High genetic diversity ofVibrio choleraein the European lake Neusiedler See is associated with intensive recombination in the reed habitat and the long-distance transfer of strains

Coastal marine Vibrio cholerae populations usually exhibit high genetic diversity. To assess the genetic diversity of abundant V. cholerae non-O1/non-O139 populations in the Central European lake Neusiedler See, we performed a phylogenetic analysis based on recA, toxR, gyrB and pyrH loci sequenced for 472 strains. The strains were isolated from three ecologically different habitats in a lake that is a hot-spot of migrating birds and an important bathing water. We also analyzed 76 environmental and human V. cholerae non-O1/non-O139 isolates from Austria and other European countries and added sequences of seven genome-sequenced strains. Phylogenetic analysis showed that the lake supports a unique endemic diversity of V. cholerae that is particularly rich in the reed stand. Phylogenetic trees revealed that many V. cholerae isolates from European countries were genetically related to the strains present in the lake belonging to statistically supported monophyletic clades. We hypothesize that the observed phenomena can be explained by the high degree of genetic recombination that is particularly intensive in the reed stand, acting along with the long distance transfer of strains most probably via birds and/or humans. Thus, the Neusiedler See may serve as a bioreactor for the appearance of new strains with new (pathogenic) properties.The study was financed by the Austrian Science Fund FWF, project nr P21625-B20. In addition, CP and ISD were partially supported by the Austrian Science Fund FWF, P25745-B20. CA thanks to the Ministerio de Educacion, Cultura y Deporte and FEDER funds for the grant AGL2014-58933-P. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.Peer Reviewe

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

Diagnostic moléculaire de l origine des contaminations fécales dans l environnement littoral (développement de marqueurs Bacteroidales spécifiques de l höte)

Les effluents d origine urbaine ou agricole sont les principales sources decontamination microbiologique participant à la dégradation des eaux et des coquillages. Les indicateurs classiques de contamination fécale, Escherichia coli et les entérocoques, permettent de mettre en évidence une contamination mais pas d en identifier l origine. L objectif principal de ce travail de thèse était de développer et de valider une approche basée sur la PCR quantitative en temps réel, pour permettre l identification de l origine des contaminations dans les eaux et les coquillages à partir de la cible Bacteroidales. L analyse phylogénétique des séquences de gène codant les ARNr 16S des Bacteroidales a permis de développer quatre marqueurs, pour identifier les contaminations fécales humaines, porcines et ruminants. L étude de la persistance des marqueurs porcs, en microcosmes d eau de rivière, a montré que sous les conditions les plus défavorables, saturation en oxygène dissous et température de 20C, les marqueurs persistaient au moins 16 jours. Ces résultats suggèrent que ces marqueurs peuvent persister assez longtemps dan les eaux pour étre identifiés: Cette hypothèse a été confirmée par leur quantification dans des eaux provenant du bassin versant de l estuaire de Daoulas et de l estuaire de l Elorn (Finistère, Bretagne). Les marqueurs Bacteroidales spécifiques de l hôte ont également été recherchés dans les coquillages naturellement contaminés où ils ont pu étre quantifiés. Cette étude, confirme l intérêt d utiliser des marqueurs développés à partir de la cible Bacteroidales, pour identifier l origine des contaminations fécales au niveau des zones de baignade et conchylicoles.Human and animal faecal pollution affects environmental water in inland and coastal areas, with negative implications for recreational uses, public safety and shellfish sanitary status. The faecal microbiological indicators used in these regulations, Escherichia coli and enteroccoci, cannot distinguish between human and animal faecal contamination. The main objective of this study was to develop and to validate a molecular approach based on quantitative real-time PCR with the Bacteroidales target, in order to identify the origin of faecal contamination in water and shellfish. Phylogenetic analysis of the partial Bacteroidales 16S rRNA gene sequences allowed the development of 4 host-specific Bacteroidales markers to identify human, porcine and ruminant faecal contaminations. The study of the pig markers persistence in river mater microcosms showed that under unfavourable conditions, aerobic and temperature of 20 C, marker persisted for at least 16 days. These results suggested that Bacteroidales markers could still ha identified after extend period of time in river waters. This hypothesis was confirmed with their detection and quantification in river waters fro the Daoulas catchment estuary and the Elorn estuary (Finistère, Brittany). Host-specific Bacteroidales markers were also quantify in naturally contaminated oysters. Thus, this study confirais the interest of the host-specific Bacteroidales markers developed from the Bacteroidales target to identify faecal contaminations in bathing waters and shellfish harvesting areas.BREST-BU Droit-Sciences-Sports (290192103) / SudocPLOUZANE-Bibl.La Pérouse (290195209) / SudocSudocFranceF

Rapport d’activités 2016. Laboratoire Santé Environnement et Microbiologie, Laboratoire National de Référence de Microbiologie des coquillages

Ce rapport présente une synthèse des actions, travaux menés par l’équipe, accompagnée par des stagiaires, doctorants, post doctorants qu’il est important de remercier pour leur contribution. Cette année 2016 a de nouveau été une année riche en résultats innovants qui permet à notre équipe d’être reconnue au niveau national et international. En effet la Microbiologie sanitaire, thème de recherche fédérant notre laboratoire, thématique à l’interface de la santé publique, de la qualité des eaux côtières et des coquillages, nous permet de développer nos activités sur le terrain ou sur une approche plus fondamentale. Les résultats obtenus nous permettent d’exercer notre activité de Laboratoire National de Référence, en apportant connaissance scientifique et expertise technique

Real-Time Reverse Transcription-PCR for Transcriptional Expression Analysis of Virulence and Housekeeping Genes in Viable but Nonculturable Vibrio parahaemolyticus after Recovery of Culturability▿

A real-time reverse transcription-PCR method was developed to determine whether the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus induced the expression of virulence genes coding for the thermostable direct hemolysin and for type III secretion system 2 (TTSS2). The culturability of clinical strain Vp5 of V. parahaemolyticus in artificial seawater at 4°C was monitored, and the VBNC state was obtained. One day after entry into the VBNC state, temperature upshifts to 20 and 37°C allowed the recovery of culturability. Standard curves for the relative quantification of expression of the housekeeping genes rpoS, pvsA, fur, and pvuA; the tdh2 gene; and the TTSS2 genes (VPA1354, VPA1346, and VPA1342) were established. The levels of expression of the pvsA and pvuA genes were stable and were used to normalize the levels of expression of the other genes. No transcriptional induction of the selected virulence genes under the temperature conditions used to recover the culturability of the VBNC bacteria was observed. The study results demonstrate that the recovery of culturability of VBNC cells of pathogenic V. parahaemolyticus is restricted to regrowth, without correlation with the induction of virulence gene expression. Disease induction would depend mainly on host-pathogen interactions that allow the expression of the virulence genes. This is the first time that the use of mRNA to detect viable cells was evaluated by computing the half-lives of multiple mRNA species under conditions inducing the VBNC state