Objetivos: Avaliar o perfil de suscetibilidade, a prevalência da produção de AmpC, β-lactamase de espectro estendido (ESBL) e Metalo-β-lactamase (M-βla) em Pseudomonas aeruginosa obtidas de dois hospitais universitários distintos (ISCMPA e HCPA) em Porto Alegre. Em adição, tipagem molecular por PFGE foi realizada entre os isolados produtores de M-βla para avaliar a relação clonal. Métodos: Foi determinada a suscetibilidade de 238 isolados de P. aeruginosa para 8 agentes antimicrobianos, através do teste de disco-difusão, usando agar Müller-Hinton (MH) de acordo com “National Committee for Clinical Laboratory Standards” (NCCLS) . Todos isolados foram avaliados para produção de AmpC com o disco de imipenem (indutor) próximo ao disco de cefepima/ceftazdima (substrato). Um achatamento no halo de cefepime/ceftazidima pela indução da enzima pelo imipenem, indicava resultado positivo para AmpC. Todos isolados forma avaliados para a presença de ESBL através do teste de aproximação de disco com ceftazidima, cefepima, cefotaxima, ceftriaxona e ticarcilina-clavulanato como inibidor de β-lactamase. A produção de M-βla foi determinada através do teste de aproximação de discos de CAZ a discos impregnados com ácido2-mercatopropiônico (2-MPA). As taxas de resistência foram comparadas através do Teste Exato de Fisher. Valor de P<0,05 foi considerado estatisticamente significativo. Análise de macrorestrição com a enzima speI foi realizada em isolados produtores de M-βla. Resultados: As taxas de resistência para todos os agentes foram superiores entre os isolados obtidos na ISCMPA em relação aos do HCPA. A ceftazidima mostrou ser o antibiótico mais efetivo contra os isolados de ambos Hospitais (ISCMPA e HCPA) com taxa de resistência de 25,7% (ISCMPA) e 6,1% (HCPA). A expressão de AmpC foi observada em 190 isolados (83,7% HCPA e 77,1% ISCMPA). Não foi possível detectar a presença de ESBL entre todos as P. aeruginosa avaliadas em ambos hospitais. Foi observada a presença de M-βla em 28 isolados (20,0%) da ISCMPA. Mas não foi detectada M-βla em nenhuma P. aeruginosa do HCPA. A análise de macrorestrição mostrou que 14 de 16 P. aeruginosa Mβla positivas pertenciam a um clone (denominado clone A), e seus subclones. Apenas dois outros clones (B e C) foram identificados em um isolado cada. / Objectives: To evaluate susceptibility profile, the prevalence of extendedspectrum β-lactamases (ESBL) production, AmpC and Metallo-β-lactamases (M-βla) in Pseudomonas aeruginosa obtained from two distinct hospitals (ISCMPA and HCPA) in Porto Alegre, Brazil. In addiction, molecular typing by PFGE was perfomed among isolates producing M-βla in order to evaluate probably clonal relatedness. Methods: The susceptibility of 238 P. aeruginosa to 8 antimicrobial agents was determined by the disk diffusion method, using Müller-Hinton agar (MH) in accordance with “National Committee for Clinical Laboratory Standards” guidelines. All isolates were evaluated for AmpC production with the imipenem disk (strong inducer) near of the cefepime/ceftazidime disk (substrate). A blunting of the cefepime/ceftazidime zone by imipenem-induced enzyme, indicated positive result for AmpC. All isolates were evaluated for ESBL production by disk approximation test with ceftazdime, cefepime, cefotaxime, ceftriaxone plus ticarcillin-clavulanate as inhibitor. M-βla production was determined by disk approximation test with disks containing CAZ and 2-mercatopropionic acid (2-MPA). The results were compared by the Fisher’s Exact Test. Macrorestriction analysis by SpeI, followed by PFGE, was perfomed in isolates M-βla positive. The resistance rates were compared by The Fisher’s Exact Test. P values < 0.05 were considered to be statistically significant. Results: The resistance rates to all antimicrobial agents were higher among isolates obtained from ISCMPA than those obtained from HCPA. The ceftazidime was the more active antibiotic against the isolates in both hospitals with resistance rates of 25,7% (ISCMPA) and 6,1% (HCPA). The derepression of AmpC was observed in 190 isolates (83,7% HCPA and 77,1% ISCMPA). It was not possible to detect the presence of ESBL among all P. aeruginosa evaluated in both hospitals. Positive results for M-βla production were observed in 28 isolates (20,0%) from ISCMPA. But none M-βla production was identified in P. aeruginosa from HCPA. The macrorestriction analysis by PFGE, showed that 14 of 16 M-βla positive P. aeruginosa beloneed to one clone (named clone A) and its subclones.Only two others clones (B and C) were identified in one isolate each.

Objetivos: Avaliar o perfil de suscetibilidade, a prevalência da produção de AmpC, β-lactamase de espectro estendido (ESBL) e Metalo-β-lactamase (M-βla) em Pseudomonas aeruginosa obtidas de dois hospitais universitários distintos (ISCMPA e HCPA) em Porto Alegre. Em adição, tipagem molecular por PFGE foi realizada entre os isolados produtores de M-βla para avaliar a relação clonal. Métodos: Foi determinada a suscetibilidade de 238 isolados de P. aeruginosa para 8 agentes antimicrobianos, através do teste de disco-difusão, usando agar Müller-Hinton (MH) de acordo com “National Committee for Clinical Laboratory Standards” (NCCLS) . Todos isolados foram avaliados para produção de AmpC com o disco de imipenem (indutor) próximo ao disco de cefepima/ceftazdima (substrato). Um achatamento no halo de cefepime/ceftazidima pela indução da enzima pelo imipenem, indicava resultado positivo para AmpC. Todos isolados forma avaliados para a presença de ESBL através do teste de aproximação de disco com ceftazidima, cefepima, cefotaxima, ceftriaxona e ticarcilina-clavulanato como inibidor de β-lactamase. A produção de M-βla foi determinada através do teste de aproximação de discos de CAZ a discos impregnados com ácido2-mercatopropiônico (2-MPA). As taxas de resistência foram comparadas através do Teste Exato de Fisher. Valor de P<0,05 foi considerado estatisticamente significativo. Análise de macrorestrição com a enzima speI foi realizada em isolados produtores de M-βla. Resultados: As taxas de resistência para todos os agentes foram superiores entre os isolados obtidos na ISCMPA em relação aos do HCPA. A ceftazidima mostrou ser o antibiótico mais efetivo contra os isolados de ambos Hospitais (ISCMPA e HCPA) com taxa de resistência de 25,7% (ISCMPA) e 6,1% (HCPA). A expressão de AmpC foi observada em 190 isolados (83,7% HCPA e 77,1% ISCMPA). Não foi possível detectar a presença de ESBL entre todos as P. aeruginosa avaliadas em ambos hospitais. Foi observada a presença de M-βla em 28 isolados (20,0%) da ISCMPA. Mas não foi detectada M-βla em nenhuma P. aeruginosa do HCPA. A análise de macrorestrição mostrou que 14 de 16 P. aeruginosa Mβla positivas pertenciam a um clone (denominado clone A), e seus subclones. Apenas dois outros clones (B e C) foram identificados em um isolado cada. / Objectives: To evaluate susceptibility profile, the prevalence of extendedspectrum β-lactamases (ESBL) production, AmpC and Metallo-β-lactamases (M-βla) in Pseudomonas aeruginosa obtained from two distinct hospitals (ISCMPA and HCPA) in Porto Alegre, Brazil. In addiction, molecular typing by PFGE was perfomed among isolates producing M-βla in order to evaluate probably clonal relatedness. Methods: The susceptibility of 238 P. aeruginosa to 8 antimicrobial agents was determined by the disk diffusion method, using Müller-Hinton agar (MH) in accordance with “National Committee for Clinical Laboratory Standards” guidelines. All isolates were evaluated for AmpC production with the imipenem disk (strong inducer) near of the cefepime/ceftazidime disk (substrate). A blunting of the cefepime/ceftazidime zone by imipenem-induced enzyme, indicated positive result for AmpC. All isolates were evaluated for ESBL production by disk approximation test with ceftazdime, cefepime, cefotaxime, ceftriaxone plus ticarcillin-clavulanate as inhibitor. M-βla production was determined by disk approximation test with disks containing CAZ and 2-mercatopropionic acid (2-MPA). The results were compared by the Fisher’s Exact Test. Macrorestriction analysis by SpeI, followed by PFGE, was perfomed in isolates M-βla positive. The resistance rates were compared by The Fisher’s Exact Test. P values < 0.05 were considered to be statistically significant. Results: The resistance rates to all antimicrobial agents were higher among isolates obtained from ISCMPA than those obtained from HCPA. The ceftazidime was the more active antibiotic against the isolates in both hospitals with resistance rates of 25,7% (ISCMPA) and 6,1% (HCPA). The derepression of AmpC was observed in 190 isolates (83,7% HCPA and 77,1% ISCMPA). It was not possible to detect the presence of ESBL among all P. aeruginosa evaluated in both hospitals. Positive results for M-βla production were observed in 28 isolates (20,0%) from ISCMPA. But none M-βla production was identified in P. aeruginosa from HCPA. The macrorestriction analysis by PFGE, showed that 14 of 16 M-βla positive P. aeruginosa beloneed to one clone (named clone A) and its subclones.Only two others clones (B and C) were identified in one isolate each.

Objetivos: Avaliar o perfil de suscetibilidade, a prevalência da produção de AmpC, β-lactamase de espectro estendido (ESBL) e Metalo-β-lactamase (M-βla) em Pseudomonas aeruginosa obtidas de dois hospitais universitários distintos (ISCMPA e HCPA) em Porto Alegre. Em adição, tipagem molecular por PFGE foi realizada entre os isolados produtores de M-βla para avaliar a relação clonal. Métodos: Foi determinada a suscetibilidade de 238 isolados de P. aeruginosa para 8 agentes antimicrobianos, através do teste de disco-difusão, usando agar Müller-Hinton (MH) de acordo com “National Committee for Clinical Laboratory Standards” (NCCLS) . Todos isolados foram avaliados para produção de AmpC com o disco de imipenem (indutor) próximo ao disco de cefepima/ceftazdima (substrato). Um achatamento no halo de cefepime/ceftazidima pela indução da enzima pelo imipenem, indicava resultado positivo para AmpC. Todos isolados forma avaliados para a presença de ESBL através do teste de aproximação de disco com ceftazidima, cefepima, cefotaxima, ceftriaxona e ticarcilina-clavulanato como inibidor de β-lactamase. A produção de M-βla foi determinada através do teste de aproximação de discos de CAZ a discos impregnados com ácido2-mercatopropiônico (2-MPA). As taxas de resistência foram comparadas através do Teste Exato de Fisher. Valor de P<0,05 foi considerado estatisticamente significativo. Análise de macrorestrição com a enzima speI foi realizada em isolados produtores de M-βla. Resultados: As taxas de resistência para todos os agentes foram superiores entre os isolados obtidos na ISCMPA em relação aos do HCPA. A ceftazidima mostrou ser o antibiótico mais efetivo contra os isolados de ambos Hospitais (ISCMPA e HCPA) com taxa de resistência de 25,7% (ISCMPA) e 6,1% (HCPA). A expressão de AmpC foi observada em 190 isolados (83,7% HCPA e 77,1% ISCMPA). Não foi possível detectar a presença de ESBL entre todos as P. aeruginosa avaliadas em ambos hospitais. Foi observada a presença de M-βla em 28 isolados (20,0%) da ISCMPA. Mas não foi detectada M-βla em nenhuma P. aeruginosa do HCPA. A análise de macrorestrição mostrou que 14 de 16 P. aeruginosa Mβla positivas pertenciam a um clone (denominado clone A), e seus subclones. Apenas dois outros clones (B e C) foram identificados em um isolado cada. / Objectives: To evaluate susceptibility profile, the prevalence of extendedspectrum β-lactamases (ESBL) production, AmpC and Metallo-β-lactamases (M-βla) in Pseudomonas aeruginosa obtained from two distinct hospitals (ISCMPA and HCPA) in Porto Alegre, Brazil. In addiction, molecular typing by PFGE was perfomed among isolates producing M-βla in order to evaluate probably clonal relatedness. Methods: The susceptibility of 238 P. aeruginosa to 8 antimicrobial agents was determined by the disk diffusion method, using Müller-Hinton agar (MH) in accordance with “National Committee for Clinical Laboratory Standards” guidelines. All isolates were evaluated for AmpC production with the imipenem disk (strong inducer) near of the cefepime/ceftazidime disk (substrate). A blunting of the cefepime/ceftazidime zone by imipenem-induced enzyme, indicated positive result for AmpC. All isolates were evaluated for ESBL production by disk approximation test with ceftazdime, cefepime, cefotaxime, ceftriaxone plus ticarcillin-clavulanate as inhibitor. M-βla production was determined by disk approximation test with disks containing CAZ and 2-mercatopropionic acid (2-MPA). The results were compared by the Fisher’s Exact Test. Macrorestriction analysis by SpeI, followed by PFGE, was perfomed in isolates M-βla positive. The resistance rates were compared by The Fisher’s Exact Test. P values < 0.05 were considered to be statistically significant. Results: The resistance rates to all antimicrobial agents were higher among isolates obtained from ISCMPA than those obtained from HCPA. The ceftazidime was the more active antibiotic against the isolates in both hospitals with resistance rates of 25,7% (ISCMPA) and 6,1% (HCPA). The derepression of AmpC was observed in 190 isolates (83,7% HCPA and 77,1% ISCMPA). It was not possible to detect the presence of ESBL among all P. aeruginosa evaluated in both hospitals. Positive results for M-βla production were observed in 28 isolates (20,0%) from ISCMPA. But none M-βla production was identified in P. aeruginosa from HCPA. The macrorestriction analysis by PFGE, showed that 14 of 16 M-βla positive P. aeruginosa beloneed to one clone (named clone A) and its subclones.Only two others clones (B and C) were identified in one isolate each.

La résistance antimicrobienne est devenue un problème majeur de santé publique. L’usage parfois abusif d’antibiotiques conduit à la sélection et à la propagation mondiale de mécanismes de résistance. Grâce à leur efficacité clinique et faible toxicité, les β-lactamines sont les antibiotiques les plus prescrits actuellement, et le mécanisme de résistance le plus répandu est l’expression de β-lactamases. Dans ces conditions, le développement de nouveaux traitements antimicrobiens, pour des cibles connues ou nouvelles, est essentiel. Plus particulièrement, le développement de nouveaux inhibiteurs des β-lactamases est très prometteur, permettant de continuer l’utilisation des antibiotiques existants. Les études biochimiques et structurales des nouvelles β-lactamases et leurs mutants synthétiques, par cristallographie aux rayons X ou par différentes techniques de modélisation moléculaire (modélisation par homologie, « docking », dynamique moléculaire, analyse du réseau des molécules d’eau) permettent une meilleure compréhension de ces enzymes. Dans ce contexte, nous avons caractérisé plusieurs β-lactamases du point de vue phénotypique, biochimique et structural.La β-lactamase CMY-136 contient une mutation inhabituelle, Y221H, par rapport à CMY-2, dans une position qui est très conservée parmi les enzymes de classe C. Les études cristallographiques et de modélisation moléculaire ont révélé des interactions stériques défavorables autour de la position mutée 221 qui peuvent affecter la conformation et la dynamique de la boucle Ω, et qui pourraient expliquer l’hydrolyse plus efficace des substrats volumineux et l’affinité plus faible de la plupart des substrats par rapport à la CMY-2.La structure cristallographique de la β-lactamase OXA-427, une nouvelle carbapénèmase de classe D, montre une Lys73 très peu carbamoylée, ce qui est très inhabituel pour cette classe d’enzyme, ainsi qu’un pont hydrophobe à proximité du site actif. Les simulations de dynamique moléculaire ont montré que la boucle β5-β6 est plus flexible et dans une conformation étendue. Ces résultats peuvent expliquer le profil d’hydrolyse unique observé expérimentalement pour cette enzyme.Les modifications dans la boucle β5-β6 de la β-lactamase OXA-48 (mutations d’alanines, délétions systematiques, remplacement par la boucle β5-β6 de la β-lactamase OXA-18) provoquent des modifications importantes dans le profil d’hydrolyse, avec une acquisition graduelle d’une activité cephalosporinase et une diminution de l’activité carbapénémase dans certains cas. Des études de cristallographie aux rayons X et modélisation moléculaire suggèrent que les différences de conformation et de flexibilité dans cette boucle et les régions adjacentes permettent une meilleure fixation des céphalosporines plus volumineuses, par rapport à l’OXA-48. L’analyse de la dynamique des molécules d’eau dans le site actif montre des changements qui sont potentiellement responsables de la diminution de l’activité par rapport aux carbapénèmes. En complément des études sur les mutants naturels, ces résultats confirment l’importance de la boucle β5-β6 pour la spécificité de substrat des enzymes de type OXA-48. La structure cristallographique du mutant 217ΔP de l’OXA-48 présente une conformation auto-inhibée inattendue, induite par la présence d’un ion nitrate, un inhibiteur auparavant inconnu des β-lactamases de classe D.La Beta-Lactamase DataBase(BLDB, http://bldb.eu) développée dans notre équipe est une ressource publique exhaustive contenant des données relatives aux β-lactamases, vérifiées et mises à jour régulièrement. Cette base de données contient tous les mutants naturels et synthétiques de β-lactamases, ainsi que toutes les structures 3D disponibles dans la PDB et la caractérisation phénotypique.Globalement, ces résultats représentent le prérequis pour une meilleure compréhension des relations structure-fonction des β-lactamases et pour le futur développement rationnel d’inhibiteurs pour ces enzymes. / Antimicrobial resistance (AMR) has become a major threat to public health nowadays. The use and abuse of antibiotics is increasingly leading to selection and spread of resistance mechanisms worldwide, greatly compromising our capacity to treat infectious diseases. AMR might ultimately result in a future without effective antimicrobial therapy. Due to their safety and clinical efficacy, β-lactams are the most utilized antimicrobial therapy, and the most common resistance mechanism is the expression of β-lactamases. Therefore, the development of new antimicrobial drugs, for novel or already known targets, is of utmost importance. In particular, the development of novel inhibitors towards β-lactamases is also quite promising, as it would allow us to continue using the effective and safe antimicrobial drugs already available today. The biochemical and structural study of novel β-lactamases or synthetic mutants, through X-ray crystallography and various molecular modelling techniques (homology modelling, docking, molecular dynamics, water network analysis), can provide valuable information. In this context, we have characterized phenotypically, biochemically and structurally several β-lactamases.The CMY-136 β-lactamase possesses an unusual mutation, Y221H, as compared to CMY-2, in a position highly conserved among class C ß-lactamases. Crystallographic and molecular modelling experiments reveal a steric impediment around the mutated position 221 that may affect the conformation and dynamics of the Ω-loop, and therefore account for an increased turnover rate for bulky substrates and a decreased affinity for most substrates as compared to CMY-2.The crystal structure of the OXA-427, a novel class D carbapenemase, shows the Lys73 only partially carbamoylated, a very unusual characteristic for this class of β-lactamases, and an unexpected hydrophobic bridge in the vicinity of the active site. Moreover, molecular dynamics simulations revealed an extended and highly flexible β5-β6 loop. Altogether, these features may explain the unique hydrolytic profile determined experimentally for this enzyme.Modifications in the β5-β6 loop of the OXA-48 β-lactamase (alanine scanning, systematic deletions, replacement with the β5-β6 loop from OXA-18) result in profound changes in the hydrolytic profile, with gradual acquisition of cephalosporinase activity and decrease of carbapenemase activity in some cases. X-ray crystallography and molecular modelling studies suggest that the altered conformation and flexibility of this loop and of adjacent regions in these mutants may allow for the better accommodation of the bulkier cephalosporins, compared to OXA-48. Additionally, water dynamics analysis highlighted changes in the water network around and inside the active site cavity that may be responsible for the lower activity towards carbapenems. Together with studies on other naturally occurring mutants, results corroborate the relevance of the β5-β6 loop on the substrate profile of OXA-type enzymes. Crystal structure of the OXA-48 217ΔP mutant reveals an unexpected self-inhibited conformation induced by the presence of a nitrate ion, a previously unknown inhibitor of class D β-lactamases.Finally, the Beta-Lactamase DataBase (BLDB, http://bldb.eu) developed in our laboratory is a comprehensive, manually curated public resource providing up-to-date structural and functional information on β-lactamases. It contains all reported naturally-occurring β-lactamases and synthetic mutants, together with all available 3D structures from the PDB and the phenotypical characterization.Overall, these results constitute an essential foundation for a better understanding of the structure-function relationship of β-lactamases, which may prove crucial for the future rational development of β-lactamase inhibitors.

β-lactam antibiotics have been the most widely used drug of choice to combat infectious disease caused by bacteria. Unfortunately, the effectiveness of these antibiotics is drastically threatened by bacterial β-lactamases. β-lactamases are currently responsible for the resistance to most β-lactam antibiotic drugs. For decades, β-lactam β-lactamases inhibitors have been used to reduce bacterial resistance, however, in this study, we will employ the use of 1,2,4-oxadiazolidinone derivatives as a non-β-lactam β-lactamases inhibitor against TEM-1 and P99 β-lactamases. The significance of oxadiazolidinone is the prominent five-membered ring in its structure, which is configurationally stable and present in other biologically active compounds such as linezolid and avibactam. Oxadiazolidinones were synthesized in two steps procedure using nitroalkanes and benzaldehyde as starting materials to produce nitrones, which in turn undergo 1,3- dipolar cycloaddition with substituted isocyanates to give the desired 1,2,4-oxadiazolidin analogs (2a, 2b, 2c and 3). Each product was purified and characterized using 1H NMR and 13C NMR, GC-MS, IR, and UV/Vis analysis. Following their successful synthesis and structural elucidation, they were tested with TEM-1 and P99 serine β-lactamase using Nitrocefin as the substrate to ascertain their effectiveness against β-lactamase. 2a, 2b, 2c and 3 showed inhibition ranging from 12-38 %.

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.

This work will look at two factors that add to the diversity of carbapenem resistant bacteria. First, it focuses on the diversity of carbapenemase resistance plasmids. 446 plasmids were characterized by size, gene content and replicon groups. We identified that on average, over 30% of the encoded proteins on each plasmid have an unknown function. Plasmid sizes ranged from 1.6kb to 500kb, with an average of around 100kb and median of 80kb. Additionally, six replicon groups account for 80% of all the carbapenemase resistance plasmids. We also highlight the lack of data available for carbapenemase carrying plasmids from bacterial genera other than Escherichia and Klebsiella, and plasmids that carry the New Delhi metallo-β- lactamase or the Verona-integron encoded metallo-β-lactamase. Second, we characterized the β-lactamase diversity of a single carbapenemase resistant Klebsiella pneumoniae. This isolate encodes six distinct β-lactamases, all of which are functional, and three of which are redundant. Additionally, we determined that the CTX-M-15 cephalosporinase imparts a greater fitness when grown in aztreonam (a monobactam) than ceftazidime (a cephalosporin). Finally, we show that individually, these β-lactamases do not account for the elevated levels of resistance seen in the parent strain, indicating that the passive resistance mechanisms (i.e. efflux pumps, altered membrane porins) may play a larger role than originally thought.

The use of reporter gene fusions to assess cellular processes such as protein targeting and regulation of transcription or translation is established technology in archaeal, bacterial, and eukaryal genetics. Fluorescent proteins or enzymes resulting in chromogenic substrate turnover, like β-galactosidase, have been particularly useful for microscopic and screening purposes. However, application of such methodology is of limited use for strictly anaerobic organisms due to the requirement of molecular oxygen for chromophore formation or color development. We have developed β-lactamase from Escherichia coli (encoded by bla) in conjunction with the chromogenic substrate nitrocefin into a reporter system usable under anaerobic conditions for the methanogenic archaeon Methanosarcina acetivorans. By using a signal peptide of a putative flagellin from M. acetivorans and different catabolic promoters, we could demonstrate growth substrate-dependent secretion of β-lactamase, facilitating its use in colony screening on agar plates. Furthermore, a series of fusions comprised of a constitutive promoter and sequences encoding variants of the synthetic tetracycline-responsive riboswitch (tc-RS) was created to characterize its influence on translation initiation in M. acetivorans. One tc-RS variant resulted in more than 11-fold tetracycline-dependent regulation of bla expression, which is in the range of regulation by naturally occurring riboswitches. Thus, tc-RS fusions represent the first solely cis-active, that is, factor-independent system for controlled gene expression in Archaea.

Les antibiotiques β-lactamines sont parmi les médicaments antimicrobiens les plus anciens et les plus utilisés. L'enzyme bactérienne β-lactamase hydrolyse l'antibiotique β-lactame en cassant la structure de base "anneau β-lactame". Pour identifier les nouvelles β-lactamases, une étude complète a été réalisée dans diverses bases de données biologiques telles que Human Microbiome Project, env_nr et NCBI nr. L'analyse a révélé que les séquences ancestrales putatives et les recherches de profil HMM jouaient un rôle important dans l'identification de la base de données homologue et métagénomique à distance dans l'enzyme β-lactamase existante comme matière noire. Les larges analyses phylogénétiques des β-lactamases existantes et nouvellement identifiées représentent les nouveaux clades dans les arbres. En outre, l'activité d'hydrolyse des antibiotiques β-lactamines de séquences nouvellement identifiées (provenant d'archées et d'humains) a été étudiée en laboratoire, ce qui montre l'activité de la β-lactamase. La deuxième phase de l'étude a été entreprise pour examiner l'évolution fonctionnelle des β-lactamases. Premièrement, des séquences de protéines ß-lactamase 1155 ont été extraites de la base de données ARG-ANNOT et des valeurs CMI la littérature correspondante. Les résultats ont révélé que l'activité fonctionnelle de la β-lactamase évoluait de manière convergente au sein de la classe moléculaire. La troisième phase de cette thèse représente le développement d'une base de données intégrative de β-lactamases. La base de données publique actuelle de β-lactamases a des informations limitées, par conséquence, une base de données intégrative a été développée. / The β-lactam antibiotics are one of the oldest and widely used antimicrobial drugs. The bacterial enzyme β-lactamase hydrolyzes the β-lactam antibiotic by breaking the core structure “β-lactam ring”. To identify the novel β-lactamases a comprehensive investigation was performed in different biological databases such as Human Microbiome Project, env_nr, and NCBI nr. The analysis revealed that putative ancestral sequences and HMM profile searches played a significant role in the identification of remote homologous and uncovered the existing β-lactamase enzyme in the metagenomic database as dark-matter. The comprehensive phylogenetic analyses of extant and newly identified β-lactamase represent the novel clades in the trees. Further, the β-lactam antibiotic hydrolysis activity of newly identified sequences (from archaea and human) was investigated in laboratory, which shows β-lactamase activity.The second phase of the investigation was undertaken to examine the functional evolution of β-lactamases. First, 1155 β-lactamase protein sequences were retrieved from ARG-ANNOT database and MIC values from the corresponding literature. The results revealed that the functional activity of β-lactamase evolved convergently within the molecular class.The third phase of this thesis presents development of an integrative β-lactamase database. The existing public database of β-lactamase has limited information, therefore, an integrative database was developed.

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.