Effluents d’abattoir et risque d’émergence

International audienceLes maladies émergentes, causes de crises sanitaires potentiellement dévastatrices, représentent un enjeu majeur pour la santé végétale, animale et humaine. Difficiles à anticiper en raison de leur caractère nouveau et imprévisible, elles suscitent une réflexion pluridisciplinaire et une analyse spécifique. L'originalité de cet ouvrage est d'appliquer des concepts d'épidémiologie générale à l'émergence de maladies dans les domaines du végétal, de l'animal et de l'humain. La première partie de l'ouvrage présente les facettes de l'émergence dans ces trois domaines. A partir d'études de cas concrets et de synthèses, les parties suivantes traitent de la détection et de l'analyse biologiques des émergences, de leur traitement statistique et de leur modélisation, des facteurs environnementaux qui les déterminent, et du franchissement de la barrière d'espèces par les virus. La dernière partie de l'ouvrage fait le point sur les politiques nationales et internationales de santé face à ces maladies. Destiné aux étudiants, aux professionnels et aux chercheurs intéressés par une vision globale des maladies émergentes, l'ouvrage s'adresse également à tout public concerné par les problématiques actuelles et futures en santé végétale, animale et humaine

Characterization of Shiga Toxin Gene (stx)-Positive and Intimin Gene (eae)-Positive Escherichia coli Isolates from Wastewater of Slaughterhouses in France

Wastewater samples from 12 slaughterhouses located in different regions in France were tested for the presence of stx-positive and eae-positive Escherichia coli isolates, and characteristics of the isolates obtained were determined. A total of 224 wastewater samples were collected in wastewater treatment plants at different stages of wastewater processing. Altogether, 5,001 E. coli isolates were obtained by colony counting and screened for the presence of stx and eae genes by multiplex PCR. stx-positive and eae-positive E. coli isolates were detected in 25% of the samples collected; they were found in 13% and 3% of the samples obtained from treated effluent and sludge, respectively, suggesting that they could be spread into the environment. Screening of the samples collected by immunomagnetic separation allowed us to isolate 31 additional E. coli serogroup O157 isolates. Four of these isolates harbored stx and eae genes. All stx-positive and eae-positive E. coli isolates were analyzed for eae and stx genetic variants, as well as for additional virulence factors and serotypes. Our results suggest that the majority of the stx- and eae-positive E. coli isolates from wastewater have low virulence for humans. However, the diversity of the enterohemorrhagic E. coli-associated virulence factors in the strains indicates that the environment may play an important role in the emergence of new pathogenic enterohemorrhagic E. coli strains

Suivi de la communauté microbienne d'un lisier de porc le long d'une filière d'élevage, approches moléculaire et culturale

The microbial community evolution of a swine manure was monitored within a piggery equipped with a flushing system for a period of 6 months. Samples were withdrawn over all the manure management process : breeding house pits, homogenised storage pit, inlet and outlet of the compressor screw, storage lagoon, and finally soil before and after spreading of the manure from the lagoon. The microbial community was characterised and monitored using two different methodologies. The first one used techniques from molecular microbiology which allow the dominant microorganisms from manure to be identified and monitored without a need for culture (total bacteria, Archaea, Clostridiacea, Bacteroides Prevotella, and Lactobacillus-Streptococcus). The second, based on cultural techniques, was used to enumerate indicative bacteria, usually subdominant, as enterobacteria, enterococcus, coliforms, Escherichia coli, and potentially pathogenic bacteria as Salmonella, Listeria monocytogenes, and Staphylococcus aureus. Both approaches revealed a stability of the microbial populations during time within each step of the manure management process separately. However, qualitative and quantitative microbial population changes were observed when manure was shifting from one step of the process to another one (ex. going from the storage pit to the lagoon). Finally, microorganisms present within the effluent before spreading in the fields were not detectable anymore within soil after the spreading, whatever the technique used (molecular or cultural).L'évolution de la communauté microbienne d'un lisier a été suivie dans un élevage de porcs équipé d'un système de flushage pendant une période de 6 mois. Des prélèvements ont été réalisés sur toute la filière de gestion des déjections : préfosses des bâtiments, fosse de stockage homogénéisée, entrée et sorties de vis compacteuse, lagune de stockage, et enfin sol avant et après épandage du lisier issu du lagunage. La communauté microbienne a été caractérisée et suivie par deux méthodologies différentes. La première utilisant les techniques de microbiologie moléculaire permet d'identifier et de suivre les micro-organismes dominants du lisier sans avoir à les cultiver (bactéries totales, Clostridium - Eubacterium, Bacteroides - Prevotella, et Lactobacillus-Streptococcus). La seconde, utilisant les techniques culturales, a été utilisée pour suivre les bactéries indicatrices, généralement sous-dominantes telles que les entérobactéries, entérocoques, coliformes, Escherichia coli, et des bactéries potentiellement pathogènes telles que les Salmonelles, Listeria monocytogenes, et Staphylococcus aureus. Les deux approches montrent une stabilité dans le temps des populations microbiennes rencontrées sur chaque étape de la filière prise séparément. En revanche, des changements de populations microbiennes et de numération sont observés lorsque le lisier passe d'une étape de la filière à une autre (ex. passage fosse lagune). Enfin, les micro-organismes présents dans les effluents avant épandage ne sont plus détectables dans le sol après épandage, quelle que soit l'approche utilisée (moléculaire ou culturale)

Suivi de la communauté microbienne d'un lisier de porc le long d'une filière d'élevage, approches moléculaire et culturale

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Dynamics of a Pig Slurry Microbial Community during Anaerobic Storage and Management

The microbial community of a pig slurry on a farm was monitored for 6 months using both molecular and cultural approaches. Sampling was carried out at all the different stages of effluent handling, from the rearing build-up to slurry spreading. Total DNA of each sample was extracted and analyzed by PCR-single-strand conformation polymorphism (SSCP) analysis using primers targeting the 16S rRNA genes from the archaeal and bacterial domains and also the Eubacterium-Clostridium, Bacillus-Streptococcus-Lactobacillus, and Bacteroides-Prevotella groups. A comparison of the SSCP profiles showed that there were rapid changes in the dominant bacterial community during the first 2 weeks of anaerobic storage and that the community was relatively stable thereafter. Several bacterial populations, identified as populations closely related to uncultured Clostridium and Porphyromonas and to Lactobacillus and Streptococcus cultured species commonly isolated from pig feces, remained present and dominant from the rearing build-up to the time of spreading. Enumeration of fecal indicators (enterococci and Escherichia coli) performed in parallel using cultural methods revealed the same trends. On the other hand, the archaeal community adapted slowly during pig slurry storage, and its diversity increased. A shift between two hydrogenotrophic methanogenic Methanobrevibacter populations from the storage pit to the pond was observed. Microorganisms present in pig slurry at the time of spreading could not be detected in soil after spreading by either molecular or cultural techniques, probably because of the detection limit inherent in the two techniques

Draft Genome Sequence of Enterohemorrhagic Escherichia coli O157:H7 Strain MC2 Isolated from Cattle in France

International audienceEnterohemorrhagic Escherichia coli (EHEC) with serotype O157:H7 is a major foodborne pathogen. Here, we report the draft genome sequence of EHEC O157:H7 strain MC2 isolated from cattle in France. The assembly contains 5,400,376 bp that encoded 5,914 predicted genes (5,805 protein-encoding genes and 109 RNA genes)

Factors Involved in the Persistence of a Shiga Toxin-Producing Escherichia coli O157:H7 Strain in Bovine Feces and Gastro-Intestinal Content

Healthy cattle are the primary reservoir for O157:H7 Shiga toxin-producing E. coli responsible for human food-borne infections. Because farm environment acts as a source of cattle contamination, it is important to better understand the factors controlling the persistence of E. coli O157:H7 outside the bovine gut. The E. coli O157:H7 strain MC2, identified as a persistent strain in French farms, possessed the characteristics required to cause human infections and genetic markers associated with clinical O157:H7 isolates. Therefore, the capacity of E. coli MC2 to survive during its transit through the bovine gastro-intestinal tract (GIT) and to respond to stresses potentially encountered in extra-intestinal environments was analyzed. E. coli MC2 survived in rumen fluids, grew in the content of posterior digestive compartments and survived in bovine feces at 15°C predicting a successful transit of the bacteria along the bovine GIT and its persistence outside the bovine intestine. E. coli MC2 possessed the genetic information encoding 14 adherence systems including adhesins with properties related to colonization of the bovine intestine (F9 fimbriae, EhaA and EspP autotransporters, HCP pilus, FdeC adhesin) reflecting the capacity of the bacteria to colonize different segments of the bovine GIT. E. coli MC2 was also a strong biofilm producer when incubated in fecal samples at low temperature and had a greater ability to form biofilms than the bovine commensal E. coli strain BG1. Furthermore, in contrast to BG1, E. coli MC2 responded to temperature stresses by inducing the genes cspA and htrA during its survival in bovine feces at 15°C. E. coli MC2 also activated genes that are part of the GhoT/GhoS, HicA/HicB and EcnB/EcnA toxin/antitoxin systems involved in the response of E. coli to nutrient starvation and chemical stresses. In summary, the large number of colonization factors known to bind to intestinal epithelium and to biotic or abiotic surfaces, the capacity to produce biofilms and to activate stress fitness genes in bovine feces could explain the persistence of E. coli MC2 in the farm environment

Animal and human pathogenic Escherichia coli strains share common genetic backgrounds

Escherichia coli is a versatile species encompassing both commensals of the digestive tracts of many vertebrates, including humans, and pathogenic strains causing various intra- and extraintestinal infections. Despite extensive gene flow between strains, the E. coli species has a globally clonal population structure, consisting of distinct phylogenetic groups. Little is known about the relationships between phylogenetic groups and host specificity. We therefore used multilocus sequence typing (MLST) to investigate phylogenetic relationships and evaluated the virulence gene content of 35 E. coli strains representative of the diverse diseases encountered in domestic animals. We compared these strains with a panel of 101 human pathogenic and 98 non-human and human commensal strains representative of the phylogenetic and pathovar diversity of this species. A global factorial analysis of correspondence indicated that extraintestinal infections were caused mostly by phylogenetic group B2 strains, whereas intraintestinal infections were caused mostly by phylogenetic group A/B1/E strains, with strains responsible from extraintestinal or intraintestinal infections having specific virulence factors. It was not possible to distinguish between strains of human and animal origin. A detailed phylogenetic analysis of the MLST data showed that numerous pathogenic animal and human strains are very closely related, and had a number of virulence genes in common. However, a set of specific adhesins was identified in animal non-B2 group strains of all pathotypes. In conclusion, human and animal pathogenic strains share common genetic backgrounds, but non-B2 strains of different origins seem to have different sets of adhesins that could be involved in host specificity

Comparison of the incidence of pathogenic and antibiotic-resistant <em>Escherichia coli</em> strains in adult cattle and veal calf slaughterhouse effluents highlighted different risks for public health

International audienceThe goal of this study was to investigate the involvement of bovine slaughterhouse effluents and bio-solids in the risk of environmental dissemination of pathogenic and antibiotic-resistant Escherichia coli. Several samples were collected from one adult cattle and one veal calf slaughterhouse wastewater treatment plant (WWTP). The treatment process had no impact on the percentage of Shiga toxin-producing E. coli (STEC) and on the percentage of atypical enteropathogenic E. coli (aEPEC). A STEC O157:H7 was isolated from the thickened sludge of the adult cattle slaughterhouse. As thickened sludge is intended to be spread on agricultural lands, the detection of this pathogenic strain is a public health issue. The percentage of antibiotic-resistant E. coli was 5.0% and 87.5% in wastewater from the adult cattle and the veal calf slaughterhouse, respectively. These percentages were not significantly different after treatment. Integron-bearing E. coli isolates were only detected in the veal calf slaughterhouse WWTP with percentages above 50.0% for all sampling points whatever the step of the treatment process. Taken together, these findings highlighted the fact that different public health risks might be associated with adult cattle or veal calf slaughterhouses regarding the dissemination of pathogenic and antibioticresistant E. coli isolates into the environment

Intimin gene (eae) subtypes-based real-time PCR strategy for the specific detection of Shiga toxin-producing Escherichia coli serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:H28 in cattle feces

International audienceShiga toxin-producing Escherichia coli (STEC) belonging to serotypes O157:H7, O26:H11, O103:H2, O111:H8 and O145:H28 are known to be associated with particular subtypes of the intimin gene (eae), namely γ1, β1, ɛ, θ and γ1, respectively. This study aimed at evaluating the usefulness of their detection for the specific detection of these five main pathogenic STEC serotypes in cattle feces. Using real-time PCR assays, 58.7% of 150 fecal samples were found positive for at least one of the four targeted eae subtypes. The simultaneous presence of stx, eae and one of the five O group markers was found in 58.0% of the samples, and the five targeted stx/eae/O genetic combinations were detected 143 times. However, when taking into consideration the association between eae subtypes and O group markers, the resulting stx/eae-subtype/O combinations were detected only 46 times. The 46 isolation assays performed allowed to recover 22 E. coli strains belonging to one of the five targeted STEC serogroups. By contrast, only 2 out of 39 isolation assays performed on samples that were positive for stx, eae and an O group marker, but were negative for the corresponding eae subtype, were successful. Characterization of the 24 E. coli isolates showed that 6 were STEC, including 1 O157:H7, 3 O26:H11 and 2 O145:H28. The remaining 18 strains corresponded to atypical enteropathogenic E. coli (aEPEC). Finally, the more discriminating eae subtypes-based PCR strategy described here may be helpful for the specific screening of the five major STEC in cattle feces