Search

Secondary bacterial infections contribute to the excess morbidity and mortality of influenza A virus (IAV) infection. Disruption of lung integrity and impaired antibacterial immunity during IAV infection participate in colonization and dissemination of the bacteria out of the lungs. One key feature of IAV infection is the profound alteration of lung myeloid cells, characterized by the recruitment of deleterious inflammatory monocytes. We herein report that IAV infection causes a transient decrease of lung conventional dendritic cells (cDCs) (both cDC1 and cDC2) peaking at day 7 post-infection. While triggering emergency monopoiesis, IAV transiently altered the differentiation of cDCs in the bone marrow, the cDC1-biaised pre-DCs being particularly affected. The impaired cDC differentiation during IAV infection was independent of type I interferons (IFNs), IFN-γ, TNFα and IL-6 and was not due to an intrinsic dysfunction of cDC precursors. The alteration of cDC differentiation was associated with a drop of local and systemic production of Fms-like tyrosine kinase 3 ligand (Flt3-L), a critical cDC differentiation factor. Overexpression of Flt3-L during IAV infection boosted the cDC progenitors’ production in the BM, replenished cDCs in the lungs, decreased inflammatory monocytes’ infiltration and lowered lung damages. This was associated with partial protection against secondary pneumococcal infection, as reflected by reduced bacterial dissemination and prolonged survival. These findings highlight the impact of distal viral infection on cDC genesis in the BM and suggest that Flt3-L may have potential applications in the control of secondary infections., Author summary Secondary bacterial infections constitute a leading cause of mortality during influenza epidemics and pandemics. Acute infections trigger the mobilization of myeloid cells from the bone marrow (BM) to the infected tissues, a process referred to as emergency myelopoiesis. Influenza infection leads to a profound alteration in the pulmonary myeloid cell compartment characterized by the recruitment of inflammatory myeloid cells. The consequences of IAV infection on BM myelopoiesis have yet to be described in detail. Here, we report that influenza infection leads to reduced number of conventional dendritic cells (cDC) in the lungs (with a peak at day 7) and severely impairs DC differentiation in the BM, for the benefit of monopoiesis. The altered cDC differentiation was independent of pro-inflammatory cytokines and was not due to an intrinsic dysfunction of cDC precursors. Defective DC differentiation during influenza was associated with a decrease in the production of the key cDC differentiation factor, Fms-like tyrosine kinase 3 ligand (Flt3-L). Overexpression of Flt3-L during influenza enhanced the number of BM cDC progenitors and restored lung cDC compartment. This phenomenon was associated with a lowered number of lung inflammatory monocytes and reduced lung damages. Importantly, Flt3-L overexpression partially protected against secondary pneumococcal infection characterized by reduced bacterial loads, improved pathological outcomes and prolonged survival. Our results provide new clues as to how influenza infection influences the differentiation of cDCs, and highlight the potential therapeutic value of Flt3-L in a disease in desperate need of better treatments.

International audience; Shellfish as a foodstuff must meet sanitary quality objectives for the protection of consumers and this quality is closely linked to the water. The oyster industry considered this challenge related to contaminations and currently, the major risk of disease is due to the presence of norovirus (NoV) since all oyster-consuming countries report outbreaks of gastroenteritis linked to the presence of this microorganism. Ultrafiltration has already demonstrated to be efficient for viral protection of oyster farms in previous studies. In this work, retention by ultrafiltration of Tulane virus, a NoV surrogate, was evaluated. The effect of virus concentration in the feed on the ultrafiltration efficiency has been assessed. Low retentions of about 1 log were observed at the lowest viral concentrations. At higher concentrations, an increase of retention up to 5 log was obtained. These results highlight the potential overestimation of UF efficiency during laboratory experiments realized at high concentrations, compared to low concentrations found in environmental resources. In agreement with other studies, higher retentions at high concentrations could be explained by formation of viral aggregates, which could facilitate the steric exclusion but also modify the electrostatic and hydrophobic interactions between isolated viruses/aggregates and membrane. Virus retentions with a fresh mineral water (Evian water) and seawater were compared. Seawater achieved higher retention rates for Tulane virus due to the membrane fouling.

Graphical abstract, Since many infected people experience no or few symptoms, the SARS-CoV-2 epidemic is frequently monitored through massive virus testing of the population, an approach that may be biased and may be difficult to sustain in low-income countries. Since SARS-CoV-2 RNA can be detected in stool samples, quantifying SARS-CoV-2 genome by RT-qPCR in wastewater treatment plants (WWTPs) has been carried out as a complementary tool to monitor virus circulation among human populations. However, measuring SARS-CoV-2 viral load in WWTPs can be affected by many experimental and environmental factors. To circumvent these limits, we propose here a novel indicator, the wastewater indicator (WWI), that partly reduces and corrects the noise associated with the SARS-CoV-2 genome quantification in wastewater (average noise reduction of 19%). All data processing results in an average correlation gain of 18% with the incidence rate. The WWI can take into account the censorship linked to the limit of quantification (LOQ), allows the automatic detection of outliers to be integrated into the smoothing algorithm, estimates the average measurement error committed on the samples and proposes a solution for inter-laboratory normalization in the absence of inter-laboratory assays (ILA). This method has been successfully applied in the context of Obépine, a French national network that has been quantifying SARS-CoV-2 genome in a representative sample of French WWTPs since March 5th 2020. By August 26th, 2021, 168 WWTPs were monitored in the French metropolitan and overseas territories of France. We detail the process of elaboration of this indicator, show that it is strongly correlated to the incidence rate and that the optimal time lag between these two signals is only a few days, making our indicator an efficient complement to the incidence rate. This alternative approach may be especially important to evaluate SARS-CoV-2 dynamics in human populations when the testing rate is low.

International audience; 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.

[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TED; International audience; This study identified sources of fecal contamination in three different French headwater and coastal catchments (the Justiçou, Pen an Traon, and La Fresnaye) using a combination of microbial source tracking tools. The tools included bacterial markers (three host-associated Bacteroidales) and chemical markers (six fecal stanols), which were monitored monthly over one or two years in addition to fecal indicator bacteria. 168 of the 240 freshwater and marine water samples had Escherichia coli (E. coli) or enterococci concentrations higher than “excellent” European water quality threshold. In the three catchments, the results suggested that the fecal contamination appeared to be primarily from an animal origin and particularly from a bovine origin in 52% (Rum2Bac) and 46% (Bstanol) of the samples and to a lesser extent from a porcine origin in 19% (Pig2Bac) and 21% (Pstanol) of the samples. Our results suggested a human fecal contamination in 56% (HF183) and 32% (Hstanol) of the samples. Rainfall also impacted the source identification of microbial contamination. In general, these findings could inform effective implementation of microbial source tracking strategies, specifically that the location of sampling points must include variability at the landscape scale.

The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) - released in water at the upstream pasture site - at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species can induce infections in humans. Therefore understanding the structure and dynamics of non-pandemic environmental populations in temperate regions, such as Mediterranean coastal systems, is important if we are to evaluate the risks of infection to humans.Environmental isolates of V. cholerae (n=109) and V. parahaemolyticus (n=89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity conditions for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk.

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 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 This work was funded by the European Regional Development Fund Interreg IVA Programme, as part of the collaborative project RiskManche. The thesis of CB was supported by a grant from Ifremer and the Agence de l’Eau Loire-Bretagne. We thank Clémence Balière from the University of Caen and Céline Courroux from Hôpital Robert-Debré, for their participation in the sampling and technical assistance and Dominique HervioHeath (Ifremer) and Huw Taylor (University of Brighton, UK) for their critical review of the manuscript. Work in the LRECUSC-laboratory was financed by the grant CN2012/303 from Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia) and The European Regional Development Fund,ERDF) SI