Global human health is increasingly challenged by emerging viral threats, especially those observed over the last 20 years with coronavirus-related human diseases, such as the Severe Acute Respiratory Syndrome (SARS) and the Middle East Respiratory Syndrome (MERS). Recently, in late December 2019, a novel Betacoronavirus, SARS-CoV-2, originating from the Chinese city of Wuhan, emerged and was then identified as the causative agent of a new severe form of pneumonia, COVID-19. Real-time genome sequencing in such viral outbreaks is a key issue to confirm identification and characterization of the involved pathogen and to help establish public health measures. Here, we implemented an amplicon-based sequencing approach combined with easily deployable next-generation sequencers, the small and hand-held MinION sequencer and the latest most compact Illumina sequencer, the iSeq100 TM system. Our results highlighted the great potential of the amplicon-based approach to obtain consensus genomes of SARS-CoV-2 from clinical samples in just a few hours. Both these mobile next-generation sequencers are proven to be efficient to obtain viral sequences and easy to implement, with a minimal laboratory environment requirement, providing useful opportunities in the field and in remote areas.

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.

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 shellfish-harvesting areas were consumed.

Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains may be responsible for food-borne infections in humans. Twenty-eight STEC and 75 EPEC strains previously isolated from French shellfish-harvesting areas and their watersheds and belonging to 68 distinguishable serotypes were characterized in this study. High-throughput real-time PCR was used to search for the presence of 75 E. coli virulence-associated gene targets, and genes encoding Shiga toxin (stx) and intimin (eae) were subtyped using PCR tests and DNA sequencing, respectively. The results showed a high level of diversity between strains, with 17 unique virulence gene profiles for STEC and 56 for EPEC. Seven STEC and 15 EPEC strains were found to display a large number or a particular combination of genetic markers of virulence and the presence of stx and/or eae variants, suggesting their potential pathogenicity for humans. Among these, an O26:H11 stx 1a eae-␤1 strain was associated with a large number of virulence-associated genes (n ‫؍‬ 47), including genes carried on the locus of enterocyte effacement (LEE) or other pathogenicity islands, such as OI-122, OI-71, OI-43/48, OI-50, OI-57, and the high-pathogenicity island (HPI). One O91:H21 STEC strain containing 4 stx variants (stx 1a , stx 2a , stx 2c , and stx 2d ) was found to possess genes associated with pathogenicity islands OI-122, OI-43/48, and OI-15. Among EPEC strains harboring a large number of virulence genes (n, 34 to 50), eight belonged to serotype O26:H11, O103:H2, O103:H25, O145:H28, O157:H7, or O153:H2. IMPORTANCEThe species E. coli includes a wide variety of strains, some of which may be responsible for severe infections. This study, a molecular risk assessment study of E. coli strains isolated from the coastal environment, was conducted to evaluate the potential risk for shellfish consumers. This report describes the characterization of virulence gene profiles and stx/eae polymorphisms of E. coli isolates and clearly highlights the finding that the majority of strains isolated from coastal environment are potentially weakly pathogenic, while some are likely to be more pathogenic. E scherichia coli is a commensal aerobic bacterium of the warmblooded animal intestinal microbiota and is used as a fecal indicator in the environment to classify shellfish-harvesting and bathing areas (1). However, E. coli can become pathogenic through the acquisition of mobile genetic elements such as bacteriophages, pathogenicity islands, and plasmids. Among pathogenic E. coli strains are the Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) strains.STEC can cause infections ranging from uncomplicated diarrheas to hemorrhagic colitis (HC) and hemolytic-uremic syndrome (HUS). Several STEC serotypes have been involved in numerous food-borne outbreaks worldwide (2, 3), and these strains have been identified as enterohemorrhagic E. coli (EHEC). Although E. coli O157:H7 has been the main serotype implicated in HC and HUS since the early ...

Sorbitol-fermenting Escherichia coli O157:[H7] is a particularly virulent clone of E. coli O157:H7 associated with a higher incidence of haemolytic uraemic syndrome and a higher case fatality rate. Many fundamental aspects of its epidemiology remain to be elucidated, including its reservoir and transmission routes and vehicles. We describe an outbreak of sorbitol-fermenting E. coli O157:[H7] that occurred in France in 2011. Eighteen cases of paediatric haemolytic uraemic syndrome with symptom onset between 6 June and 15 July 2011 were identified among children aged 6 months to 10 years residing in northern France. A strain of sorbitol-fermenting E. coli O157:[H7] stx2a eae was isolated from ten cases. Epidemiological, microbiological and trace-back investigations identified multiply-contaminated frozen ground beef products bought in a supermarket chain as the outbreak vehicle. Strains with three distinct pulsotypes that were isolated from patients, ground beef preparations recovered from patients' freezers and from stored production samples taken at the production plant were indistinguishable upon molecular comparison. This investigation documents microbiologically confirmed foodborne transmission of sorbitol-fermenting of E. coli O157 via beef and could additionally provide evidence of a reservoir in cattle for this pathogen.