Mycobacterium abscessus (Mabs) est un pathogène émergent entrainant de graves infections pulmonaires, notamment chez les patients mucoviscidosiques. L'expression différentielle des glycopeptidolipides (GPLs) permet de distinguer le morphotype rugueux (R), présentant un défaut de synthèse des GPLs, du morphotype lisse (S) exprimant les GPLs. Différents modèles ex vivo et in vivo rapportent que le variant R est impliqué dans des manifestations plus sévères associées à une réponse inflammatoire intense. Cependant, ces modèles d'étude restent particulièrement limités pour élucider les caractéristiques de cette infection. L'embryon de zebrafish (ZF) offre de nombreux avantages motivant et validant son utilisation pour une meilleure compréhension des maladies infectieuses. Ce travail de thèse a pour objectif de développer un modèle d'infection de Mabs dans l'embryon de ZF.Pour étudier la physiopathologie de l'infection de Mabs dans ce modèle, l'élaboration d'un protocole de microinjection des bactéries et des méthodes de suivi de la charge bactérienne ont été réalisés. Les techniques d'imagerie ont été employées pour déterminer la chronologie de l'infection au sein des embryons infectés. Les techniques de qRT-PCR, l'utilisation de lignées de ZF transgéniques et la technologie antisens (morpholinos) ont été utilisées pour déterminer le rôle du système immunitaire (Si) inné et de l'inflammation dans la physiopathologie infectieuse. Par ailleurs, le potentiel du ZF en tant qu'organisme modèle en pharmacologie a été mis à profit pour étudier l'activité in vivo d'antibiotiques (ATB) sur Mabs.Le variant R induit une infection létale plus robuste que le S, caractérisée par le développement d'abcès au niveau du système nerveux central (SNC) associés à une réponse inflammatoire intense et au recrutement de neutrophiles. Le suivi des infections a révélé que les bactéries étaient rapidement phagocytées par les macrophages au niveau du site d'injection. Une fois infectés, ces derniers traversent la barrière endothéliale et transportent les mycobactéries dans les tissus du SNC, soulignant leur rôle clé dans la dissémination du pathogène. Des expériences menées dans des embryons dépourvus de macrophages ont validé ces observations en montrant que les bactéries étaient incapables de rejoindre le SNC et restaient confinées dans le système vasculaire. Implanté au sein du tissu nerveux, le macrophage infecté entre en apoptose, libérant ainsi le pathogène dans le milieu extracellulaire. Une fois libéré, à la différence du variant S, la morphotype R forme des cordes augmentant rapidement de taille et capables d'initier le développement d'abcès volumineux. La taille démesurée de ces cordes par rapport à celle des phagocytes professionnels représenterait une stratégie permettant au variant R d'échapper à la phagocytose et donc de promouvoir sa multiplication extracellulaire et d'assurer la progression létale du processus infectieux. Enfin, ce modèle nous a permis de déterminer, en temps réel, l'efficacité thérapeutique de plusieurs ATBs sur les embryons infectés,qui s'accompagne d'une forte réduction de mortalité des embryons et d'une importante diminution des signes physiopathologiques au niveau du SNC. Ces résultats indiquent que l'embryon de ZF représente un modèle d'infection prometteur et pertinent pour 1) l'étude de la virulence de Mabs 2) l'évaluation de la contribution du SI innée au cours de l'infection et 3) le suivi directe de l'effet d'ATBs. Ce nouveau modèle, combiné aux outils déjà disponibles chez le ZF, devrait permettre de mieux caractériser la relation entre Mabs et mucoviscidose, notamment l'implication éventuelle de la protéine CFTR dans la résistance à cette bactérie. Par ailleurs, l'embryon étant particulièrement propice au criblage à haut débit, l'optimisation de ce système biologique pourrait être exploitée dans le cadre d'approches thérapeutiques innovantes pour identifier de nouveaux agents anti-infectieux contre Mabs. / The emerging pathogen Mycobacterium abscessus causes severe lung infections particularly in cystic fibrosis (CF) patients. The Smooth (S) morphotype displays surface expression of glycopeptidolipids (GPL) while the Rough (R) morphotype is characterized by the loss of surface-associated GPL. Previous studies suggested that the R variant is involved in more severe clinical forms, associated with a hyper-proinflammatory response. However, the molecular mechanisms responsible for the virulence and physiopathology associated to the Rvariant remain unknown. The zebrafish embryo offers many advantages that motivated and validated its use for a better understanding of infectious diseases. In this study, a zebrafish model of infection was developed toinvestigate and compare the pathogenesis of R and S variants.A microinjection protocol was first developed and the fate and progression of the infection was monitored at a spatiotemporal level by videomicroscopy. A transcriptomic approach by qRT-PCR, an antisense technology using morpholinos and transgenic zebrafish lines were used to evaluate the contribution of theinnate immune system and the role of inflammation during infection. In addition, the potential of the embryo has been used to study the in vivo pharmacological activity of antibiotics during M. abscessus infection. In contrast to the S variant, the R morphotype induced a more robust and lethal infection in zebrafish embryos, characterized by the rapid development of bacterial foci within the central nervous system (CNS). The use of a mpx:GFP zebrafish transgenic line, exhibiting green fluorescent granulocytes, indicated that neutrophils are actively recruited to CNS infection foci. An intense pro-inflammatory response with production of TNFα was measured by qRT-PCR. Next, the use of a mpeg1:mCherry transgenic zebrafish line, exhibiting red fluorescent macrophages, demonstrated the presence of isolated or small aggregated bacilli within macrophages during early infection. In contrast, later stages were characterized by the presence of large aggregates of extensively replicating extracellularly that enables mycobacteria to induce a strong inflammatoryresponse, leading to rapid tissue damage (abscess) and to larval death. In addition, the high propensity of the R variant to form cords in vivo may, represent a strategy evolved by the R (but not S) M. abscessus, to escape themacrophage or avoid being phagocytosed by macrophages or granulocytes. The role of macrophages in the diffusion of bacteria to the CNS was evaluated in macrophage-depleted embryos. Here, M. abscessus failed to disseminate from vasculature to CNS as shown by infections performed in KDR:GFP transgenic line, exhibiting green vascular endothelium. In addition, we also showed that the activity of antibiotics on infected-embryos is associated with a strong reduction of embryonic mortality, reduction in the bacterial burden and a significant decrease in physiopathological signs in the CNS, which could be imaged in real-time and at high resolution.These results propose the zebrafish embryo as a suitable model, particularly relevant to 1) the study of M. abscessus virulence, 2) the evaluation of role of innate immune system during infection process and to 3)monitor, at spatiotemporal level, the effects of antibiotics in an infected vertebrate. In addition, the antisense technology allowing knocking-down cftr expression can now be optimized to mimic a CF environment. This should greatly help to define the relationship between M. abscessus in CF patients. Moreover, the embryo isparticularly conducive to high-throughput screening, thus allowing this biological system to be exploited in the search for new therapeutic molecules against M. abscessus and other CF-associated patients

A Mycobacterium abscessus subspécie abscessus é um pesadelo quando envolvida em infecção pulmonar que são incuráveis, a despeito do uso de antimicrobianos com atividade in vitro, caso o tratamento não inclua a ressecção cirúrgica da área afetada. É a micobactéria patogência de crescimento rápido mais frequentemente isolada de culturas de sítios pulmonares. Há um número reduzido de opções terapêuticas para o tratamento dessas infecções, e é ainda mais reduzido o número de antimicrobianos que atingem concentrações terapêuticas no compartimento intracelular, em particular no fagossomo. O número limitado de antimicrobianos disponíveis para tratamento apontam a necessidade de determinação do perfil de susceptibilidade frente a antimicrobianos isolados e em combinação, nos compartimentos intra e extracelular. Os objetivos deste estudo foram avaliar: a sensibilidade de M. abscessus estruturadas em biofilmes e presentes no interior dos macrófagos; a ocorrência de sinergismo quando da associação entre fármacos, inibidores de betalactamase e o anti-inflamatório. As combinações entre os antimicrobianos foram apenas indiferente quanto ao FIC e a atividade dos fármacos em biofilme e em macrófagos é bacteriostático. / Mycobacterium abscessus subspecies abscessus is a nightmare when involved in lung infection that is incurable, despite the use of antibiotics with in vitro activity, if the treatment does not include surgical resection of the affected area. It is a MCR - rapidly growing mycobacteria pathogenic most frequently isolated from cultures of lung sites. There are a small number of therapeutic options for the treatment of such infections is further reduced and the number of drugs that reach therapeutic concentrations in the intracellular compartment, particularly in the phagosome. The limited number of antimicrobials available for treatment indicate the need for determining the susceptibility profile against antimicrobials alone and in combination, in the intra and extracellular compartments. The objectives of this study were sensitivity of MCR structured biofilms and present in macrophages, the occurrence of synergism when the association between drugs, beta-lactamase inhibitors and anti-inflammatory. Combinations of antimicrobials were just indifferent and the activity of drugs on biofilms and macrophages was bacteriostatic.

A Mycobacterium abscessus subspécie abscessus é um pesadelo quando envolvida em infecção pulmonar que são incuráveis, a despeito do uso de antimicrobianos com atividade in vitro, caso o tratamento não inclua a ressecção cirúrgica da área afetada. É a micobactéria patogência de crescimento rápido mais frequentemente isolada de culturas de sítios pulmonares. Há um número reduzido de opções terapêuticas para o tratamento dessas infecções, e é ainda mais reduzido o número de antimicrobianos que atingem concentrações terapêuticas no compartimento intracelular, em particular no fagossomo. O número limitado de antimicrobianos disponíveis para tratamento apontam a necessidade de determinação do perfil de susceptibilidade frente a antimicrobianos isolados e em combinação, nos compartimentos intra e extracelular. Os objetivos deste estudo foram avaliar: a sensibilidade de M. abscessus estruturadas em biofilmes e presentes no interior dos macrófagos; a ocorrência de sinergismo quando da associação entre fármacos, inibidores de betalactamase e o anti-inflamatório. As combinações entre os antimicrobianos foram apenas indiferente quanto ao FIC e a atividade dos fármacos em biofilme e em macrófagos é bacteriostático. / Mycobacterium abscessus subspecies abscessus is a nightmare when involved in lung infection that is incurable, despite the use of antibiotics with in vitro activity, if the treatment does not include surgical resection of the affected area. It is a MCR - rapidly growing mycobacteria pathogenic most frequently isolated from cultures of lung sites. There are a small number of therapeutic options for the treatment of such infections is further reduced and the number of drugs that reach therapeutic concentrations in the intracellular compartment, particularly in the phagosome. The limited number of antimicrobials available for treatment indicate the need for determining the susceptibility profile against antimicrobials alone and in combination, in the intra and extracellular compartments. The objectives of this study were sensitivity of MCR structured biofilms and present in macrophages, the occurrence of synergism when the association between drugs, beta-lactamase inhibitors and anti-inflammatory. Combinations of antimicrobials were just indifferent and the activity of drugs on biofilms and macrophages was bacteriostatic.

Mycobacterium abscessus is an emerging pathogen with infections increasing worldwide, especially among Cystic Fibrosis (CF) patients. During my PhD, I studied key aspects of the biology of M. abscessus spp.; particularly, I studied host-pathogen interactions, antimicrobial resistance mechanisms, and genetic determinants of virulence. First, I performed phenotypic characterization of M. abscessus spp. clinical isolates obtained from CF patients classified according to subspecies and clustering. I found clustered isolates, representing probable transmission events, were phenotypically distinct from sporadic isolates and showed adaptation phenotypes associated with chronic lung infection, such as enhanced intracellular survival, increased antibiotic resistance, and metabolic adaptations to the host environment. Second, I assessed the role of an inserted element containing an active methyltransferase in M. a. massiliense. Infection experiments with an isolate containing the inserted element (BIR1049wt) and a knockout strain (BIR1049Δ1809078_1815649) showed decreased survival of BIR1049Δ1809078_1815649 within macrophages. RNAseq analysis showed a distinct gene expression pattern between both isolates, with a number of mycobacterial virulence factors upregulated in BIR1049wt. Third, I studied heritable non-mutational antibiotic resistance mechanisms in M. abscessus to linezolid and clofazimine. For both antibiotics, I found clonal isolates of M. abscessus spp. with varying susceptibilities and different gene expression patterns, suggesting transcriptional regulation of antibiotic resistance. Mutation- mediated resistance to clofazimine was also found due to mutations in two transcriptional regulators predicted to regulate efflux pumps. Last, I evaluated the potential of repurposing a kinase inhibitor (compound H) in clinical trials for the treatment of cancer and CF, to treat M. abscessus infection. I found compound H enhanced killing of intracellular M. abscessus in macrophages through stimulation of autophagy and lysosomal function. I further studied over 60 chemical analogues of compound H in order to find a more active and specific compound for M. abscessus infection.

<p><i>Mycobacterium abscessus </i>is a nontuberculous mycobacterium found in the environment that is becoming an emerging infectious pathogen capable of causing numerous types of infections. It is more antibiotic resistant than <i>Mycobacterium tuberculosis</i> and is becoming more prevalent in developed nations. Current treatments are not standardized and have poor success records and there is no definitive method in properly treating these infections. We tested an <i>in vitro </i>model that mimics the reducing environment that <i>M. abscessus</i> experiences during an infection by subjecting it to thiol-reductive stress. We observed that thiol reductive stress stabilized biofilms formed by <i>M. abscessus</i>. We found that <i>M. abscessus </i>in biofilms became tolerant to the antibiotics: clarithromycin, amikacin, and streptomycin. We postulated that efflux pumps might be involved in transport of the precursors of lipids associated with biofilm formation. Therefore, we investigated whether the efflux pump inhibitors affected biofilm formation and found that verapamil, CCCP and reserpine inhibited the formation of biofilms. We investigated the biosynthesis of lipids during biofilm formation by metabolic radiolabeling with ¹⁴C-acetate, the building block of fatty acids. We found that the biosynthesis of several phospholipids were elevated during biofilm formation and that the efflux pump inhibitor verapamil and exogenous fatty acids inhibited their biosynthesis. Further studies are needed to understand the roles of these lipids in biofilm formation.</p>

<p><i>Mycobacterium abscessus</i> is an emerging infectious pathogen capable of causing pulmonary disease similar to tuberculosis, but has many intrinsic and extrinsic properties making it more drug-resistant than <i>Mycobacterium tuberculosis</i>. Current treatments, including those used for <i>M. tuberculosis</i> infection, have had poor results. Although <i>in vitro</i> studies have shown promise with drug treatment for this microorganism, clinical trials have been mostly unsuccessful. An <i>in vitro </i>model that mimics the physiological stresses encountered within the human body is likely to enable the discovery of mechanisms of antibiotic resistance used by <i>M. abscessus</i> during infection. Therefore, we subjected <i>M. abscessus</i> to a combination of stresses thought to be encountered by mycobacteria inside the human body. We subjected the pathogen to low oxygen, low pH, and nutrient starvation. This is the first report on subjecting <i>M. abscessus</i> to such a combination of stresses. It is also the first to investigate the effect of the combination of stresses on the tolerance of the pathogen to antibiotics, and the effect of efflux pump inhibitors under such conditions. We found that under these conditions, <i>M. abscessus</i> entered a non-replicating state. We investigated whether the multiple-stressed <i>M. abscessus</i> displayed altered tolerance to antibiotics commonly used to treat infection, and whether efflux pump inhibitors affected the antibiotic resistance under such conditions.<i> </i>We found that when subjected to our multiple stress model, <i>M. abscessus</i> in the reactivating phase had higher tolerance to erythromycin in combination with efflux pump inhibitors verapamil and reserpine compared to non-replicating <i>M. abscessus</i>. Reactivating phase cells had a higher tolerance to antibiotic erythromycin than non-replicating cells. Reactivating phase cells also showed antibiotic tolerance in the presence of ATP. This physiologically-relevant experimental model for <i>M. abscessus</i> could potentially be used in discovering the mechanisms of antibiotic resistance in the pathogen.</p>

La mycobactérie à croissance rapide Mycobacterium abscessus est un pathogène opportuniste de l’homme, particulièrement des poumons, capable de se multiplier au sein de macrophages (MФ) mais aussi d’amibes environnementales. Nous avons pu montrer que l’environnement amibien est propice à l’adaptation de M. abscessus à cette survie intramacrophagique. Par des analyses transcriptomiques comparant les adaptations de M. abscessus au sein d’amibes ou de MФ, nous observons des enrichissements de voies biologiques démontrant des adaptations au stress oxydatif et des adaptations métaboliques telle que la béta-oxydation des lipides et l’assimilation des sulfates. Ces adaptations ont également été observées chez Mycobacterium tuberculosis, mycobactérie pathogène pulmonaire stricte de l’homme responsable de la tuberculose. Parmi les gènes induits par M. abscessus au sein des amibes figurent des gènes impliqués dans le transport de polyamines, la biosynthèse du MoCo (molybdopterin cofactor) ainsi que l’assemblage des centres fer-soufre (Fe-S). L’induction de tels gènes, décrits comme des facteurs de virulence chez certaines bactéries intracellulaires, contribuerait à la virulence de M. abscessus en permettant sa survie intramacrophagique. Quarante-cinq gènes ont été identifiés comme fortement induits en amibes chez M. abscessus. Mycobacterium chelonae, appartenant au même complexe génomique que M. abscessus et responsable exclusivement d’infections cutanées, ne présente pas de telles inductions après analyse de son transcriptome intracellulaire, ce qui pourrait expliquer son absence de survie en MФ. Cinq opérons recouvrant 10 gènes ont été délétés au sein du génome de M. abscessus par recombinaison homologue. Ces gènes sont requis pour la survie de M. abscessus en amibes et en MФ. La surexpression chez M. chelonae de deux de ces gènes, MAB_1517c et MAB_2649, codant respectivement une protéine TcmP (tetracenomycin polyketide synthesis O-methyltransferase) et une MmpS (mycobacterial membrane protein small), a conféré une survie intra-macrophagique à M. chelonae, suggérant que l’induction de ces gènes en amibes favorise la survie intracellulaire de M. abscessus en MФ. Enfin, l’analyse du transcriptome de M. abscessus en MФ révèle des adaptations propres à la vie intramacrophagique. Différents gènes particulièrement induits sont impliqués dans le métabolisme de la proline, la sécrétion de protéine par le système de sécrétion de type II ou appartiennent la voie MEP (méthylérythritol phosphate), des voies biologiques contribuant à la virulence de pathogènes. De plus, parmi les gènes induits, certains correspondent à des activités de N-acétylation, d’oxydoréduction, de liaison à l’oxygène ou de détoxification de l’oxyde nitrique par des dioxygénases qui sont enrichies. Comparé à 5 autres opérons délétés, sélectionnés selon leur niveau d’induction et leur activité biologique, le gène eis2 (MAB_4532c), codant une N-acétyltransférase, est essentiel à la survie de M. abscessus en MФ.L’utilisation d’une approche complémentaire, le criblage d’une banque de mutants par transposition chez M. abscessus en amibes, a révélé le rôle essentiel du gène mmpl8 (mycobacterial membrane protein large 8) parmi la famille de protéines MmpL, impliquées dans le transport et/ou la synthèse de lipides mycobactériens. Chez M. abscessus, l’absence de production de cette protéine est corrélée à un défaut d’export d’un nouveau glycolipide (GDND, glycosyl diacetylated nonadecyl diol) et à un phénotype délétère pour la bactérie en MФ.En conclusion, notre travail a montré le rôle fondamental de l’amibe dans l’apprêtement de M. abscessus à la survie intramacrophagique. Trois gènes ayant fait l’objet d’études approfondies, mmpL8, eis2 ainsi que le gène eccB4 (MAB_3759c) mis en évidence par le crible amibien de la banque de mutants de transposition, participent à ce phénotype, confirmant le caractère pathogène de M. abscessus. / Mycobacterium abscessus is a rapidly growing mycobacterium, causing opportunistic infections in humans, and notably pulmonary infections. M. abscessus is able to multiply inside macrophages (MФ) and environmental amoebae. Here we demonstrate that M. abscessus undergoes adaptations in amoebae allowing its survival in MФ. Intracellular adaptations of M. abscessus to amoebae and MФ were assessed by RNAseq. We observed a significant enrichment of biological pathways reflecting adaptations to oxidative stress and metabolic adaptations illustrated by the consumption of fatty acids and activation of the sulfate assimilation pathway. These adaptations have been described in intramacrophagic Mycobacterium tuberculosis, a strictly pathogenic mycobacteria infecting the lung of humans and causing tuberculosis. Among the set of genes induced by M. abscessus during the amoebal co-culture are genes implicated in polyamine transport, MoCo (molybdopterin cofactor) biosynthesis and iron-sulfur (Fe-S) cluster assembly. The induction of such genes, described as virulence factors from intracellular bacteria, might enhance M. abscessus virulence and thus allow its survival in MФ. Forty-five genes are highly induced along the amoebal co-culture. In comparison, the amoebal co-culture with Mycobacterium chelonae, a mycobacterium that belongs to the same genomic complex as M. abscessus and causing solely extrapulmonary infections, does not elicit the same adaptations; potentially explaining M. chelonae inability to persist in macrophages. Five operons, representing a total of 10 genes, were deleted from M. abscessus genome by homologous recombineering. These genes are required for both M. abscessus survival in amoebae and MФ. Overexpression of two of these genes in M. chelonae, MAB_1517c and MAB_2649, encoding a TcmP (tetracenomycin polyketide synthesis O-methyltransferase) protein and an MmpS (mycobacterial membrane protein small) protein respectively, enhances M. chelonae survival in MФ, suggesting that the induction of these genes favors M. abscessus survival in MФ. Analyses of M. abscessus transcriptome in MФ also shed light on adaptations specific to the bacterium intramacrophagic life. Several genes highly induced in macrophages are implicated in biological pathways known to contribute to bacteria virulence, including proline metabolism, protein secretion by the type II secretion system and the MEP (methylerythritol phosphate) pathway. Among the set of induced genes selected according to their level of induction and their biological activity, N-acetylation and redox activities, bounding to oxygen and detoxification from nitric oxide by dioxygenases are significantly enriched. Among operons from this set of genes, it appears that M. abscessus eis2 gene (MAB_4542c), encoding a N-acetyltransferase, is essential for M. abscessus survival in MФ.In addition, a complementary approach to RNAseq, the screening of a transposon (Tn) mutant library of M. abscessus inside amoebae, revealed important roles of the mmpL8 gene encoding a mycobacterial membrane protein large belonging to a family of proteins implicated in lipid biosynthesis and export to the cell surface. When this protein was no longer produced by M. abscessus, a lower amount of a new glycolipd family (GDND, glycosyl diacetylated nonadecyl diol) was observed as well as a deleterious phenotype in MФ.To conclude, our work has shown a fundamental role of amoebae in triggering the virulence of M. abscessus, further allowing its survival in macrophages. Besides, three genes that have been studied more extensively – mmpL8, eis2 and eccB4 (revealed by the Tn library screening) – are required for M. abscessus survival in macrophages and confirmed its pathogenic behavior.

Le complexe Mycobacterium abscessus (MABSC) fait partie des mycobactéries non tuberculeuses. C’est un pathogène opportuniste environnemental pouvant causer des infections pulmonaires chroniques, en particulier chez les patients atteints de mucoviscidose, et extra-pulmonaires sévères. Il est également résistant à la plupart des antibiotiques, et ne connait pas de traitement efficace. Il se divise en trois sous-espèces : Mycobacterium abscessus subsp. abscessus, Mycobacterium subsp. massiliense et Mycobacterium subsp. bolletii, Ces trois sous espèces possèdent des différences cliniques et de résistance aux antibiotiques, une identification sûre et rapide est don essentielle. De plus, son épidémiologie reste peu connue, notamment au sein des patients atteints de mucoviscidose.Dans un premier temps, nous avons évalué la performance d’identification au sein du MABSC et la caractérisation des principales résistances du kit Genotype NTM-DR. Celui-ci s’est révélé extrêmement efficace, avec une réussite de 100% d’identification de la sous-espèce mais aussi pour la caractérisation des résistances à la clarithromycine et à l’amikacine, qui sont les antibiotiques recommandés pour le traitement d’une infection à MABSC. Ce kit est donc une alternative rapide et robuste aux principales méthodes d’identification et de caractérisation de la résistance chez MABSC.Dans un second temps, nous avons testé l’efficacité de deux molécules différentes, la clofazimine et la tigecycline, en synergie. Une synergie a pu être mise en évidence sur 42 % des isolats, et la présence de clofazimine a permis d’abaisser la concentration minimale inhibitoire de la tigecycline pour 52 % des isolats. Cette association pourrait constituer une alternative thérapeutique efficace et mieux supportable pour le patient.Enfin, nous avons réalisé deux études épidémiologiques : au Vietnam, chez des patients possédant une condition pulmonaire inflammatoire, et en France, chez des patients atteints de la mucoviscidose dans cinq centres hospitaliers différents. En France, un suivi longitudinal a pu être réalisé sur 7 patients de deux centres différents.Le Vietnam présentait un grand nombre de Sequence Type (ST) nouveaux (29/38) ainsi qu’une grande diversité génotypique (0.72 pour 53 isolats). Une majorité de M. massiliense a été mis en évidence. De plus, des ST responsables d’épidémies dans différents pays ont aussi été retrouvés (ST 23 et ST 117). Trois complexes clonaux ont été identifiés, reflétant la grande diversité du complexe M. abscessus dans ce pays. Deux de ces complexes clonaux étaient constitués de M. abscessus subsp. massiliense contenaient les ST 23 et 117. Le grand nombre de nouveaux génotype est dû au manque de données au Vietnam, il s’agit là de la première étude épidémiologique dans ce pays.En France, on retrouvait un plus grand nombre de sequence type connus (20/31), certainement dû au nombre d’études existantes dans cette région. Une majorité de M. abscessus subsp. abscessus a été retrouvé. Sept sequence type d’importance épidémiologique ont été retrouvés, dont le ST 23. Le suivi longitudinal montre que : le risque d’infection exogène est grand ; le pool de génotypes à Brest est plus important qu’à Montpellier, même si les ST persistent chez un même patient, on ne peut exclure le risque d’une infection exogène. Enfin, l’analyse VNTR des isolats des cinq centres suggère une possible transmission nosocomiale ainsi que la diffusion de génotypes en France.En conclusion, ce travail de thèse a permis d’améliorer l’approche de l’identification et du traitement des infections du complexe M. abscessus, en plus d’explorer et de comparer son épidémiologie dans deux populations différentes. Nous montrons la présence ubiquitaire de certains génotypes d’importance cliniques, une grande diversité génotypique, une variabilité du portage chez le patient mucoviscidose ainsi qu’un risque d’infections exogènes et de transmission chez cette population. / The Mycobacterium abscessus complex is a nontuberculous mycobacteria. It is an environmental opportunistic pathogen that can cause chronic lung infections, especially in patients with cystic fibrosis, and severe extra-pulmonary infections. It is also resistant to most antibiotics, and does not have an effective treatment. It is divided into three subspecies: Mycobacterium abscessus subsp. abscessus, Mycobacterium subsp. massiliense and Mycobacterium subsp. bolletii, These three subspecies have clinical and antibiotic resistance differences, Thus, a safe and rapid identification is essential. In addition, little is known about its epidemiology, particularly among patients with cystic fibrosis.First, we assessed the identification performance within the M. abscessus complex and the characterization of the main resistances of a commercial assay (Genotype NTM-DR). The kit was highly efficient, with 100% success in identifying the subspecies and also in characterizing resistance to clarithromycin and amikacin, which are the recommended antibiotics for the treatment of complex M. abscessus infection. This kit is therefore a fast and robust alternative to the main methods for identifying and characterizing resistance in the M. abscessus complex.Secondly, we tested the efficacy of two different molecules, clofazimine and tigecycline, in synergy. Synergy was demonstrated in 42% of the isolates, and the presence of clofazimine reduced the minimum inhibitory concentration of tigecycline in 52% of the isolates. This combination could be an effective and more tolerable therapeutic alternative for the patient.Finally, we carried out two epidemiological studies: in Vietnam, in patients with inflammatory lung disease, and in France, in patients with cystic fibrosis in five different hospitals. In France, longitudinal follow-up was carried out on 7 patients from two different centres, Brest and Montpellier. Two different typing tools were used: MLST and VNTR.Vietnam had a large number of new Sequence Type (ST) (29/38) and a high genotypic diversity (0.72 for 53 isolates). A majority of M. abscessus subsp. massiliense was found. In addition, STs responsible for epidemics in different countries have also been found (ST 23 and ST 117). Three clonal complexes have been identified, reflecting the great diversity of the M. abscessus complex in this country. Two of these clonal complexes were composed of M. abscessus subsp. massiliense containing ST 23 and 117. The large number of new genotypes is due to the lack of data in Vietnam, this is the first epidemiological study in this country.In France, there was a greater number of known sequence type (20/31), certainly due to the number of existing studies in this region. A majority of M. abscessus subsp. abscessus was found. Seven sequence type of epidemiological importance were found, including ST 23. Longitudinal monitoring shows a high variability of the ST in Brest compared to Montpellier, which is confirmed by the VNTR. This follow-up shows that: the risk of exogenous infection is high; the genotype pool in Brest is greater than in Montpellier, even if STs persist in the same patient, the risk of exogenous infection cannot be excluded. Finally, the VNTR analysis of isolates from the five centres suggests possible nosocomial transmission and the spread of genotypes in France.In conclusion, this thesis work has improved the approach to the identification and treatment of M. abscessus complex infections, as well as exploring and comparing its epidemiology in two different populations. We show the ubiquitous presence of certain clinically important genotypes, a high genotypic diversity, a variability in carrying in the cystic fibrosis patient as well as a risk of exogenous infections and transmission in this population

Premièrement, nous avons analysé 14 génomes publiés de M. abscessus montrant quece taxon comprend au moins cinq taxons différents spécifiés par des caractéristiques microbiologiques d’intérêt médical. Au cours d'un deuxième travail, nous avons développé une technique d’identification et de génotypage de M. abscessus qui a permis de distinguer sans ambiguïté M. massiliense de M. bolletii et M. abscessus.Nous avons ensuite analysé le bactériophage de M. bolletii que nous avons nommé Araucaria. La résolution de sa structure 3D a montré une capside et un connecteur similaires à ceux de plusieurs bactériophages de bactéries à Gram négatif et positif; et une queue hélicoïdale décorée par des pointes radiales. La partie basale (baseplate) du phage Araucaria présente des caractéristiques observées dans les phages se liant à des récepteurs de protéines. Araucaria se lie à son hôte en deux temps, un premier par liaison de la queue aux saccharides de l'hôte puis un deuxième par liaison de la baseplate aux protéines de la paroi cellulaire.Nous avons analysé la présence de séquence de phages dans 48 génomes disponibles de M. abscessus. Notre analyse phylogénétique suggère que les espèces de M. abscessus ont été infectées par différents mycobactériophages et ont une histoire évolutive différente de celle des hôtes mycobactériens et contiennent aussi des protéines acquises par transfert horizontal.Enfin, nous avons séquencé et analysé deux mycobactéries non-tuberculeuses responsables d’infections opportunistes, Mycobacterium simiae et Mycobacterium septicum. / In a first step, we reviewed the published genomes of 14 M. abscessus strains showing that M. abscessus sensu lato comprises of five different taxons specified by particular characteristics of microbiological and medical interests. In a second step, based on sequencing of eight intergenic spaces, we developed a Multispacer Sequence Typing technique (MST) for M. abscessus group sub-species identification and strain genotyping. MST clearly differentiates formerly “M. massiliense” organisms from other M. abscessus subsp. bolletii organisms. We also analyzed a bacteriophage from M. bolletii that we named Araucaria. We resolved Araucaria 3D structure, its capsid and connector share close similarity with several phages from Gram- or Gram+ bacteria. The helical tail decorated by radial spikes, possibly host adhesion devices. Its host adsorption device, at the tail tip, assembles features observed in phages binding to protein receptors. All together, these results suggest that Araucaria may infect its mycobacterial host using a mechanism involving adhesion to cell wall saccharides and protein, a feature that remains to be further explored. We also analysed 48 M. abscessus sequenced genomes for encoding prophages. Our phylogenetic analyses suggested that M. abscessus species were infected by different mycobacteriophages and have a different evolutionary history than the bacterial hosts and some proteins that are acquired by horizontal gene transfer mostly mycobacteriophages’ proteins and hypothetical proteins. Finally, we sequenced and analyzed two non-tuberculosis mycobacterium causing human infections, Mycobacterium simiaie and Mycobacterium septicum.

Mycobacterium abscessus est une mycobactérie responsable principalement d’infections pulmonaires, en particulier chez les patients atteints de mucoviscidose ou de dilatation des bronches. M. abscessus est naturellement résistante aux antituberculeux, laissant peu d’options thérapeutiques. Le traitement de référence associait classiquement un aminoside, un macrolide (clarithromycine) et une β-lactamine (céfoxitine ou imipénème), avec un taux de succès d’environ 50 %. Cependant, des souches résistantes à la clarithromycine sont fréquemment isolées, remettant en cause l’utilisation de cet antibiotique. M. abscessus produit naturellement une β-lactamase à large spectre (BlaMab) mais les mécanismes d’action des β-lactamines n’ont pas été étudiés chez cette espèce, ce qui constitue une entrave à l’optimisation des traitements par cette classe d’antibiotiques. Le premier objectif était d’identifier et de caractériser les cibles des β-lactamines chez cette espèce. Inhibant la dernière étape de polymérisation du peptidoglycane, les cibles potentielles des β-lactamines sont trois familles d’enzymes : les D,D-transpeptidases et les D,D­carboxypeptidases appartenant à la famille des protéines de liaison à la pénicilline (PLP), ainsi que les L,D-transpeptidases qui sont majoritairement responsables de cette dernière étape chez cette espèce. Pour identifier les cibles, des mutants résistants aux β-lactamines ont été sélectionnés à partir de la souche de référence M. abscessus CIP104536 et d’un dérivé portant une délétion du gène blaMab (∆blaMab). Pour les deux souches, l’émergence de la résistance aux β-lactamines a requis de multiples étapes, ce qui constitue un atout pour leur utilisation thérapeutique. Pour les mutants obtenus à partir de la souche CIP104536, les analyses phénotypiques ont montré que la résistance aux β-lactamines n'est pas due à une augmentation de l’efficacité catalytique de BlaMab, à une surproduction de cette enzyme, ou à une diminution de la perméabilité. Le séquençage des génomes de mutants résistants n’a pas révélé de mutations dans les gènes codant pour les L,D-transpeptidases, mais des mutations ont été trouvées dans des gènes codant pour deux PLP. D’autres mutations se situent dans des gènes codant en particulier pour des protéines non caractérisées. L’acquisition de la résistance pourrait donc dépendre de mutations affectant des facteurs essentiels à l’activité des cibles des β­lactamines. Le deuxième objectif était d’étudier et de comparer l’activité in vitro des β-lactamines sur M. abscessus. Des expériences de bactéricidie et d’activité intracellulaire chez le macrophage infecté ont été effectuées pour les souches CIP104536 et ∆blaMab. Parmi les antibiotiques étudiés (amikacine, céfoxitine, imipénème, ceftaroline, et amoxicilline), l’imipénème est le plus efficace sur les deux souches. Sur la souche ∆blaMab, l’association d’imipénème et d’amikacine est bactéricide. En l’absence de BlaMab, l’amoxicilline est aussi efficace que l’imipénème. L’avibactam augmente l’activité de la ceftaroline mais l’inhibition de BlaMab est seulement partielle en intracellulaire. Les résultats obtenus in vitro montrent que l’imipénème est supérieur à la céfoxitine pour des concentrations atteignables dans le sérum. L’inhibition de BlaMab pourrait augmenter l’efficacité de l’imipénème et d’autres composés utilisés pour traiter les infections pulmonaires à M. abscessus. / Mycobacterium abscessus is an important pathogen responsible for pulmonary infections in cystic fibrosis patients or in patients suffering from bronchiectasis. The treatment of infections due to M. abscessus is complicated since this bacterium is naturally resistant to the anti­tuberculous agents. The recommended treatment includes an aminoglycoside, a macrolide (clarithromycin) and a β-lactam (cefoxitin or imipenem), with a success rate of about 50 %. However, strains resistant to clarithromycin are frequently isolated, questioning the use of this antibiotic. M. abscessus naturally produces a broad spectrum β-lactamase (BlaMab) but the mechanisms of action of the β-lactams have not been studied in this species, impairing the optimization of the treatment by these antibiotics. The first objective was to identify and characterize the targets of β-lactams antibiotics in this species. Inhibiting the final stage of the peptidoglycan polymerization, the potential targets of β-lactams are three families of enzymes: the D,D-transpeptidases and D,D­carboxypeptidases belonging to the family of penicillin-binding proteins (PBP), and the L,D-transpeptidases which are mainly responsible for this final stage in this species. To identify the targets, mutants resistant to β-lactams have been selected from the reference strain M. abscessus CIP104536 and from its β-lactamase-deficient derivative ΔblaMab. For both strains, the emergence of resistance to β­lactams has required multiple steps, which is an advantage for the therapeutic use of these antibiotics. For the mutants derived from the strain CIP104536, phenotypic analyzes showed that the resistance to β-lactams is not due to an increase in the catalytic efficiency of BlaMab, to an overproduction of this enzyme, or to a decrease in permeability. Genomes sequencing of the resistant mutants did not reveal mutations in the genes encoding the L,D-transpeptidases, but mutations have been found in genes encoding two PBPs. Other mutations have been detected in genes encoding uncharacterized proteins. Acquisition of resistance could therefore depend on mutations affecting key factors essential for the activity of β-lactams targets. The second objective was to study and compare the in vitro activities of β-lactams against M. abscessus. Bactericidal experiments and intracellular activity in the infected macrophage were performed for the strains CIP104536 and ΔblaMab. Among the antibiotics tested (amikacin, cefoxitin, imipenem, ceftaroline, and amoxicillin), imipenem is the most effective agent against the two strains. Combination of imipenem and amikacin was bactericidal against the ΔblaMab mutant. In the absence of BlaMab, amoxicillin was as active as imipenem. Avibactam increased the intracellular activity of ceftaroline but inhibition of BlaMab was only partial intracellularly. Evaluation of the killing and intracellular activities of β-lactams indicates that imipenem is superior to cefoxitin at clinically achievable drug concentrations. Inhibition of BlaMab could improve the efficacy of imipenem and extend the spectrum of drug potentially useful to treat pulmonary infections.