Les processus de différenciation et la résistance des kystes aux traitements de désinfection chez l'amibe libre Vermamoeba vermiformis

V. vermiformis est une amibe libre répandue dans l'environnement et les milieux artificiels comme les réseaux d'eau chaude sanitaire (RECS). Il est maintenant bien établi qu'elle joue un rôle de réservoir pour des bactéries pathogènes, comme L. pneumophila. Le contrôle de V. vermiformis dans les RECS représente donc un enjeu sanitaire important. Les amibes libres peuvent passer d'une forme métaboliquement active (trophozoïte) à une forme de résistance, le kyste, lorsque les conditions sont défavorables ce qui leur confère une résistance aux traitements. Malgré la haute prévalence de V. vermiformis dans les RECS, les processus de différenciation et la résistance de ses kystes aux traitements n'ont été que peu étudiés. Nous avons donc investigué les changements morphologiques et ultrastructuraux qui s'opèrent lors de l'enkystement et désenkystement de V. vermiformis. Il en ressort que l'enkystement est un phénomène rapide (9 h) qui conduit à la formation de kystes entourés d'une paroi double couche. Lors du désenkystement, les trophozoïtes n'émergent pas à travers un ostiole comme c'est le cas chez Acanthamoeba. Puis, nous avons étudié l'effet des conditions environnementales et de la concentration cellulaire sur l'enkystement. Nous avons observé que plus la concentration cellulaire est élevée plus l'enkystement est rapide, ce qui suggère l'existence de mécanismes de communication intercellulaire. Enfin, nous avons étudié la résistance des kystes aux traitements utilisés dans les RECS et aux protéases. Ces traitements étaient efficaces, in vitro, pour inactiver les kystes de V. vermiformis. Ces travaux ont permis d'apporter des connaissances de bases sur les processus de dVermamoeba vermiformis is a free-living amoeba (FLA) widespread in the environment and artificial environments such as hot water networks. It is now well established that it acts as a reservoir for many pathogenic bacteria, such as Legionella pneumophila. The control of V. vermiformis in artificial environments represents an important health issue. FLA can turn from a metabolic active form (trophozoite) to a resistance form, called cyst, when conditions are unfavorable. Cysts are more resistant to treatments. Despite the high prevalence of V. vermiformis in hot water networks, the processes of differentiation and the resistance of cysts to disinfection treatments have been poorly studied. Therefore we investigated morphological and ultrastructural changes occurring during encystment and excystment of V. vermiformis. It appears that encystment is a fast process (9 h) which leads to the formation of cysts surrounded by a double-layered wall. During excystment, trophozoites do not emerge through an ostiole as is the case with Acanthamoeba. Then, we studied the effect of environmental conditions and cell concentration on encystment. We observed that the higher cell concentration was, the faster the encystment was, which suggests the existence of intercellular communication. Finally, we studied the resistance of cysts to conventional disinfection treatments used in hot water networks and to innovative treatment with proteases. These treatments were effective, in vitro, to inactivate V. vermiformis cysts. This work provides new finding regarding differentiation processes and cysts resistance of V. vermiformis, a free-living amoeba poorly studied.POITIERS-SCD-Bib. électronique (861949901) / SudocSudocFranceF

Global Transcriptional Analysis of Spontaneous Sakacin P-Resistant Mutant Strains of Listeria monocytogenes during Growth on Different Sugars

Subclass IIa bacteriocins have strong antilisterial activity and can control the growth of Listeria monocytogenes in food. However, L. monocytogenes may develop resistance towards such bacteriocins. In this follow-up study, the transcriptomes of a high level (L502-1) and a low level (L502-6) spontaneous sakacin P-resistant mutant strain of L. monocytogenes were compared to the wild-type (L502). The growth of the resistant strains was reduced on mannose but not affected on cellobiose and the transcriptomics was performed during growth on these sugars. The mannose phosphotransferase system (PTS) encoded by the mptACD operon (mpt) is known for transporting mannose and also act as a receptor to class IIa bacteriocins. The mpt was repressed in L502-1 and this is in accordance with abolition of the bacteriocin receptor with resistance to class IIa bacteriocins. In contrast, the mpt was induced in L502-6. Despite the induction of the mpt, L502-6 showed 1,000 times more resistance phenotype and reduced growth on mannose suggesting the mannose-PTS may not be functional in L502-6. The microarray data suggests the presence of other transcriptional responses that may be linked to the sakacin P resistance phenotype particularly in L502-6. Most of commonly regulated genes encode proteins involved in transport and energy metabolism. The resistant strains displayed shift in general carbon catabolite control possibly mediated by the mpt. Our data suggest that the resistant strains may have a reduced virulence potential. Growth sugar- and mutant-specific responses were also revealed. The two resistant strains also displayed difference in stability of the sakacin P resistance phenotype, growth in the presence of both the lytic bacteriophage P100 and activated charcoal. Taken together, the present study showed that a single time exposure to the class IIa bacteriocin sakacin P may elicit contrasting phenotypic and transcriptome responses in L. monocytogenes possibly through regulation of the mpt

The Transcriptome of the Nosocomial Pathogen Enterococcus faecalis V583 Reveals Adaptive Responses to Growth in Blood

gains access to the bloodstream and establishes a persistent infection is not well understood.. infections

Comparative Genomic Analysis of Pathogenic and Probiotic Enterococcus faecalis Isolates, and Their Transcriptional Responses to Growth in Human Urine

Urinary tract infection (UTI) is the most common infection caused by enterococci, and Enterococcus faecalis accounts for the majority of enterococcal infections. Although a number of virulence related traits have been established, no comprehensive genomic or transcriptomic studies have been conducted to investigate how to distinguish pathogenic from non-pathogenic E. faecalis in their ability to cause UTI. In order to identify potential genetic traits or gene regulatory features that distinguish pathogenic from non-pathogenic E. faecalis with respect to UTI, we have performed comparative genomic analysis, and investigated growth capacity and transcriptome profiling in human urine in vitro. Six strains of different origins were cultivated and all grew readily in human urine. The three strains chosen for transcriptional analysis showed an overall similar response with respect to energy and nitrogen metabolism, stress mechanism, cell envelope modifications, and trace metal acquisition. Our results suggest that citrate and aspartate are significant for growth of E. faecalis in human urine, and manganese appear to be a limiting factor. The majority of virulence factors were either not differentially regulated or down-regulated. Notably, a significant up-regulation of genes involved in biofilm formation was observed. Strains from different origins have similar capacity to grow in human urine. The overall similar transcriptional responses between the two pathogenic and the probiotic strain suggest that the pathogenic potential of a certain E. faecalis strain may to a great extent be determined by presence of fitness and virulence factors, rather than the level of expression of such traits

Barriers to Dissemination of Antibiotic Resistance Genes from Wastewater

Antibiotic resistance genes can spread between environments and eventually reach pathogenic bacteria through horizontal gene transfer. Wastewater treatment plants (WWTPs) receive water from various sources and contain a mixture of human pathogens and antibiotic waste. Therefore, WWTPs are believed to be a major source of antibiotic resistant gene dissemination. However, it has been demonstrated that even if resistance genes are abundant in WWTPs, only a few are disseminated to other environments. In this study, we expressed 58 genes from a WWTP in Denmark. The genes are known to give resistance towards different antibiotics in Escherichia coli. The level of resistance to antibiotics as well as the fitness cost was analysed to evaluate if these factors could constitute barriers to gene dissemination. We found that both the level of resistance and the fitness cost conferred by the antibiotic resistance WWTP genes were highly variable. Moreover, an increased level of resistance was not necessarily correlated with a fitness cost. This study could not conclude that the level of resistance or the fitness cost could explain the barrier to dissemination of these genes.

Barriers to Dissemination of Antibiotic Resistance Genes from Wastewater

Antibiotic resistance genes can spread between environments and eventually reach pathogenic bacteria through horizontal gene transfer. Wastewater treatment plants (WWTPs) receive water from various sources and contain a mixture of human pathogens and antibiotic waste. Therefore, WWTPs are believed to be a major source of antibiotic resistant gene dissemination. However, it has been demonstrated that even if resistance genes are abundant in WWTPs, only a few are disseminated to other environments. In this study, we expressed 58 genes from a WWTP in Denmark. The genes are known to give resistance towards different antibiotics in Escherichia coli. The level of resistance to antibiotics as well as the fitness cost was analysed to evaluate if these factors could constitute barriers to gene dissemination. We found that both the level of resistance and the fitness cost conferred by the antibiotic resistance WWTP genes were highly variable. Moreover, an increased level of resistance was not necessarily correlated with a fitness cost. This study could not conclude that the level of resistance or the fitness cost could explain the barrier to dissemination of these genes.

Vaccination ADN contre la chlamydiose abortive ovine (évaluation de la protection des vaccins ADN MOMP, DnaK et GroEL dans un modèle murin d'infection)

Chlamydophila abortus est une bactérie intracellulaire obligatoire qui infecte principalement les brebis mais peut-être également transmise à l'homme. Elle est responsable d'avortements tardifs sans signe avant-coureur ou de la mise bas de petits chétifs, difficiles à élever. Les avortements ovins occasionnent des pertes économiques importantes dans les élevages. Dans une stratégie de vaccination ADN, les séquences codant pour des protéines immunogènes de C. abortus ont été clonées dans le vecteur d'expression eucaryote pcDNA3.1. L'effet protecteur des vaccins ADN contenant les séquences codant pour la protéine majeure de la membrane externe (MOMP) et les protéines de choc thermique, DnaK et GroEL ont été évalués dans un modèle murin de gestation. Les injections intramusculaires des ADN vaccinaux ont protégé partiellement les souris des avortements induits par l'infection. La charge bactérienne des rates des souris gestantes ou non, et des placentas n'a pas été réduite par la vaccination ADN. Toutefois, nous avons observé une protection fœtale probablement due à l'effet barrière du placenta. La réponse humorale générée par la vaccination ADN s'est avérée spécifique mais faible. Elle implique des anticorps, d'isotype majoritaire IgG2a, mais qui n'ont pas de pouvoir neutralisant in vitro sur l'infectivité de C. abortus. Afin d'optimiser la réponse immunitaire induite par la vaccination ADN avec le gène codant pour la protéine DnaK, un rappel protéique a été réalisé avec la protéine DnaK exprimée chez E. coli. Bien que cette stratégie ait considérablement augmentée la réponse humorale, aucune modification de la protection induite par le vaccin ADN seul n'a été obtenue. Il semblerait donc que, dans ce cas, l'antigène ait un faible pouvoir protecteur. Compte tenu que les antigènes immunogènes connus pour être protecteurs dans d'autres modèles n'ont donné que des résultats modérés en vaccination ADN contre C. abortus, nos prochains travaux consisteraient à identifier de nouveaux antigènes protecteurs en vaccination ADN. Dans ce but, nous pourrions utiliser une nouvelle stratégie prometteuse, consistant au criblage d'une banque génomique d'ADN bactérien, en fonction de son pouvoir protecteur in vivo, après immunisation génique.TOURS-BU Sciences Pharmacie (372612104) / SudocSudocFranceF

Barriers to Dissemination of Antibiotic Resistance Genes from Wastewater

Antibiotic resistance genes can spread between environments and eventually reach pathogenic bacteria through horizontal gene transfer. Wastewater treatment plants (WWTPs) receive water from various sources and contain a mixture of human pathogens and antibiotic waste. Therefore, WWTPs are believed to be a major source of antibiotic resistant gene dissemination. However, it has been demonstrated that even if resistance genes are abundant in WWTPs, only a few are disseminated to other environments. In this study, we expressed 58 genes from a WWTP in Denmark. The genes are known to give resistance towards different antibiotics in Escherichia coli. The level of resistance to antibiotics as well as the fitness cost was analysed to evaluate if these factors could constitute barriers to gene dissemination. We found that both the level of resistance and the fitness cost conferred by the antibiotic resistance WWTP genes were highly variable. Moreover, an increased level of resistance was not necessarily correlated with a fitness cost. This study could not conclude that the level of resistance or the fitness cost could explain the barrier to dissemination of these genes.

Etude du régulon sigma-54 et de son implication dans la sensibilité aux bactériocines de sous-classe IIa chez Listeria monocytogenes

Les bactériocines de sous-classe IIa sont des peptides à activité anti-Listeria synthétisés par des bactéries lactiques. La sensibilité de Listeria monocytogenes implique le facteur de transcription s54, pour lequel trois activateurs associés ont été identifiés dans le génome, ManR, LacR et CotR. Le facteur s54 et ManR régulent l'expression d'une perméase PTS de la famille mannose, EIItMan, qui joue un rôle crucial dans la sensibilité aux bactériocines IIa. EIItMan est constituée d'une sous-unité cytoplasmique IIABtMan et d'un complexe membranaire IICtMan-IIDtMan. Sa localisation membranaire a suggéré son rôle putatif de récepteur à ces peptides anti-bactériens. Afin d'approfondir, à l'échelle moléculaire, l'implication de s54 et de EIItMan dans la sensibilité aux bactériocines IIa, nous avons développé deux axes de recherche. Dans un premier temps, une étude globale des gènes régulés par s54 a été réalisée. Nous avons mené une analyse comparative transcriptomique et protéomique entre un mutant s54 et la souche sauvage EGDe. Dans nos conditions d'étude, mptACD, codant EIItMan, est l'unique opéron directement contrôlé par s54. Le facteur s54 a un effet principal sur le métabolisme des sucres, probablement orchestré par la répression de mptACD. Dans un second temps, nous avons étudié le rôle de EIItMan dans le mode d'action des bactériocines IIa. L'analyse par RT-PCR de l'expression de mptACD montre une relation entre son niveau d'expression et le degré de sensibilité des mutants. L'expression hétérologue de l'opéron mptACD chez une bactérie insensible, Lactococcus lactis, conduit à une sensibilité de la souche aux bactériocines IIa. Nous avons aussi montré que seule l'expression du gène mptC (codant IICtMan) est nécessaire pour sensibiliser L. lactis. Ainsi, la sous-unité membranaire IICtMan joue un rôle crucial dans la sensibilité et pourrait être la cible privilégiée des bactériocines IIa.Subclass IIa bacteriocins produced by lactic acid bacteria, are peptides active against Listeria. The s54 transcriptional factor is involved in the sensitivity of Listeria monocytogenes to these peptides. Three s54 associated activators were identified in L. monocytogenes genome, ManR, LacR and CotR. The s54 factor and ManR regulated the EIItMan synthesis, a mannose PTS permease, which is crucial in sensitivity to IIa bacteriocins. EIItMan is composed of a cytoplasmic subunit, IIABtMan, and a membrane complex IICtMan-IIDtMan. Its membrane localization led our group to suggest that it could be a receptor to antibacterial peptides. The role of s54 and EIItMan in sensitivity to IIa bacteriocins was investigated through two approaches. First, we realised a global analysis of genes regulated by s54, by the comparison of the transcriptome and proteome profiles of a s54 mutant and the wild type EGDe strain. In our conditions study, mptACD, encoding EIItMan, is the only operon shown to be directly regulated by s54. The s54 mutation has a main influence on sugars metabolism, may be due to the repression of mptACD. Second, we study the implication of EIItMan in sensitivity mechanism to IIa bacteriocins. The analysis by RT-PCR of the mptACD expression suggested a link between its level expression and the level of sensitivity of L. monocytogenes mutants. Besides, the heterologous expression of mptACD in an insensitive species, Lactococcus lactis, led to sensitize the strains to IIa bacteriocins. We also show that only the mptC expression (encoding IICtMan) is necessary to induce sensitivity in L. lactis. Thus, the membrane subunit, IICtMan is crucial for the sensitivity and could be the particular target of IIa bacteriocins.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Diagnostic et traitement du syndrôme obstructif des voies respiratoires supérieures chez le chien brachycéphale, avec support audiovisuel

Les particularités anatomiques des races brachycéphales sont à l'origine d'affections obstructives respiratoires. Ces affections doivent être traitées chirurgicalement. Ce travail, à visée pédagogique, a pour but de présenter, à l'aide d'un document vidéo, les diverses techniques chirurgicales actuellement utilisées pour traiter ce syndrome.MAISONS-ALFORT-Ecole Vétérin (940462302) / SudocSudocFranceF