La tuberculose est la neuvième cause de mortalité dans le monde. Les progrès pour contrôler cette maladies ont été ralentis par plusieurs facteurs, notamment la diffusion de souches de tuberculose à bacilles multirésistants aux antibiotiques (MDR). Le traitement de la tuberculose MDR est long, toxique, et souvent inefficace. Après plus de 40 ans de pénurie, deux nouveaux médicaments ont été approuvé pour le traitement de la tuberculose MDR : la bédaquiline et le delamanide. Globalement, il y eu des délais considérables dans l'introduction de ces nouveaux médicaments dans la pratique clinique, comme souligné par le faible nombre d'études présentes en littérature. Les études présentés dans ce travail ont décrit des cohortes de patients atteints de tuberculose MDR et traités par bédaquiline et/ou delamanide en France, en montrant d'abord une tolérance satisfaisante, même pour des durées prolongées de traitement et pour l'association des deux nouveaux médicaments. En outre, l'efficacité microbiologique de la bédaquiline apparait comparable à celle des fluoroquinolones, la classe d'antibiotiques la plus efficace pour la tuberculose MDR. Dans une autre étude, l'on a mis en évidence l'apparition rapide de la résistance à la bédaquiline en France, ce qui était inattendu, en particulier pour les cas de résistance primaire. Malgré les limitations méthodologiques associées aux études observationnelles et rétrospectives, ce travail a permis d'augmenter les connaissances sur la tolérance et l'efficacité du traitement avec les nouvelles molécules pour la tuberculose MDR. Une suite des recherches est envisagée avec la mise en place d'une cohorte national prospective des cas MDR. / Tuberculosis is the ninth leading cause of death worldwide. Progress in controlling this disease has been slowed down by multiple factors, including the emergence of multidrug-resistant (MDR) strains. MDR tuberculosis treatment is long, toxic, and often not effective. After more than 40 years of draught, two new drugs have been approved for the treatment of MDR tuberculosis: bedaquiline and delamanid. Globally, there have been considerable delays in the introduction of these new drugs in clinical practice, as shown by the little number of studies which are available in literature. The studies described in this manuscript have described cohorts of patients affected by MDR tuberculosis and treated with bedaquiline and/or delamanid in France: the main finding was a satisfying safety, even for prolonged treatment durations and for the association of the two new drugs. In addition, the microbiological efficacy of bedaquiline was shown to be comparable to the one of the fluoroquinolones, the most effective antibiotic class to treat MDR tuberculosis. In another study, we have shown the rapid appearance of bedaquiline resistance in France, an unexpected finding in particular for the cases of primary resistance. Notwithstanding the methodologic weaknesses associated with observational and retrospective studies, this work has increased the evidence on safety and efficacy of MDR tuberculosis treatment with the new drugs. The next step will be represented by the establishment of a prospective national research cohort of MDR tuberculosis cases.

Due to the misuse of antibiotics, multi-drug resistant (MDR) bacteria have become more rampant in our society; these MDR have given rise to diseases that are not readily curable. One such agent is the Mycobacterium tuberculosis complex, which is a causative agent of tuberculosis (TB). Timely diagnostics of the bacterial infection and detection of bacterial drug-susceptibility profiles helps to initiate the necessary treatment in a timely fashion and to limit transmission of the disease. For more affordable detection of bacterial diseases, such as TN, tag-free split aptamer probes are advantageous. This proposal aims at designing split aptamer probes for detection of point mutations in the rpoB and katG genes of M. tuberculosis that are associated with resistance to two front-line antibiotics – rifampin and isoniazid, respectively, which causes MDR-TB. The probes will be designed and tested with synthetic oligonucleotide mimics of the bacterial genes in terms of their limit of detection and selectivity in discriminating the targets with single-nucleotide substitutions.

Avian pathogenic Escherichia coli (APEC) causes a wide range of diseases in chickens called colibacillosis, resulting a substantial economic loss to the poultry industry. This dissertation aims at addressing this disease by exploring key virulence-associated genes (VAGs), swarming motility (SM), and multi-locus sequence types (MLST) of E. coli isolates obtained from colibacillosis-infected or asymptomatic commercial hens. Secondly, by classifying the E. coli isolates into different virulence categories based on the presence of five key VAGs [iroN, ompT, hlyF, iutA, and iss]. Finally, by performing embryo lethality assays and experimental infection studies to establish the effect of these VAGs. Our findings showed a significantly higher proportion of E. coli isolates obtained from colibacillosis lesions possessed the ompT gene compared to the isolates of asymptomatic commercial hens. A trend of a higher occurrence of the iutA gene was also observed in the isolates of colibacillosis cases. Based on the presence of all five VAGs, we categorized 87.5% of the isolates obtained from colibacillosis lesions into the virulent category and 64.71% of the isolates obtained from asymptomatic commercial hens into the avirulent category. During the embryo lethality assay, we found an interaction effect of virulence categories and SM on embryo mortality. Motile and hyper-motile isolates of virulent and moderately virulent categories caused significantly higher embryo mortality than the non-motile isolates of the same categories. Isolates of the avirulent category significantly reduced the relative embryo weight of the remaining live embryos. The MLST of the isolates did not have any influence on embryo lethality, or relative embryo weight. Yolk sac retention of the remaining live embryo was unaffected by virulence category, MLST, and SM of the isolates. During the experimental infection studies, we observed higher mortality and lesion scores in layer chicks inoculated intratracheally by virulent isolates compared to avirulent isolates. These two classes of isolates also had a different colonization pattern in the extra-intestinal tissues. The avirulent isolates preferably colonized deeper respiratory tracts such as airsacs whereas, the virulent isolates colonized systemic organs such as the liver. Overall, we expect this dissertation will establish the contribution of five key VAGs on embryo and chick mortality, lesion development, and colonization pattern of E. coli isolates. These findings will facilitate the selection of VAGs for field diagnosis of APEC.

No description available.

L’émergence et l’augmentation continue de la résistance aux médicaments chez Mycobacterium tuberculosis (MTB) constituent un défi majeur pour la lutte antituberculeuse. Pour résoudre le problème de temps (2 à 8 semaines) posé par les tests classiques de sensibilité aux médicaments (DST), des tests moléculaires ont été développés pour la détection précoce des mutations associées à la résistance. Jusqu'à présent, à l'exception du séquençage complet (incompatible avec un diagnostic de routine dans les pays en développement), aucun test ne détecte simultanément les différents types de résistance majeurs en une seule réaction. Sur la base de la littérature et de travaux antérieurs menés au Vietnam, au Laos et au Cambodge, une puce à ADN capable de détecter 184 mutations liées à la résistance aux médicaments de première et de deuxième lignes a été mise au point. En comparaison avec les données de DST, la puce a montré une sensibilité (84,3%-100%) et une spécificité élevées (89,2%-100%). Par rapport au séquençage, la puce a donné des résultats comparables, avec une sensibilité entre 90% et 100% et une spécificité entre 98,2% et 100%. Elle offre, de plus, une meilleure couverture que les tests moléculaires approuvés par l'OMS, car elle permet la détection des résistances aux médicaments de première et de deuxième lignes dans un seul test. Le temps de traitement des isolats issus de la culture est d’environ 6 à 7 heures, ce qui réduit considérablement le temps de diagnostic par rapport au DST. En conclusion, la puce à ADN a été développée avec succès. Certains aspects techniques doivent maintenant être améliorés pour en faire un outil de diagnostic abordable et facile à utiliser. / The emergence and continuous increase of drug resistance in Mycobacterium tuberculosis (MTB) is a major challenge for tuberculosis (TB) control. To overcome the time-consuming problem of conventional drug susceptibility testing (DST), many molecular-based tests have been recently developed for early detection of drug resistance-associated mutations. Up to now, except whole genome sequencing (not ready for routine diagnostic in low and middle-income countries), no test has the capacity to simultaneously detect the different types of drug resistance to first- and second-line drugs in one reaction. Based on the literature and previous works carried out in Vietnam, Laos and Cambodia, in this study, a DNA chip was developed able to detect 184 main mutations conferring resistance to both first- and second-line drugs. Compared to DST, the DNA chip showed high sensitivity (between 84.3% and 100%) and high specificity (between 89.2% and 100%). Compared to sequencing, the DNA chip showed comparable accuracy, with sensitivity between 90% and 100% and specificity between 98.2% and 100%. The DNA chip showed a better coverage than the WHO endorsed molecular tests since it enables the detection of both first- and second-line drug resistances in one test. The turn-around time is about 6-7h from cultured isolates reducing considerably the diagnostic time compared to cultured-based DST. Finally, the DNA chip has been successfully developed, even if certain technical aspects need to be improved to make an affordable and easy-to-use diagnostic tool.

Introduction: Nosocomial infections remain a major cause of mortality and morbidity. The problem is most apparent in intensive care units (ICUs). Most ICU patients are compromised and vulnerable as a result of disease or severe trauma. One in ten people admitted to hospital is given an antibiotic for infection. The risk of acquiring a nosocomial infection in a European ICU is approximately 20%. It is vitally important that ways are found to prevent transmission between patients and personnel, and that local hygiene routines and antibiotic policies are developed. This thesis is a holistic work focused particularly on antimicrobial antibiotic resistance, antibiotic consumption and to some extent on hygiene in Swedish ICUs. Aims: The general aim of this thesis was to investigate bacterial resistance and antibiotic consumption in Swedish ICUs and to try to correlate ICU demographic data with antibiotic consumption and antibiotic resistance. Additional aims were to investigate on which clinical indications antibacterial drugs are prescribed in the ICU, and to investigate the emergence of resistance and transmission of Pseudomonas aeruginosa in the ICU using cluster analysis based on antibiograms and genotype data obtained by AFLP. Material and methods: In paper 1-3, antibiotic consumption data together with bacterial antibiotic resistance data and specific ICU-demographic data were collected from an increasing number of ICUs over the years 1997-2001. Data from ICUs covering up to six million out of Sweden’s nine million inhabitants were included. In paper 4, the indications for antibiotic prescribing were studied during two weeks in 2000. Paper 5 investigated Pseudomonas aeruginosa isolates in order to detect cross-transmission with genotype obtained by AFLP, and antibiogram-based cluster analysis was also performed in order to see if this could be a quicker and easier substitute for AFLP. Results: This thesis has produced three important findings. Firstly, antibiotic consumption in participating ICUs was relatively high during the study period, and every patient received on average more than one antimicrobial drug per day (I-IV). Secondly, levels of antimicrobial drug resistance seen in S. aureus, E. coli and Klebsiella spp remained low when data were pooled from all ICUs throughout the study period, despite relatively high antibiotic consumption (I-V). Thirdly, the prevalence of antibiotic resistance in CoNS and E. faecium, cefotaxime resistance in Enterobacter, and ciprofloxacin and imipenem resistance in P. aeruginosa was high enough to cause concern. Conclusion: For the period studied, multidrug resistance in Swedish ICUs was not a major problem. Signs of cross-transmission with non-multiresistant bacteria were observed, indicating a hygiene problem and identifying simple improvements that could be made in patient care guidelines and barrier precautions. A need for better follow up of prescribed antibiotics was evident. With further surveillance studies and monitoring of antibiotics and bacterial resistance patterns in the local setting as well as on a national and international level, some of the strategic goals in the prevention and control of the emergence of antimicrobial-resistant microbes may be achievable.

To combat the ever-growing clinical burden imposed by antibiotic-resistant pathogens, multiple-antibiotic treatments are increasingly being considered as promising treatment options. The impact of multiple-antibiotic treatments on the evolution of resistance is not well understood however, and debate is ongoing about the effectiveness of various multiple-antibiotic treatments. In this thesis, I investigate how aspects of multiple-antibiotic treatments impact the rate of evolution of antibiotic resistance in the opportunistic human pathogen Pseudomonas aeruginosa. In particular, I look at the impact of interactions between antibiotics in combination on the evolution of resistance, and how creating heterogeneity in the antibiotic environment by rotating the antibiotics used may change the rate of evolution of resistance. I characterise the interactions present in 120 combinations of antibiotics and find that the type of interaction can be predicted by the mechanism of action of the antibiotics involved. I investigate the effect of a subset of these combinations on the evolution of antibiotic resistance. My results refute the influential but poorly-evidenced hypothesis that synergistic combinations accelerate the evolution of resistance, even when synergistic combinations have the same inhibitory effect on sensitive bacteria as additive or antagonistic antibiotic combinations. I focus on a combination of the antibiotics ceftriaxone and sulfamethoxazole and test whether it is more effective in preventing the evolution of resistance than predicted by the inhibitory effect of the combination on sensitive bacteria. I do not find the combination to be more effective than predicted. Finally, I create heterogeneous antibiotic environments by rotating the antibiotic present at different rates. For the first time in a laboratory setting, I test how varying the rate of fluctuation in the antibiotics present in a heterogeneous antibiotic environment impacts the rate of evolution of resistance. Unexpectedly, I find the rate of evolution of resistance increases with increasing levels of antibiotic heterogeneity.

No description available.

No description available.

En raison de leur résistance aux agents chimiothérapeutiques, les transporteurs ABC de phénotype MDR ont attiré l'attention de la communauté scientifique. Notre projet vise à trouver des conditions dans lesquelles les transporteurs ABC restent fonctionnels en solution pour aboutir à la cristallisation de ces protéines dans une conformation active. Dans ce but, nous avons conçu et développé une nouvelle classe de détergents, à base de calix[4]arène, qui stabilisent ces protéines. Afin de résoudre la structure 3D à résolution atomique du transporteur ABC bactérien "BmrA", responsable de la résistance aux antibiotiques, nous avons utilisé une approche classique utilisant des détergents commerciaux en parallèle à nos détergents innovants. En présence de la Foscholine 12, nous avons obtenu des cristaux diffractant jusqu’à 5 Å de résolution. Cependant, les données de diffraction n’étaient pas suffisantes pour déterminer la structure tridimensionnelle complète de la protéine, seuls les domaines transmembranaires ont été résolus. D'autre part, nous avons atteint l'objectif de l'extraction, la purification et la stabilisation de ce transporteur à l'aide des détergents à base de calix [4] arène. Nous avons également montré que ces détergents promeuvent et améliorent la cinétique de cristallisation de BmrA, une étape que nous sommes en train d’optimiser, pour obtenir des cristaux de meilleure résolution, pour résoudre la structure 3D de BmrA qui sera utilisé pour concevoir des inhibiteurs adaptés / Due to their preponderance in the resistance to chemotherapies, the MDR ABC transporters have drawn the attention of the scientific community. Our project aimed at finding conditions in which ABC transporters are active in solution to lead the crystallization of these proteins in an active conformation. In this purpose, we conceived and developed a new class of detergents, based on calix[4]arene ring, that stabilize these proteins. In order to solve the 3D-structure to atomic resolution of bacterial ABC transporter “BmrA” responsible for antibiotic resistance, we used a classical approach with commercial detergents in addition to the innovative ones. We have crystallized the protein in presence of Foscholine 12 with a diffraction resolution up to 5 Å. The data was incomplete; solving partially the structure of the transmembrane domains. On the other hand, we have reached the objective of extraction, purification and stabilization of this transporter by using calix[4]arene-based detergents. We have also shown that these detergents promote and enhance the kinetics of crystallization of BmrA, a step that we are improving, to get crystals of better resolution, for resolving the BmrA 3D-structure which will be used to design adapted inhibitors