Les Escherichia Coli potentiellement pathogenes dans l’environnement littoral : cas des STEC et des EPEC

The contamination of coastal areas by potentially pathogenic enteric bacteria is of concern for the sustainability of some uses, such as shellfish farming, recreational shellfish harvesting and bathing. The contamination of these environments may occur through the land-spreading of livestock wastes, animal feces deposited on pastures, wastewaters from slaughterhouses or from municipal wastewater treatment plants. The presence of these bacteria in coastal environment may present a potential risk to human health. In fact, shellfish-borne outbreaks or human infections may occur by the consumption of shellfish from contaminated areas or the ingestion of water during bathing in contaminated waters, respectively. To date, few studies focusing on the presence and the diversity of enteric bacteria, such as pathogenic Escherichia coli (E. coli) in coastal environments and shellfish have been reported. The PhD project aimed to (i) evaluate the presence of pathogenic E. coli, more precisely, Shiga toxinproducing E. coli (STEC) and enteropathogenic E. coli (EPEC) in shellfish-harvesting areas and their upstream catchments, (ii) characterize the diversity of isolated STEC and EPEC, (iii) estimate the molecular risk assessment by screening 75 E. coli virulence-associated gene targets and then (iv) evaluate the contamination and depuration of oysters in contact with STEC. For this purpose, during a 2-year study (February 2013 to January 2015), shellfish batches (n=238), freshwater (n=216), seawater (n=12), and surface sediment (n=39) samples from three selected shellfish-harvesting areas and their upstream catchments, were monthly analyzed to evaluate the presence of STEC and EPEC strains. Five percent of samples were positive for the isolation of at least one STEC and 14% for the isolation of at least one EPEC. Twenty-eight STEC and 89 EPEC strains were isolated representing 1% of the total E. coli (n=12 016). The isolated STEC and EPEC strains belonged to a high diversity with 75 different serotypes, 79 distinguishable PFGE patterns and 46 distinguishable sequence types. STEC and EPEC strains were distributed into to 15 and 55 virulence profiles, respectively. One STEC strain isolated from a mussel batch, belonging to the serotype O26:H11 stx1+eae+ displayed 45 additional virulence genes among the 75 investigated genes. Seventyfive percent of EPEC strains displayed between one and 19 virulence genes associated with pathogenicity islands specific to pathogenic E. coli involved in human infections. No difference in the kinetics of the contamination and depuration of oysters by STEC and non-STEC E. coli was found. To our knowledge, this study is the first to focus on the diversity of STEC and EPEC strains isolated from coastal environments. This study highlights the weak presence of STECs and EPECs in the French shellfish-harvesting areas studied and a potential pathogenicity of some strains. The low prevalence of STEC and EPEC strains in shellfish from B- and C-categories (depuration of shellfish before commercialization), as well as the decontamination of shellfish by STEC, fast and similar to that of non-pathogenic E. coli, is rather in favor of a limited risk of contamination of shellfish by STEC in the studied areas. The results obtained during this study are important to better understand the health risk associated with STEC and EPEC in coastal areas.La contamination des zones littorales par des bactéries entériques potentiellement pathogènes pour l’Homme constitue un problème majeur pour la pérennité de certains usages tels que la conchyliculture, la pêche à pied ou la baignade. Ces bactéries provenant de rejets urbains ou agricoles peuvent atteindre les zones conchylicoles ou de baignade et être impliquées dans des toxi-infections alimentaires collectives (TIAC) lors de la consommation de coquillages contaminés ou dans des infections par contact avec l’eau de baignade contaminée. Actuellement, très peu de données sont disponibles sur la présence et la diversité des bactéries entériques telles que les Escherichia coli (E. coli) pathogènes dans les coquillages en France. Ce sujet de thèse a donc eu pour objectifs : i) d’évaluer la présence de E. coli pathogènes de type E. coli producteurs de Shiga-toxines (STEC) et de E. coli entéropathogènes (EPEC) en zone littorale et plus précisément dans des zones conchylicoles et leurs bassins versants en amont, ii) de caractériser la diversité des souches STEC et de EPEC isolées, iii) d’estimer le risque pathogène de ces souches par la recherche de gènes de virulence et enfin iv) d’évaluer le comportement des souches STEC dans les coquillages par rapport à l’indicateur E. coli, vis-à-vis de la cinétique de contamination de décontamination des coquillages. La présence des E. coli pathogènes a été recherchée sur une période de deux ans (Février 2013 à Janvier 2015) dans les coquillages (n=238) de trois zones conchylicoles situées en Bretagne et en Normandie ainsi que dans les eaux aux exutoires des bassins versants (n=216), dans le sédiment (n=39) et l’eau de mer (n=12). Cinq pourcents des échantillons ont permis l’isolement d’au moins une souche STEC et 14 % l’isolement d’au moins une souche EPEC. Un total de 28 souches STEC et 89 souches EPEC différentes ont été isolées dans ces échantillons, représentant 1 % de la totalité des souches E. coli isolées (n = 12 016). Les souches STEC et EPEC isolées présentaient une grande diversité avec 75 sérotypes identifiés, 79 profils phylogénétiques différents et 46 profils ST (Sequence Type). Les souches STEC et EPEC isolées étaient réparties respectivement au sein de 15 et 55 profils de virulence différents. Une souche STEC de sérotype O26 :H11 stx1+eae+ présentant 45 gènes de virulence sur les 75 gènes recherchés, a été isolée dans un lot de moule. Soixante-quinze pourcents des souches EPEC présentaient entre 1 et 19 gènes de virulence associés à des îlots de pathogénicités caractéristiques de souches E. coli pathogènes responsables d’infection grave chez l’Homme, révélant le potentiel pouvoir pathogène de certaines souches. Enfin, l’étude de la cinétique de contamination de décontamination d’huîtres au contact de souches STEC isolées au cours de cette étude, n’a pas montré de différence de comportement comparé à un E. coli non STEC. Les travaux réalisés au cours de cette thèse sont à notre connaissance les premiers de ce genre. Ils ont permis de mettre en évidence la faible présence de STEC et de EPEC au niveau de zones conchylicoles françaises ainsi que le potentiel pouvoir pathogène de certaines souches. La faible prévalence de souches STEC et EPEC dans les coquillages issus des sites conchylicoles suivis de catégorie B ou C (purification des coquillages avant commercialisation) ainsi que la décontamination des coquillages par les STEC, rapide et similaire à celle des E. coli non pathogènes, est plutôt en faveur d’un risque faible de contamination par les STEC des coquillages dans les zones étudiées. Les résultats acquis au cours de cette thèse sont des éléments importants pour mieux appréhender le risque sanitaire lié aux STEC et aux EPEC en zone littorale

Successful detection of pathogenic Shiga-toxin-producing Escherichia coli in shellfish, environmental waters and sediment using the ISO/TS-13136 method

The presence of highly pathogenic Shiga-toxin-producing Escherichia coli (STEC) in shellfish, upstream waters and sediment from coastal shellfish sites was evaluated using the ISO/TS-13136 method. Shellfish (oysters, mussels and cockles), water and sediment samples were collected monthly over a period of 1 year. The method used real-time PCR detection of stx1, stx2 and eae genes and genetic markers corresponding to the five major serogroups (O157, O26, O103, O111 and O145) on enrichment broths and the identification of STEC when these genes and markers were detected. stx genes were detected in the broth of 33% of shellfish batches (n = 126), 91% of water samples (n = 117) and 28% of sediment (n = 39). One stx1+, eae+ O26:H11 strain was isolated from a shellfish batch, and O26:H11, O145:H28 and O103:H2 strains without the stx gene (n = 9) were isolated from shellfish and waters. In conclusion, this study shows the suitability of the ISO/TS-13136 method to assess the presence of highly pathogenic E. coli strains in shellfish farming areas. It also highlights a low prevalence of STEC and consequently suggests a reduced corresponding human health risk

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

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 stx1a eae-β1 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 (stx1a, stx2a, stx2c, and stx2d) 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

Rapid genotyping protocol to improve dengue virus serotype 2 survey in Lao PDR

International audienceDengue fever is one of the major public health problems in Lao PDR. Over the last decade, dengue virus (DENV) epidemics were characterized by a novel predominant serotype accompanied by at least two other serotypes. Since 2008, DENV-2 circulated at a low level in Lao PDR but its epidemiologic profile changed at the end of 2018. Indeed, the number of confirmed DENV-2 cases suddenly increased in October 2018 and DENV-2 became predominant at the country level in early 2019. We developed a Genotype Screening Protocol (GSP) to determine the origin(s) of the Lao DENV-2 and study their genetic polymorphism. With a good correlation with full envelope gene sequencing data, this molecular epidemiology tool evidence the co-circulation of two highly polymorphic DENV-2 genotypes, i.e. Asian I and Cosmopolitan genotypes, over the last five years, suggesting multiple introductions of DENV-2 in the country. GSP approach provides relevant first line information that may help countries with limited laboratory resources to reinforce their capabilities to DENV-2 and to follow the epidemics progresses and assess situations at the regional level

Molecular Profiling of Shiga Toxin-Producing Escherichia coli and Enteropathogenic E. coli Strains Isolated from French Coastal Environments

International audienceABSTRACT 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 -β1 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. IMPORTANCE The 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

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

International audienceSTEC/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

Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017

International audiencePneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic