes by genetic mobile elements such as bacteriophages, pathogenicity islands, and plasmids (Touchon et al., 2009). Pathogenic E. coli are distributed into diarrheagenic E. coli pathotypes including enterotoxigenic E. coli (ETEC), Shigella/enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC), enteropathogenic E. coli (EPEC), Shiga toxinproducing E. coli (STEC; for review, Croxen et al., 2013)andinto extra-intestinal E. coli pathotypes (Russo and Johnson, 2000). Escherichia coli occurrence in seafood is considered a sanitary case and may represent a risk to the consumers if related to diarrheagenic E. coli (for review, Costa, 2013). The study presented here focuses on EPEC (one of the main causes of diarrhea in infants) and STEC (an emerging zoonotic pathogen). Enteropathogenic E. coli is an important cause of infantile watery diarrhea, which is more frequently encountered in lowincome countries than in the industrialized world (Nataro and Kaper, 1998). They are known to create distinctive lesions on the surface of intestinal epithelial cells, called attaching and effacing (A/E) lesions. This property is encoded by genes, including eae, grouped together in a pathogenicity island referred to the ‘locus of enterocyte effacement’ (LEE; Paton and Paton, 1998). EPEC is transmitted from host to host via the fecal-oral route through contaminated surfaces, waters and food and human carriers. Humans, including symptomatic and asymptomatic children and asymptomatic adults, are the most likely source (Levine and Edelman, 1984). Animals, such as cattle and wildlife species, have been found to be additional sources (Singh et al., 2015). Twelve O serogroups have been recognized as EPEC by the World Health Organization: O26, O55, O86, O111, O114, O119, O125, O126, O127, O128, O142, and O158 (WHO, 1987). Shiga-toxin producing Escherichia coli areresponsiblefor the mucoid-bloody diarrhea that can progress to hemolytic uremic-syndrome (HUS), especially in children. One of the most important pathogenicity factors produced by STEC strains is the Shiga toxin (Stx), encoded by a lambdoid bacteriophage (O’Brien et al., 1984). Shiga toxins can be divided into two types, Stx1 (almost identical to Shiga toxin produced by Shigella dysenteriae type 1) and Stx2, encoded by stx1 and stx2 genes, respectively (Scheutz et al., 2012). In addition, the STEC strains are often able to produce the A/E lesions as a result of the presence of the LEE pathogenicity island, as in EPEC. This subset of STEC strains is also known as enterohemorrhagic E. coli (EHEC; McDaniel et al., 1995). Instead of this LEE pathogenicity island, they can also possess the auto-agglutinating adhesin factor designated Saa (STEC autoagglutinating adhesin; Paton et al., 2001). Adhesion to the intestinal mucosa is an essential step in the infection cycle of E. coli, which contributes to pathogenesis in humans. Other factors are involved in the virulence of STEC but also of EPEC, such as enterohemolysin A, encoded by the ehxA gene and associated with cytotoxic effects on endothelial cells that may contribute to the development of HUS (Jiang et al., 2015). STEC infections have been reported following the ingestion of contaminated food or water, after bathing in contaminated waters or contact with animals (for review, Croxen et al., 2013). The principal reservoir of STEC is the digestive tract of animals, particularly of cattle that are healthy carriers (Bibbal et al., 2015). Other animals, such as sheep, goats, swine, birds, and other wild animals, as well as humans, can also harbor STEC (Mora et al., 2012;Chandran and Mazumder, 2013). Most human illness is caused by the serotype STEC O157:H7 (Paton and Paton, 1998). However, it is becoming evident that non-O157 isolates belonging to the serogroups O26, O45, O91, O103, O111, O113, O121, O145 also cause significant human illness (Mellmann et al., 2009;USDA, 2011). In Europe, O157:H7 and the four serotypes: O26:H11, O103:H2, O111:H8, and O145:H28 are the most widely implicated in human STEC infections, constituting the five highly pathogenic serotypes (EFSA, 2013). Shiga-toxin producing Escherichia coli and EPEC contamination of the environment may occur through the spreading of livestock manure, animal waste on pastures, via wastewaters from slaughterhouses or from treatment plant effluents and by wildlife (Muniesa et al., 2006;Singh et al., 2015). In such environments, STEC and EPEC strains are exposed to various stresses, such as low temperature or nutrient depletion and the ability to form biofilm could be an advantage to increase persistence (Vogeleer et al., 2014). Frontiers in Microbiology | www.frontiersin.org 2December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment To date, very few studies have focused on the detection and isolation of pathogenic E. coli belonging to the STEC and EPEC pathovars in coastal environments (Gourmelon et al., 2006; Bennani et al., 2011). The aim of the study presented here was to detect and characterize STEC and EPEC strains from French shellfish-harvesting areas and their upstream watershed in order to assess the diversity of these pathogenic E. coli potentially present in this type of hostile environment. For this purpose, during a 2-years study, shellfish batches, freshwater, seawater, and surface sediment samples from three selected shellfish-harvesting areas and their upstream watersheds, the location of intensive livestock activities (cattle, swine, poultry, and/or sheep), were analyzed monthly to evaluate the presence of STEC and EPEC strains. MATERIALS AND METHODS Sampling Locations and Sample Description Shellfish, water, and surface sediment samples were collected from three shellfish-harvesting sites on the French coast of within the Eastern English Channel and their watersheds. One of these sites, located in Brittany (site 1), corresponded to a 121 km2watershed, characterized by intensive livestock farming (cattle, swine, and poultry), with a human population of about 9,000 inhabitants. The two others were situated in Normandy; site 2 was characterized by a 1,000 km2catchment, with large livestock farming (cattle, sheep, swine, and poultry) and about 40,000 inhabitants, while the second site (site 3) corresponded to a 50 km2watersheds with large livestock farming (cattle, sheep, and swine) and about 7,000 inhabitants. These two latter watersheds are geographically closer together than the Brittany site (location of the sites Supplementary Figure S1). The three shellfish-harvesting areas are classified as category B for oysters (Crassostrea gigas) and mussels (Mytilus edulis) and as category C for common cockles (Cerastoderma edule) according to European regulation (European Directive 91/492/EEC; Anonymous, 2004). Shellfish from category B shellfish-harvesting areas must be depurated before being sold and shellfish from category C areas must be relayed at least 2 months prior to sale for consumption. Shellfish [oyster, mussel, and common cockle batches (site 1, n=120; site 2, n=72; and site 3, n=46)] and freshwater samples from nine sampling sites upstream of shellfish-harvesting areas (site 1, n=96; site 2, n=72; site 3, n=48) were collected monthly from February 2013 to January 2015, whereas surface sediment samples (site 1, n=13; site 2, n=13; site 3, n=13) were collected from February 2013 to January 2014 and seawater samples (site 1, n=12) from February 2014 to January 2015. Isolation of STEC and EPEC Strains Samples were transported in insulated cooler boxes to the laboratory and analyzed within 24 h. After opening, total shellfish flesh, including shellfish flesh and intravalvular liquid, were homogenized in a commercial blender (Waring Products Division, Torrington, CT, USA) for 60 s at high speed. Twenty-five grams of homogenized total shellfish flesh were inoculated into 225 ml of buffered peptone water (BPW). For surface sediments, 10 g were introduced into the same volume of BPW. For water samples, 1 L was filtered using 0.45 μm cellulose membranes (Pall Gelman GN-6 Metricel; Pall Corporation, St Germain-en-Laye, France) and the filter was placed in 225 ml of BPW. Incubation was performed at 37◦C for 24 h. Shiga-toxin producing Escherichia coli and EPEC strains were isolated from the environmental samples using three additional protocols. The first one, described by Balière et al. (2015), involves application of the ISO/TS-13136 method, which focuses on isolation of strains belonging to the five highly pathogenic serotypes and was applied to samples collected from February 2013 to February 2014. The two other protocols involve the isolation of STEC and EPEC with or without an enrichment step and independently from the serotype. These were applied to samples collected from February 2013 to January 2015. For the protocol with an enrichment step (described in Balière et al., 2015), DNA was extracted from 500 μLof each BPW enrichment broth using NucliSENS Nucleic Acid Extraction Reagents for miniMAG (BioMérieux, Marcy l’Etoile, France), according to the manufacturer’s instructions. The stx and eae genes were detected by real-time PCR (Agilent MX3000P, Waldbronn, Germany), using primers and probes published previously (Nielsen and Andersen, 2003;Perelle et al., 2007), according to the ISO/TS-13136: 2012 technical specification, with slight modifications concerning the PCR cycles [denaturation for 10 s at 95◦C, primer annealing for 5 s at 55◦C, and extension for 25 s at 60◦C (45 cycles)]. BPW broths identified positive for stx and eae were screened for STEC and EPEC isolates by streaking 1 μL of these broths onto Tryptone-Bile-Xglucuronide agar (TBX; AES chemunex, Bruz, France) and onto chromIDTM agar (BioMérieux), followed by incubation at 44◦C for 24 h. The final protocol to be used involves the screening of E. coli isolated directly from the water and shellfish samples without an enrichment step. For this protocol, 1, 10, and 100 ml of water were filtered through 0.45 μm cellulose membranes and the filters were placed onto TBX agar. For shellfish, 10 g of blended total shellfish flesh were distributed onto five empty and sterile plates with overlay super-cooled TBX agar. All TBX plates were incubated at 44◦C for 24 h. Presumptive STEC and EPEC isolates were confirmed by real-time PCRs targeting stx1,stx2,andeae genes, as described above after a DNA extraction of each isolate by boiling at 100◦C, for 15 min. The STEC and EPEC isolates were characterized using several protocols as described below. Serotyping The serotypes of the STEC and EPEC strains were characterized using the serotyping method by agglutination, as described by Blanco et al. (2003). More precisely, determination of O and H antigens was carried out by agglutination as previously described (Guinée et al., 1981), employing all available O (O1-O185) and Frontiers in Microbiology | www.frontiersin.org 3December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment H (H1-H56) antisera. All antisera were absorbed with the corresponding cross-reacting antigens to remove the nonspecific agglutinins. The O and H antisera were produced in the Laboratorio de Referencia de E. coli (USC, Lugo, Spain). Isolates that did not react with O antisera were considered as non-typeable (ONT) and those non-motile were HNM. Detection of Enterohemolysin and Adhesin The presence of ehxA (encoding enterohemolysin A) and saa (encoding STEC autoagglutining adhesin) genes in these isolates was investigated by conventional PCR using primers previously described by Paton and Paton (2002). Phylogenetic Group Isolates were classified into the four main E. coli phylogenetic groups (A, B1, B2, or D) using a conventional triplex PCR method based on the detection of two genes, chuA, required for heme transport in enterohemorrhagic O157:H7 E. coli, yjaA, initially identified in the recent complete genome sequence of E. coli K12, for which the function is unknown, and of an anonymous DNA fragment designated TSPE4.C2 using primers described previously by Clermont et al. (2000). Pulsed-field Gel Electrophoresis The genetic relatedness of the isolates was studied by the pulsed-field gel electrophoresis method (PFGE) according to Bidet et al. (2005). Isolated strains were inoculated in nutrient broth containing 1.3% NaCl (Bio-Rad, Marnes-la-coquette, France) and incubated at 37◦C for 24 h. Bacterial DNA was extracted from 400 μl of the enrichment broth using the CHEF Bacterial Genomic DNA Plug Kit (Bio-Rad) according to the manufacturer’s recommendations. Bacterial DNA was digested for between 16 and 20 h at 37◦C with the restriction endonuclease XbaI (Roche Diagnostic, Meylan, France) according to the manufacturer’s recommendations. Each electrophoresis was performed using a lambda ladder molecular mass marker (BioRad) for the normalization of gel images. The migration was performed on a 1% agarose gel using the CHEF-DRIII apparatus (Bio-Rad) at 6 V cm−1for 27 h, with pulse times varying linearly between two and 49 s. The bacterial DNA restriction patterns were analyzed using the Bionumerics software 7.5 (Applied Maths, Kortrijk, Belgium). The similarity of PFGE profiles was compared and a dendrogram was created using the band-based Dice unweighted-pair group method, using average linkages (UPGMA), based on 1% position tolerance and 0% position optimization. Branch quality was evaluated using Cophenetic correlation. PFGE patterns were considered clonally related when they had a similarity coefficient higher than 80%. Multilocus Sequencing Typing The genetic relatedness of the isolates was also studied using the multilocus sequencing typing method (MLST). Fragments of seven housekeeping genes, i.e., adk (adenylate kinase), fumC (fumarate hydratase), gyrB (DNA gyrase), icd (isocitrate/isopropylmalate dehydrogenase), mdh (malate dehydrogenase), purA (adenylosuccinate dehydrogenase), and recA (ATP/GTP binding motif) were amplified and sequenced using suitable primers (Wirth et al., 2006) with minor modifications for the recA primers (recAR 5-TCG-TCGAAA-TCT-ACG-GAC-CGG-A-3;recAF15 -ACC-TTT-GTAGCT-GTA-CCA-CG-3). The PCR cycle included denaturation for 60 s at 95◦C, primer annealing for 60 s at 56◦C(for adk,purA, recA, and icd), at 65◦C(formdh and gyrB), or at 68◦C(forfumC), and extension for 60 s at 72◦C(35 cycles) in MJ Research PTC-200 (DNA Engine, Waltham, MA, USA). Sequencing was performed in both directions with the fluorescent dye terminator Sanger method on ABI3730 (Applied Biosystem) by Eurofins Genomics (Ebersberg, Germany). The alleles and sequence types (ST) were assigned in accordance with the E. coli MLST database (http://mlst.warwick.ac.uk/mlst/ dbs/Ecoli). Static Biofilm Formation Assay A biofilm formation assay was performed as previously described by Tremblay et al. (2015). In addition to the incubation temperature of 30◦C, the ability to form biofilms was also tested at 18◦C, in order to reproduce marine temperate environmental conditions (Moldoveanu, 2012). Briefly, overnight cultures at 37◦C in LB media were diluted (1:100) in 5 ml of M9 medium with glucose (0.4% wt/vol) and minerals (1.16 mM MgSO4, 2μM FeCl3,8μMCaCl 2,and16μMMnCl 2) and incubated for 24 h at 37◦C. These cultures were diluted (1:100) in M9 medium supplemented with glucose and minerals and were inoculated in triplicate into microtitre plates (Costar 3370; Corning, NY, USA). After 24 h of incubation at 18 or 30◦C, unattached cells were removed by washing three times with phosphate-buffered saline (PBS). Plates were dried at 37◦Cfor 15 min and biofilms were stained with crystal violet (0.1% wt/vol) for 2 min. After removal of crystal violet solution, the biofilms were washed three times with PBS and dried at 37◦C for 15 min. The biofilm stain was dissolved with 150 μl of 80% (vol/vol) ethanol and 20% (vol/vol) acetone and biofilms were quantified by measuring the absorbance at 590 nm (OD590) with a microplate reader (Powerwave; BioTek Instruments, Winooski, VT, USA). The results for the static biofilms formed at 18 and 30◦C were compared using two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc comparison using GraphPad Prism, version 4.02 (GraphPad Software, San Diego, CA, USA). Strains were divided into three groups based on the OD590 of bacterial biofilm: strong (A590 >0.6), medium (0.6 ≥A590 ≥0.3) and weak or none (A590 <0.3). Antibiotic Resistance Antimicrobial susceptibility testing based on the disk diffusion method was performed on a selection of STEC and EPEC isolates. Sixteen antibiotics were tested: Tobramycin (10 μg), Fosfomycin (50 μg), Cefalotin (30 μg), Imipenem (10 μg), Tigecyclin (15 μg), Gentamycin (15 μg), Cefotaxim (30 μg), Cefoxitin (30 μg), Doxycyclin (30 μg), Ciprofloxacin (5 μg), Augmentin (20 μg Frontiers in Microbiology | www.frontiersin.org 4December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment amoxicilin; 10 μg clavulanic acid), Ticarcillin (75 μg), Bactrim (1.25 μg trimethoprim; 23.75 μg sulfamethoxazol), Nalidixic acid (30 μg), Amikacin (30 μg), Amoxicillin (25 μg) on MuellerHinton medium (AES chemunex, Bruz, France). Plates were incubated at 37◦Cfor24h. Environmental Data and Statistical Analysis Rainfall data (2-days cumulative rainfall before sampling date) were provided by the meteorological stations from Meteo France at Pleurtuit (site 1) and at Coutances (sites 2 and 3). The water temperature was measured manually at each sampling. The data on temperature and precipitation were categorized into three groups whose boundaries were defined so that they are likely to categorize the data significantly for the studied sites and they allow to have in each category a number of sample consistent with a reliable statistical analysis. Comparisons of STEC and EPEC prevalence between the type of samples, the site, the season, the temperature and the precipitation were analyzed by the chi-square test. A p-value of <0.05 was considered statistically significant. RESULTS Detection and Isolation of STEC and EPEC Strains The stx gene was detected in 30.3, 85.9, 41.7, and 28.2% of shellfish, freshwater, seawater and surface sediment enrichment broths, respectively (Table 1). The eae gene was detected in 74.8, 100, 100, and 43.6% of shellfish, freshwater, seawater, and surface sediment enrichment broths, respectively (Table 1). STEC were isolated from 5.0% of the 238 shellfish, 5.6% of the 216 freshwater, 8.3% of the 12 seawater, and 2.6% of the 39 surface sediment samples analyzed, whereas EPEC were isolated from 8.0, 21.3, and 33.3% of the shellfish, freshwater, and seawater samples, respectively. No EPEC were isolated from surface sediments (Table 2). A total of 57 STEC and 117 EPEC isolates were obtained from these samples. However, as 29 STEC and 28 EPEC had identical serotypes, PFGE and MLST patterns, virulence gene profiles, and phylogroups to other isolates cultivated from the same samples, they were considered to be replicates and not retained. The remaining 28 STEC and 89 EPEC isolates represented 0.2 and 0.7% of the total E. coli (n=12,016), respectively (Table 3). TABLE 2 | Isolation of STEC and EPEC as regard to sample parameters. Sample parameter No. of samples No. of samples with at least one STEC isolate (%)a No. of samples with at least one EPEC isolate (%)a Type Shellfish 238 12 (5.0)19 (8.0) Freshwater 216 12 (5.6)46 (21.3) Seawater 12 1 (8.3)4(33.3) Superficial sediment 39 1 (2.6)0 χ2test p=0.845 p=3.09 10−5 Site Site 1 241 10 (4.1)34 (14.1) Site 2 157 7 (4.5)20 (12.7) Site 3 107 9 (8.4)15 (14.0) χ2test p=0.244 p=0.931 Season Fall 146 4 (2.7)11 (7.5) Summer 126 17 (13.5)22 (17.5) Autumn 124 3 (2.4)24 (19.4) Winter 109 2 (1.8)12 (11.0) χ2test p=4.44 10−5p=0.0314 Temperature (◦C) 0–<10 172 5 (2.9)15 (8.7) 10–<15 189 14 (7.4)29 (15.3) 15–>15 144 7 (4.9)25 (17.4) χ2test p=0.167 p=0.086 Precipitation (mm)b 0–<1 253 17 (6.7)30 (11.9) 1–<10 144 5 (3.5)21 (14.6) 10–>10 108 4 (3.7)18 (16.7) χ2test p=0.296 p=0.495 Total 505 26 (5.1) 69 (13.7) aPercentage calculated based on the total of no. samples for each type of sample. bPrecipitation: 2-days cumulative rainfall before sampling date. Shiga-toxin producing Escherichia coli strains represented 0.2, 0.2, and 0.3%, of the isolated E. coli from sites 1–3, respectively. EPEC strains represented 0.8, 0.6, and 0.7% of the isolated E. coli from sites 1–3, respectively (Table 3). For the three sites, the majority of STEC strains derived from freshwater samples (50, 57.1, and 44.4% of samples from sites 1–3, respectively) and from shellfish batches (41.7, 42.9, and 44.4% in the sites 1–3, respectively; Table 3). Only two STEC strains were isolated from seawater and surface sediment samples from sites TABLE 1 | Prevalence of stx and eae genes in shellfish, freshwater, seawater, and superficial sediment enrichment broths. Type of sample No. of samples No. of stx-positive broth (%)aNo. of eae-positive broth (%)aNo. of stx-andeae-positive broth (%)a Shellfish 238 72 (30.3)178 (74.8)64 (26.9) Freshwater 216 196 (85.9)216 (100)196 (85.9) Seawater 12 5 (41.7)12 (100)5(41.7) Superficial sediment 39 11 (28.2)17 (43.6)8(20.5) Total 505 284 (56.2)423 (83.7)273 (54.1) aPercentage calculated based on the total of no. samples for each type of sample. Frontiers in Microbiology | www.frontiersin.org 5December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment TABLE 3 | Number of STEC and EPEC strains isolated from shellfish, freshwater, seawater, and superficial sediment samples collected in the three shellfish-harvesting areas and their watersheds, as regard to the total number of E. coli isolates. Type of sample Total (%)aShellfish (%) Freshwater (%) Seawater (%) Superficial sediment (%) Site 1 No. E. coli isolates 5,676 1,343 3,410 225 30 No. STEC strains 12 (0.2) 5 (41.7)b6 (50.0)b1 (8.3)b0 No. EPEC strains 47 (0.8) 8 (17.4)b35 (76.1)b4 (8.5)b0 Site 2 No. E. coli isolates 3,682 757 2,925 ndc4 No. STEC strains 7 (0.2) 3 (42.9)b4 (57.1)bnd 0 No. EPEC strains 23 (0.6) 10 (43.5)b13 (56.5)bnd 0 Site 3 No. E. coli isolates 2,658 678 2,036 nd 83 No. STEC strains 9 (0.3) 4 (44.4)b4 (44.4)bnd 1 (11.1)b No. EPEC strains 19 (0.7) 5 (26.3)b14 (73.7)bnd 0 Total of the three sites No. E. coli isolates 12,016 2,778 8,371 225 117 No. STEC strains 28 (0.2) 12 (42.9)b14 (50.0)b1 (3.6)b1 (3.6)b No. EPEC strains 89 (0.7) 23 (25.8)b62 (69.7)b4 (4.5)b0 aPercentage calculated based on the total of no. E. coli isolates from each sites. bPercentage calculated based on the total of no. STEC or EPEC strains isolated from the three sites. cnd: non-determined. 1 and 3, respectively. The majority of EPEC strains derived from the freshwater samples (76.1, 56.5, and 73.7% of samples from sites 1–3, respectively) and the remaining EPEC derived from shellfish batches (17.4, 43.5, and 26.3%, respectively) and seawater samples (8.5% only in the site 1; Table 3). Nearly one third of the STEC strains were obtained from samples collected in May 2013 (32.1%, 9/28), whereas the EPEC strains were mostly obtained from samples collected in November 2013 (21.3%, 19/89) and August 2014 (12.4%, 11/89; Table 4). The entire sample set demonstrated a seasonal effect with potential pathogenic E. coli as STEC strains were significantly more frequently isolated in Summer and EPEC strains in Summer and Autumn (p<0.05; Table 2). However, no correlation between the prevalence of both STEC and EPEC and pluviometry nor temperature was observed (Table 2). VirulenceGeneProfiles By considering the presence of a single virulence gene or a combination of the four virulence genes investigated (i.e., stx, eae, ehxA, and saa) in the 117 STEC or EPEC strains, eight virulence gene profiles were found. The most frequent profile presented the eae gene only (70.1% of the strains) followed by the profile presenting the stx2 gene only (11.1%) and the profile presenting the stx1 gene only (7.7%). Seven strains (6.0%) were shown to possess the eae and ehxA genes. The stx1-stx2-ehxA-saa profile was found in three strains and three other virulence gene profiles were observed only once, i.e., the stx1-eae-ehxA,thestx1-stx2-saa, and the stx2-ehxA-saa profiles (Table 4). Seven STEC strains carrying stx1, three carrying stx2,andtwo carrying both stx1 and stx2 geneswereisolatedfromthesite1 whereas three STEC strains harboring the stx1 gene, 11 the stx2 gene, and two presenting both stx1 and stx2 genes were recovered from sites 2 and 3. Phylogroups The STEC strains (n=28) were mainly distributed among the phylogroups A, B1, and D (39.3, 35.7, 21.4%, respectively). Only one strain belonged to phylogroup B2. The EPEC strains (n=89) belonged to all the phylogroups; the strains from phylogroup B1 and B2 (38.2 and 38.2%, respectively) being more prevalent than those from phylogroups A and D (18.0 and 5.7%, respectively; Table 5). More precisely, at site 1, the STEC strains were mainly divided between phylogroups B1 and D. At sites 2 and 3, the STEC strains were divided between phylogroups A and B1. At sites 1–3, the EPEC strains belonged to all the investigated phylogroups, with a majority belonging to phylogroups B1 and B2. Serotyping The 117 (STEC or EPEC) strains selected in this study belonged to 44 O antigens and 24 H antigens and presented 75 distinguishable serotypes (Table 4). Among all strains, 13 strains were nontypable (NT) for the O antigen [ONT:H2 (n=1), ONT:H31 (n=1), ONT:H34 (n=1), ONT:H6 (n=6), ONT:H8 (n=2), ONT:H10 (n=1), ONT:H11 (n=1)] and 24 strains were uncharacterized for the H antigen (HNM: non-motile or HNT: non-typable).Two strains were non-typable for either antigens (ONT:HNT). Eighteen different serotypes (O:H) were identified among the STEC strains. Only one STEC belonging to one of the five highly pathogenic serotypes was isolated: an O26:H11 stx1+, eae+,and ehxA+strain, from a mussel batch. One strain from serotype O91:H21 and carrying stx1,stx2,ehxA, and saa genes was also identified among the STEC strains. The most detected serotype among the STEC strains was the O100:HNM (n=9). Fifteen additional serotypes (O149:H31/HNM, O154:H31/HNM, O130:H11, O15:H16, O185:H28, O2:H32, O28:H1, O63:H11, O76:H19, O8:H12, O88:H25, ONT:H10, and ONT:H11) were Frontiers in Microbiology | www.frontiersin.org 6December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment TABLE 4 | Characteristics of STEC and EPEC strains isolated from the three French shellfish-harvesting sites from the Eastern English Channel coastal area and their watersheds. Serotype (no. of isolate) Virulence gene (no. of isolate) Sample origin (no. of isolate) Sampling site (no. of isolate) Sampling month-year Precipitation (mm) STEC O100:HNM (9) stx2 SFm(1), SFc(1), 2 May-13 0.1 FW(1), SFo(1), SFm(1) 3 May-13 0.1 SFm(1), S(1) 3 June-13 6.1 SFm 2 March-14 0.1 SFo 3 June-14 0 O15:H16(1) stx2 SFm 1 June-13 0.2 O2:H32(1) stx2 SW 1 February-14 0 O8:H19(2) stx2 FW 3 June-14 0 FW 3 July-14 1.4 O149:H1(1) stx1 FW 1 April-13 1.6 O149:HNM(1) stx1 FW 1 April-13 1.6 O154:H31(2) stx1 SFo(1), FW(1) 1 May-13 0.1 O154:HNM(1) stx1 SFm 1 November-14 15.4 O28: H1(1) stx1 SFc 1 March-14 0.1 O76:H19(1) stx1 FW 2 August-14 26.0 O88:H25(1) stx1 FW 2 September-14 0.4 ONT:H10(1) stx1 FW 2 April-14 0.3 O26:H11(1) stx1+eae+ehxA SFm 1 November-13 15.4 O63:H6(1) stx2+ehxA+saa FW 1 November-14 24.0 ONT:H11(1) stx1+stx2+saa FW 1 May-13 1.8 O185:H28(1) stx1+stx2+ehxA+saa FW 2 Apr-13 1.6 O130:H11(1) stx1+stx2+ehxA+saa FW 1 December-13 0 O91:H21(1) stx1+stx2+ehxA+saa FW 3 May-13 1.8 EPEC O103:H25(1) eae FW 3 March-14 0.1 O103:HNM(1) eae FW 1 February-14 0 O108:H21(3) eae FW 3 November-13 0.1 SFc 2 January-14 6.5 FW 3 July-14 1.4 O113:H6(4) eae SFm(1), SFc(1) 1 November-13 15.4 FW 3 November-13 0.1 SFo 3 August-14 26 O116:H20(1) eae FW 1 November-13 15.4 O125:H6(2) eae FW 1 November-13 15.4 FW 2 September-14 0.4 O128:H2(1) eae FW 2 November-13 0 O137:H6(2) eae FW 1 August-13 1.0 FW 3 January-15 2.3 O145:H34(1) eae FW 1 July-14 6.5 O146:H21(1) eae SFm 3 August-14 26.0 O146:H6(1) eae SFc 2 September-14 0.4 O15:H2(2) eae FW 3 August-14 26.0 SFm 2 January-15 2.3 O153:H21(1) eae FW 1 January-14 7.2 O157:H16(1) eae FW 1 July-14 6.5 O159:H7(1) eae FW 1 November-14 24.0 O167:H3(1) eae FW 1 August-13 2.8 O179:H31(2) eae FW 1 January-14 7.2 FW 3 June-14 0 O2:H45(1) eae FW 2 December-13 0 O20:HNT(1) eae SW 1 April-14 0 (Continued) Frontiers in Microbiology | www.frontiersin.org 7December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment TABLE 4 | Continued Serotype (no. of isolate) Virulence gene (no. of isolate) Sample origin (no. of isolate) Sampling site (no. of isolate) Sampling month-year Precipitation (mm) O23:H8(2) eae FW 2 August-13 0.7 SFc 2 August-14 26.0 O25:H2(1) eae FW 1 September-14 0 O28:H16(1) eae SFm 1 November-13 15.4 O29:H19(1) eae FW 2 October-13 22.8 O33:H6(1) eae FW 1 February-14 0 O39:HNM(1) eae FW 1 January-14 7.2 O40:HNM(1) eae FW 1 February-14 0 O42:H37(1) eae FW 3 March-13 0 O5:H40(1) eae SFm 2 February-13 0.8 O51:HNM(1) eae FW 2 October-13 22.8 O63:H6(4) eae FW 1 November-13 15.4 FW 2 November-13 0.1 FW 1 January-14 7.2 FW 3 October-14 0.5 O63:HNM(1) eae FW 3 October-14 0.5 O71:H49(2) eae SFm(1), SFc(1) 1 October-13 0.4 O71:HNM(1) eae FW 1 August-14 1.0 O8:H14(1) eae FW 1 November-13 15.4 O85:H31(1) eae FW 1 October-14 8.3 O85:HNM(1) eae FW 1 August-14 1.0 O86:H31(1) eae SFc 2 August-14 26.0 O88:H25(1) eae SFm 3 September-14 0.4 O88:H8(1) eae SFo 3 August-14 26.0 O9:HNM(1) eae FW 2 November-13 0.1 O91:H10(1) eae SFc 1 July-14 6.5 O98:H56(1) eae FW 1 December-14 0 O98:H8(1) eae SFm 1 November-13 15.4 O98:HNM(2) eae SW 1 February-14 0 FW 1 March-14 0 O98:HNT(1) eae FW 1 December-14 0 ONT:H2(1) eae FW 1 August-13 2.8 ONT:H31(1) eae FW 1 October-14 8.3 ONT:H34(1) eae FW 3 December-14 10.6 ONT:H6(6) eae FW 1 September-13 1.0 FW 1 August-14 1.0 FW 3 September-14 0.4 FW 3 October-14 0.5 SFm 2 December-14 0 SW 1 December-14 10.6 ONT:H8(2) eae FW 1 April-14 0 FW 1 August-14 1 ONT:HNT(2) eae SW 1 November-14 24.0 FW 3 October-14 10.6 O28:HNM(1) eae+ehxA FW 1 June-14 0.2 O145:H28(2) eae+ehxA SFm(1), SFc(1) 2 June-13 6.1 O177:H11(1) eae+ehxA FW 2 July-14 1.4 O26:H11(6) eae(5), eae+ehxA(1) FW 2 August-13 0.7 FW(3), SFm(1), SFc(1) 1 November-13 15.4 O103:H2(3) eae(2), eae+ehxA(1) FW 2 February-13 0.8 FW 2 June-13 6.1 FW 1 November-13 15.4 O153:H2(3) eae(2), eae+ehxA(2) SFm(1), FW(1) 2 (1), 3 (1) March-14 0.1 SFc 2 July-14 1.4 NT, non-typable; NM, non-motile. Sample origin: SF: shellfish, o: oyster, m: mussel, c: cockle, FW: freshwater, SW: seawater, S: superficial sediment. ( ): number of strain and when it is not specified the no. of strain is one. (1) Brittany site, (2) first Normandy site, and (3) second Normandy site. Precipitation: 2-days cumulative rainfall before sampling date. Frontiers in Microbiology | www.frontiersin.org 8December 2015 | Volume 6 | Article 1356 Balière et al. STEC/EPEC in Coastal Environment TABLE 5 | Distribution of phylogroup A, B1, B2, and D among STEC and EPEC strains isolated in the three French shellfish-harvesting sites from the Eastern English Channel coastal area and their watersheds. Phylogroup Total A (%) B1 (%) B2 (%) D (%) Site 1 No. STEC strains (%)a12 2 (16.7)4(33.3)06(50.0) No. EPEC strains (%)b47 13 (27.7)13 (27.7)19 (40.4)2(4.4) Site 2 No. STEC strains (%)a73(42.9)3(42.9)1(14.2)0 No. EPEC strains (%)b23 2 (8.7)13 (56.5)5(21.7)3(13.0) Site 3 No. STEC strains (%)a96(66.7)3(33.3)00 No. EPEC strains (%)b19 1 (5.3)8(42.1)10 (52.6)0 Total of the three sites No. STEC strains (%)c28 11 (39.3)10 (35.7)1(3.6)6(21.4) No. EPEC strains (%)c89 16 (18.0)34 (38.2)34 (38.2)5(5.7) aPercentage calculated based on the total no. of STEC strains isolated from each site. bPercentage calculated based on the total no. of EPEC strains isolated from each site. cPercentage calculated based on the total no. of STEC and EPEC strains isolated from the three sites. identified within the STEC strains and contained one or two individual isolates each. Fifty-seven serotypes were identified among the EPEC strains. Eleven strains belonged to the highly pathogenic serotypes: O26:H11 (n=6), O103:H2 (n=3), and O145:H28 (n=2). The remaining EPEC strains belong to a large diversity of serotypes listed in Table 4. It should be noted that some serotypes were isolated at different months and in different types of samples. For example, the nine strains of serotype O100:HNM stx2+were isolated from seven shellfish batches (n=7; oyster, mussel, and common cockle batches), from one freshwater (n=1) and from one surface sediment sample (n=1). Three O154:H31 stx1+and their immotile form were isolated from two shellfish batches (n=2; oyster and mussel batches) and from one freshwater sample (n=1). The EPEC serotypes, O108:H21 (n=3), O113:H6 (n=4), O15:H2 (n=2), O153:H2 (n=3), O23:H8 (n=2), O26:H11 (n=6), and O71:H49/HNM (n=3) were all isolated from shellfish batches and also from freshwater samples (Table 4). The same serotypes were sometimes isolated from geographically independent sites. For example, serotypes O103:H2, O125:H6, and O26:H11 were isolated from sites 1 and 2 and serotypes O113:H6, O137:H6, and O179:H31 from sites 1 and 3. Finally, the O63:H6 and ONT:H6 serotypes were isolated from all the three sites (Table 4). PFGE and MLST Profiles The genetic relatedness of 26 STEC and 79 EPEC strains was investigated by PFGE and MLST analysis (Supplementary Figure S2). Seventy-nine distinguishable PFGE patterns (PT) and 46 distinguishable sequence types (STs) were obtained. Seven other STs (seven strains, one STEC and six EPEC) were obtained but have not as yet been described. These results demonstrate a high level of genetic diversity among the strains isolated. The highest diversity was observed for the EPEC strains, which represented 8.1% of the PTs identified (64/79) and 71.7% of the STs (33/46). The STEC serotype O100:HNM (n=9) presented identical PT (D) and ST (ST933; Figure 1A) despite of their three specificities: isolated (a) from oyster, mussel, and common cockle batches, freshwater, and superficial sediment samples, (b) from the sites 2 and 3, (c) during the sampling campaigns of May 2013, June 2013, March 2014, and June 2014. With regards to the major serotypes (Figure 1B), the six EPEC O26:H11 strains presented three additional PTs (i.e., L, BV, and AL) and two STs (i.e., ST29 and ST48) isolated from mussel and common cockle batches and freshwater samples. One of those belonging to the ST29 was isolated from the same mussel batch from which the STEC O26:H11 belonging to the ST21 was isolated. A unique PT (I) was observed for the two EPEC O145:H28 (ST not yet described), isolated from mussel and common cockle batches and sampled during the same campaign (June 2013). Both O103:H2 isolated during two different months (i.e., June 2013 and November 2013) had distinguishable PTs (i.e., M and N) and STs (i.e., ST1146 and ST343). Additionally, identical PTs and STs were found among the other STEC and EPEC strains isolated from different types of sample (i.e., shellfish vs. freshwater) or between shellfish batches (i.e., mussel vs. common cockle) and between freshwater samples, often from samples taken from the same sites on the same date (e.g., O153:H2, O108:H21 serotypes; Supplementary Figure S2). Biofilm Formation Biofilm formation by a subset of 13 EPEC and nine STEC strains was evaluated at 18 and 30◦C on plastic surface. At both temperatures, strains varied in their ability to form biofilm (OD590 =0.03 for the lowest, OD590 =1.9 for the highest). In general, a large number of strains were strongly to moderately adherent and more strains formed a biofilm at 18 than at 30◦C (Figure 2). Indeed, 11 of the 22 strains formed strong biofilms [serotypes O2:H32, O149:H1, ONT:H11, O91:H21, O185:H28, O26:H11 (n=2), O145:H28 (n=1), O103:H2 (n=3)], six formed medium biofilms [serotypes O26:H11 (n=3), O145:H28 (n=2) and O125:H6] and five formed weak biofilms or no biofilm at all [serotypes O100:HNM, O154:H31, O15:H16, and O26:H11 (n=2)]. At 30◦C, 11 strains formed strong biofilms [serotypes O2:H32, O149:H1, ONT:H11, O91:H21, O185:H28, O145:H28 (n=3), O103:H2 (n=2) and O125:H6] but Frontiers in Microbiology | www.frontiersin.org 9December 2015 | Volume 6 | Article 1356