La tuberculose (TB) reste parmi les 10 premières causes de décès dans le monde ; l’émergence/réémergence de la TB résistante aux antituberculeux aggrave la situation et représente un défi majeur pour l’éradication de la TB. Le Laos est entouré par des pays fortement touchés par la TB et la TB multi-résistante (MDR) et cette maladie représente une priorité en termes de santé publique dans ce pays. Il n’existe encore aucune donnée sur la structure génétique et la résistance aux antibiotiques de la population de M. tuberculosis au Laos.Dans ce contexte, ce travail avait pour but d’analyser la diversité génétique et la structure des populations de M. tuberculosis ainsi que les déterminants génétiques associés à la résistance à partir d’échantillons collectés lors de l’enquête de prévalence nationale de la Tuberculose (TBPS) 2010-2011, l’enquête de résistance aux antituberculeux (DRS) 2016-2017 et chez les cas suspects de MDR-TB au Laos (2010-2014). Plusieurs techniques d’analyses ont été utilisées, comprenant les tests de sensibilité aux médicaments (phénotypique et génotypique), le séquençage et le génotypage par spoligotypage et MIRU-VNTR. Les données ont été analysées par des méthodes statistiques et phylogénétiques.Premièrement, ce travail s’est focalisé sur la diversité des familles de M. tuberculosis circulant au Laos. Les familles EAI et Beijing (76.7% et 14.4% respectivement) ont été principalement observées dans les échantillons de TBPS, alors que la famille Beijing était plus fréquente dans les échantillons de DRS et chez les patients suspectés de MDR-TB (35% et 41% respectivement). La transmission récente était non-négligeable avec un taux de « clustering » global de 11.9%, et des taux pour Beijing de 20 % et EAI de 11 %. Deuxièmement, les résultats ont révélé des profils de résistance très diverses allant de la mono-résistance jusqu’à la pré-XDR (ultrarésistance). Les mutations associées aux profils de résistance ont montré une grande diversité, avec cependant certaines mutations majeures dans les gènes rpoB, katG, et rpsL. Le gène pncA a montré un pattern différent avec de la diversité sans mutations prééminentes. En plus des mutations détectées, des délétions et insertions de bases ont été également observées. Le séquençage a montré son utilité pour la détection de la résistance aux antibiotiques dans les trois échantillons à l’étude. Enfin, la famille Beijing, famille la plus problématique au niveau mondial en termes de résistance et de transmissibilité, a été identifiée de manière significative dans le groupe de patients <35 ans, principalement dans les provinces du Nord, dans les cas de transmissions récentes et chez les isolats très résistants. Tous ces points suggèrent un risque d’émergence de la MDR-TB accrue au Laos dû à la famille Beijing.En conclusion, cette étude permet d’avoir pour la première fois un aperçu de la structure des populations de M. tuberculosis au Laos. Les résultats soulignent le risque d’augmentation du nombre de cas infectés par la famille Beijing et donc des cas de résistance. Pour empêcher une dégradation de la situation, il est essentiel d’améliorer les stratégies pour le dépistage des résistances et de développer des tests moléculaires capables de couvrir un large nombre de mutations qui soit simple à implémenter dans les pays à ressources limités. Les résultats de ce travail serviront de base en termes de famille/sous-famille/génotype et de mutations associées à la résistance au Laos. Ces données pourront être comparées avec de futures études/analyses pour étudier l’évolution de la TB et de la TB résistante et ainsi d’évaluer l’efficacité des politiques de contrôle mises en place. La description des mutations associées aux résistances est utilisée pour créer une base de données régionale en collaboration avec le Vietnam et le Cambodge pour développer un outil de diagnostic basé sur la technologie des puces à ADN pour améliorer la détection de la résistance dans la région. / Tuberculosis (TB) is still one of the top 10 leading causes of death worldwide; the emergence/re-emergence of drug resistant TB aggravates the situation globally and challenges the prospect of ending TB by 2035. Lao PDR is surrounded by TB and MDR-TB high burden countries and TB continues to be one of the priority infection diseases in this country. The prevalence of TB in 2010 was almost twice as high than previous estimates and little is known about drug resistance. Up to now, M. tuberculosis population data regarding drug resistance and genetic structure are totally absent. In this context, we aimed to study the diversity and the structure of M. tuberculosis population and the genetic determinants associated to drug resistance using clinical samples collected from the TB prevalence survey (TBPS), 2010-2011; from the Drug resistance survey (DRS), 2016-2017 and from presumptive MDR-TB cases in Lao PDR (2010-2014). Various methods and analyses were used, including drug susceptibility testing (phenotypic and genotypic), DNA sequencing and genotyping of M. tuberculosis using spoligotyping and MIRU-VNTR. The data were analyzed by statistical and phylogenetic analyses.Firstly, this work was focused on the diversity of M. tuberculosis families circulating in Lao PDR. According to the result form TBPS, EAI and Beijing family (76.7% and 14.4% respectively) were mainly observed, while Beijing family was more observed in DRS, and presumptive MDR-TB cases (35% and 41% respectively). The level of recent transmission in Lao PDR was non-negligible with a global clustering rate of 11.9% and in Beijing and EAI of 20% and 11%, respectively. Secondly, the results demonstrated the diversity of drug resistant patterns from mono-resistance to pre-extensively drug resistance (pre-XDR). A high diversity of mutations associated with drug resistance was also observed, however common mutations were mainly found (e.g: mutations in rpoB gene, katG and rpsL). The pattern was different for pncA gene, we observed a diversity of mutations without preeminent ones. Besides the number of known and unknown mutations associated with anti-TB drug resistance, deletion and insertion of bases were also observed. The sequencing showed its usefulness for drug resistance detection. Lastly, Beijing family, which is the more problematic family in the world in terms of resistance and transmissibility, was observed on a significant manner in young age group, mainly in the northern provinces, in recent transmission cases and among highly drug resistant isolates, suggesting an increasing risk of highly drug resistance TB due to highly transmissible Beijing strains in Lao PDR.In conclusion, this study provides the first genetic insights into the M. tuberculosis population in Lao PDR. The results underline the risk of increase of Beijing and drug resistant TB in the country. In order to prevent a more serious situation in the future regarding drug resistance as observed in neighboring countries, there is an urgent need of effective strategy improvement for drug resistance screening and the development of rapid molecular tests that cover a large number of drug resistance simultaneously with a feasible implementation in the limited resource countries. The results of genotyping from our study will be the baseline of families/subfamilies/genotype of M. tuberculosis population and of the mutations associated with drug resistance in Lao PDR. These data will be compared with further study/analysis to evaluate the trend of TB and drug resistant TB in the country and to determine if the drug resistance is under control after the set-up of new policies. The data of drug resistance associated mutations are used to build a regional database in collaboration with Vietnam and Cambodia in order to develop a diagnostic tool based on DNA chip technology to improve the drug resistance detection in the region.

Pseudomonas aeruginosa est un pathogène opportuniste responsable de pneumonies. Il est particulièrement impliqué dans la mortalité des patients sous ventilation mécanique et des patients atteints de la mucoviscidose. Ces infections sont difficiles à traiter en raison de l’existence de nombreuses résistances aux antibiotiques chez cette bactérie et des alternatives thérapeutiques s’avèrent donc nécessaires. Nous avons ainsi émis l’hypothèse qu’une application oropharyngée de lactobacilles pourrait permettre de limiter les infections à P. aeruginosa et leurs effets chez les patients concernés. L’objectif principal de ce travail était d’évaluer les effets d’un mélange de lactobacilles dans un modèle murin de pneumonie à P. aeruginosa. Les effets de lactobacilles isolés dans les cavités orales de volontaires sains sur la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1 ont été mesurés in vitro. Les effets des lactobacilles sélectionnés ont ensuite été évalués dans un modèle d’infection de cellules épithéliales respiratoires(A549) par P. aeruginosa PAO1 puis dans un modèle murin de pneumonie à P. aeruginosa PAO1. Les effets de 87 lactobacilles sur la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1 ont été déterminés in vitro,aboutissant à la sélection de 3 et 5 souches ayant respectivement inhibé la formation de biofilm et l’activité élastolytique de P. aeruginosa PAO1. Parmi ces souches, L. fermentum K.C6.3.1E, L. paracasei ES.D.88 et L. zeae Od.76,qui étaient les souches les plus actives contre la formation de biofilm ou l’activité élastolytique et les plus acidifiantes lors de croissances dans une salive artificielle, ont été associées dans un mélange testé dans un modèle cellulaire d’infection à P. aeruginosa PAO1. Ce mélange n’a pas eu d’effet cytotoxique et a démontré un effet cytoprotecteur vis-à-vis de l’infection à P. aeruginosa PAO1. In vivo, l’administration intratrachéale de ces mêmes bactéries de façon prophylactique a permis d’une part de réduire les charges pulmonaires en P. aeruginosa PAO1 et d’autre part de réduire ses effets pro-inflammatoires au niveau pulmonaire (IL-6, TNF-α). Ces résultats prometteurs laissent entrevoir la possibilité de nouvelles applications thérapeutiques pour les probiotiques. / Pseudomonas aeruginosa is an opportunistic pathogen that causes pneumonia and which is involved in themortality of mechanically-ventilated or cystic fibrosis patients.These infections are difficult to treat because of the existence of many antibiotic resistances in P. aeruginosa and therapeutic alternatives are needed. Our hypothesis was that the use of probiotics could be an alternative to antibiotic therapy in order to reduce P. aeruginosa infections and its injurious and pro-inflammatory effects in lungs.The main goal of this work was to evaluate the effects of lactobacilli in a murine model of P. aeruginosa pneumonia.The first step of this work was to screen lactobacilli isolated from oral cavities of healthy volunteers against biofilmformation and elastolytic activity of P. aeruginosa PAO1. The effects of selected lactobacilli were then evaluated in amodel of infection of lung epithelial cells by P. aeruginosa PAO1 and in a murine model of P. aeruginosa PAO1pneumonia. Eighty-seven lactobacilli were tested in vitro, leading to the selection of 3 and 5 strains respectively active against biofilm formation and elastolytic activity. The most active strains (L. fermentum K.C6.3.1E, L. paracasei ES.D.88and L. zeae Od.76) toward biofilm formation and elastolytic activity were chosen to be tested in vitro, in a cell model of P. aeruginosa PAO1 infection. This mix showed cytoprotective effect against P. aeruginosa PAO1. Finally, the prophylactic intratracheal administration of the mix of lactobacilli in mice allowed to reduce the pulmonary loads in P.aeruginosa PAO1. In the same time, the pro-inflammatory effects(IL-6 and TNF- α) of the infection were reduced. These promising results suggest the possibility of new therapeutic applications for probiotics.

SoxR est un régulateur transcriptionnel à centre [2Fe-2S] qui induit une réponse adaptative permettant à E. coli de résister aux composés redox actifs, générateurs de stress superoxyde. En présence de composés redox actifs, le centre [2Fe-2S] de SoxR est oxydé ce qui lui permet d’activer l’expression du gène soxS codant pour un régulateur transcriptionnel activant l’expression d’une centaine de gènes. Parmi les gènes du régulon SoxRS on trouve ceux permettant de résister au superoxyde mais aussi aux antibiotiques. J’ai montré qu’en présence de phénazine méthosulfate (PMS), un composé redox actif, la machinerie de biogénèse des centres Fe-S utilisée pour la maturation de SoxR est différente suivant les conditions environnementales. En effet, en aérobie la maturation de SoxR est assurée par la machinerie SUF, alors qu’en anaérobie c’est la machinerie ISC qui intervient. J’ai également étudié l’importance de SoxR, et des machineries ISC et SUF, dans la résistance aux antibiotiques induite par la présence de PMS. J’ai montré qu’en présence de PMS, E. coli peut résister à la norfloxacine, par un mécanisme SoxR dépendent, et ceci quelque soit la machinerie de biogénèse des centres FeS présente. D’autre part, j’ai étudié l’impact des conditions environnementales, comme la teneur en CO2 dans l’atmosphère sur la capacité d’ E. coli à résister au stress oxydant. J’ai testé, expérimentalement les prédictions obtenues par un modèle d’équations différentielles permettant de simuler la concentration des ROS dans la cellule. J’ai montré que le CO2 a un effet de protection lors d’un stress au H2O2 probablement en capturant les HO• produits par la réaction de Fenton. / SoxR is a [2Fe-2S] cluster-containing transcriptional regulator that mounts the adaptive response allowing E. coli to tolerate superoxide-propagating compounds. When cells are exposed to redox cycling drugs the Fe-S cluster of SoxR undergoes a reversible univalent oxidation to yield the oxidized active protein. The only known target of SoxR is the soxS gene that is itself a transcriptional regulator activating the expression of more than 100 genes including those for superoxide and antibiotic resistance. I showed that the machinery used to mature SoxR under phenazine methosulfate (PMS) exposition, a redox cycling drug, was different depending on the environmental conditions used. In aerobiosis, the SUF machinery ensured SoxR maturation, while in anaerobiosis the ISC machinery was required. I also monitored the implication of SoxR, the ISC and SUF machineries, in antibiotic resistance induced by PMS exposition. I showed that E. coli can resist to norfloxacin under PMS exposition in a SoxR-dependent manner whatever the Fe-S cluster biogenesis machinery available. Last, I studied the impact of environmental conditions, such as atmospheric CO2 concentration, on the ability of E. coli to cope with oxidative stress. I have experimentally tested the predictions obtained by a mathematical model that simulates ROS dynamics. I showed that carbon dioxide has a protective effect on hydrogen peroxide stress likely by scavenging the radical hydroxyl produced by the Fenton reaction.

Les bactéries pathogènes opportunistes de l'Homme (bpo) sont retrouvées dans le milieu hospitalier où elles sont responsables d'infections nosocomiales ainsi que dans les milieux naturels terrestres et aquatiques. Elles présentent souvent des résistances intrinsèques aux antibiotiques élevées. En milieu clinique, l'usage intensif d'antibiotiques peut conduire à l'émergence de souches dites « Multi Drug Resistant ». L'anthropisation des milieux naturels peut également influencer la prévalence et les propriétés de résistance des bpo. Mes travaux ont porté sur l'impact de l'épandage d'amendements organiques sur la prévalence de bpo dans les sols, leur diversité génétique et leurs propriétés de résistance aux antibiotiques. Une étude des espèces Stenotrophomonas maltophilia, Pseudomonas aeruginosa et Burkholderia du « cepacia complexe » (Bcc) réalisée sur des sites du Burkina-Faso amendés ou non en déchets urbains bruts a mis en évidence des différences dans les propriétés de résistance des 3 modèles. S. maltophila présente fréquemment des phénotypes MDR contrairement à P. aeruginosa et aux Bcc. Une approche de génomique comparative entre souches de S. maltophilia d'origine environnementale ou clinique et de phénotypes sensibles à MDR a été réalisée afin d'élucider l'origine génétique de l'hétérogénéité des phénotypes de résistance. Une variation dans le contenu en pompes à efflux et la présence de pompes souche spécifique chez des souches environnementales ont été observées. L'étude de l'expression d'une de ces pompes confirme son implication dans la résistance aux antibiotiques et dans l'adaptation à des paramètres environnementaux tels que la température / Opportunistic bacterial pathogens (obp) of Man are found in hospital setting where they are responsible for nosocomial infections as well as in terrestrial and aquatic natural environments. Obp often show high intrinsic antibiotic resistance level. Moreover, the intensive use of antibiotics in clinical settings can lead to the emergence of "Multi Drug Resistant" strains. The anthropisation of the natural environment leads to modifications in bacterial diversity of these environments and can affect the prevalence and the antibiotic resistance properties of obp. My research focused on the impact of organic amendments on the prevalence, genetic diversity and antibiotic resistance properties of obp. A study on the species Stenotrophomonas maltophilia, Pseudomonas aeruginosa and the “Burkholderia cepacia complex" (Bcc) was conducted on sites in Burkina Faso amended or not with raw urban wastes. This study showed differences in antibiotic resistance properties between the 3 models. S. maltophila frequently showed MDR phenotypes unlike P. aeruginosa and Bcc. A comparative genomics study between S. maltophilia strains from environmental or clinical origin showing sensitive or MDR phenotypes was performed to elucidate the genetic origins of heterogeneity in the resistance phenotypes. A variation in the efflux pumps content was observed between strains. The expression of an efflux pump specific to an environmental MDR strain was then evaluated and confirmed its likely involvement in antibiotic resistance and adaptation to environmental parameters such as temperature

Atividades antropogênicas relacionadas ao uso massivo de antibacterianos tem favorecido para que ambientes aquáticos sejam importantes locais para seleção e/ou disseminação de bactérias multirresistentes (MR). O objetivo do estudo foi monitorar a ocorrência de bactérias MRs em ambientes aquáticos públicos no estado de SP, caracterizando genótipos de resistência adquirida. Foram analisados 50 isolados de bactérias gram-negativas, recuperadas de amostras de água, sendo que 70% dos isolados apresentaram perfil de MR, identificando-se os genes blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2), blaKPC-2 (n= 3), qnrB (n= 1), oqxA (n= 3), oqxB (n= 3) e, aac(6)1b-cr (n= 7). Destaca-se a primeira detecção de A. calcoaceticus produtora de KPC-2. Ambientes aquáticos de acesso público podem ser importantes fontes para a disseminação e/ou transmissão de uma ampla variedade de espécies bacterianas MRs tanto para seres humanos como para ecossistemas associados, sendo que a presença de genótipos endêmicos sugere contaminação por esgoto doméstico e/ou hospitalar. / Anthropogenic activities related to the massive use of antibacterial has contributed to the selection and spread of multidrug-resistant (MDR) into the aquatic environment. This study aimed to monitor the occurrence of MDR bacteria in public aquatic environments, in the state of São Paulo, characterizing genotypes of acquired resistance. Of the 50 gram-negative isolates, recovered of water samples, 70% exhibited a MDR profile. Indeed, blaCTX-M-2 (n= 5), blaCTX-M-9 (n= 1), blaCTX-M-15 (n= 2)-, blaKPC-2 (n= 3)-, qnrB (n= 1)-, oqxA (n= 3)-, oqxB (n= 3)- and aac(6)-1b-cr (n = 7) genes were identified. Noteworthy is the first the detection of KPC-2-producing Acinetobacter calcoaceticus. Aquatic environments, with public access, may be important sources for the dissemination and/or transmission of a wide variety of MDR bacterial species to both humans and associated ecosystems. Moreover, the presence of endemic genotypes suggests contamination by domestic and/or hospital sewage.

Introdução: Bactérias do gênero Aeromonas estão presentes em ambientes de água doce, salgada e salobra. O isolamento destes microrganismos já foi relatado em água de abastecimento público e alimentos. Algumas espécies podem ser patogênicas ao homem, causando gastrenterites e outras infecções. Isolados de Aeromonas de fontes diversas expressam resistência a antimicrobianos, especialmente a -lactâmicos, devido à presença de enzimas -lactamases. A patogenicidade das espécies se deve à virulência multifatorial, que compreende a produção de enterotoxinas (Act, Alt e Ast), de elastase, presença de flagelo, entre outros. Objetivo: Isolar, identificar e quantificar Aeromonas hydrophila isoladas de esgoto e lodo tratado; pesquisar a ocorrência dos genes de virulência e resistência a -lactâmicos. Material e Métodos: A detecção e quantificação de Aeromonas hydrophila foram realizadas por meio da técnica de membrana filtrante e meio de cultura específico; a identificação foi realizada por meio da PCR utilizando um par de primers específicos para a espécie. Após a confirmação da espécie foi realizado o antibiograma para conhecer o perfil de resistência aos antibióticos; a pesquisa dos genes de virulência act, alt, ast, ela, lip e fla e genes de resistência a -lactâmicos foi realizada por meio da PCR e seqüenciamento. Resultados: Foram analisadas 15 amostras (seis de esgoto tratado e nove de lodo tratado). Destas, somente nove foram positivas para A. hydrophila, obtendo-se 441 colônias típicas, das quais 348 foram positivas, por PCR para identificação do gênero e 209 para identificação da espécie. Os 209 isolados, sendo 92 do esgoto tratado e 117 do lodo tratado, apresentaram os seguintes valores na pesquisa dos genes de virulência: 36 por cento (act), 40 por cento (ast), 78 por cento (alt), 82 por cento (fla), 86 por cento (lip) e 87 por cento (ela) e 100 por cento dos isolados apresentaram pelo menos um dos genes. Para os testes de sensibilidade aos antibióticos todos os isolados foram resistentes a pelo menos um dos antibióticos. A produção de enzimas MBL, ESBL e AmpC foi detectada em isolados. Também foram encontrados genes de resistência cphA, bla TEM e bla MOX, enquanto que os genes bla VIM, bla IMP, bla e bla não foram detectados. Conclusão: Os resultados sugerem que A. hydrophila pode resistir ao processo de tratamento de esgoto e lodo, além disso, pode apresentar diversos genes de virulência e resistência a antibióticos, motivos pelos quais A. hydrophila pode ser uma ameaça a Saúde Pública, uma vez que estas amostras são reutilizadas para fins urbanos ou agrícolas / Introduction: Bacteria of the genus Aeromonas are present in fresh, brackish and salty waters. The isolation of these microorganisms has been reported in public water supplies and foods. Some species can be pathogenic to humans, causing gastroenteritis and other infections. Aeromonas isolates from different sources express resistance to antimicrobials, especially -lactams, due to the presence of lactamase enzymes. The pathogenicity of the species is due to the multifactorial virulence, wich includes the production of enterotoxins (Act, Alt and Ast) of Elastase and presense of flagello, among others. Objectives: Identify and quantify Aeromonas hydrophila isolated from treated wastewater and sludge, to investigate the occurrence of virulence genes and resistance to -lactams. Material and methods: The detection and quantification of A. hydrophila were made through the membrane filter technique and specific culture medium, the identification was performed by PCR using a pair of primers specific for the species. After confirming the species sensitivity was performed to know the profile of antibiotic resistance, the survey of virulence genes act, alt, ast, ela, lip, fla and resistance to -lactams gene was performed by PCR and sequencing. Results: We analyzed 15 samples (six of nine treated wastewater and sludge). Of these only nine were positive for A. hydrophila, resulting in 441 typical colonies, of wich 348 were positive by PCR to identify the genus and 209 for species identification. The 209 isolates, being 92 and 117 of treated wastewater and treated sludge showed the following values in the study of the virulence genes: 36 per cent (act), 78 per cent (alt), 82 per cent (fla), 86 per cent (lip), 87 per cent (ela) and 100 per cent of the isolates had at least one of the genes. For antibiotic susceptibility testing all isolates were resistant to at least one antibiotic. The production of MBL, ESBL and AmpC enzyme was detected in isolates. It was also found resistance genes cphA, bla TEM and bla MOX, while genes bla VIM , bla IMP , bla and bla FOX CTX-M were not detected. Conclusion: The results suggest that A. hydrophila can resist the process of treating of wastewater and sludge, moreover, may have different virulence genes and antibiotic resistance, which is why A. hydrophila can be a threat to public health, since these samples are reused for agricultural or urban purposes. , bla SHV

Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this work, a summary is provided of our current understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria (Chapter 1). The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities. Although this thesis primarily explores structure and function, it also elucidates the hidden regulatory mechanism (post-translational) behind the association of a small protein called AcrZ with the tripartite complex AcrAB/TolC, in connection with the lipid environment, and the resulting changes in the latter's functionality (Chapter 2). A regulatory role of the native membrane lipid environment as well as of small proteins for efflux pump activity have previously been hypothesised. I present the first example of a function-regulating role of the lipid cardiolipin in combination with a small protein binding partner (AcrZ) for the substrate selectivity and transport activity of an efflux pump protein (AcrB). This regulation happens through induced structural changes which have remained unseen so far. Alongside with these results, a nanodisc reconstitution method was experimentally adapted for a structure-function investigation of an efflux pump (complex) using cryo-EM (Chapter 2). Beyond some fundamental regulatory insights, hidden intrinsic transport mechanisms for some transporters have also remained to be explored and studied. The discovery of a mechanism for active influx by a prominent efflux pump model system (Chapter 3) provides hope that this phenomenon is more common amongst multidrug transporters and that it could be utilised for drug discovery purposes. This novel feature explains the contradictory findings on this transporter in the past and raises new questions about the little-known physiological role and evolution of efflux pumps. The development and evolution of antimicrobial resistance has frequently shown to be a multifactorial and fast-moving process. One of these factors is the evolution of pumps itself towards an altered functionality (e.g. towards a broader or altered substrate spectrum or higher efflux rates). Against this background, the role of key carboxylate residues for efflux-energising proton trafficking was investigated for a prominent study model of a secondary-active transporter (Chapter 4). The re-allocation and/or addition of acidic residues was demonstrated to result in the preservation of wild type activity or the generation of hyper-efflux activity, respectively. These findings suggest that rapid emergence of antimicrobial resistance could be enhanced by the 'plasticity' in the location of key carboxylate residues with a role in proton coupling. It also demonstrates the necessity of antimicrobial drug design programmes to anticipate possible trajectories of an adaptive evolution of efflux pump. The 'cryo-EM revolution' has boosted the pace at which new structural and functional insights into multidrug efflux pumps are gained. Nevertheless, in order to derive the structure of individual pump components or of a full assembly, it is sometimes necessary to identify and characterise homologues and mutants, which would allow the application of cryo-EM for obtaining near-atomic maps. Functional analyses presented in this work helped to characterise a homologue and mutants of the MacAB/TolC tripartite complex to justify the obtained protein structures and strategies for further functional characterisation (Chapter 5). Given (1) the unusual stoichiometry of a MacB dimer in complex with a hexameric membrane-fusion protein (MacA), which leads to a seeming leakiness of the assembly, and (2) the fact that substrate has to pass through a narrow aperture in the membrane-fusion protein for extrusion, it is rather surprising that MacB was previously shown to transport an entire toxin. An experimental approach was developed that could enable the structure determination of a toxin-bound full assembly of MacAB/TolC (Chapter 5). Finally, the role of multidrug efflux pumps for the evolution of multidrug resistance is yet to be studied and better explored. For instance, evolutionary trajectories of pump overexpression, as compared to those of regular expression or no expression, are unknown yet could have the potential to reveal useful insights for spread prevention and drug design. The outline of an experimental design with some preliminary validating data is presented in Chapter 6.

Instead of planktonic growth in nature, many species of bacteria form biofilm to survive in harsh conditions. Although many chronic bacterial infections are caused by bacterial species in a biofilm lifestyle, previous research has focused on studying antibiotic resistance in planktonic growth. Here we used a modified MBEC assay, i.e. biofilm growth on pegs, to determine Escherichia coli biofilm inhibitory concentrations (BIC) of ciprofloxacin, streptomycin and rifampicin and to study the minimal selective concentration (MSC) for ciprofloxacin in E. coli biofilm. We could observe high inhibitory concentrations for all antibiotics in the biofilm pre-formed in media without antibiotics compared to the biofilm formed in antibiotics. We also show preliminary result indicating that sublethal concentrations of ciprofloxacin lead to the selection of ciprofloxacin resistant mutants in biofilm and that the selection level is lower than what was observed in planktonic growing E. coli. With more knowledge in how the biofilm formation precedes in different antibiotic settings, the treatment for chronic biofilm infections used today could be evaluated and changed so that the infections could be eradicated.

Dans une perspective comparative entre la France et les États-Unis, ce travail analyse le processus de publicisation des liens entre l’antibiorésistance et l’alimentation, ainsi que ses implications en termes de contribution au développement de la production appelée sans antibiotique et de l’approche One Health. En partant de la prise de conscience des conséquences de l’usage des antibiotiques dans l’élevage, la recherche s’inscrit dans une réflexion pragmatiste de constitution des problèmes publics et s’appuie sur un corpus hybride composé de documents publiés entre 1980 et 2016 (presse écrite, littérature institutionnelle et entretiens semi-directifs). La méthode développée s’enrichit des outils de textométrie issus de l’analyse de discours et s’intéresse à l’émergence des dénominations et des formules qui nomment le problème, ses causes et ses solutions. La comparaison montre que le processus de publicisation de liens entre l’antibiorésistance et l’alimentation dévoile une trajectoire opposée dans les deux pays. Dans le cas français, ce processus s’inscrit dans un schéma top-down et se caractérise par une publicisation tardive faisant suite aux démarches des instances sanitaires européennes et internationales. L’appropriation du problème par des associations de consommateurs, ainsi que l’investissement des acteurs agroalimentaires dans le développement de la production sans antibiotique, n’émergent que récemment. En revanche, aux États-Unis, ce processus s’inscrit dans un modèle bottom-up suite à la constitution d’un public d’organisations non gouvernementales autour du problème. Leur mobilisation a contribué significativement au développement de programmes d’élevage sans antibiotique ainsi qu’à la mise à l’agenda gouvernemental du problème et le lancement d’un plan national dans une approche One Health. / In a cross-country perspective between France and the United States, this research analyses the process of publicizing the links between antibiotic resistance and food, as well as its contribution to the development of the antibiotic free production and the implementation of the One Health approach. Starting with the awareness of the antibiotic use in livestock consequences, the study relies on the pragmatist approach of the constitution of the public problems. It is based on wide corpora composed by documents published between 1980 and 2016 (written press, institutional literature and semi-directive interviews). The analysis method uses textometric tools derived from discourse analysis and focuses on the emergence of formulas that name the problem, its causes and its solutions. The comparison uncovers an opposite process between the two countries. In France, this process is part of a top-down approach and is characterized by a late publicization following the European and international health authorities’ initiatives. The consumer associations taking over the problem, as well as the agri-food actors’ commitment to the antibiotic free production, is very recent. In the United States, this process reveals a bottom-up model following a non-governmental organizations public constitution taking over the problem. Their mobilization has contributed to the development of the antibiotic free breeding programs, as well as to place the problem on the government agenda that launched a national plan in a One Health approach.

Chez les bactéries à Gram-négatif, l’imperméabilité de la membrane externe est un facteur majeur contribuant au développement de la résistance. Chez Escherichia coli, les porines OmpF et OmpC sont des protéines de la membrane externe qui forment des canaux pour la diffusion de petites molécules hydrophiles tels que les antibiotiques. L’expression des porines est soumise à une régulation fine, et des petits ARN non-codants (sRNAs, small RNAs) jouent un rôle important au niveau post-transcriptionnel. Dans ce cadre, et en utilisant E. coli comme bactérie modèle, les objectifs de mon travail de thèse étaient : (1) de caractériser la régulation du sRNA MicC et la co-régulation putative de la porine quiescente OmpN; (2) d’examiner l'effet global de MicC sur le transcriptome; (3) d’analyser l'impact de l'expression de MicC sur la sensibilité aux antibiotiques. Les résultats obtenus montrent l’induction de MicC en présence d'antibiotiques de la famille des β-lactamines, ou en l’absence du facteur sigma de réponse au stress de l’enveloppe sigmaE. Ces mêmes conditions activent aussi l'activité d'une fusion ompN-lacZ, indiquant une régulation transcriptionnelle commune de micC et ompN. Etant donnée la conservation de MicC chez les entérobactéries, nous avons effectué une étude par RNASeq pour déterminer l'impact de la surexpression de MicC sur le transcriptome d’E. coli et identifié 60 ARNm régulés par MicC en plus de sa cible initiale ompC. L'identification des spectres cibles globaux des sRNAs est importante pour comprendre leur importance dans la physiologie bactérienne, ici celui de MicC dans la résistance aux antibiotiques. / A major factor contributing to antimicrobial resistance is the inability of antibiotics to penetrate the bacterial outer membrane to reach their target. In Escherichia coli, the two abundantly expressed porins OmpF and OmpC form channels for diffusion of small hydrophilic molecules including antibiotics. The expression of porins is under complex regulation and the small regulatory RNAs (sRNAs) fine tune the porin expression level at post-transcriptional level. MicF and MicC are the two major sRNAs that negatively regulate expression of OmpF and OmpC, respectively. Interestingly, these two sRNAs are encoded next to porin gene, i.e. micF-ompC and micC-ompN, suggesting a dual regulation. Our goals in this work were: (1) to characterize the regulation of the sRNA MicC and the putative co-regulation of the quiescent porin OmpN in E. coli; (2) to examine the global effect of MicC on the E. coli transcriptome; (3) to analyze the impact of MicC expression on antibiotic susceptibility. Our work shows that the expression of micC was increased in the presence of carbapenems and cephalosporins and in an rpoE depleted mutant. The same conditions enhanced the expression of OmpN, suggesting a dual regulation of micC and ompN. We also performed RNA sequencing to determine the impact of MicC overexpression on E. coli transcriptome. This identified 60 mRNA targets negatively regulated by MicC apart from its original target. Identification of the global target spectra of MicC is of importance to understand its importance on the overall bacterial physiology, and more specifically on AMR.