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Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis (Mtb) that kills around 1.3 million people annually. Multi-drug resistant TB (MDR-TB) strains are increasingly encountered, in part resulting from shortcomings of current TB drug regimens that last between six to nine months. Patients may stop taking the antibiotics during their allotted regimen, leading to drug resistant TB strains. Novel drug screening platforms are therefore necessary to find drugs effective against MDR-TB. In order to discover compounds that target under-exploited pathways that may be essential only in vivo, the proposed screening platform will use a novel approach to drug discovery by blocking essential protein-protein interactions (PPI). In Mtb, PPI can be monitored by mycobacterial protein fragment complementation (M-PFC). This project will re-engineer the M-PFC assay to include the red fluorescent mCherry reporter for increased efficiency and sensitivity in high-throughput screening applications. To optimize the mCherry assay, we have developed fluorescent M-PFC reporter strains to monitor distinct PPI required for Mtb virulence: homodimerization of the dormancy regulator DosR. A drug screen will then identify novel compounds that inhibit this essential PPI. The screen will involve positional-scanning combinatorial synthetic libraries, which are made up of chemical compounds with varying side chains. This work will develop novel tools for TB drug discovery that could identify new treatments for the emerging world threat of MDR-TB.

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose. / Background: Global efforts to control tuberculosis are currently being hampered by a continuing rise in the prevalence of HIV/AIDS. Although outbreaks of multidrug resistant tuberculosis (MDR-TB) are commonly reported among AIDS populations, the link between HIV/AIDS and the development of drug-resistance remains unclear. Objectives: This thesis aimed to: (1) build a knowledge foundation regarding underlying factors associated with outbreaks of MDR-TB among HIV/AIDS patients; (2) use this knowledge framework to develop preliminary health measures for controlling pulmonary tuberculosis among HIV/AIDS patients; and (3) in an effort to better implement these health measures, refine existing culture-based diagnostics for Mycobacterium tuberculosis. Methods: Four studies were conducted: (1) a longitudinal study to identify the underlying factors associated with an epidemic of MDR-TB among AIDS patients receiving Directly- Observed Therapy Short-course (DOTS) for pulmonary tuberculosis in Lima, Peru between 1999 and 2005; (2) a cross-sectional study to characterize the prevalence and factors associated with gastrointestinal shedding with mycobacteria among AIDS patients at different stages in the natural history of tuberculosis; (3) a pilot study to develop screening strategies for pulmonary tuberculosis among hospitalized HIV/AIDS patients using the Microscopic Observation Drug Susceptibility (MODS) assay; and (4) a laboratory-based study to define the optimal critical concentrations needed for detecting drug resistance in M. tuberculosis using MODS. Results: Study 1 revealed that an epidemic of MDR-TB among AIDS patients receiving DOTS for pulmonary tuberculosis was due to super-infection with a specific clone of M. tuberculosis rather than the development of secondary drug-resistance. Although this epidemic clone was more common among patients in the AIDS cohort, risk of superinfection did not differ between AIDS and non-AIDS patients after adjusting for baseline risk of exposure, suggesting that another factor possibly associated with diarrhea may be contributing to the strain’s high prevalence among AIDS patients. Study 2 showed that the majority of AIDS patients in the later stages of pulmonary tuberculosis exhibited gastrointestinal shedding with mycobacteria. Stool shedding was rare in the absence of pulmonary tuberculosis. AIDS patients were also less likely to shed mycobacteria if they had been hospitalized during the previous two years for another medical condition. Study 3 confirmed that pulmonary tuberculosis was common among hospitalized AIDS patients but frequently misdiagnosed using currently recommended diagnostic algorithms. The MODS assay detected most cases and was equally effective when targeted to patients clinically suspicious for tuberculosis. Study 4 demonstrated that low grade drug resistance in M. tuberculosis to ethambutol and streptomycin was difficult to detect with MODS using currently recommended drug-concentration standards in broth. Its diagnostic utility could be improved by modifying drug-concentration standards, and including two versus one critical concentration well for standardized testing. Conclusion: Our studies underscore the need to improve the diagnosis and treatment of tuberculosis among AIDS patients living in resource-constrained settings, all in an effort to prevent morbidity, mortality and the transmission of drug-resistant strains. They also highlight the indirect effect that general health care among HIV-infected patients can have on the development of tuberculosis.

Após o surgimento e disseminação das β-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos β-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de β-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a β-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica. / After emergence and broad dissemination of extended spectrum β-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against β-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other β-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure.

Introduction: Les efforts globaux pour contrôler la tuberculose sont présentement restreints par la prévalence croissante du VIH/SIDA. Quoique les éclosions de la tuberculose multi résistante (TB-MDR) soient fréquemment rapportées parmi les populations atteintes du SIDA, le lien entre VIH/SIDA et le développement de résistance n’est pas clair. Objectifs: Cette recherche visait à : (1) développer une base de connaissances concernant les facteurs associés à des éclosions de la TB-MDR parmi les patients atteints du VIH/SIDA; (2) utiliser ce cadre de connaissances pour accroître des mesures préliminaires pour mieux contrôler la tuberculose pulmonaire chez les patients atteints du VIH/SIDA; et (3) afin d’améliorer l’application des ces mesures, affiner les techniques bactériologiques existantes pour Mycobacterium tuberculosis. Méthodologie: Quatre études ont été réalisées : (1) Une étude longitudinale pour identifier les facteurs associés avec une éclosion de la TB-MDR parmi les patients atteints du SIDA qui ont reçu le traitement directement supervisé de courte durée (DOTS) pour la tuberculose pulmonaire au Lima et au Pérou entre 1999 et 2005; (2) Une étude transversale pour décrire différentes étapes de l’histoire naturelle de la tuberculose, la prévalence et les facteurs associés avec la mycobactérie qu’on retrouve dans les selles des patients atteints du SIDA; (3) Un projet pilote pour développer des stratégies de dépistage pour la tuberculose pulmonaire parmi les patients hospitalisés atteints du SIDA, en utilisant l’essaie Microscopic Observation Drug Susceptibility (MODS); et (4) Une étude laboratoire pour identifier les meilleures concentrations critiques pour détecter les souches MDR de M. tuberculosis en utilisant l’essaie MODS. Résultats : Étude 1 démontre qu’une épidémie de TB-MDR parmi les patients atteints du SIDA qui ont reçu DOTS pour la tuberculose pulmonaire ait été causée par la superinfection du clone de M. tuberculosis plutôt que le développement de la résistance secondaire. Bien que ce clone ait été plus commun parmi la cohorte de patients atteints du SIDA, il n’avait aucune différence de risque pour superinfection entre les patients avec ou sans SIDA. Ces résultats suggèrent qu’un autre facteur, possiblement associé à la diarrhée, peu contribuer à la prévalence élevée de ce clone chez les patients atteints du SIDA. Étude 2 suggère que chez la plupart des patients atteints du SIDA il a été retrouvé une mycobactérie dans leurs selles alors qu’ils étaient en phase terminale au niveau de la tuberculose pulmonaire. Or, les patients atteints du SIDA ayant été hospitalisés pendant les deux dernières années pour une autre condition médicale sont moins à risque de se retrouver avec une mycobactérie dans leurs selles. Étude 3 confirme que la tuberculose pulmonaire a été commune à tous les patients hospitalisés atteints du SIDA, mais diagnostiquée incorrectement en utilisant les critères cliniques présentement recommandés pour la tuberculose. Or, l’essaie MODS a détecté pour la plupart de ces cas. De plus, MODS a été également efficace quand la méthode a été dirigée aux patients soupçonnés d’avoir la tuberculose, à cause de leurs symptômes. Étude 4 démontre les difficultés de détecter les souches de M. tuberculosis avec une faible résistance contre ethambutol et streptomycine en utilisant l’essai MODS avec les concentrations de drogue présentement recommandées pour un milieu de culture. Cependant, l’utilité diagnostique de MODS peut être améliorée ; modifier les concentrations critiques et utiliser deux plaques et non une, pour des tests réguliers. Conclusion: Nos études soulèvent la nécessité d’améliorer le diagnostic et le traitement de la tuberculose parmi les patients atteints du SIDA, en particulier ceux qui vivent dans des régions avec moins de ressources. Par ailleurs, nos résultats font ressortir les effets indirects que les soins de santé ont sur les patients infectés par le VIH et qu’ils peuvent avoir sur le développement de la tuberculose. / Background: Global efforts to control tuberculosis are currently being hampered by a continuing rise in the prevalence of HIV/AIDS. Although outbreaks of multidrug resistant tuberculosis (MDR-TB) are commonly reported among AIDS populations, the link between HIV/AIDS and the development of drug-resistance remains unclear. Objectives: This thesis aimed to: (1) build a knowledge foundation regarding underlying factors associated with outbreaks of MDR-TB among HIV/AIDS patients; (2) use this knowledge framework to develop preliminary health measures for controlling pulmonary tuberculosis among HIV/AIDS patients; and (3) in an effort to better implement these health measures, refine existing culture-based diagnostics for Mycobacterium tuberculosis. Methods: Four studies were conducted: (1) a longitudinal study to identify the underlying factors associated with an epidemic of MDR-TB among AIDS patients receiving Directly- Observed Therapy Short-course (DOTS) for pulmonary tuberculosis in Lima, Peru between 1999 and 2005; (2) a cross-sectional study to characterize the prevalence and factors associated with gastrointestinal shedding with mycobacteria among AIDS patients at different stages in the natural history of tuberculosis; (3) a pilot study to develop screening strategies for pulmonary tuberculosis among hospitalized HIV/AIDS patients using the Microscopic Observation Drug Susceptibility (MODS) assay; and (4) a laboratory-based study to define the optimal critical concentrations needed for detecting drug resistance in M. tuberculosis using MODS. Results: Study 1 revealed that an epidemic of MDR-TB among AIDS patients receiving DOTS for pulmonary tuberculosis was due to super-infection with a specific clone of M. tuberculosis rather than the development of secondary drug-resistance. Although this epidemic clone was more common among patients in the AIDS cohort, risk of superinfection did not differ between AIDS and non-AIDS patients after adjusting for baseline risk of exposure, suggesting that another factor possibly associated with diarrhea may be contributing to the strain’s high prevalence among AIDS patients. Study 2 showed that the majority of AIDS patients in the later stages of pulmonary tuberculosis exhibited gastrointestinal shedding with mycobacteria. Stool shedding was rare in the absence of pulmonary tuberculosis. AIDS patients were also less likely to shed mycobacteria if they had been hospitalized during the previous two years for another medical condition. Study 3 confirmed that pulmonary tuberculosis was common among hospitalized AIDS patients but frequently misdiagnosed using currently recommended diagnostic algorithms. The MODS assay detected most cases and was equally effective when targeted to patients clinically suspicious for tuberculosis. Study 4 demonstrated that low grade drug resistance in M. tuberculosis to ethambutol and streptomycin was difficult to detect with MODS using currently recommended drug-concentration standards in broth. Its diagnostic utility could be improved by modifying drug-concentration standards, and including two versus one critical concentration well for standardized testing. Conclusion: Our studies underscore the need to improve the diagnosis and treatment of tuberculosis among AIDS patients living in resource-constrained settings, all in an effort to prevent morbidity, mortality and the transmission of drug-resistant strains. They also highlight the indirect effect that general health care among HIV-infected patients can have on the development of tuberculosis.

The spread of healthcare-associated infections (HAIs) and multi-drug resistance in healthcare networks is a major public health issue. Evaluating the role of inter-facility patient transfers that form the structure of these networks may provide insights on novel infection control measures. Identifying novel infection control strategies is especially important for multi-drug resistant pathogens such as Carbapenemase-producing Enterobacteriaceae (CPE) due to limited treatment options. The increasing use of inter-individual contact and inter-facility transfer network data in mathematical modelling of HAI spread has helped these models become more realistic; however, they remain limited to a few settings and pathogens. The main objectives of this thesis were two-fold: 1) to better understand the structure of the healthcare networks of France and their impact on HAI spread dynamics; and 2) to assess the role of transfers on the spread of CPE in France during the 2012 to 2015 period. The French healthcare networks are characterized by centralized patient flows towards hubs hospitals and a two-tier community clustering structure. We also found that networks of patients with HAIs form the same underlying structure as that of the general patient population. The number of CPE episodes have increased over time in France and projections estimate that the number of monthly episodes could continue to increase with seasonal peaks in October. The general patient network was used to show that, since 2012, patient transfers have played an increasingly important role over time in the spread of CPE in France. Multiple spreading events of CPE linked to patient transfers were also observed. Despite subtle differences in the flows of patients with an HAI and the general patient population, the general patient network may best inform novel infection control measures for pathogen spread. The structure of healthcare networks may help serve as a basis for novel infection control strategies to tackle HAIs in general but also CPE in particular. Key healthcare hubs in large metropoles and key patient flows connecting hospital communities at the local and regional level should be considered in the development of coordinated regional strategies to control pathogen spread in healthcare systems. / La propagation des infections nosocomiales (IN), notamment liées aux bactéries multi-résistantes, au sein du réseau des hôpitaux, est un grand enjeu de santé publique. L’évaluation du rôle joué par les transferts inter-établissements des patients sur cette propagation pourrait permettre l’élaboration de nouvelles mesures de contrôle. L’identification de nouvelles mesures de contrôle est particulièrement importante pour les bactéries résistantes aux antibiotiques comme les entérobactéries productrices de carbapenemase (EPC) pour lesquelles les possibilités de traitement sont très limitées. L’utilisation des données de réseaux de contact inter-individus et de transferts inter-établissement dans la modélisation mathématique ont rendu ces modèles plus proches de la réalité. Toutefois, ces derniers restent limités à quelques milieux hospitaliers et quelques pathogènes. La thèse a eu pour objectifs de 1) mieux comprendre la structure des réseaux hospitaliers français et leur impact sur la propagation des IN ; et 2) évaluer le rôle des transferts sur la propagation des EPC.Les réseaux hospitaliers français sont caractérisés par des flux de patients vers des hubs et par deux niveaux de communautés des hôpitaux. La structure du réseau de transfert des patients présentant une IN n’est pas différente de celle du réseau général de transfert des patients. Au cours des dernières années, le nombre d’épisode d’EPC a augmenté en France et les prédictions prévoient une poursuite de cette augmentation, avec des pics de saisonnalité en octobre. Ce travail a également montré que, depuis 2012, les transferts de patients jouent avec les années un rôle de plus en plus important sur la diffusion des EPC en France. Des évènements de propagation multiple liée aux transferts sont également de plus en plus souvent observés.En conséquence, la structure du réseau des hôpitaux pourrait servir de base pour la proposition des nouvelles stratégies de contrôles des IN en général, et des EPC en particulier. Les hôpitaux très connectés des grandes métropoles et les flux des patients entre les communautés locale et régionale doivent être considérés pour le développement de mesures de contrôle coordonnées entre établissements de santé.

Após o surgimento e disseminação das β-lactamases de amplo espectro em membros da família Enterobacteriaceae, os antibióticos carbapenêmicos (imipenem, meropenemeertapenem) têm sido considerados a terapia de escolha devido à estabilidade apresentada contra estas enzimas. A desvantagem destes antibióticos é a sua capacidade de induzir resistência aos β-lactâmicos e a outros antibióticos quimicamente não relacionados. O imipenem tem favorecido a indução de cefalosporinases cromossômicas (AmpC) e também tem sido relacionado, in vivo, com a seleção de mecanismos intrínsecos de resistência, contribuindo com o perfil multi -droga resistente (MDR). Esse perfil é freqüentemente associado à diminuição da permeabilidade por alteração na síntese de porinas em conjunto com um aumento da atividade de bombas de efluxo, as quais não permitem o estabelecimento de uma concentração ativa do antibiótico no interior da célula bacteriana. O presente trabalho teve como objetivo avaliar o estabelecimento do perfil MDR em enterobactérias provenientes de isolados clínicos em função da exposição a diferentes concentrações de imipenemin vitro. A seleção do grupo das amostras estudadas foi feito por meio da determinação do perfil de sensibilidade dos isolados, tipagem molecular e ensaio de hidrólise de Imipenem. Nos isolados selecionados para a indução foi realizada numa etapa inicial (etapa basal) a análise de porinas de membrana externa por SDS-PAGE e o estudo de genes codificadores de β-lactamases pela técnica de PCR. O estudo do estabelecimento do perfil MDR foi feito por meio de passagens sucessivas das amostras em meio contendo concentrações sub-inibitórias de imipenem seguido de análise fenotípica (CIM e acúmulo do antibiótico intracelular e SDS-PAGE), e a análise da expressão gênica de genes associados a permeabilidade de membrana (ompC, ompF eAcrA) e genes reguladores(marA e ompR). Após a indução com o imipenem, 77% dos isolados induzidos aumentaram a CIM para os carbapenêmicos, mudando assim o perfil de resistência observado na etapa basal Também foi afetado o perfil de resistência para outros antibióticos não relacionados a β-lactámicos, porém numa percentagem menor. Com relação à alteração da permeabilidade, a perda de porina foi observada apenas para um isolado, no entanto a diminuição na expressão gênica de Omp36 foi significativa desde o começo da indução. A expressão da bomba de efluxoAcrAB foi afetada pela indução com imipenem, aumentando significativamente a expressão de AcrA, enquanto os reguladores estudados, MarA e OmpR tiveram a sua expressão induzida pelo imipenem. Foi possível observar também associação do nível de expressão gênica do regulador MarA com a expressão de AcrA,porém não foi possível observar uma associação estatisticamente significativa deste regulador com o perfil de expressão de OMPs. A indução de OmpR foi associado com um aumento da expressão de RNAm de Omp35, já para Omp36 foi possível observar apenas uma tendência na repressão deste gene. O estudo da resposta destes genes reguladores e determinantes de resistência, em resposta à exposição ao com o imipenem in vitro, permitiu reportar o comportamento molecular da bactéria numa resposta adaptativa no estagio inicial do estabelecimento do fenótipo MDR. A utilização de isolados clínicos com diversos determinantes de resistência permitiu observar a variabilidade nas respostas adaptativas das enterobacterias, o que é fundamental para a compreensão dos mecanismos de adaptação da bactéria e sua contribuição na falha terapêutica. / After emergence and broad dissemination of extended spectrum β-lactamases into the Enterobacteriaceae family, the carbapenemic antibiotics (imipenem, meropenem and ertapenem) have been considered the chosen therapy in the treatment of nosocomial infections by the stability that these antibiotics show to these enzymes. The disadvantage of carbapenems is theirs capacity to induce resistance against β-lactamics and to other chemically unrelated antibiotics. The imipenem has been shown to induce chromosomal cephalosporinases (AmpC) and it was also related, in vivo, with the selection of intrinsic mechanism leading to multi-drug resistance profile (MDR). This profile is usually associated with membrane impermeability due to reduced outer membrane porin synthesis with an incremented activity of efflux pumps, which results in a reduced concentration of antibiotics inside the bacteria. This study aimed to evaluate the establishment of the MDR profile in Enterobacteriaceae from clinical isolates by exposure to different concentrations of imipenem in vitro. The selection of the study group was performed by determination of antibiotic susceptibility profile,molecular typing and hydrolysis assay of imipenem. In the selected isolates submitted to induction, in an initial step (baseline), was performed the outer membrane porin analysis by SDS-PAGE and the gene-specific amplification of B-lactamase enzymes by PCR. The study of the establishment of MDR was performed by progressive passages with subclinical concentrations of imipenem, followed each one by the evaluation of phenotypic profile (MIC, accumulation antibiotic in celland SDS-PAGE) and gene expression analysisof genes related to membrane permeability (ompC, ompF and acrA) and regulatory genes(MarA and ompR). After induction with imipenem, 77 % of the isolates increased the MIC for the carbapenems, changing the resistance profile at the baseline. In a lesser percentage, the resistance profile to other β-lactams-unrelated antibiotics was also affected. Loss of porin was observed only for an isolated, however a significantly decreased Omp36 mRNA expression was observed from the start of induction. The expression of the efflux pump AcrAB ,was also affected by the imipenem induction, significantly increasing the AcrA gene expression, whereas the studied regulatory genes,MarA and OmpR,were induced by the imipenem. It was also possible to observe an association between the expression of the regulator MarA and the expression of AcrA, nevertheless no association was observed between this regulator and OMPs . OmpR induction was associated with an increased Omp35mRNA expression, however only a trend for the repression of Omp36was observed. The study of the response of these regulatory genes and genetic determinants of resistance, in response to the imipenem exposure in vitro, allowed to report the molecular behavior of the bacteria in an adaptive response in the initial stage of the establishment of a MDR phenotype. The use of clinical isolates with diverse resistance determinants allowed observing the variability in adaptive responses in enterobacteria, which is important to understand the adaptive mechanisms of bacteria to this antibiotic, the involvement in the emergence of the MDR profile and its contribution to the treatment failure.

Einleitung: Das Auftreten von multiresistenten Bakterien in der Bevölkerung und in Kran-kenhäusern sowie in der Tierhaltung hat in den letzten Jahrzehnten stark zugenommen. Die weltweite Zunahme multiresistenter gramnegativer Bakterien, insbesondere Entero-bakterien wie Klebsiella (K.) pneumoniae und Escherichia (E.) coli, gibt Anlass zu wach-sender Besorgnis und ist Gegenstand zahlreicher Studien. Ziele der Untersuchungen: Im Rahmen der vorliegenden Studie sollte die Prävalenz von Extended-Spectrum-Beta-Lactamase (ESBL)-produzierenden Enterobakterien (ESBL-E) bei Milchkälbern untersucht und Risikofaktoren für deren Auftreten unter Verwendung von Daten zu Antibiotikaeinsatz, Betriebshygiene und Tiergesundheit identifiziert werden. Tiere, Material und Methoden: Zehn Betriebe mit einem Median von 781 Milchkühen (319-1701) nahmen an der Studie Teil. Die Betriebe wurden zweimal im Abstand von 7-11 Monaten besucht und Kotproben von jeweils 10 neugeborenen Kälbern gesammelt. Alle untersuchten Kälber waren jünger als zwei Wochen mit einem Durchschnittsalter von 6,8 (±3,9) Tagen. Die Kotproben wurden 1:10 verdünnt und im Doppelansatz auf Brilli-anceTM ESBL-Agar plattiert. Nach 24 Stunden bei 37 °C wurden die Kolonien gezählt und die Gesamtanzahl der koloniebildenden Einheiten (cfu)/ml berechnet. Die Bakterien-spezies wurde biochemisch identifiziert. Die ESBL-Produktion wurde mittels MICRONAUT-S β-Lactamase-Platten phänotypisch bestätigt. Zusätzlich wurden weitere Resistenztest mit der VITEK® 2 Technologie durchgeführt. Die Bestimmung der Phy-logruppen der E. coli-Isolate und das Screening auf bla-Gene wurde mittels PCR durch-geführt. Der Hygienestatus der Betriebe wurde mit Hilfe eines standardisierten Fragebo-gens erfasst und bewertet und Daten zu Tiergesundheit und Antibiotikaeinsatz wurden über Tier-Scoring und das Herdemanagementprogramm gesammelt. Ergebnisse: ESBL-E konnten in allen Betrieben und 96,5 % der Kotproben nachgewiesen werden. Der dominierende Anteil der ESBL-produzierenden Isolate waren E. coli (92,9 %), gefolgt von Enterobacter (E.) cloacae (5,1 %) und K. pneumoniae subsp. pneumoniae (2,0 %). Die Mehrheit der E. coli-Isolate wurde eindeutig der Phylogruppe C zugeordnet (25,0 %), gefolgt von den Phylogruppen A (15,2 %) und E (14,1 %). Die CTX-M-Gruppe 1 wurde am häufigsten nachgewiesen (80,4 %). Neben der Resistenz gegenüber Penicillinen und Cephalosporinen war die Mehrheit der Isolate zusätzlich ge-genüber einer oder mehrerer weiterer Substanzklassen resistent, wobei ein hoher Anteil gegenüber Fluorchinolonen resistent war. 52,5 % der Isolate wurden außerdem als drei-fach multiresistente gramnegative Bakterien (3MRGN) gemäß der Kommission für Kran-kenhaushygiene und Infektionsprävention charakterisiert. Keines der Isolate war 4MRGN, d.h. keines zeigte eine Carbapenem-Resistenz. Penicilline wurden bei Kälbern in den meisten Betrieben am häufigsten verabreicht und stellten auf Herdenebene in al-len Betrieben die vorherrschende Substanzklasse dar. Insgesamt war die Anzahl der Kälber, die vor der Probenahme behandelt wurden, eher gering (11,7 %). Analysen der Daten zum Betriebsmanagement ergaben Schwächen bei der Biosicherheit und der Rei-nigung und Desinfektion. Neben Beta-Laktam-Antibiotika als den am häufigsten verwen-deten Antibiotika konnten keine weiteren Risikofaktoren identifiziert werden. Schlussfolgerungen: Die Prävalenz von ESBL-E in unserer Studie war außergewöhnlich hoch. Obwohl die Prävalenz mit zunehmendem Alter der Rinder nachgewiesenermaßen abnimmt, sollten unsere Ergebnisse Anlass zur Entwicklung von Strategien sein, die den Eintrag von ESBL-E in das Kälberaufzuchtsystem frühzeitig verhindern. Dies können beispielsweise der verantwortungsbewusste Einsatz antibiotischer Trockensteller und ei-ne sorgfältige Hygiene in Abkalbeboxen und Kälberställen sein. Weitere Untersuchungen sind erforderlich, um den/die Eintrittspunkt(e) von ESBL-E in die Kälberaufzucht zu defi-nieren.:1 Einleitung 2 Literaturübersicht 2.1 Antimikrobielle Resistenzen 2.1.1 Entstehung antimikrobieller Resistenzen 2.1.2 Resistenzmechanismen 2.1.3 Verbreitung von Resistenzen 2.2 Beta-Laktamantibiotika 2.2.1 Penicilline 2.2.2 Cephalosporine 2.2.3 Carbapeneme und Monobactame 2.2.4 ß-Laktamasehemmer 2.2.5 Bakterielle Resistenzmechanismen gegenüber Beta-Laktamen 2.3 Beta-Laktamasen 2.3.1 Klassifikation 2.3.2 Extended-Spectrum-Beta-Laktamasen 2.4 Vorkommen und Verteilung von ESBL bei Nutztieren und ihr zoonotisches Potenzial 2.5 Nachweis und Identifikation von ESBL 2.5.1 Phänotypischer Nachweis 2.5.2 Genotypische Identifikation 3 Veröffentlichung 4 Diskussion 5 Zusammenfassung 6 Summary 7 Literaturverzeichnis 8 Danksagung / Introduction: The occurrence of multidrug-resistant bacteria in the community and in hospi-tals as well as in animal husbandry has increased rapidly over the last decades. The in-creasing occurrence of multidrug-resistant gram-negative bacteria, especially enterobac-teria such as Klebsiella (K.) pneumoniae and Escherichia (E.) coli, is of growing concern and has been subject to many studies worldwide. Study aims: We studied the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in dairy calves as part of a routine health check protocol. In addition, data regarding antimicrobial use (AMU), farm hygiene, and farm manage-ment were collected in order to identify possible risks for ESBL occurrence. Animals, material and methods: Ten farms participated in the study with a median of 781 milking cows (319-1701). All calves investigated were younger than two weeks with an average age of 6.8 (±3.9) days. The farms were visited and samples were collected twice at an interval of 7-11 months. Faecal samples diluted 1:10, were plated onto Bril-lianceTM ESBL agar in duplicates. After 24 hours at 37 °C, colonies were counted and to-tal colony forming units (cfu)/ml calculated. Bacteria species were identified biochemical-ly. ESBL-production was phenotypically confirmed using the MICRONAUT-S β-Lactamases system. Additionally, antimicrobial susceptibility was tested using VITEK® 2 technology. Phylotyping of E. coli isolates and screening for bla genes was performed by PCR. The hygienic status of the farms was recorded and rated using a standardized questionnaire developed for dairy cattle and data on animal health and antimicrobial treatment were collected through animal scoring and the herd management program. Results: ESBL-producing enterobacteria were detected on all farms and 96.5 % of calves investigated shed ESBL-positive bacteria. Of all ESBL-producing isolates, the majority were E. coli (92.9 %), followed by Enterobacter cloacae (5.1 %) and Klebsiella pneu-moniae subsp. pneumoniae (2.0 %). The majority of E. coli isolates was clearly assigned to phylogroup C (25.0 %), followed by phylogroups A (15.2 %) and E (14.1 %). CTX-M group 1 was most frequently detected (80.4 %). Besides resistance to penicillins and cephalosporins, the majority of isolates was also resistant to one or more antibiotic clas-ses, with a high proportion being resistant against fluoroqinolones. 52.5 % of isolates were further characterised as threefold multidrug resistant gram-negative bacteria (3MDR-GNB) according to the German Commission for Hospital Hygiene and Infection Prevention. None of the isolates were 4MDR-GNB, i.e. none revealed carbapenem-resistance. Penicillins were the most frequently administered antibiotics to calves on most farms and were the predominant substance class at herd level on all farms. Over-all, the number of calves treated prior to sampling was rather low (11.7 %). Analyses of data regarding the farm management identified weaknesses in biosecurity and cleaning and disinfection. Besides beta-lactam antibiotics being the most commonly used antibiot-ics no other risk factors could be identified. Conclusions: The prevalence of ESBL-carriers in dairy calves in our study was exceptional-ly high. Although ESBL-E-prevalence has been described to decrease with increasing age of cattle, our findings should be motivation to develop strategies to prevent the entry of ESBL-E into the calf rearing system at an early stage such as prudent use of antimi-crobials during drying off and diligent hygiene in calving pens and calf housing. Further investigation is needed, to define the entry point(s) of ESBL-E into calf rearing.:1 Einleitung 2 Literaturübersicht 2.1 Antimikrobielle Resistenzen 2.1.1 Entstehung antimikrobieller Resistenzen 2.1.2 Resistenzmechanismen 2.1.3 Verbreitung von Resistenzen 2.2 Beta-Laktamantibiotika 2.2.1 Penicilline 2.2.2 Cephalosporine 2.2.3 Carbapeneme und Monobactame 2.2.4 ß-Laktamasehemmer 2.2.5 Bakterielle Resistenzmechanismen gegenüber Beta-Laktamen 2.3 Beta-Laktamasen 2.3.1 Klassifikation 2.3.2 Extended-Spectrum-Beta-Laktamasen 2.4 Vorkommen und Verteilung von ESBL bei Nutztieren und ihr zoonotisches Potenzial 2.5 Nachweis und Identifikation von ESBL 2.5.1 Phänotypischer Nachweis 2.5.2 Genotypische Identifikation 3 Veröffentlichung 4 Diskussion 5 Zusammenfassung 6 Summary 7 Literaturverzeichnis 8 Danksagung

Clarireedia spp. (formerly Sclerotinia homoeocarpaF.T. Bennett) is the causal agent dollar spot, the most economically important turfgrass disease impacting golf courses in North America. The most effective strategy for dollar spot control is repeated application of multiple classes of fungicides. However, reliance on chemical application has led to resistance to four classes of fungicides as well as multidrug resistance (MDR). Fungi are known to detoxify xenobiotics, like fungicides, through transcriptional regulation of three detoxification phases: modification, conjugation and secretion. Little is known, however, of the protein-protein interactions that facilitate these pathways. Following next-generation RNA sequencing of Clarireedia spp., a fungus-specific transcription factor, XDR1, was determined to play a role in constitutive and induced overexpression of phases I and III genes of xenobiotic detoxification. Further, a novel activation domain (AD) on XDR1 that does not directly bind with xenobiotics was confirmed to be highly conserved among fungal species. Therefore, we hypothesize that XDR1 must be activated by interacting with other binding partners at this AD in order to regulate downstream xenobiotic detoxification pathways. The main objective of this study is to identify additional proteins/ co-repressors that activate XDR1 in order to gain a better understanding of how transcriptional regulation of xenobiotic detoxification pathways leads to MDR. In order to test the hypothesis, fungicide sensitive strain (HRS10) and fungicide resistant strain (HRI11) were transformed and tagged with xdr1/XDR1 and the 3xFLAG tag. As a result, four fungal transformants were generated and those are HRS10-XDR1-3xFLAG, HRS10-xdr1- 3xFLAG, HRI11-XDR1-3xFLAG, and HRI11-xdr1-3xFLAG. The total protein extractions (whole cell lysates) were subjected to co-immunoprecipitation and the samples were analyzed using LC-MS/MS. According to the set of results, more than 50 proteins were detected with HRS10-XDR1-3xFLAG with and most of these binding partners having functions related to post translational modification, protein turnover, intracellular trafficking, secretion and vascular transport. Going forward, information gained from this experiment could be used to explore how XDR1 interacts with its binding partners to facilitate the transcription of drug metabolizing genes responsible for multidrug resistance. This information could also help identify additional fungicide metabolism pathways in filamentous fungi.

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