Occurrence of Bacterial Pathogens and Human Noroviruses in Shellfish-Harvesting Areas and Their Catchments in France

During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish.


Detection, isolation and characterization of Salmonella
Salmonella spp. were investigated using ISO-6579-1:2017 method and detection of invA and ttrBCA genes in selective enrichments by qPCR (Gonzáles-Escalona et al., 2012). Briefly, 25 g of homogenized blended SF, 10 g of sediment or membranes corresponding to the filtration of 1 liter of water were placed in buffered peptone water (BPW). The suspensions were incubated at 37°C for 18 hours. One hundred microliters of the BPW pre-enrichment were then transferred into 10 ml of Rappaport-Vasilliadis Soya peptone Broth (RVS) for selective enrichment at 41.5°C for 24 hours. In addition, 1 ml of the BPW pre-enrichment was transferred into 10 ml of Muller-Kauffman broth with tetrathionate-novobiocin (MKTTn) for selective enrichment at 37°C for 24 hours. Then, streaking onto selective agar plates: XLD, xylose desoxycholate agar (VWR) and Brilliance Salmonella (Oxoid) and incubation at 37°C of the plates for 24 hours were performed. Presumptive colonies (up to five by selective liquid media RVS and MKTTn) were streaked onto nutritive agar plates and incubated at 37°C for 24 hours.
Nucleic acids of these strains were released by heating at 100°C for 10 min and isolates were confirmed as Salmonella by detecting invA and ttrCBA genes specific of the Salmonella genus by qPCR (Gonzáles-Escalona et al., 2012).

Detection, isolation and characterization of Campylobacter
Campylobacter spp. were investigated using ISO-10272:2016 method and qPCR detection of 16S rRNA genes from enrichment broth (Leblanc-Maridor et al., 2011). Briefly, 25 g of homogenized blended SF, 10 g of sediment or membranes corresponding to the filtration of 1 liter of water were placed in Bolton broth under microaerobic conditions (CampyGen; Oxoid). Then, streaking onto selective agar plates: Casa medium (BioMérieux, Lyon, France) and mCCDA (Oxoid) and incubation at 41.5°C of the plates for 48-72 hours were performed. Up to eight presumptive colonies from these plates were streaked onto Karmali plates (Oxoid) and incubated at 41.5°C for 48-72 hours under microaerobic conditions.
In addition, Campylobacter were isolated from water samples using a passive migration method. In this purpose, 50 ml of water were filtered on a 0.2 µm nitrocellulose membrane. The filter was then placed retentate-down on a 0.45 µm cellulose membrane previously deposited on Casa agar. After incubation under microaerobic conditions at 41.5°C for 18 hours, filters were removed from the agar surface and plates were reincubated another 48 h.

Detection, isolation and characterization of Vibrio
Presence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in a selection of samples including shellfish and seawater was evaluated using ISO-21872:2017 method and detection of toxR gene, 16S-23S intergenic spacer (IGS) region and vvhA gene in selective enrichments by qPCR or PCR (Hervio-Heath et al., 2012, Chun et al., 1999, Lee et al., 1997. Primers specific for tdh and trh genes were used for the identification of haemolysin-producing and potentially pathogenic V. parahaemolyticus (Hervio-Heath et al., 2002). Briefly, 25 g of homogenized blended SF or membranes corresponding to the filtration of 1 liter of seawater were placed in alkaline peptone water 2% NaCl (APW, pH 8.6) and incubated at 41.5°C for 6 hours. One ml of the APW pre-enrichment was transferred in 10 ml APW and incubated at 41.5°C for 18 hours. The APW-enriched cultures were used for DNA extraction and V. parahaemolyticus, V. cholerae and V. vulnificus isolations.