Salmonella spp. é reconhecida como uma das bactérias que mais causam doenças de origem alimentar no mundo. Dentre as diversas sorovariedades de Salmonella, a Typhimurium é uma das sorovariedades de maior ocorrência no mundo. Várias metodologias de tipagem fenotípicas e genotípicas foram desenvolvidas com o intuito de se delinear a epidemiologia e diversidade genotípica de Salmonella Typhimurium. Entretanto, a tipagem fenotípica é muitas vezes limitada por sua baixa capacidade de diferenciação de subtipos pertencentes a uma mesma sorovariedade de Salmonella, um problema minimizado pelos métodos genotípicos. No Brasil, foram realizados poucos estudos que genotiparam linhagens de S. Typhimurium. Os objetivos deste estudo foram caracterizar linhagens de S. Typhimurium isoladas de humanos, alimentos, animais e ambiente do animal no Brasil quanto ao seu potencial patogênico, perfil de resistência a antimicrobianos e diversidade genotípica. Foram estudadas 119 linhagens de S. Typhimurium, isoladas de material clínico de humanos (43), alimentos diversos (49), material clínico de suínos (22) e do ambiente de suínos (5), entre 1983 e 2013, provenientes de várias Estados do Brasil. A presença de 12 genes de virulência foi pesquisada por PCR. O perfil de resistência a 13 antimicrobianos foi realizado pelo método de discodifusão. A tipagem molecular foi realizada por Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variablenumber tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) e sequenciamento do genoma completo para 92 linhagens de S. Typhimurium isoladas de humanos (43) e alimentos (46). As metodologias PFGE, ERIC-PCR e MLVA foram realizadas para 70 linhagens de S. Typhimurium isoladas de humanos (43), animais (22) e ambiente do animal (5). Todas as 119 linhagens apresentaram os genes sipA, flgK, flgL e invA. O gene sipD e o gene sopE2 foram encontrados em 118 (99,2%) linhagens. O gene fljB foi encontrado em 117 (98,3%) linhagens. O gene sopD foi presente em 114 (95,8%) linhagens, o gene sopB em 111 (93,3%) linhagens, o gene ssaR em 102 (85,7%) linhagens, o gene sifA em 86 (72,3%) linhagens e 45 (37,8%) linhagens apresentaram o gene plasmidial spvB. De um total de 119 linhagens, 64 (62,2%) linhagens foram resistentes a pelo menos um dos 13 antimicrobianos testados, sendo que 36 (30,3%) linhagens foram multi-droga resistentes (MDR). Na comparação dos isolados de humanos e alimentos, as linhagens isoladas de humanos antes de meados 1990, ficaram alocadas nos grupos PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 e G1, G2, H para CRISPRMVLST. As linhagens isoladas de humanos após esse período ficaram alocadas nos grupos PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 e G2. As linhagens isoladas de alimentos ficaram alocadas nos grupos PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVAB2, G1 e G2. Por MLST, do total de 92 linhagens isoladas de humanos e alimentos, 77 linhagens foram tipadas como ST19. Pelo sequenciamento do genoma completo, as linhagens isoladas de alimentos e humanos ficaram alocadas no grupos I e J independente das datas de isolamento. Na comparação dos isolados de humanos e animais, as linhagens das duas origens ficaram alocadas nos grupos PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 e MLVA-D. ii Conclui-se que a grande prevalência de genes de virulência nas linhagens de S. Typhimurium estudadas reforça o potencial das mesmas causarem doenças em humanos, bem como, os riscos de sua presença em alimentos, animais para consumo humano e ambiente. A ocorrência de S. Typhimurium multi-droga resistentes isoladas de alimentos diversos e de suínos para consumo é um alerta para o possível risco de humanos ingerirem alimentos contaminados por tais linhagens. Em conjunto os resultados de PFGE, ERIC-PCR, MLVA, CRISPR-MVLST sugerem que as linhagens de S. Typhimurium isoladas de humanos eram geneticamente mais diversificadas antes de meados de 1990, o que pode sugerir a seleção de um subtipo de S. Typhimurium mais adaptado, depois que Salmonella Enteritidis tornou-se a sorovariedade de maior ocorrência no Brasil após esse período. Com relação às linhagens isoladas de alimentos, os resultados de PFGE, ERIC-PCR, MLVA e CRISPR-MVLST sugerem que durante o período estudado houve a circulação de mais de um subtipo no país. Os resultados de MLST sugerem que tais linhagens tenham uma origem filogenética comum. Os resultados do sequenciamento do genoma completo sugerem que houve a circulação de mais de um subtipo de S. Typhimurium no país, com relação às linhagens de humanos e alimentos. Também alerta para o possível risco de linhagens MDR isoladas de alimentos contaminarem humanos e/ou disseminarem genes de resistência a antibióticos para linhagens de origem clínica e não clínica. Na comparação dos isolados de humanos e animais, os resultados de PFGE, ERICPCR e MLVA sugerem que algumas linhagens isoladas de suínos e humanos podem descender de um subtipo comum. Ademais, as linhagens MDR isoladas de suínos e do ambiente de suínos alertam para o possível risco de porcos usados para consumo contaminarem humanos, o ambiente e outros porcos. / Salmonella spp. is recognized as one of the most involved bacteria that cause food-borne diseases in the world. Among the various serovars of Salmonella, Typhimurium is one of the most frequent serovars worldwide. Several phenotypic and genotypic typing methods have been developed in order to delineate the epidemiology and genotypic diversity of Salmonella Typhimurium. However, phenotypic typing is often limited by its low capacity to differentiate subtypes belonging to the same serovar of Salmonella, a problem minimized by genotypic methods. In Brazil, few studies have been conducted that genotyped S. Typhimurium strains. The aims of this study were to characterize S. Typhimurium strains isolated from humans, food, animals and animal\'s environment in Brazil regarding its pathogenic potential, antimicrobial resistance and genotypic diversity. We studied 119 S. Typhimurium strains isolated from human clinical material (43), different foods (49), clinical material from pigs (22) and pigs environment (5), between 1983 and 2013 from various States of Brazil. The presence of 12 virulence genes was investigated by PCR. The resistance profile against 13 antimicrobial was performed by the disk diffusion method. Molecular typing was performed by Pulsed-field gel electrophoresis (PFGE), Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), Multiple-locus variable-number tandem-repeats analysis (MLVA), Clustered regularly interspaced short palindromic repeats - Multi-virulence locus sequence typing (CRISPR-MVLST), Multilocus sequence typing (MLST) and whole genome sequencing for 92 S. Typhimurium strains isolated from humans (43) and food (46). PFGE, ERIC-PCR and MLVA methods were performed for 70 S. Typhimurium strains isolated from humans (43), animals (22) and the animal\'s environment (5). All 119 strains showed the sipA, flgK, flgL and invA genes. The sipD and sopE2 genes were found in 118 (99.2%) strains. The fljB gene was found in 117 (98.3%) strains. The sopD gene was present in 114 (95.8%) strains, the gene sopB in 111 (93.3%) strains, the ssaR gene in 102 (85.7%) strains, the gene sifA in 86 (72.3%) strains and 45 (37.8%) strains showed the plasmid gene spvB. From a total of 119 strains, 64 (62.2%) strains were resistant to at least one of the 13 antimicrobials tested, and 36 (30.3%) strains were multi-drug resistant (MDR). In the comparison of isolates from humans and food, the strains isolated from humans before mid-1990s were allocated in PFGE-A, PFGE-B1, PFGE-B2, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2 and G1, G2, H for CRISPR-MVLST. The strains isolated from humans after this period were allocated in PFGE-B1, ERIC-A, MLVA-B1, MLVA-B2 and G2 clusters. The strains isolated from food were allocated in PFGE-A, PFGE-B1, ERIC-A, ERIC-B, MLVA-A, MLVA-B1, MLVA-B2, G1 and G2 clusters. By MLST, of the total of 92 strains isolated from humans and food, 77 strains were typed as ST19. By whole genome sequencing, the strains isolated from food and humans were allocated in I and J clusters independently of its isolation date. In the comparison of isolates from humans and animals, strains of the two origins were allocated in PFGE-D1, PFGE-D2, ERIC-C1, MLVA-C1 and MLVA-D clusters. In conclusion, the high frequency of virulence genes in the S. Typhimurium strains studied reinforces their potential hazard to cause disease in humans, as well as the risk of its presence in food, animals for human consumption and the environment. The occurrence of S. Typhimurium multi-drug iv resistant isolated from various food and pigs for consumption is an alert of the possible risk for humans to ingest contaminated food with those strains. Together the results of PFGE, ERIC-PCR, MLVA e CRISPR-MVLST suggest that S. Typhimurium strains isolated from humans were genetically more diverse before mid-1990s, which might indicate the selection of a more adapted S. Typhimurium subtype after Salmonella Enteritidis became the most prevalent serovar in Brazil. Regarding the strains isolated from food, the results of PFGE, ERIC-PCR, MLVA and CRISPR-MVLST suggest that during the studied period there was circulation of more than one subtype in the country. The MLST results suggest that these strains have a common phylogenetic origin. The results of the whole genome sequencing suggest that there may be more than one subtype circulating in the country, with respect to the strains of human and food origins. Also, alerts for the possible risk of MDR strains isolated from food to contaminate humans and/or disseminate antibiotic resistance genes for strains of clinical and non-clinical origin. In the comparison of isolates from humans and animals, the results of PFGE, ERIC-PCR and MLVA suggest that some strains isolated from pigs and humans may descend from a common subtype. In addition, the MDR strains isolated from pigs and pig environment warn for the possible risk of pigs used for human consumption to contaminate humans, the environment and other pigs.

Les espèces bactériennes exhibent plusieurs états de structure de populations pouvant varier de clonale à panmictique selon l'importance des transferts horizontaux et la nature de leur écosystème. Dans mon travail de thèse, je me suis intéressé à trois espèces bactériennes, Staphylococcus aureus, Legionella pneumophila et Pseudomonas aeruginosa qui reflètent trois situations différentes. Afin de pouvoir décrire de façon rapide de grandes collections de souches, j'ai utilisé comme marqueurs de diversité le polymorphisme de séquences répétées en tandem appelées VNTRs, pour Variable Number Tandem Repeat. La méthode MLVA, ou Multiple Loci VNTR Analysis, est une méthode de typage moléculaire qui s’appuie sur l’étude concomitante du polymorphisme de plusieurs loci VNTRs. Dans un premier temps, j'ai conçu des protocoles de typage automatisés pour les trois espèces considérées, puis j'ai appliqué ces outils pour traiter de questions d'épidémiologie. S. aureus, espèce à structure clonale, est un pathogène majeur responsable notamment de toxi-infections alimentaires collectives (TIAC). Les travaux réalisés ont permis de démontrer la spécificité d’hôte de certains complexes clonaux et l’origine humaine des cas de TIAC. L. pneumophila est un pathogène de l’environnement dont la structure de population est atypique : présumée panmictique dans la nature, la bactérie semble connaitre une évolution clonale lorsque son écosystème est restreint, dans un milieu anthropique par exemple. L’étude épidémiologique menée sur la population de L. pneumophila dans la ville de Rennes a mis en évidence la présence d’un écotype, non impliqué dans les cas cliniques épidémiques, particulièrement adapté aux réseaux d’eau. P. aeruginosa, modèle de bactérie panmictique, colonise les bronches de patients atteints de mucoviscidose. Le suivi longitudinal de patients indique que les souches installées sont persistantes et quasi-exclusive de la niche qu’elles occupent. L’exploration de cette diversité du monde bactérien est un préalable à l’investigation épidémiologique des maladies infectieuses. Avec un même outil moléculaire de première intention, cette thèse retrace l’épidémiologie et la structure de trois espèces bactériennes très différentes. L’adaptation à un nouvel environnement (hôte animal, niche écologique, organe) est l'occasion d'expansions clonales. / Bacterial species exhibit diversity in their population structure varying from clonal to panmictic according to the abundance of horizontal transfer and the nature of their ecosystem. During my PhD, I focused on three bacterial species, Staphylococcus aureus, Pseudomonas aeruginosa and Legionella pneumophila, which reflect three different situations. To perform the characterisation of large strain collections, I studied the polymorphism of molecular markers called VNTRs for Variable Number Tandem Repeat. MLVA (Multiple Loci VNTR Analysis) is a PCR based typing method that relies on the concomitant analysis of several VNTRs loci. Initially, I designed automated typing protocols for the three species, then I applied these tools to address issues of epidemiology. S. aureus, a clonal species, is a major cause of food poisoning. The present work confirmed the existence of host-specific clonal complexes and demonstrated the predominantly human origin of foodborne disease cases. L. pneumophila is an environmental pathogen whose population structure is atypical: it is presumed panmictic in the environment but the bacterium expands clonally when the ecosystem is restricted, in an anthropogenic habitat for instance. A long-term epidemiological monitoring of L. pneumophila populations in the city of Rennes highlighted the presence of an ecotype, not involved in epidemic cases, particularly adapted to hot water supply systems. P. aeruginosa, a well-described panmictic bacterium, colonizes CF patients’ airways. The longitudinal monitoring of patients provided evidence that the settled strains were persistent and exhibited strong exclusivity for the occupied niche. Exploring the bacterial world diversity is a prerequisite for epidemiological investigation of infectious diseases. Using a first-line molecular tool, these works trace the epidemiology and the population structure of three bacterial species. The adaptation to a new environment (animal host, ecological niche, organ) generally results in clonal expansions.

Nos travaux ont cherché à mieux comprendre les mécanismes à l’origine de la virulence de Staphylococcus lugdunensis, espèce au pouvoir pathogène proche de celui de Staphylococcus aureus. Nous avons procédé à la première caractérisation fonctionnelle chez cette espèce d’un système à deux composants, LytSR. Ce système s’est révélé impliqué dans le contrôle de processus métaboliques majeurs et dans la virulence. Il est en effet impliqué dans la formation de biofilm, probablement en lien avec le contrôle exercé sur la mort cellulaire. LytSR est de plus impliqué dans la pathogenèse des infections à S. lugdunensis, comme démontré dans le modèle d’infection du nématode Caenorhabditis elegans. Pour mieux caractériser et suivre la diffusion de clones prédominants chez cette espèce, nous avons dans un deuxième volet développé trois nouvelles méthodes de typage (MLVA, TRST et fbl-typing), reposant sur le polymorphisme de séquences répétées en tandem. Ces méthodes se sont révélées très discriminantes, permettant la définition de nouveaux génotypes chez cette espèce clonale. Ces outils sont à ce titre très prometteurs pour des études micro- comme macro-épidémiologiques chez S. lugdunensis, le fbl-typing apparaissant à bien des égards comme l’outil utilisable en première ligne (http://fbl-typing.univ-rouen.fr/). Enfin, nous avons montré que la variabilité de la liaison de S. lugdunensis au fibrinogène in vitro peut être en partie expliquée par des variations génétiques de fbl. / Our work has sought to better understand mechanisms involved in Staphylococcus lugdunensis virulence, a species whose pathogenicity is close to that of Staphylococcus aureus. We performed the first functional characterization of a two-component regulatory system, LytSR, in this species. This system has been shown to be involved in the control of major metabolic processes and in virulence. It is indeed involved in biofilm formation, probably in connection with the control of cell death. LytSR is furthermore implicated in the pathogenesis of S. lugdunensis infections, as demonstrated in the infection model of the nematode Caenorhabditis elegans. To better characterize and monitor the diffusion of predominant clones in this species, we have in a second part developed three new typing methods (MLVA, TRST and fbl-typing), based on the polymorphism of variable number of tandem repeats. These methods were highly discriminant, allowing the definition of new genotypes in this clonal species. These tools are very promising for micro- and macro-epidemiological studies in S. lugdunensis, fbl-typing appearing in many ways as the frontline tool (http://fbl-typing.univ-rouen.fr/). Finally, we have shown that the variability of the binding of S. lugdunensis to fibrinogen in vitro can be partly explained by some fbl genetic variations.

Made available in DSpace on 2016-06-21T13:43:16Z (GMT). No. of bitstreams: 2 833.pdf: 2723781 bytes, checksum: a830784ddefc542106be74ce8aa431fa (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Made available in DSpace on 2016-07-05T22:16:54Z (GMT). No. of bitstreams: 3 833.pdf.txt: 159741 bytes, checksum: 9e6065c4897ceb926534eb7e10453293 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 833.pdf: 2723781 bytes, checksum: a830784ddefc542106be74ce8aa431fa (MD5) Previous issue date: 2009 / Fundação Oswaldo Cruz. Centro de Pesquisas Aggeu Magalhães. Recife, PE, Brasil / A Yersinia pestis é o agente etiológico da peste, uma doença primária de roedores, transmitida por pulgas infectadas e que pode infectar o homem e outros mamíferos. O objetivo do trabalho foi realizar a tipagem de 63 cepas de Y. pestis de três focos de peste do PE. As cepas foram isoladas de diferentes fontes e períodos. Das 63 cepas, 20 foram isoladas de um epizootia, em agosto de 1967, na Chapada do Araripe-PE. Também foram estudadas oito cepas de Y. pestis isoladas em outros países, cinco cepas de Y. pseudotuberculosis e nove de Y. enterocolitica. Foram utilizados onze VNTRs pela técnica do MLVA. Dos onze VNTRs para as cepas da epizootia apenas um revelou-se polimórfico apresentando diferentes alelos. Os demais VNTRs revelaram-se monomórficos. Entre os onze VNTRs analisados para as 51 cepas de Y. pestis (43 brasileiras e 8 estrageiras) dois se revelaram monomórficos gerando amplicons com 7 e 2 unidades repetitivas (UR). Os outros nove VNTRs analisados revelaram-se polimórficos gerando dois a oito alelos. As cepas de Y. pseudotuberculosis apresentaram-se polimórficas para 10 VNTRs gerando amplicons de tamanhos diversos, o VNTR ms09 foi o único monomórfico gerando um amplicon de 700 pb com 28 UR. Das nove cepas de Y. enterocolitica analisadas com os onze locos, sete apresentaram-se monomórficos com amplicons de 700, 250, 270, 690, 231 e 379 pb. Os outros quatro VNTRs analisados apresentaram um padrão de amplificação polimórfico com amplicons de tamanhos diferentes para o mesmo loco. O padrão de amplificação gerado com as cepas de Y. pestis possibilitou distribui-las em 35 perfis genotípicos. A análise das cepas pelo dendrograma permitiu agrupá-las em cinco clados, onde no clado I ficaram agrupadas a maioria das cepas brasileiras de Y. pestis, as cepas estrangeiras de Y. pestis ficaram agrupadas nos clados II e IV, enquanto que Y. enterocolitica e Y. pseudotuberculosis ficaram nos clados III e V respectivamente. Diante dissso pode-se considerar que o MLVA mostrou-se uma ferramenta útil em estudos filogenéticos e epidemiológicos das cepas brasileiras de Y. pestis, além de estudos intraespecíficos com as espécies de Y. enterocolitica e Y. pseudotuberculosis. As análises revelaram diversidade genética entre as cepas de Y. pestis isoladas de diferentes fontes e períodos e sua continuação poderá gerar dados importantes para estabelecer relações filogenéticas entre as cepas, contribuindo para um melhor entendimento da disseminação e transmissão do agente etiológico da peste na natureza e a dinâmica da epidemiologia no Brasil

Clostridium difficile is a major cause of infectious diarrhea in hospitalized patients. Clostridium difficile-associated disease (CDAD) is of gaining importance now due to its increasing incidence and severity. However, little is known about the C. difficile infections in the Czech Republic. The aim of the study was to characterize C. difficile strains recently isolated (2008 to 2011) from patients hospitalized with gastrointestinal disease in four Prague health care institutions using molecular typing methods; PCR toxinotyping, PCR ribotyping and MLVA (multilocus variable number tandem repeat analysis). Among 273 C. difficile strains, we identified 8 toxinotypes (0, III, IV, V, VI, VIII, IX a XXIII) and 63 ribotypes, of which ribotypes 596 (23,4 % patient), 017 (13,9 %) and 176 (7 %) were the most frequent. According to PCR ribotyping, the situation in the Czech Republic is the most similar to the situation in Poland. Within ribotypes 017, 017/1 and 017/2 and ribotypes 596 and 596/1, 5 and 4 distinct clusters were identified by MLVA, none of which was institution-specific. Additionally, pathogenic C.difficile were isolated from piglet faeces (63,3 %) in a single piglet farm, evaluating the role of C. difficile as an emerging animal pathogen. All piglet isolates belonged to the toxinotype 0 and the ribotype...

Le Staphylococcus aureus résistant à la méthicilline (SARM) est un pathogène important qui a été identifié comme agent d‟infection chez les animaux d‟élevage et les travailleurs exposés à ces animaux. Au Canada, très peu d‟informations sont disponibles concernant les SARMs d‟origine porcine. L‟objectif de cette étude était de déterminer la prévalence des SARMs provenant de porcs à l‟abattoir, de caractériser leur résistance aux antibiotiques ainsi que d‟évaluer le niveau de séroconversion des porcs envers le S. aureus chez les animaux porteurs ou non du SARM. Un total de 107 isolats ont été identifiés positifs aux SARMs sur 660 échantillons. La prévalence de SARMs à l‟abattoir A était de 30,8% et de 23,8% à l‟abattoir B. La susceptibilité aux antibiotiques a été déterminée en utilisant la méthode de micro-dilution de Sensititre. Tous les isolats ont démontré une sensibilité envers la ciprofloxacine, la gatifloxacine, la gentamicine, la lévofloxacine, le linézolide, la quinupristine/dalfopristine, la rifampicine, la streptomycine, le triméthoprime/sulfaméthoxazole et la vancomycine. De la résistance a été observée envers la daptomycine (0,93%), l‟érythromycine (29%), la clindamycine (29%), la tétracycline (98,1%). De plus, 30% des SARMs isolés étaient résistants à plus de deux antibiotiques autres que les β-lactamines. Par typage, deux clones prédominants ont été obtenus ainsi que deux types de SCCmec (type V et possiblement un nouveau type comprenant les cassettes III et IVb). 15 clones ont été identifiés par typage MLVA, comprenant les clones prédominants VI (40.1%; 43/107) et XI (17.7%; 19/107). Deux souches de SARMs ont été caractérisées par biopuce à ADN et des gènes d‟antibiorésistance, de typage (SCCmec et MLST) et de virulence ont été identifiés. Sans considération pour le site de colonisation, les porcs SA-/MRSA- (n=34) et les porcs SA+ (n=194) montrent, respectivement, des taux de séroconversion de 20.6% et 32.5%. Les porcs colonisés par un SARM à un site de iv prélèvement et non colonisés par un SA à l‟autre site (n=18) montrent une séroconversion (5.6%) significativement (P < 0.05) plus faible comparativement aux porcs colonisés par SA à un ou deux sites de prélèvement et n‟ayant pas de SARM. Nos résultats démontrent que les porcs provenant d‟abattoir peuvent être colonisés par des SARMs multi-résistants aux antibiotiques. De plus, ces SARMs sont possiblement capable de coloniser leurs hôtes sans stimuler la production d‟anticorps et ce par l‟atténuation de la réponse immunitaire ou par la colonisation de porcs qui sont moins immunocompétents. / Methicillin-resistant Staphylococcus aureus (MRSA) found in food producing animals is a major public health concern. Transmission to humans has been reported and MRSA represents a reservoir of antimicrobial resistance genes. Little is known on how MRSA successfully establishes colonization and how it is able to persist in the host. This study was conducted to determine the occurrence and the antimicrobial resistance profile of MRSA from abattoir pigs and their level of seroconversion toward S. aureus (SA). A total of 107 isolates were identified as MRSA from 660 samples. Antimicrobial susceptibilities were determined by broth microdilutions. Fifteen clones were identified by MLVA with clones VI (40.1%; 43/107) and XI (17.7%; 19/107) being the most predominant. All MRSA isolates were pvl-, tst-, eta- and etb-negative. Most isolates were SCCmec type V (70.1%; 75/107). All MRSA isolates were susceptible to ciprofloxacin, gatifloxacin, gentamicin, levofloxacin, linezolid, quinupristin/dalfopristin, rifampin, streptomycin, trimethroprim/sulfamethoxazole and vancomycin. However, resistance was observed toward clindamycin (29%), daptomycin (0.9%), erythromycin (29%) and tetracycline (98.1%). Multi-resistance was confirmed in MRSA since 28% of all isolates were resistant toward three antimicrobials other than β-lactams. The effect of MRSA carriage on seroconversion was examined to see whether the host responded differently to MRSA or SA colonization. The presence of SA-specific antibodies in pig serums was measured for each animal using indirect ELISA and a mixture of two widespread SA antigens (IsdH [HarA] and IsdB). Regardless of the colonization site, SA-/MRSA- pigs (n=34) and SA+ pigs (n=194) showed 20.6% and 32.5% seroconversion, respectively. Notably, pigs colonized by MRSA at one body site and no SA at the other sampling site (n=18) showed a significantly lower (5.6%) seroconversion (P < 0.05) compared to pigs colonized by SA at one or both vi sites without MRSA. The findings of the study show that the nares and axillae of abattoir pigs can harbor MRSA strains with multiple antimicrobial resistances. In addition, these MRSA were possibly able to colonize the host either without stimulating antibody production, by attenuating the immune response or by colonizing pigs that are less immunocompetent. This may explain the success of MRSA colonization and persistence in pigs. Further studies are required to better elucidate MRSA colonization in abattoir pigs and their public health risk.

Les espèces bactériennes exhibent plusieurs états de structure de populations pouvant varier de clonale à panmictique selon l'importance des transferts horizontaux et la nature de leur écosystème. Dans mon travail de thèse, je me suis intéressé à trois espèces bactériennes, Staphylococcus aureus, Legionella pneumophila et Pseudomonas aeruginosa qui reflètent trois situations différentes. Afin de pouvoir décrire de façon rapide de grandes collections de souches, j'ai utilisé comme marqueurs de diversité le polymorphisme de séquences répétées en tandem appelées VNTRs, pour Variable Number Tandem Repeat. La méthode MLVA, ou Multiple Loci VNTR Analysis, est une méthode de typage moléculaire qui s'appuie sur l'étude concomitante du polymorphisme de plusieurs loci VNTRs. Dans un premier temps, j'ai conçu des protocoles de typage automatisés pour les trois espèces considérées, puis j'ai appliqué ces outils pour traiter de questions d'épidémiologie. S. aureus, espèce à structure clonale, est un pathogène majeur responsable notamment de toxi-infections alimentaires collectives (TIAC). Les travaux réalisés ont permis de démontrer la spécificité d'hôte de certains complexes clonaux et l'origine humaine des cas de TIAC. L. pneumophila est un pathogène de l'environnement dont la structure de population est atypique : présumée panmictique dans la nature, la bactérie semble connaitre une évolution clonale lorsque son écosystème est restreint, dans un milieu anthropique par exemple. L'étude épidémiologique menée sur la population de L. pneumophila dans la ville de Rennes a mis en évidence la présence d'un écotype, non impliqué dans les cas cliniques épidémiques, particulièrement adapté aux réseaux d'eau. P. aeruginosa, modèle de bactérie panmictique, colonise les bronches de patients atteints de mucoviscidose. Le suivi longitudinal de patients indique que les souches installées sont persistantes et quasi-exclusive de la niche qu'elles occupent. L'exploration de cette diversité du monde bactérien est un préalable à l'investigation épidémiologique des maladies infectieuses. Avec un même outil moléculaire de première intention, cette thèse retrace l'épidémiologie et la structure de trois espèces bactériennes très différentes. L'adaptation à un nouvel environnement (hôte animal, niche écologique, organe) est l'occasion d'expansions clonales.

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