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1
Zavala, Agustin. "Structure-function studies of β-lactamases." Thesis, Université Paris-Saclay (ComUE), 2018. http://www.theses.fr/2018SACLS567.
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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 enzymesAntimicrobial 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
2
Hammond, David Scott. "SHV β-lactamases : DNA diagnostics and evolution." Thesis, Queensland University of Technology, 2006. https://eprints.qut.edu.au/16194/1/David_Hammond_Thesis.pdf.
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TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors.
It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels.
In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels.
Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype.
To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions.
A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.
3
Hammond, David Scott. "SHV β-lactamases : DNA diagnostics and evolution." Queensland University of Technology, 2006. http://eprints.qut.edu.au/16194/.
Full textAbstract:
TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors. It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels. In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype. To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions. A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.
4
Robin, Frédéric Gérard Jean Michel. "Exploration moléculaire, biochimique et structurale de β-lactamases à spectre étendu de sensibilité diminuée aux inhibiteurs des β-lactamases." Clermont-Ferrand 1, 2007. http://www.theses.fr/2007CLF1MM16.
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L'utilisation intensive de nouvelles β-lactamines comme les céphalosporines de 3ème génération (C3G) ou les inhibiteurs des β-lactamases a entraîné l'évolution des β-lactamases. Ainsi au cours des années 80-90 ont émergé les β-lactamases de spectre étendu (BLSE), capables d'hydrolyser les C3G, et les pénicillinases résistantes aux inhibiteurs (PRI). Enfin, depuis la fin des années 90, 4 enzymes associant des substitutions observées chez les BLSE et chez les PRI ont été décrites. Ainsi, des souches produisant de telles enzymes pourraient devenir plus difficilement détectables par les tests utilisés en routine et donc conduire à des échecs thérapeutiques. Notre travail s'inscrit dans l'étude de ces nouvelles enzymes, encore rarement rapportées. Cette étude nous a permis de décrire 4 nouveaux Mutants Complexes dérivés de la β-lactamase TEM-1 (TEM-109, TEM-125, TEM-151 et TEM-152) à partir de souches d'E. Coli isolées entre 2001 et 2004. Parmi ces enzymes, toutes présentaient une activité sur les C3G, mais seuls TEM-125 et TEM-152 avaient un niveau de résistance aux inhibiteurs élevé proche de celui d'enzymes de type PRI. Enfin, l'étude structurale et fonctionnelle de la BLSE résistante au tazobactam BES-1 a permis de mettre en évidence un certain nombre d'originalités structurales expliquant son activité atypique, comme un élargissement des parois du site actif et des interactions réalisées par le résidu Arg220, notamment avec la Thr237. Notre étude suggère donc l'émergence de ces nouvelles enzymes et fournit des données structurales nécessaires à la mise au point de nouvelles molécules antibiotiques ou inhibitrices.
5
Guillon, Hélène. "Activité carbapénémase des β-lactamases de type AmpC." Amiens, 2013. http://www.theses.fr/2013AMIED003.
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Les β-lactamases de type AmpC (céphalosporinases) semblent fréquemment responsables de la résistance aux carbapénèmes chez les entérobactéries, ce que permettent d’évoquer de nombreuses descriptions cliniques. L’objectif de cette thèse était d’effectuer une caractérisation phénotypique, biochimique et moléculaire de l’activité carbapénémase des AmpC. Tout d’abord, les gènes des cinq principales céphalosporinases plasmidiques ont été clonés puis transformés dans la souche imperméable Escherichia coli HB4. Les comparaisons phénotypiques et structurales des clones recombinants ont montré que les céphalosporinases CMY-2, ACT-1, et DHA-1 se distinguent des autres enzymes par leur capacité à conférer une résistance aux carbapénèmes et par la présence d’une asparagine en position 346 (Asn 346), située à proximité du site catalytique. Des expériences de mutagénèse dirigée, consistant à substituer l'Asn 346 par des résidus de taille, de charge et de polarité différentes dans la céphalosporinase CMY-2, ont démontré le rôle de cet acide aminé dans l’activité carbapénémase des céphalosporinases. La caractérisation biochimique de trois variants a révélé que l'Asn 346 contribue à l’affinité de CMY-2 pour l'imipénème. L'étude de l’activité carbapénémase des AmpC chromosomiques à spectre étendu (ACSE) a constitué le second volet de la thèse. Le séquençage, le clonage et la caractérisation biochimique d'une nouvelle ACSE produite par une souche clinique de E. Coli résistante à l'ertapénème ont démontré que l'extension du spectre d'hydrolyse des céphalosporinases, qui est due à une augmentation de leur affinité, peut être aussi responsable d'une résistance aux carbapénèmesOwing to several clinical reports, it appears that AmpC-type β-lactamases (cephalosporinases) account frequently for carbapenem resistance in Enterobacteriaceae. The aim of this study was to perform a phenotypic, biochemical, and molecular characterization of the carbapenem-hydrolyzing activity of AmpC-type β-lactamases. First of all, the genes encoding the five main plasmid-mediated AmpC β-lactamases were cloned and transferred into the porin-deficient Escherichia coli HB4 strain. Phenotypic and molecular comparison of the recombinant strains revealed that only CMY-2, ACT-1, and DHA-1 conferred resistance to carbapenems and had an asparagine residue at position 346 (Asn 346), located in the vicinity of the active site. Site-directed mutagenesis experiments were performed to replace the Asn 346 residue of CMY-2 β-lactamase by amino acids differing in size, charge, and polarity. It confirmed the contribution of Asn 346 to the carbapenem-hydrolysing activity of cephalosporinases. Biochemical characterization of three variants revealed that Asn 346 assisted the binding of imipenem. The analysis of the carbapenem-hydrolyzing activity of chromosomal extended-spectrum AmpC β-lactamases (ESAC) constitutes the second part of this thesis. Sequencing, cloning and biochemical characterization of a novel ESAC produced by an ertapenem-resistant E. Coli clinical isolate demonstrated that the extension of the hydrolysis spectrum of cephalosporinases, which was due to increased affinity, may also contribute to carbapenem resistance
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Bertoncheli, Claudia de Mello. "Identificação de metalo-β-lactamases em bacilos gram-negativos não fermentadores isolados no Hospital Universitário de Santa Maria." Universidade Federal de Santa Maria, 2008. http://repositorio.ufsm.br/handle/1/5877.
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Conselho Nacional de Desenvolvimento Científico e TecnológicoIn recent years, the isolation of bacteria producing β-lactamases has caused concern around the world, due to the fact these enzymes hydrolysis the ring β-lactam antimicrobials used in the main clinic. This aim of this study was asses the prevalence metallo-β-lactamases (MbL) in isolates of Pseudomonas aeruginosa and Acinetobacter baumannii obtained from patients admitted at the University Hospital of Santa Maria (HUSM). The profile of susceptibility for all isolates was evaluated by the disk diffusion method standardized by CLSI. The antimicrobial disks were distributed in a way that allows the identification of strains producers of AmpC and ESBL. For the identification of the producers of MbL the test of disk approximation with EDTA 0.1 M, EDTA 0,5M and acid 2-mercaptopropionic were performed. Isolates that did not have any of the mechanisms of resistance search were classified as multiresistant (MDR). The minimum inhibitory concentration (MIC) for ceftazidima, imipenem and polymyxin B was assessed by broth method microdilution for all isolated, according to CLSI. From January to June 2006, were obtained 32 isolates the P.aeruginosa and 41 the A. baumannii, the those 17 (23.29%) were β-lactamase AmpC-type producers, 11 (15.07%) were MbL producers, and 45 (61,64%) were classified as MDR. All strains producing MbL were Pseudomonas aeruginosa. The sensitivity of the isolates according to the CIM for antimicrobial evaluated were: 90,28% for polymyxin B, 36,11% for imipenem and 18% for ceftazidima. There was a high prevalence of MDR isolates and producers of β-lactamase-type AmpC and MbL in HUSM, this is extremely worrying once there is limiting therapy available. This situation becomes even more worrying with the find of isolates resistant the polymyxin B, witch is one of the last options of treatment for MDR isolates and producers of MbL. The detection of microorganisms is extremely important for the committees of infection hospital with the goal of preventing outbreaks, as well as guide the medical team on the conduct therapy, since there are few effective antimicrobial clinically for these pathogens and no prospects for development the new antimicrobial in the near future.
Nos últimos anos, o isolamento de bactérias produtoras de β-lactamases tem causado preocupação em todo o mundo, devido ao fato dessas enzimas hidrolisarem o anel β- lactâmico dos principais antimicrobianos utilizados na clínica. Este trabalho teve por objetivo avaliar a prevalência de metalo-β-lactamases (MbL) em isolados de Pseudomonas aeruginosa e Acinetobacter baumannii obtidos de pacientes atendidos no Hospital Universitário de Santa Maria (HUSM). O perfil de sensibilidade para todos os isolados foi avaliado pelo método de disco difusão padronizado pelo CLSI. Os discos de antimicrobianos utilizados foram distribuídos de forma que permitisse a identificação dos isolados produtores de AmpC e ESBL. Para a identificação dos produtores de MbL utilizou-se o teste de disco aproximação com os seguintes agentes quelantes: EDTA 0,1M, EDTA 0,5 M e ácido 2-mercaptopropiônico. Os isolados que não possuíam nenhum dos mecanismos de resistência pesquisados foram classificados como multirresistentes (MDR). A concentração inibitória mínima (CIM) para ceftazidima, imipenem e polimixina B foi avaliada pelo método de microdiluição em caldo para todos os isolados, de acordo com o CLSI. Durante o período de janeiro a junho de 2006 foram obtidos 32 isolados de P.aeruginosa e 41 de A. baumannii, destes 17 (23,29%) foram produtores de β-lactamase do tipo AmpC, 11 (15,07%) foram produtores de MbL e 45 (61,64%) foram classificados como MDR. Todas as cepas produtoras de MbL foram de Pseudomonas aeruginosa. A sensibilidade dos isolados de acordo com a CIM para os antimicrobianos avaliados foram as seguintes: 90,28% para polimixina B, 36,11% imipenem e 18% ceftazidima. Observou-se uma alta prevalência de isolados MDR no HUSM, além de isolados produtores de β-lactamase do tipo AmpC e MbL, o que é extremamente preocupante devido limitar a terapia a poucos antimicrobianos. Esta situação torna-se ainda mais preocupante com a detecção de isolados resistentes a polimixina B, a qual é uma das últimas opções de tratamento para infecções causadas por isolados de P. aeruginosa e Acinetobacter baumannii MDR e produtores de MbL. A detecção desses microrganismos é de grande importância para as comissões de controle de infecção hospitalar com o objetivo de prevenir surtos, bem como orientar a equipe médica sobre a conduta terapêutica, uma vez que há poucos antimicrobianos efetivos clinicamente para esses patógenos e as perspectivas para o desenvolvimento de novos antimicrobianos em um futuro próximo são mínimas.
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Makena, Anne. "Structural and biochemical characterisation of bacterial metallo-β-lactamases." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:c6129257-5d92-4dd3-9a47-d0dbbcb361d9.
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One of the most prominent ways by which bacteria develop resistance to the widely used β-lactam antibiotics is by production of β-lactamases. Metallo-β-lactamases (MBLs) in particular pose an increasing public health risk due to the wide dissemination and broad substrate profile. The successful development of novel MBL inhibitors for clinical use is also dependent on sufficient understanding of the structural and functional properties of the target MBLs. From a medicinal chemistry point of view, the combination of biological diversity and structural similarity of MBLs presents a rather challenging task for the development of selective small molecules for inhibitors for bacterial MBLs. The progress made in this quest thus far is discussed at length in the introduction (Chapter 1). Robust and affordable high throughput screening platforms are indispensable in drug development. Chapter 2 of the thesis describes the identification of a novel cephalosporinbased substrates, CLS405, for MBL activity and inhibition assays. The CLS405-based assay is used in an exemplary screening of potential MBL inhibitors, resulting in the identification of N-hydroxythiazoles as novel MBL inhibitor scaffolds. An umbelliferone-derived cephalosporin substrate (FC-5) is also investigated for application in screening of MBL activity in vivo. Clinically useful MBL inhibitors must have a sufficient breadth of selectivity against multiple variants MBLs; this is particularly challenging due to the rapid MBL evolution. In Chapters 3 and 4, comparative studies of the activity, stability and structural properties of some of the clinically reported variants of the New Delhi metallo-β-lactamases (NDMs) are described. Nominal differences are noted in the kinetic parameters of the recombinant proteins, except for the apparent substrate inhibition observed with nitrocefin for variants containing the M154L substitution. Further investigations reveal significant differences in thermal stability of the NDM variants with the double mutants (NDM-5, NDM-7, NDM-8 and NDM-12) being the most stable variants, suggesting that protein stability may be a driver of MBL evolution. The Verona imipenemase metallo-β-lactamases (VIM) enzymes are among the most widely distributed MBLs with >40 variants reported (Chapter 5). Comparative studies on the biochemical and biophysical properties were carried out for five VIM variants: VIM-1, VIM- 2, VIM-4, VIM-5 and VIM-38. Clear differences in the thermal stabilities of the variants are observed, despite their similar β-lactamase activities, as previously shown for NDM variants. Interestingly, the variants show marked differences in the inhibition profile, with isoquionolone-based inhibitors selectively inhibiting VIM-5 and VIM-38 compared to VIM-1 and VIM-2. Overall, the work supports the proposal that protein stability may be an important factor in MBL evolution. Crucially, the study highlights the importance of screening MBL variants during inhibitor development programs.
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Keshri, Vivek. "Evolutionary analysis of the β-lactamase families." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0250.
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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 développéeThe 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
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Bellais, Samuel. "β-lactamases à large spectre chez les flavobacteriaceae et résistance naturelle aux β-lactamines." Paris 11, 2002. http://www.theses.fr/2002PA114805.
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Arlet, Guillaume. "Bêta-lactamases à spectre élargi : découverte et analyse épidémiologique." Paris 11, 1992. http://www.theses.fr/1992PA114835.
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Fisette, Olivier. "Propriétés dynamiques et catalytiques des β-Lactamases de classe A." Doctoral thesis, Université Laval, 2012. http://hdl.handle.net/20.500.11794/23975.
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Tableau d’honneur de la Faculté des études supérieures et postdoctorales, 2012-2013.Les β-lactamases sont le principal mécanisme bactérien de défense contre les β-lactamines. Elles catalysent l’inactivation de ces antibiotique par le clivage de leur noyau β-lactame. Les β-lactamases les plus communes sont celles de la classe A, qui rassemble une grande variété d’enzymes aux spécificités de substrat variées. Ces protéines ont été l’objet de nombreuses recherches expérimentales et théoriques. Plusieurs études de dynamique par spectroscopie RMN ont été réalisées dans notre laboratoire sur les enzymes modèles TEM-1 et PSE-4. Le présent projet continue l’investigation de ces deux β-lactamases par des méthodes théoriques. Un protocole de simulation de DM a été établi et validé par comparaison avec des données de relaxation RMN. Une nouvelle technique d’analyse conjointe DM/RMN a également été développée, permettant de limiter les problèmes de sous et sur-ajustement présents dans l’analyse « model-free ». Pour comparer la dynamique des β-lactamases de classe A en présence et en absence de leur substrat, un potentiel pour les β-lactamines a été développé, en tenant compte de la géométrie et du potentiel chimique particuliers du noyau β-lactame. Ce champ de forces est transférable, permettant la construction d’une variété d’antibiotiques. Nos simulations, couplées aux précédentes études par RMN, démontrent qu’il existe une dualité dynamique dans les β-lactamases de classe A : elles sont hautement structurées à l’échelle ps-ns, mais aussi le siège de mouvements lents (µs-ms) aux abords du site actif et particulièrement dans la boucle qui borde le site catalytique. La rigidité ps-ns favorise un positionnement optimal des résidus du site actif pour une catalyse efficace, et permet la tolérance de mutations potentiellement déstabilisantes. Les mouvements à l’échelle µs-ms les plus intéressants sont localisés dans la boucle Ω et confirment son rôle régulatoire : elle permet l’ouverture du site actif pour l’entrée du substrat et le largage du produit. La liaison du substrat a des effets à longue portée rigidifiant TEM-1. On observe également un mouvement accru de la boucle Ω dans TEM-1 et PSE-4. Les interactions spécifiques menant à cette plus grande flexibilité varient toutefois d’une enzyme à l’autre : il y conservation des propriétés dynamiques.
β-Lactamases are the main bacterial mechanism of resistance against β-lactams. They inactivate these antibiotics by cleaving their β-lactam ring. Class A enzymes are the most prevalent, with a broad variety of substrate specificities. These proteins were studied by numerous experimental and theoretical studies. NMR spectroscopy measurements were performed in our laboratory on model enzymes TEM-1 and PSE-4. This project continues the investigation of the dynamic properties of these two β- lactamases using theoretical methods. An MD simulation protocol was established and validated using NMR relaxation data. A new joint MD/NMR analysis technique was developped, allowing a reduction of under- and over-fitting problems in model-free analysis. To compare class A β-lactamase dynamics in presence and absence of substrate, a potential was developped to describe β-lactams, taking into account the particular geometry and chemical potential of the β-lactam cycle. This force field is transferable, allowing the construction of a variety of antibiotics. Our simulations, along with past NMR studies, prove the existence of a dynamical duality in class A β-lactamases : they are highly structured on the ps-ns timescale, but also subjected to slow motions (µs-ms) in the vicinity of the active site, particularly in the Ω loop that borders the catalytic site. Ps-ns rigidity favors an optimal positioning of active site residues for an efficient catalysis, and allows the protein to tolerate potentially destabilizing mutations. The most interesting µs-ms motions are located in the Ω loop, confirming its regulatory role : it opens the active site for substrate entry and product release. Substrate binding has structuring long-range effects on TEM-1. Increased loop motions are also observed in both TEM-1 and PSE-4. However, specific interactions responsible for this higher flexibility vary between the two enzymes : protein dynamics properties are conserved.
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Gonçalves, Ana Lúcia Saraiva. "Avaliação da produção de β-lactamase em pseudomonas aeruginosa obtidas de dois Hospitais de Porto Alegre." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2005. http://hdl.handle.net/10183/24617.
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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.
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Zarnayova, Martina. "Mécanismes de résistance aux beta-lactamines chez des isolats cliniques de la famille des enterobacteriaceae provenant de l'hôpital Ruzinov à Bratislava, Slovaquie." Dijon, 2006. http://www.theses.fr/2005DIJOMU15.
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Soroka, Daria. "Rôle du motif SDN dans l'inhibition et l'activité des β-lactamases des mycobactéries." Thesis, Paris 6, 2016. http://www.theses.fr/2016PA066432/document.
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Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaCMycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC
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Evans, Benjamin. "Significance of the OXA-51-like β-lactamases of Acinetobacter baumannii." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4481.
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The genus Acinetobacter currently contains 34 species, the vast majority of which are not regularly implicated in causing infection. However, incidences of hospitalacquired infection with Acinetobacter species are increasing, mainly due to the rise in the number of infections caused by the species Acinetobacter baumannii in immunocompromised patients particularly in intensive care units (ICUs). Due to high levels of resistance in A. baumannii to many classes of antibiotic, the carbapenems have been portrayed as the ‘drugs of choice’ for treating infections with this species. However, the activity of the carbapenems against A. baumannii has come under threat with the identification of four groups of class D β-lactamases carried by members of the species. Of these, the OXA-51-like enzymes have been suggested to be ubiquitous and intrinsic enzymes within A. baumannii. This presents the worrying scenario of the potential for all A. baumannii to become carbapenem resistant, leaving few treatment options available for this species. This project aimed to investigate the epidemiological spread of the OXA-51-like β-lactamases, examine the diversity within these enzymes, and whether this diversity may have implications for their ability to confer resistance to the carbapenems. A functional map showing the amino-acid similarities between the OXA-51-like enzymes demonstrated that the enzymes fall into distinct closely-related groups, with notable clusters surrounding OXA-66, OXA-69 and OXA-98. PCR and sequencing analysis of a geographically diverse group of 64 A. baumannii isolates demonstrated that isolates forming specific sequence groups (SGs) as defined by Turton et al (2007) also contained the same or closely related blaOXA-51-like gene. Higher minimum inhibitory concentrations (MICs) of carbapenems were found in association with acquired carbapenemases, or with the presence of ISAba1 upstream of the blaOXA-51- like gene. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates did not demonstrate relatedness between isolates which formed the same sequence group. Multilocus sequence typing (MLST) of a subset of 44 isolates grouped isolates more consistently with the SGs and blaOXA-51-like alleles, indicating that PFGE is unreliable for use with A. baumannii unless studying a short time period, and that blaOXA-51-like alleles are a good epidemiological marker. Mutation studies using meropenem with five A. baumannii isolates encoding different OXA-51-like enzymes, while resulting in an increase in meropenem MICs of between 8- and 128-fold, did not result in a nucleotide substitution in the blaOXA- 51-like genes or a change in the upstream region of the genes in any isolate suggesting that the carbapenems may not be producing a strong selective pressure on the blaOXA- 51-like genes. Analysis of πN/πS ratios for the blaOXA-51-like genes, MLST genes and the TEM, SHV and CTX-M β-lactamase families showed the blaOXA-51-like genes to be under less positive selection than these other β-lactamases, though under less purifying selection than the MLST genes. Phylogenetic analysis of the MLST genes and the blaOXA-51-like genes indicates that the blaOXA-51-like genes have been evolving within A. baumannii since its speciation, and that different groups of blaOXA-51-like genes have been evolving at different rates corresponding to different rates of evolution within their parent lineages. Structural modelling studies based upon the published crystal structure for OXA-40 indicated that amino-acid variation at particular sites in the OXA-51-like enzymes are likely to have an effect of enzyme function. Alterations at amino-acid position 167 change the shape of the entrance to the active site which may affect hydrolysis by accommodating the antibiotic differently, or may affect the substrate profile of the enzyme. The substitution of glutamine for proline at position 194 may significantly alter the shape of the enzyme thereby affecting substrate hydrolysis. This project found that specific groups of blaOXA-51-like genes are associated with specific A. baumannii lineages and that these genes could serve as convenient epidemiological markers. Most of the diversity within the OXA-51-like enzymes is due to their continued evolution within A. baumannii since the species’ emergence. However, certain amino-acid changes may play a role in altering the rate of hydrolysis or substrate profile of these enzymes.
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Kalp, Matthew Douglas. "Raman Crystallographic Studies of Inhibitor Reactions in Class A β-Lactamases." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1228504500.
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Drawz, Sarah Michel. "Inhibition of Class A and C β-Lactamases: Challenges and Promise." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1270163923.
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Walker, Rachel Anne. "The role of β-lactamases in carbapenem-resistant Gram-negative bacteria." Thesis, University of Edinburgh, 1999. http://hdl.handle.net/1842/22715.
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In this thesis sixty-one clinical Pseudomonas aeruginosa isolates were acquired from hospitals within Japan and fifty-one of these strains were resistant to imipenem and/or meropenem (MIC >4mg/l). Neither IMP-1 nor a novel carbapenemase could be detected in any of these strains; instead synergism between a cephalosporinase and lowered outer membrane permeability was found to be the most prevalent mechanism of imipenem resistance. The carbapenem-hydrolysing metallo-β-lactamase produced by members of the genus Aeromonas have in the past few years demanded attention from a clinical and enzymological point of view. Two imipenem-resistant Aeromonas veronii biovar sobria strains 13 and 99 were isolated from a water source in South India. An imipenem-based detection method applied after isoelectric focusing revealed that a β-lactamase with a pI of 5.84 was responsible for carbapenem hydrolysis in strains 13 and 99 and unlike previously reported Aeromonas metallo-β-lactamases this enzyme could be detected with nitrocephin. Purification of this novel enzyme, nominated AVS-1, further demonstrated the unusual properties of this carbapenemase, most notably its insensitivity to EDTA. A metallo-β-lactamase gene was amplified from A. veronii bv. sobria strains 13 and 99 by PCR. Sequencing of the PCR product revealed that these two strains possess a metallo-β-lactamase gene that is closely related to the metallo-β-lactamase gene imiS previously identified in an isolate of A. veronii bv. sobria. Therefore, minor amino acid substitutions may account for the extended substrate specificity and unusual inhibitor profile of AVS-1. Two non-carbapenem-hydrolysing β-lactamases were also cloned from A. veronii bv. sobria strain 13. One of these β-lactamases a clavulanic acid sensitive β-lactamase was found to be an ampS-like penicillinase. The other cloned β-lactamase could unfortunately not be sequenced.
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Bou, Kallaba Malek. "Etude des sites métalliques et modélisation de la réactivité des métallo-β-lactamases par des calculs de chimie quantique." Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTT173/document.
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Les métallo-β-lactamases sont des enzymes qui confèrent aux bactéries qui les synthétisent une résistance aux antibiotiques. La classe B représente les β-lactamases, dans lesquelles le site actif contenant un ou deux atomes de Zn favoriserait l'hydrolyse des antibiotiques. La dégradation des antibiotiques par les enzymes bactériennes est un mécanisme majeur de résistance. L’objectif de ce travail de thèse est de mettre en œuvre des outils de modélisation fondés sur des méthodes de mécanique quantique en vue de déterminer les structures de métallo-β-lactamases avec des inihibiteurs, étape nécessaire pour comprendre ultérieurement quels sont les mécanismes de réaction favorisant la dégradation d’un inhibiteur par des métallo-β-lactamases et fournir des informations qui serviront à mieux interpréter les phénomènes biologiques.Nous avons tout d’abord déterminé les géométries et la stabilité de complexes de coordinations métalliques de systèmes modèles contenant du Zn, comme dans les sites métalliques des métallo-β-lactamases, ou du Cu, complexés à des histidines coordonnées par les Nπ ou Nτ, afin de voir s’il y a une préférence géométrique pour l’une ou l’autre des deux coordinations et voir l’influence de ces différentes coordinations possibles sur les paramètres géométriques au niveau du site métallique. Enfin la présence d’eau et l’influence du solvant aqueux a été étudiée. Grâce à ces méthodes de chimie quantique fondées sur la théorie de la fonctionnelle de la densité, nous avons montré comment ces méthodes permettent d’avoir des informations structurales sur la symétrie adoptée par les centres métalliques, du Zn2+ et du Cu2+. Cette étude structurale nous a permis de mettre en évidence des différences structurales entre ces deux ions métalliques et de déterminer les spectres vibrationnels. Ces investigations nous ont permis de mettre en évidence la nature des liaisons métal-ligand grâce à des approches topologiques. Nous avons montré que ces études préliminaires nous ont permis de choisir la meilleure méthode de calculs DFT pour étudier des centres à zinc dans des structures de β-lactamases.Pour compléter l’étude de structures de métallo-β-lactamases, nous avons déterminé la structure de l’enzyme native L1 (β-lactamase) qui a permis de reproduire les paramètres géométriques des structures expérimentales de L1. Nous avons montré que l’approche combinant des études quantiques et classiques (QM/MM) permet de reproduire avec une très bonne confiance les paramètres structuraux de sites actifs de l’enzyme L1.Enfin, nous avons déterminé les structures de certains sites actifs de la famille B3 des Métallo-β lactamases (Enzyme L1) pour comparer les affinités de différentes familles d’inhibiteurs synthétisées à l’IBM de Montpellier (Institut des Biomolécules de Montpellier) et prédire la structure possible de L1 avec différents inhibiteurs par des méthodes QM/MM pour voir si cette stratégie pourra être appliquée à d’autres inhibiteurs pour des métallo-β-lactamasesMetallo-β-lactamases are enzymes that give the bacteria that synthesize them antibiotic resistance. B Class represents the beta-lactamases, wherein one or two Zn atom(s) promote(s) β-lactams (antibiotics) hydrolysis. The major resistance mechanism is the degradation of the β-lactams by bacterial enzymes called β-lactamases. One major approach to overcome this resistance deals with combination therapy in which a β-lactam drug is given along with a β-lactamase inhibitor, which protects the former from inactivation. The objective of this thesis is to implement modeling tools based on quantum mechanical methods to determine metallo-β-lactamase structures with inhibitors, a step necessary to understand at a later stage the mechanisms of response to the degradation of the inhibitor by β-lactamases and to provide information that will serve to better interpret biological phenomena.We have first determined the geometries and the stability of metal coordination complexes of model systems containing Zn, as in the metallo-β-lactamase metal sites, or Cu, complexed to histidines coordinated by Nπ or Nτ, in order to see if there is a geometric preference for one or the other of the two coordination’s and to see the influence of these different possible coordination’s on the geometrical parameters at the metallic site. Finally, the presence of water and the influence of the aqueous solvent were studied. Using these methods of quantum chemistry based on the density functional theory, we have shown how these methods provide structural information on the symmetry adopted by the metallic centers of Zn2 + and Cu2 +. This structural study allows us to demonstrate structural differences between these two metal ions and to determine the vibrational spectra. These investigations were able to demonstrate the nature of the metal-ligand bonds through topological approaches. We have shown that these preliminary studies have conducted us to choose the best method of DFT calculations for studying zinc centers in β-lactamase structures.To complete the study of metallo-β-lactamase structures, we have determined the structure of the native enzyme L1 (β-lactamase) which permitted to reproduce the geometric parameters of the experimental structures of L1. We have shown that the combination of quantum and classical approaches (QM/MM) allows to reproduce with very good confidence the structural parameters of the L1 enzyme active sites.Finally, we have determined the structures of certain active sites in the B3 family of Metallo-β lactamases (Enzyme L1) to compare the affinities of different families synthesized at IBM in Montpellier (Institute of Biomolecules of Montpellier) and to predict the possible structure of L1 with different inhibitors by QM / MM methods to see if this strategy can be applied to other inhibitors for metallo-β-lactamases
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Verdet, Charlotte. "Organisation génétique des céphalosporinases acquises." Paris 11, 2007. http://www.theses.fr/2007PA114808.
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Les b-lactamases de classe C (AmpC) acquises contribuent à la résistance des entérobactéries aux céphalosporines de 3ème génération. Parmi les six clusters de céphalosporinases acquises décrits dans le monde, trois émergent en région parisienne, principalement chez les espèces Klebsiella pneumoniae et Escherichia coli : les céphalosorinases DHA-1 provenant du chromosome de Morganella morganii, ACC-1 de Hafnia alvei, et CMY de Citrobacter freundii. Les gènes ampC qui codent ces céphalosporinases sont le plus souvent en position plasmidique. Plusieurs structures génétiques sont impliquées dans la mobilisation des gènes blaDHA-1, blaACC-1 et blaCMY : les séquences d’insertion ISCR1 au sein d’un intégron complexe, ou ISEcp1. De nombreux remaniements ultérieurs à la mobilisation de ampC expliquent la variabilité de son organisation génétique : la séquence d’insertion IS26, notamment, semble avoir un rôle important dans la stabilisation de ampC sur un support génétique mobileAcquired class C b-lactamases (AmpC) are responsible for the resistance to third-generation cephalosporins in Enterobacteriaceae. Among the six clusters of acquired cephalosporinases reported in the world, three are emerging in the Paris area, mostly in Klebsiella pneumoniae and in Escherichia coli : the enzymes DHA-1 originating from Morganella morganii, ACC-1 originating from Hafnia alvei and CMY originating from Citrobacter freundii. Acquired cephalosporinases are encoded by ampC gene, mostly plasmid-mediated. Several genetic elements are involved in the mobilization of blaDHA-1, blaACC-1 and blaCMY : insertions sequences ISCR1 associated to a complex integron, or ISEcp1. Then, different genetic rearrangements around ampC may explain the variability of ampC organization : insertion sequence IS26, notably, could have been involved in the stabilization of ampC on a mobile genetic element
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Deschaseaux, Marie-Louise. "Contribution à l'étude de ß-Lactamases : Intérêt comparé des méthodes biochimiques et de l'hybridation moléculaire." Besançon, 1987. http://www.theses.fr/1987BESA2019.
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L'hybridation moléculaire (avec une sonde spécifique de la bêta -lactamase tem) et l'isoélectrofocalisation sont mises en œuvre pour étudier les bêta -lactamases chez 328 bactéries a gram négatif résistant a l'ampicilline ou à la ticarcilline et représentant onze genres différents. L'hybridation moléculaire présente une faisabilité supérieure a celle de l'isoelectrofocalisation et permet la détection de génotypes non exprimes ainsi que l'identification de nouvelles enzymes
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Menif, Chaabene Basma. "Systèmes toxines-antitoxines plasmidiques chez Escherichia coli producteurs de bêta-lactamases à spectre élargi." Paris 6, 2012. http://www.theses.fr/2012PA066663.
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Pour élucider certains facteurs ayant pu favoriser le succès des CTX-M-15, nous avons caractérisé les systèmes TA plasmidiques (pemKI, ccdAB, hok-sok, vagCD, pndAC, parDE, relBE et srn-B) de souches cliniques de 2 collections d’Escherichia coli producteurs de BLSE l’une tunisienne et l’autre française et nous avons montré que dans ces 2 collections, les plasmides IncF hébergeant les CTX-M-15 sont significativement plus riches en système TA (en moyenne plus de 2 systèmes TA) et particulièrement en vagCD, système TA identifié dans un plasmide de virulence de Salmonella. Ce système vagCD était significativement associé aux plasmides IncF hébergés par les souches appartenant au clone mondial O:25 H4 ST131. Nous avons montré aussi que les souches hébergeant les CTX-M-15 de la collection tunisienne sont assez riches en gènes de virulence particulièrement en gènes codant la cytotoxicité (hylA et cnfI) ce qui pourraient contribuer à leur diffusion massive dans nos hôpitaux. D'autre part, nous avons trouvé une forte prévalence des BLSE (30 %) chez les souches d’E. Coli commensales du tube digestif des volailles d’élevage intensif en Tunisie. La quasi-totalité de ces BLSE était plutôt de type CTX-M-1 que CTX-M-15 comme celles isolées chez l’homme et hébergées par des plasmides de groupe d’incompatibilité IncI1 tous renferment le système TA pndAC et rarement vagCD. Finalement, on a obtenu des simples mutants des systèmes TA (ccdAB, pemK et vagCD) du plasmide CTX-M-15 de la souche clonale O25, ST131 (TN03) en utilisant la technique de mutagenèse dirigée de Datsenko et al mais nous n’avons pas trouvé de perte significative des plasmides mutés au cours de la division cellulaireTo better understand the success of CTX-M, and particularly of CTX-M-15, we characterized the plasmid addiction systems (pemKI, ccdAB, hok-sok, vagCD, pndAC, parDE, relBE and srn-BC) in two collections of ESBL-producing Escherichia coli strains, one French and the other Tunisian. We have demonstrated that in both collections the addiction systems, mainly vagCD, were significantly more prevalent in IncF CTX-M-15-carrying plasmids. The vagCD system, which is associated to Salmonella virulence plasmids, was significantly associated to the pandemic ST131 clone. Moreover, the CTX-M-15 producing E. Coli isolates of the Tunisian collection had a high frequency of virulence factors particularly hylA and cnfI. On the other hand, we have detected a high prevalence of ESBLs in faecal samples of healthy poultry in Tunisia. The most common type of ESBLs identified was CTX-M-1, harboured by self conjugative IncI1 plasmids which all comprised the pndAC system and rarely vagCD. Finally, deletion mutants of the TA systems ccdAB, pemK and vagCD harboured by the CTX-M-15 plasmid of a French ST131 strain (TN03) were created using the method by Datsenko et al, but we didn’t observe a significant loss of mutant plasmids during cell division. Therefore, we plan to construct double or triple system TA mutants so that we could evaluate the role of the association of multiple addiction systems in plasmid stabilization
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Lefebvre, Anne-Laure. "Etude de l’activité in vitro des β-lactamines sur Mycobacterium abscessus et recherche de leurs cibles." Thesis, Sorbonne Paris Cité, 2015. http://www.theses.fr/2015USPCB107.
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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. abscessusMycobacterium 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
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Almarghi, Norya. "Characterisation of CTX-M-β-lactamases in Enterobacteriaceaeae in hospitals in Kuwait." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8102.
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Introduction: In this decade, the CTX-M family of enzymes are considered to be the most common type of extended-spectrum β-lactamases (ESBLs). The production of these Class A β-lactamases are noted to be most prevalent in the Enterobacteriaceaeae family. Many global reports indicated that CTX-M-15, of the CTX-M-1 group, is a growing concern, causing resistance to different classes of antibiotics. Worrisome trends of the spread of this enzyme have been indicated in nosocomial and community settings worldwide. Moreover, the same predicament is faced along the Middle East area, especially in the absence of restricted antibiotic usage policies. Many reports from Kuwait indicated the spread of a multi-drug resistant (MDR) blaCTX-M-15 gene in different hospitals. blaCTX-M-15 genes are often known to be carried on large transferable plasmids. Usually, the mobilization of these plasmid-encoded enzymes is carried out by insertion sequence like ISEcp1. Aims: This work aims to investigate the distribution of blaCTX-M genes in five major hospitals in Kuwait and to study and analyse the genetic environment of the described blaCTX-M genes. Materials and methods: One hundred and seven isolates of E. coli (84) (78.50%) and K. pneumoniae (23) (21.49%) were collected between 2006 and 2010 from five distantly located hospitals in Kuwait. All of the collected isolates were identified as ESBL-producers using Vitek 2 system. The production of cefotaximases was detected using disc diffusion with cefotaxime and clavulanic acid according to Clinical and Laboratory Standards Institute (CLSI) criteria. Conformation of CTX-M production was maintained by PCR amplification and further sequencing. The minimum inhibitory concentrations (MICs) of the collected isolates were determined by the double dilutions agar method described by the CLSI. Four different classes of antibiotics were used (aminoglycosides, different generations of cephalosporins, fluoroquinolones, and 3 different carbapenems). The genotypic relatedness of the collected strains was assessed by the use of an enhanced Pulsedfield gel electrophoresis (PFGE) method. Further amplifications with primer walking and simplex PCR were done to seek the genetic context of the MDR strains. S1 nuclease was used to size plasmids carrying the described blaCTX-M genes and conjugation studies were implemented to detect the transferability of the plasmids carrying the reported blaCTX-M genes. Results: All of the collected strains showed to be ESBL-producers and in particular cefotaximases-producers. Upon amplification, CTX-M-1 group was the only CTX-Mgroup present in the collected strains. When sequenced, blaCTX-M-15 was found to be the most prevalent. In addition, strains carrying the blaCTX-M-3 gene were identified, these have previously been found in the Middle East; however, this thesis has the first descriptions of blaCTX-M-28, blaCTX-M-55, and blaCTX-M-117 in this region. After the determination of the MICs of the collected strains, 94 (87.85%) were resistant to cefepime, 107 (100%) to cefotaxime, 48 (44.85%) to cefoxitin, 78 (72.89%) ciprofloxacin, and 71 (66.35%) to gentamicin. All of the strains were susceptible to carbapenems. Twenty-eight strains (26.2%) showed MDR pattern. With the enhanced PFGE method, only 22 isolates exhibited banding patterns that allowed grouping them into 10 distinct PFGE clusters. Notably, strains sharing ≥85% were from the same hospitals (isolates 1 with 2, 21 with 22, and 91 with 92 from the maternity hospital (M), 52 with 53 from Kuwait Oil Company hospital (KOC), 78 with 79 and 83 with 84 from Infectious Diseases Hospital (IDH), 97 with 98 and 95 with 96 from Al-Amiri hospital(A) ). Primer walking and simplex PCR experiments used for the genetic environment studies yielded 7 different genetic constructions for the described blaCTX-M genes. All of the described blaCTX-M genes were carried on plasmids ranging in size from 60 – 271 kb. Only 3 of the selected strains were of IncFII and the rest wereindeterminate. Possibly, two blaCTX-M-15 genes are likely to be carried on the chromosome. All of the described blaCTX-M genes are considered to be transferable except for blaCTX-M-28. The sizes of the conjugative plasmids and incompatibility groups are the same as their parental plasmids. Conclusion: In conclusion, blaCTX-M-15 is the most common ESBL gene found in Kuwaiti hospitals. It is also causing a MDR pattern with resistance to 3 different generations of cephalosporins and to two other classes (aminoglycosides and gentamicin), but sensitive to carbapenems. This led to restricting the treatment option into carbapenems. Antibiotic selective pressure could have played a major role in the development of blaCTX-M-15 derivatives such as blaCTX-M-28, blaCTX-M-55, and blaCTX-M-117. The probable explanation of the spread of blaCTX-M-15 is horizontal gene transfer carried by ISEcp1 and the conjugative properties of the plasmids carrying blaCTX-M-15. Variability of the genetic environments obtained explains the nonconditional existence of ISEcp1 to the ‘’W’’ region. Absence of the ISEcp1 in one of the reported structures of blaCTX-M-15 genetic contexts is noted. Therefore, the existence of blaCTX-M-15 could be due to the presence of another insertion sequences downstream or as a part of a larger gene cassette.
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Alqurashi, Maher Sulaiman M. "Characterization of CTX-M β-lactamases in Enterobacteriaceae from major teaching hospitals." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/8820.
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Escherichia coli and Klebsiella pneumoniae cause a wide range of infections. Multidrug-resistance strains carrying extended-spectrum β-lactamases (ESBLs) has become a growing problem worldwide. The CTX-M type ESBLs has emerged distinctly, especially in Escherichia coli and Klebsiella pneumoniae. CTX-M type has been associated with many outbreaks of infections both in the hospitals and community. CTX-M-15 is now identified as the most predominantly distributed CTX-M enzyme. Clonal outbreaks of CTX-M-15 producing Enterobacteriaceae have been described in many countries including the United Kingdom, and Escherichia coli is the most commonly involved species. A total of 100 isolates were received in 2010 from London St George’s hospital, England, 50 Escherichia coli, 17 Klebsiella spp, 9 Enterobacter spp, 13 Proteus spp, 6 Lactose fermenting coliforms, 2 Pantoea spp, one Serratia marcescens, one Morganella morganii, and one Hafnia alvei. The antimicrobial susceptibility results showed that 5 Escherichia coli and one Klebsiella pneumoniae isolates were found to be resistance to cefotaxime, ceftazidime, ceftriaxone, cefotaxime, ciprofloxacin, and gentamicin, making them multi-drug resistant bacteria. None of the isolates showed resistance to imipenem, ertapenem, or morepenem, thus making carbapenems the drug of choice for the treatment of these infections due to multi-resistant isolates. The overall frequency of CTX-M-15 type ESBL-producers detected in this study was 6 (6%) most of them 5/6 (83%) were from Escherichia coli and one was (17%) Klebsiella pneumoniae isolates. The 6 CTX-M-positive isolates were typed by PFGE, only two strains of Escherichia coli showed more than 85% similarity, owing to clonal homology for both strains. The rest strains showed less than 85% similarity. S1 nuclease plasmid profiles were obtained for ESBL-producers isolates. A total of one to three plasmids per isolate, ranging from approximately 78.0 to 152.0 kb, were observed. The plasmids from most isolates were assigned to be IncFA and IncFB replicons. Analysis of phylogenetic groups showed group A and group B2. The method of phylogenetic classification of exteraintestinal pathogenic Escherichia coli depends on examine and combination of two preserved genes (chuaA and yjaA) and the DNA fragment TSP. Primer walking and PCR experiments were used for the genetic environment studies which showed 5 different genetic constructions for the described blaCTX-M-15 genes. Conjugation studies were used to detect the transferability of the plasmids harbouring the reported blaCTX-M-15 genes. Three isolates were found transferable by conjugation. In conclusion, this study reports the presence of hospital highly resistant blaCTX-M-15 in St George’s hospital. The spread of blaCTX-M-15 is probably due to horizontal gene transfer harbouring ISEcp1 and the conjugative properties of plasmids carrying blaCTX-M-15.
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Oliveira, Filho José Carlos de. "Resistência a antimicrobianos e diversidade de β-lactamases em Escherichia coli de origem aviária." Universidade Federal de Viçosa, 2006. http://locus.ufv.br/handle/123456789/5368.
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The diversity of resistance mechanisms to antimicrobial compounds was investigated in E. coli strains isolated from poultry. All 30 isolates were resistant to ampicillin and 26 of them were resistant to at least two antibiotics. Multi-resistance profiles were confirmed, including intermediate levels. Forty percent of the strains presented four resistance markers and 93% of the isolates were resistant to tetracycline, an antibiotic commonly used in poultry farms. The β-lactamase diversity among the strains was probed against seven compounds. Among strains bearing the same resistance profile for other groups of antimicrobial drugs, it was possible to observe different β-lactamases activity against the tested substrates. This fact suggests either a pool of resistance genes or regulatory differences. The isolate that displayed confirmed activity against six of the β-lactamic antibiotics bears a large molecular mass plasmid that confers the AmpR phenotype. A fragment of this plasmid with approximately 5 kb was subcloned in the pCCR9 vector which is commonly used to detect β-lactamase genes. The amino acid deduced from nucleotide sequencing displayed 100% of identity to TEM-1, a class A serine β-lactamase. Next to the gene encoding TEM-1 was found putative transposon related to the Tn3 family, albeit with some differences in the order and direction of transcription of the genes.
A diversidade dos mecanismos de resistência a antimicrobianos foi investigada em Escherichia coli, originadas de frangos de corte. Dos 30 isolados, todos resistentes à ampicilina, 26 apresentaram mais de uma marca de resistência. Foram confirmados modelos de multirresistência, incluindo níveis intermediários de resistência. Em 40% dos casos, ocorreram quatro marcas e 93% dos isolados resistiram à ação de tetraciclina, um promotor de crescimento usual na agropecuária. A diversidade de β-lactamases entre os isolados foi demonstrada pela ação direta contra sete β-lactâmicos. Os isolados com mesmo modelo de resistência possuíam, sob mesmas condições, espectro de ação e de atividade diferentes, mostrando que há amplo pool de genes de resistência, ou diferenças regulatórias nessas bactérias. O isolado com maior espectro de ação, confirmado sobre seis β-lactâmicos, contém um plasmídeo de alta massa molecular, que confere resistência a ampicilina. Um fragmento deste plasmídeo, com aproximadamente 5 kb, foi clonado em pCCR9, vetor usado para detecção de genes de resistência a β-lactâmicos. As análises das seqüências obtidas revelaram 100 % de identidade com TEM-1, uma serina β-lactamase da classe A. Próximo ao gene codificador da TEM-1, foi encontrado um transposon putativo relacionado com os da família Tn3, porém com particularidades na ordem e direção de transcrição dos genes componentes.
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Madec, Stéphanie. "Résistance des bactéries aux antibiotiques à noyau β-lactame : mécanismes et incidences." Brest, 2001. http://www.theses.fr/2001BRES3105.
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Vernet-Garnier, Véronique. "Recherche des facteurs de virulence (aérobactine et caractère mucoi͏̈de) chez "Klebsiella pneumoniae" : étude "in vivo" de souches isogéniques de K-Pneumoniae et validation d'un modèle septicémique murin à visée thérapeutique." Paris 11, 1992. http://www.theses.fr/1992PA114805.
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Bareyt, Sébastian. "Synthèses et applications de composés hybrides polyoxométallate-substrat biologique." Paris 6, 2005. http://www.theses.fr/2005PA066266.
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Soroka, Daria. "Rôle du motif SDN dans l'inhibition et l'activité des β-lactamases des mycobactéries." Electronic Thesis or Diss., Paris 6, 2016. https://accesdistant.sorbonne-universite.fr/login?url=https://theses-intra.sorbonne-universite.fr/2016PA066432.pdf.
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Mycobacterium tuberculosis et Mycobacterium abscessus produisent les β-lactamases BlaC et BlaMab qui contribuent à la résistance intrinsèque de ces bactéries aux β-lactamines. Notre objectif est de caractériser l’inhibition de ces β-lactamases par l’avibactam et le clavulanate pour contribuer au développement de nouveaux traitements. Nous avons déterminé le profil de substrat et d’inhibition de BlaMab ainsi que sa structure cristalline, révélant trois différences majeures avec BlaC. BlaMab a une activité supérieure à celle de BlaC pour toutes les β-lactamines sauf la céfoxitine qui est utilisée pour les infections dues à M. abscessus. BlaC est inhibée irréversiblement par le clavulanate et inefficacement par l’avibactam alors que BlaMab présente le comportement inverse impliquant une hydrolyse du clavulanate et une inhibition très rapide par l’avibactam. La structure de BlaMab diffère de celle de BlaC principalement par le remplacement du motif conservé SDN par SDG. L’introduction de SDG dans BlaMab et de SDN dans BlaC a montré que cette différence détermine le profil d’inhibition des β-lactamases. Une seule mutation peut donc entraîner l’émergence d’une résistance aux combinaisons d’une β-lactamine avec le clavulanate ou l’avibactam mais pas avec les deux inhibiteurs. L’avibactam et le clavulanate offrent donc des alternatives thérapeutiques en cas de résistance à l’un des inhibiteurs. Nous nous sommes également intéressés aux β-lactamines partenaires du clavulanate, pour le traitement de la tuberculose et montrer que la structure des carbapénèmes pouvait être optimisée pour améliorer l’inactivation des cibles et diminuer l’hydrolyse par BlaCMycobacterium tuberculosis and Mycobacterium abscessus produce the β-lactamases BlaC and BlaMab that contribute to the intrinsic resistance of those bacteria to β-lactams. Our objective was to characterize the inhibition of these β-lactamases by avibactam and clavulanate in order to contribute to the development of new treatments. We have determined the inhibition and substrate profiles of BlaMab, as well as its crystal structure, revealing three major differences with BlaC. BlaMab is more active than BlaC with respect to hydrolysis of all β-lactams except cefoxitin, which is used for the treatment of infections due to M. abscessus. BlaC is inhibited irreversibly by clavulanate and inefficiently by avibactam. In contrast, BlaMab shows the opposite behavior involving hydrolysis of clavulanate and a rapid inhibition by avibactam. Structurally BlaC differs from BlaMab mainly by the replacement of the conserved motif SDN by SDG. The introduction of SDG in BlaMab and of SDN in BlaC revealed that this difference determines the inhibition profile of the β-lactamases. A single mutation can therefore lead to the emergence of resistance to the association of β-lactam with clavulanate or avibactam, but not to both associations. Thus, avibactam and clavulanate offer therapeutic alternatives in case of resistance to one of the two inhibitors. We have also investigated the β-lactam partners of clavulanate for the treatment of tuberculosis and showed that the structure of carbapenems could be optimized to enhance the inactivation of the targets and to reduce hydrolysis by BlaC
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Dimude, Juachi Uzochukwu. "CTX-M β-lactamases and associated integrons : their dissemination in Gram-negative bacteria." Thesis, University of Edinburgh, 2013. http://hdl.handle.net/1842/11729.
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Gram-negative bacteria are able to cause many infections including blood stream infections (BSI).These bacteria may become resistant to antibiotics, often by acquiring genes in the presence of antibiotic selection pressure. Multi drug resistant Gram-negative bacteria have become an increasing problem worldwide. A study of antibiotic resistance in Gram-negative bacteria isolated from blood cultures from patients in the New Royal Infirmary of Edinburgh (NRIE) was performed. In addition, a study was performed on isolates from patients in an intensive care unit in Egypt. All isolates were investigated for susceptibility to an extensive range of antibiotics. Gram-negative bacteria from Edinburgh found to be resistant to either cefotaxime or ceftazidime were investigated further. Among the cefotaxime/ceftazidime resistant isolates, Polymerase Chain Reaction (PCR) analysis revealed the presence of CTX-M- β-lactamases. Seven E.coli isolates were found to have CTX-M-15 β-lactamases while the CTX-M-14 β-lactamase was detected in six Enterobacter cloacae. The insertion sequence ISEcp1 was detected upstream of the blaCTX-M-15 gene in some isolates while IS26 was found truncating the ISEcp1 in other isolates. Conjugation experiments found the blaCTX-M-15 gene was transferable to E. coli J62-2. All the isolates had detectable plasmids, a plasmid ~260kb carried the blaCTX-M-15 gene. Analysis of the -containing isolates by PFGE shows that those carrying the CTX-M-14 β-lactamase were identical indicating cross infection within the hospital. The CTX-M- 15 β-lactamase-containing isolates showed four isolates had ≥85% similarity but the others were diverse. Class 1 integrons were found in eight of the CTX-M β-lactamase containing isolates with the associated gene cassette and sul1 gene. The isolates from Egypt were found to be resistant to carbapenem, which is the final mainstream antibiotic option in the treatment of multidrug resistant Gram-negative bacteria. Further analysis revealed all carried the CTX-M-14 β-lactamase and two additionally carried the VIM-4 metallo β-lactamase, which accounted for the resistance to the carbapenems. Furthermore, the insertion sequence ISEcp1 was found upstream of the blaCTX-M-14 gene in two of the isolates. The blaVIM-4 gene was found to be part of the gene cassette in the class 1 integron associate with complex ISCR1. Two of the Egyptian isolates had a detectable plasmid, ~300kb in size, which carried both blaCTX-M-14 and blaVIM-4 genes. All the blood culture isolates were examined to ascertain the persistence of sulphonamide resistance despite the long-term prescribing reduction on this antibacterial. PCR was performed to detect sul1, sul2 and sul3 genes in all the isolates. Of the sulphonamide resistant isolates 25 carried the sul1, 27 carried the sul2 and none carried the sul3 genes. Eight isolates had both the sul1 and sul2 genes. Most of the isolates carried sul1 had Int1 as part of the same class 1 integron. Interestingly three isolates were PCR negative for sul1 but positive for sul2 and int1. Int2 and 3 were found in 3 and 2 isolates respectively. The class 1 integron contained different insert gene cassettes; dfrA (dfrA17, dfrA16, dfrA15), aadA (aadA5, aadA2, aadA1) and blaOXA-1 families in addition to the resident sul gene. In conclusion this thesis shows the diversity of the genetic environment and carriers of the CTX-M β-lactamases within the same hospital. Sulphonamide resistance in Gram-negatives persists despite the prescribing reduction of this antibacterial in a Scottish hospital and the recommended constraint on the use of sulphonamide.
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Osazee, Joseph Osamudiamen. "Molecular Docking, Synthesis and Evaluation of Pyrrolo[2,1-c][1,4]benzodiazepines Derivatives as Non-β-lactam β-lactamases Inhibitors." Digital Commons @ East Tennessee State University, 2016. https://dc.etsu.edu/etd/3082.
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Our research aim was to design, synthesize, and study the competitive enzyme inhibition kinetics of pyrrolo[2,1-c][1,4]benzodiazepine (PBD) derivatives as potential non-²-lactam ²-lactamase inhibitors. All compounds (1-13) passed the Lipinski’s rule of 5 test and were docked into the active site of TEM-1 ²-lactamase. PBD derivatives 1-7 were synthesized in high yields and tested for their potency against TEM-1 and P99 ²-lactamases. Kinetic data showed that compounds 1, 4, 5, and 7 possessed inhibitory activity against TEM-1 ranging from 4-34 %. Docking results revealed significant interactive spanning of the active site of TEM-1 by PBDs. The limited inhibitory activity of the compounds, 1-7 could be attributed to the lack of solubility and bulky nature of the molecules, thus limiting the optimal ligand-enzyme interactions. 1,2,4- Oxadiazolinones (8-13) were further synthesized to reduce the steric hindrance of the PBD scaffolds while promoting the electrophilicity of the potentially active lactam and also evaluated for potency.
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Correia, Joana Alves Veloso Domingues. "Colonização nosocomial por bactérias de Gram negativo resistentes produtoras de β-lactamases em cães." Master's thesis, Universidade de Lisboa. Faculdade de Medicina Veterinária, 2015. http://hdl.handle.net/10400.5/8492.
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Dissertação de Mestrado Integrado em Medicina VeterináriaOs antibióticos β-lactâmicos são os mais usados, tanto na medicina veterinária como humana. O mecanismo de resistência mais comum a esta classe de antibióticos é a produção de β-lactamases (β-lactamases de espectro alargado - ESBLs, cefalosporinases - AmpCs e carbapenemases) por bactérias de Gram negativo. Os objectivos deste trabalho foram: detectar a presença e quantificar a colonização nosocomial do trato gastrointestinal por bactérias de Gram negativo produtoras de β-lactamases, em cães submetidos a tratamento cirúrgico e identificar quais os factores de risco responsáveis pela colonização e pelo aumento da carga bacteriana. Recolheram-se amostras fecais a 43 cães pertencentes ao grupo de controlo do ambiente (C1ca, n=43) e a 25 cães do grupo de cirurgia: na admissão ao hospital no dia da cirurgia (C1cx, n=25) e após a cirurgia (C2cx, n=22). As recolhas foram realizadas no Hospital Escolar da FMV-ULisboa, entre Fevereiro e Julho de 2014. As amostras foram processadas no Laboratório de Resistência aos Antibióticos e Biocidas (LRAB). Foram isoladas bactérias resistentes às cefalosporinas de 3ª geração (3CG) a partir do meio de MacConkey com 2μg/ml de cefotaxima e bactérias resistentes aos carbapenemos a partir do meio de MacConkey com 2μg/ml de meropenem. Foram realizados testes fenotípicos (Testes de Susceptibilidade aos Antibacterianos [TSA] pelo método de difusão em disco) e testes genotípicos (Polymerase Chain Reactions [PCRs]), de forma a identificar qual o mecanismo de resistência presente nas bactérias isoladas. Nas estirpes resistentes às 3CG, verificou-se um aumento significativo de animais colonizados em C2cx (73%, n=16/22), relativamente a C1cx (P=0,007) e a C1ca (P=0,017). O número de animais colonizados foi também significativamente superior no grupo C1ca (P=0,030) relativamente aos cães em C1cx. As contagens foram significativamente superiores em C2cx comparativamente a C1cx(P=0,004) e a C1ca(P<0,0001), e em C1ca relativamente a C1cx (P=0,0006). Entre os possíveis factores de risco analisados o factor residente (P=0,030) foi o único significativo para o aumento do número de animais colonizados com estirpes resistentes às 3CG e o factor tempo de contacto com a FMV(P=0,001) para o aumento da carga bacteriana. A resistência aos carbapenemos em C1cx foi de 4% (n=1), em C2cx de 23% (n=5) e no grupo C1ca foi de 2% (n=1). As β-lactamases mais prevalentes foram as ESBLs, foram detectadas apenas duas AmpCs plasmídicas e não foram detectadas Carbapenemases. Este trabalho vem reforçar a importância da aplicação de sistemas de controlo de colonização e de infecção nosocomial, para assim melhorar os cuidados de saúde animal e proteger a Saúde Pública.
ABSTRACT – NOSOCOMIAL COLONIZATION BY RESISTANT β-LACTAMASES PRODUCERS GRAM NEGATIVE BACTERIA IN DOGS - β-Lactam antibiotics are the most frequently used both in human and veterinary medicine. The most common resistant mechanism to this class of antibiotics is the production of β- lactamases (Extended Spectrum β-lactamases - ESBLs, Cephalosporinases - AmpCs and Carbapenemases) by Gram negative bacteria. The aim of this study was to detect the presence and quantify nosocomial colonization of the gastrointestinal tract by β-lactamase producing Gram negative bacteria on dogs treated surgically and identify the risks factors responsible for the colonization and the increased bacterial quantification. Fecal samples were collected from 43 dogs belonging to the environmental control group (C1ca, n=43) and 25 dogs from the surgery group: on admission to the hospital before surgery (C1cx, n=25) and after surgery (C2cx, n=22). The samples were collected at the FMV-ULisboa Veterinary Teaching Hospital between January and July 2014. The samples were treated in the FMV-ULisboa Laboratory of Resistance to Antibiotics and Biocids. Bacteria resistant to 3rd generation cephalosporins (3CG) were isolated from MacConkey medium with 2μg/μl cefotaxime and carbapenem resistant bacteria were isolated from MacConkey medium with 2μg/μl of meropenem. Phenotypic tests (Antibacterial Susceptibility Testing using the disk diffusion method) and genotypic tests (Polymerase Chain Reactions) were used in order to identify the resistant mechanism in isolated bacteria. There was a significant increase of 3CG resistant strains colonized dogs in C2cx (73%, n=16/22) comparing to C1cx (P=0,007) and C1ca (P=0,017). The number of colonized dogs was also significantly higher in C1ca group (P=0,030) comparing to the C1cx. The bacteria number were significantly higher in C2cx comparing to C1cx(P=0,004) and C1ca group (P<0,0001), and in C1ca group comparing to C1cx (P=0,0006). The resident risk factor was the only one significant (P=0,030) for the increase of colonized animals with 3CG resistant strains and FMV contact time (since when dogs come to FMV) risk factor was the only one significant (P=0,001) for the increased bacterial quantification. The carbapenem resistance bacteria colonization in the dogs of C1cx was 4% (n=1) and in C2cx was 23% (n=5) and in C1ca group was 2% (n=1). The most prevalent β-lactamases were the ESBLs, only two plasmid mediated AmpC were detected and none Carbapenemases. This study enhances the importance of nosocomial colonization and infection control systems, in order to improve animal health and safeguard Public Health.
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Decamps, Sophie. "Synthèse d'inhibiteurs fluorés de carbapénémases : combattre la résistance aux antibiotiques des bactéries à Gram négatif." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114807.
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Le phénomène de résistance des bactéries à Gram négatif aux antibiotiques est aujourd’hui un problème sanitaire mondial majeur. La production de β-lactamases, et plus particulièrement de carbapénémases, enzymes capables d’hydrolyser la plupart des agents antibactériens de la classe des β-lactames, est le mécanisme de résistance le plus répandu. Dans ce mémoire, nous décrivons la conception et la synthèse de nouveaux inhibiteurs fluorés de carbapénémases. Notre objectif a été la synthèse de monobactames trifluorométhylés en position C4.Nous avons développé une nouvelle voie de synthèse diastéréosélective par expansion de cycle d’aziridines pour l’accès à des 3-bromo-4-CF3-azétidin-2-ones. Ces composés ont été fonctionnalisés par substitution nucléophile, réactions radicalaires et organométalliques en position C3. Dans une seconde partie, des essais de cyclisation de β-hydroxyaminoesters et acides ainsi que de β-hydroxy-hydroxamates ont été entrepris. Pour obtenir ces intermédiaires β-hydroxyaminoesters et acides, nous avons étudié la réactivité des hydroxylamines sur des accepteurs de Michael trifluorométhylés.Enfin, l’évaluation biologique des composés a été réalisée par des tests enzymatiques en suivi par spectroscopie UV. L’évaluation par RMN 19F a également été entreprise, et a permis le développement d’outil de diagnostique et de screening, toujours en cours d’optimisationMultidrug resistant gram-negative pathogens are emerging worldwide. β-lactamases production, especially carbapenemases, enzymes with broad hydrolytic capabilities towards β-lactams, is a global spread mechanism of resistance among gram-negative bacteria. We report here the design and the synthesis of new fluorinated inhibitors of carbapenemases. Our aim was to synthesize trifluoromethylated monobactams in C4 position. We have developed a new diastereoselective pathway by ring expansion of aziridines to access to 3-bromo-4-CF3-azetidin-2-ones. These compounds have been successfully functionalized in C3 position via nucleophilic substitution, radical and organometallic reactions.In a second part, cyclisation attempts of β-hydroxyaminoesters and acids, as well as β-hydroxy-hydroxamates have been conducted. A study of Michael addition of hydroxylamines on trifluoromethylated Michael acceptors have been achieved in order to obtain the β-hydroxyaminoesters and acids derivatives.Finally, biological evaluations of synthesized compounds have been realized through enzymatic tests. 19F NMR evaluation have been accomplished and led to development of diagnostic and screening tools, and it is still in under optimisation
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Foucault, Bruno. "Evolution de la résistance des entérobactéries aux B-lactamines : étude des mécanismes conférant la résistance à l'imipénème dans une souche clinique d'Escheria coli." Paris 5, 1999. http://www.theses.fr/1999PA05P102.
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Kalu, Chimdi Eke. "Synthesis and Evaluation of 1,2,4-oxadiazolidinones: The Search for A Potential Non-β-lactam β-lactamase Inhibitors." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etd/3578.
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β-lactam antibiotics have been the most widely used drug of choice to combat infectious disease caused by bacteria. Unfortunately, their effectiveness is drastically threatened by bacterial β-lactamases. β-lactamases is responsible for the resistance to most antibiotic drugs. For decades, β-lactam β-lactamases inhibitors have been used to reduce bacterial resistance; however, in this study 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 scaffold in its structure, which is configurationally stable and present in other biologically active compounds such as linezolid and avibactam. Oxadiazolidinones were synthesized by treating nitrones with isocyanates. The synthesized compounds were characterized using 1H and 13C NMR, GC-MS, and FTIR. Afterward, they were tested using Nitrocefin as substrate to determine their effectiveness against TEM-1 and P99 serine β-lactamase. Compound 2a-2c, and 3 showed inhibition ranging from 12-38%.
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Fosse, Thierry. "Contribution à l'étude biochimique et génétique des β-lactamases des bactéries anaérobies et des Aeromonadaceae." Nice, 2009. http://www.theses.fr/2009NICE4113.
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Le but de ce travail a été dans une première partie d’effectuer la caractérisation biochimique et génétique des ß-lactamases des bactéries anaérobies du biofilm de la flore buccale : Prevotella spp. (CfxA2), Capnocytophaga spp. (CfxA3 et SPU-1) et Fusobacterium spp. (FUS-1). La mobilité du gène CfxA2 chez Prevotella sp. Est due à un transposon et au plasmide pCAP01 chez Capnocytophaga ochracea. La deuxième partie a permis de caractériser les ß-lactamases produites par Aeromonas spp. Bactérie ubiquitaire de l’environnement : A. Caviae (CAV-1 progéniteur de la ß-lactamase de type ampCplasmidique FOX-1), A. Enteropelogenes (TRU-1), A. Hydrophila (TEM-24 BLSE plasmidique) et aeromonas media (PER-6 nouvelle BLSE). La distribution des trois types de β-lactamases endogènes (classes B, C et D) était dépendante de l’espèceThe aim of the first part of this work was to determine the biochemical and genetic characteristics of β-lactamases of anaerobic bacteria isolated from oral flora biofilm : Prevotella spp. (CfxA2), Capnocytophaga spp. (CfxA3 and SPU-1) and Fusobacterium spp. (FUS-1). The mobility of the CfxA2 gene is due to a transposon in Prevotella species gene and to the pCAP01 plasmid in Capnocytophaga ochracea. In contrast, F. Nucleatum polymorphum (FUS-1) and C. Sputigena (SPU-1) β-lactamases seem non transferable and species related. In the second part, we characterized β-lactamases produced by Aeromonas spp. , an ubiquitous environmental bacterium : a. Caviae (CAV-1 the progenitor of FOX-1 a plasmidic ampC type β-lactamase), A. Enteropelogenes (TRU-1), A. Hydrophila (TEMM-24 a plasmidic ESBL) and aeromonas media (PER-6 a new ESBL). The distribution of the 3 types of endogenous β-lactamases (Classes B, C and D) was species dependent
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Champs, de Saint Léger Christophe de. "Surveillance épidémiologique des entérobactéries productrices de bêta-lactamases à spectre élargi de 1984 à 1990 au CHRU de Clermont-Ferrand." Paris 11, 1991. http://www.theses.fr/1991PA114809.
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Choi, Hwanho. "Investigation on the molecular mechanism of 2OG-oxygenases and serine β-lactamases through computational chemistry." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:871c9408-aa61-4ca2-9eac-42cbb18fbfaa.
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This thesis describes studies on the molecular mechanisms of 2OG-oxygenases and serine β-lactamases, and work towards the decomposition of the thermodynamics of solvation processes using Quantum Mechanics (QM), Molecular Dynamics (MD), and Free Energy Perturbation (FEP) methods. The first chapter gives a brief introduction to quantum chemistry and free energy perturbation methods as applied to the concept of potential energy surface and transition state theory. Chapter 2 describes QM and MD calculations to investigate recyclisation and hydrolysis in avibactam mediated serine β-lactamase inhibition. β-Lactams inhibit penicillin-binding proteins (PBPs) and serine β-lactamases by acylation of a nucleophilic active site serine. Avibactam is approved for clinical use in combination with a cephalosporin, and is a breakthrough non β-lactam inhibitor, which also inhibits via serine acylation. QM and MD calculations on the avibactam-mediated inhibition of a clinically relevant cephalosporinase reveal recyclisation of the avibactam derived carbamoyl complex is favored over hydrolysis. In contrast, analogous recyclization in β- lactam mediated inhibition is disfavored. Avibactam recyclization is promoted by a proton shuttle, a 'structural' water protonating the nucleophilic serine, and stabilization of negative charge on the avibactam derived carbonyl oxygen. The results reveal the potential of calculations for distinguishing between bifurcating pathways and in generating hypotheses for predicting resistance. The inability of β- lactams to undergo recyclization may be an Achilles heel, but one that might be addressed by reversibly binding inhibitors. Chapter 3 describes work using DFT calculation on the selectivity of the hydroxylation of ankyrin repeat domain substrates as catalyzed by a 2-oxoglutarate (2OG) dependent oxygenase. Factor inhibiting hypoxia inducible factor (FIH) is a promiscuous protein hydroxylase that typically catalyses hydroxylations at the C-3 position of protein residues. Amongst residues hydroxylated by FIH, leucine has drawn special interest from a stereochemical perspective. Based on ab initio density functional calculations at the B3LYP/def2-TZVP level of theory, structural and energetic features of the FIH catalysed hydroxylation of L- and D-leucine were investigated. The DFT and experimental results show that L- and D-leucine are oxidized to (3R)-β-hydroxy-L-leucine and (3S)-β- hydroxy-D-leucine, respectively. D-, but not L, -leucine undergoes a double hydroxylation to form β,γ-dihydroxy-D-leucine. The results imply that the difference in the catalytic actions of FIH on L- and D-leucine stems from the different complexation modes in the active site caused by a change in C-α stereochemistry. X-ray crystallography analyses provide evidence for the proposed catalytic mechanisms. Despite the importance of molecular hydration entropy (ΔShyd) in chemical and biological processes, the accurate calculation of ΔShyd is difficult due to the complexities of solute-water interactions. The studies in chapter 4 concerned a novel method for accurately estimating ΔShyd based on a modified thermodynamic decomposition approach. The key feature of the method is that solute-water interactions are decomposed into hydrophobic and electrostatic parts to calculate their respective contributions to ΔShyd. Although free energy perturbation (FEP) methods have been employed widely, the poor convergent behavior of the van der Waals interaction term in the potential function limits its accuracy and robustness. The new method combines the FEP approach and the scaled particle theory (or information theory) to separately calculate the electrostatic solute-water interaction term (ΔSelec) and the hydrophobic contribution (the cavity formation) entropy (ΔScav). The method appears to be effective with a substantial accuracy enhancement in ΔShyd estimation compared to conventional FEP calculations. ΔScav appears to dominate over ΔSelec in magnitude even in the case of polar solutes, implying that the major contribution to the entropic cost for hydration comes from the formation of a solvent-excluded volume. The method thus enhances the accuracy of ΔShyd prediction by complementing the conventional full FEP method.
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Phichith, Denis. "Caractérisation fonctionnelle d'un heptapeptide cyclique inhibiteur d'activités β-lactamases et de protéines liant la pénicilline." Compiègne, 2005. http://www.theses.fr/2005COMP1575.
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A partir d'une enzyme, la β-Iactamase, nous avons généré un anticorps antiidiotypique à activité β-Iactamase que nous avons nommé 9G4H9. En comparant les résidus essentiels à l'activité catalytique de la β-Iactamase, nous avons proposé un modèle de site actif pour l'anticorps 9G4H9 dans lequel nous avons identifié les résidus arginine 24, sérine 26, lysine 27, sérine 28 et acide glutamique en position 98 susceptible d'être impliqués dans l'activité catalytique. Nous avons montré l'implication de tous les résidus identifiés dans l'activité catalytique hormis le résidu lysine en position 27. L'anticorps 9G4H9 a servi de cible pour la sélection d'un peptide inhibiteur par phage-display, nommé Pep90, d'activités β-Iactamases et d'activités parentes. Au cours de cette étude, nous avons caractérisé les modalités d'interaction de Pep90 respectivement avec l'anticorps catalytique 9G4H9, le scFv 9G4H9, différentes classes de β-Iactamases et DD-peptidases9G4H9, a catalytic antibody displaying β-lactamase-Iike activity, has been elicited by the anti-idiotypic approach using β-Iactamase as first antigen. We proposed an active site model for antibody 9G4H9 in which we find residues arginine 24, serine 26, lysine 27, serine 28 and glutamic acid 98 that could be involved in β-Iactamase activity. We showed that ail the residues are involved in catalysis except residue lysine 27. Ln the second part of the work, antibody 9G4H9 was used as target to screen β-Iactamase activity inhibitor among cyclic heptapeptide bank displayed on bacteriophage M13. One of the phage-displayed peptide (pep90) issued from the selection procedures was shown to be a competitive inhibitor of the β-Iactamase activity of the anti-idiotypic antibody, with a Ki = 38uM. We showed that Pep90 interact with several class of penicillin-binding protein, thus opening routes to the design of antibiotic-like molecules
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Cao, Thi Bao Van. "Etude de la résistance acquise aux β-lactamines chez les entérobactéries." Paris 11, 2002. http://www.theses.fr/2002PA114801.
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Siebor, Eliane. "Caractérisation physicochimique et immunologique de ß-lactamases chez les entérobactéries : un modèle d'étude chez le genre Serratia." Lyon 1, 1989. http://www.theses.fr/1989LYO10004.
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Cette etude a porte sur les cephalosporinases chez le genre serratia. Elles sont inductibles et presentent une grande heterogeneite dans leurs constantes cinetiques et leurs points isoelectriques. Dans un but prospectif, des variants resistants synthetisant une cephalosporinase constitutive ont ete obtenus chez differentes enterobacteries, apres exposition a un agent mutagene. La frequence de ces mutants est la plus elevee pour enterobacter cloacae et citrobacter freundii, aussi bien in vitro qu'in vivo. Ils entrainent une resistance plus ou moins importante a l'ensemble des beta-lactamines, a l'exception du mecillinam, de la nf-thienamycine et du cm 40874. La selection d'un mutant constitutif a haut niveau de serratia liquefaciens a permis de determiner les constantes cinetiques de toutes les beta-lactamines. Les antibiotiques non hydrolyses ont ete testes comme inhibiteurs. Le processus d'inactivation de la cephalosporinase par l'aztreonam et le latamoxef est caracteristique des inhibiteurs suicides. L'etude des inhibiteurs de penicillinases a montre que le sulbactam etait egalement un inhibiteur irreversible des cephalosporinases. Contrairement a l'acide clavulanique, il n'est pas inducteur de cephalosporinase et presente une fixation differente sur les proteines de liaison a la penicilline. Dans le cas des etudes immunologiques, seules les methodes de neutralisation de l'activite enzymatique presentent une haute specificite. L'obtention d'anticorps monoclonaux apporte un nouvel interet dans la comparaison phylogenique de beta-lactamases
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Bihan-Avalle, Bérangère. "Utilisation des propriétés du réseau idiotypique pour la genèse d'anticorps catalytiques : production et caractérisation d'anticorps monoclonaux anti-idiotypiques à activité B lactamase." Compiègne, 1995. http://www.theses.fr/1995COMPD841.
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Les propriétés d'image interne des anticorps anti-idiotypiques ont permis d'envisager la génération d'anticorps catalytiques (abzymes) à partir d'un site actif enzymatique. C'est l'activité beta-lactamase qui a été choisie pour modéliser l'approche allant de l'enzyme à l'abzyme. Dans un premier temps, l'anticorps porteur de l'idiotype (Ab1), image en négatif du site actif, est produit. Parmi tous les anticorps monoclonaux anti beta-lactamase caractérises, l'un, l'Ab1 7AF9, est apparu dirigé contre une partie au moins du site actif de la beta-lactamase, puisque l'inhibant partiellement. L'activité inhibitrice de l'anticorps 7AF9 a été caractérisée comme étant de type mixte, à la fois compétitif et non compétitif. C'est cet anticorps qui a été choisi pour induire, chez les souris Biozzi, des anticorps anti-idiotypiques (Ab2), porteurs de l'image interne du site actif de la beta-lactamase et par conséquent, potentiellement catalytiques. Une première évaluation de la capacité de la souris à produire des anticorps à activité beta-lactamase est faite sur les anticorps polyclonaux. L'activité beta-lactamase des antisérums augmente au fur et à mesure de l'immunisation par 7AF9, laissant supposer un lien entre l'apparition d'Ab2 et l'activité enzymatique. La production d'anticorps monoclonaux Ab2 a permis de mettre en évidence une IgG, 9G4H9, au comportement michaelien, présentant une activité d'hydrolyse des céphalosporines (kcat = 2,3 x 10"3 min-1) et des pénicillines (kcat - 0,9 m-1).
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Boutal, Hervé. "Développement et validation de tests de détection rapide de la résistance aux antibiotiques." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS499/document.
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Les béta-lactamines sont les antibiotiques préférentiellement utilisés contre les bactéries Gram négatif responsables d’infections. La dissémination mondiale d’organismes produisant des béta-lactamases à spectre élargi (BLSE) ou des carbapénémases est une préoccupation générale ainsi qu’une menace économique.Parmi ces organismes, les Entérobactéries jouent un rôle important dans les infections nosocomiales (ainsi que les infections communautaires pour E. coli). L’émergence et la dissémination d’Entérobactéries productrices de BLSE (E-BLSE), exprimant principalement des béta-lactamases de la famille des CTX-Ms, et dans une mesure plus inquiétante de carbapénémases (EPC), principalement les enzymes NDM, KPC, IMP, VIM et OXA-48, sont sans le moindre doute un problème de santé publique majeur.Les CTX-Ms hydrolysent les céphalosporines à large spectre et sont les BLSEs principalement rencontrées chez les Entérobactéries lors d’infections urinaires communautaires, mais aussi les bactériémies à E. coli qui peuvent en découler. Ces infections sévères sont traitées avec des carbapénèmes, considérés comme les antibiotiques de dernier recours. Malheureusement, leur utilisation croissante à soumis les entérobactéries à une pression de sélection conduisant à de plus en plus de souches montrant une sensibilité réduite aux carbapénèmes pouvant aboutir à un échec thérapeutique.Si l’on considère les possibilités de traitement limitées pour les E-BLSEs, que les EPCs sont souvent résistantes à plusieurs si ce n’est toutes les classes d’antibiotiques, et que pour certaines peu ou pas de traitements antibiotiques sont disponibles, leur rapide détection et identification sont essentielles. Des tests fiables sont nécessaires pour aider les cliniciens à, rapidement mettre en place des mesures de contrôle de ces infections, adapter les traitements antibiotiques et optimiser les stratégies de soins et leur issue favorable.Lors de la détection des E-BLSEs et des EPCs, il est aussi crucial d’identifier la béta-lactamase impliquée pour la mise en place d’une thérapie adaptée. Les méthodes basées sur la spécificité des anticorps sont sans aucun doute parmi les plus appropriées pour atteindre cet objectif.Pour répondre aux besoins actuels, les méthodes de détection des résistances ont antibiotiques doivent être peu coûteuses (coûts réduits des consommables et des équipements) et facile à mettre en place (technicité faible) pour l’utilisateur. C’est pourquoi nous avons décidé de développer des tests immunochromatographiques qui répondent parfaitement à ce cahier des charges. Pour atteindre cet objectif, nous avons produit des anticorps monoclonaux dirigés contre les CTX-Ms, et les familles de carbapénémases NDM, KPC, et OXA-48. Les tests immuno-chromatographiques correspondants ont été développés et validés. Nos tests sont robustes, facilement transférables dans une version commerciale et stables pour 24 mois sans réfrigération. Ils sont conviviaux, performants en termes de spécificité et sensibilité, et peu couteux, de 7€ (pour un mono-test) à moins de 15€ (pour un multiplex). De plus les résultats sont obtenus dans un court délai sans la nécessité d’un équipement particulier pour la lecture. Nous avons validé un mono-test pour la détection des CTX-Ms du groupe 1, et évalué la détection des groupes 1, 2, 8 et 9 directement dans des échantillons cliniques comme les hémocultures ou l’urine. Des mono-tests pour la détection des NDMs, KPCs et des OXA-48 et un multiplex pour la détection simultanée des cinq principales carbapénémases ont également été validés. Pour ce faire, nous avons utilisés 180 souches isolées sur boites, provenant du Centre National de Référence pour la résistance aux carbapénémases chez les Entérobactéries, dont le contenu en béta-lactamase est caractériséBeta-lactams are antibiotics preferentially used against gram-negative bacilli infections. The worldwide spread of extended spectrum beta-lactamases (ESBL) or carbapenemase-producing organisms is a global concern and also an economic threat.Within those organisms, Enterobacteriaceae have a major role as causes of nosocomial infections (and, for E. coli, also of community-acquired infections). The emergence and dissemination of ESBL-producing Enterobacteriaceae (ESBL-E), mainly expressing beta-lactamases from the CTX-M family, and in a worrier aspect of carbapenemase-producing Enterobacteriaceae (CPE), mainly NDM, KPC, IMP, VIM and OXA-48 like enzymes, are undoubtedly a matter of great public health concern.CTX-Ms hydrolyze broad-spectrum cephalosporins and are the most encountered BLSE in Enterobacteriaceae, and CTX-Ms producers have been reported as the most prevalent ESBL producers in community-onset urinary tract infections (UTIs). Moreover, CTX-M-producing E.coli are a major cause of bloodstream infections that are often secondary to UTIs. These severe infections are treated with carbapenems, considered as last resort antibiotics. Unfortunately, their increasing use put a selective pressure on Enterobacteriaceae, leading to more and more strains showing decreased susceptibility to carbapenems and potentially leading to therapeutic failure.Considering the limited treatment options for ESBL-E and that CPE are often resistant to several if not all classes of antibiotics, and for which very few (or no) antibiotic options remain available, their rapid detection and identification are essential. Reliable tests are needed to help physicians, to quickly provide appropriate infection control measures, to adapt rapidly antibiotic treatment and optimize care strategies and outcomes.While detecting ESBL-Es or EPCs, it is also crucial to identify the implicated beta-lactamase for accurate therapy implementation. To do so, the antibody-specificity based methods are undoubtedly appropriate. To respond to the current needs, antimicrobial drug resistance detection methods must be cheap (reduced costs of consumables and equipment) and easy to use (reduced technical complexity) for the end user, and LFIAs respond to this requirements. Our objective was to develop such tests, and this led us to produce monoclonal antibodies against CTX-Ms, NDM, KPC, IMP, VIM and OXA-48 carbapenemase families and to develop and validate the corresponding LFIAs. Our tests are robust assays, easily transferable in a commercialized version, stable for more than 24 months without refrigeration, user-friendly (no requirement of trained staff), high performance (sensitive and specific), low cost, from 7€ (monotest) to less than 15€ (multiplex). Moreover, the detection results are obtained in short delay without the need for highly technical equipment for the readout.Here, we validated a LFIA for the detection of CTX-Ms (from group 1) and to a wider extent evaluated the direct detection of CTX-Ms from groups 1, 2, 8 and 9 in clinical samples such as blood culture and urine. Mono-tests to detect NDMs and OXA-48-like, and a multiplex for the simultaneous detection of the five main carbapenemases were also validated. These validations were conducted using 180 well characterized isolates in terms of their -lactamase content from the French National Reference Centre for carbapenem-resistant Enterobacteriaceae
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Pérez-García, Pablo [Verfasser], and Wolfgang R. [Akademischer Betreuer] Streit. "Enzyme promiscuity at the origin of metallo-β-lactamases and within the α/β-hydrolase superfamily / Pablo Pérez-García ; Betreuer: Wolfgang R. Streit." Hamburg : Staats- und Universitätsbibliothek Hamburg, 2020. http://d-nb.info/1214370268/34.
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Wackenaer-Descleves, Estelle. "Les β-lactamases chromosomiques des Raoultella spp : support pour la résistance aux antibiotiques et outils de diagnostic étiologique." Paris 5, 2008. http://www.theses.fr/2008PA05T037.
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Les espèces de Raoultella (anciennement Klebsiella). R. Planticola (Rp), R. Ornithinolytica (Ro) et R. Terrigena (Rt) sont difficilement différentiables phénotypiquement des espèces de Klebsiella en routine. Après avoir (i) clone les p-Iactamases des Raoultella (PLA, ORN et TER), (ii) déterminé le niveau d'identité entre elles (94% entre PLA et ORN et 78 % avec TER) et avec les autres p-lactamases de la classe A (70% avec TEM-1, 68% avec SHV-1 et 38% avec KOXY) et (iii) démontré des différences notables entre les activités enzymatiques de PLA et de TER tant entre elles qu'avec TEM-1, la place du gène bla pour l'identification de Rp et Ro a été évaluée sur une large collection d'isolats en comparaison aux gènes rpoB et ADNr 16S. Cette approche diagnostique a permis de découvrir que 70% des isolats Ro sont négatifs pour le test sur lequel a été fondé l'espèce Ro à savoir l'ornithine décarboxylase et que le gène bla via son analyse par RFLP permet de distinguer Ro de Rp sans ambiguïtéThe three species of Raouliellu (formerly Klebsiella). R. Planticola (Rp), R. Ornithinolytica (Ro) and R. Terrigena (Rt) cannot be distinguished from the species of Klebsiella spp. By the tests used in the routine by microbiological laboratories. After having (i) cloned the p-lactamases of the 3 Raoultella species (PLA, ORN and TER), (ii) evaluated the percentage of identity between each other (94% between PLA and ORN, and 78% with TER) and with other class A P-lactamases (70% with TEM-1, 68% with SHV-1 and 38% with KOXY), and (iii) studied the p-lactamase activity of PLA and TER, the reliability of the bla gene for Rp and Ro identification was determined in comparison with that of the 16S rDNA and rpoB genes in 35 Raoultella spp. Isolates. This study allowed us to discover that 70% of the isolates identified as Ro were negative for the ornithine decarboxylase test, meaning negative for the biochemical character on which Ro definition was based, and to develop a new test, bla RFLP. To unambiguously identify Ro and Rp
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Saves, Isabelle. "Evolution de la béta-lactamase TEM-1." Toulouse 3, 1995. http://www.theses.fr/1995TOU30206.
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Les beta-lactamines (penicillines et cephalosporines) representent soixante pour-cent des antibiotiques administres. Le mecanisme majeur de resistance a ces antibiotiques est la production d'enzymes periplasmiques, les beta-lactamases, inactivant les antibiotiques par hydrolyse du cycle beta-lactame. La beta-lactamase tem-1 est tres largement repandue dans les differentes especes bacteriennes. Elle hydrolyse tres efficacement les penicillines, mais pas les cephalosporines de troisieme generation, et elle est sensible a l'inhibition par l'acide clavulanique. Cependant, elle evolue rapidement. La substitution d'un petit nombre d'acides amines peut lui permettre d'hydrolyser les cephalosporines, ou de devenir resistante aux inhibiteurs. Le mecanisme d'hydrolyse des substrats comprend une etape d'acylation de la serine catalytique par le cycle beta-lactame, puis une etape de deacylation liberant l'enzyme native et l'antibiotique degrade. Dans un premier temps, une methode de determination des constantes de vitesses elementaires d'acylation et de deacylation a ete mise au point, en couplant la spectrophotometrie et la spectrometrie de masse par ionisation electrospray. Dans un deuxieme temps, cette methode, combinee a l'etude cinetique classique, a permis la comparaison des vitesses d'hydrolyse des penicillines et des cephalosporines par l'enzyme sauvage et des mutants naturels. Cette demarche combinee a la determination cristallographique de la structure tridimensionnelle de mutants, et a la modelisation moleculaire, a partir de la structure de tem-1, a permis de mettre en evidence le role essentiel de la boucle omega, formant un cote du site actif, dans l'evolution de tem-1 vers l'hydrolyse des cephalosporines de troisieme generation. D'une facon similaire, l'etude cinetique de tem-1 et de mutants resistants a l'inhibition, la spectrometrie de masse, la modelisation moleculaire et le calcul electrostatique, ont ete combines afin de confirmer differentes hypotheses sur le mecanisme d'inhibition de tem-1, puis d'expliquer le mecanisme d'evolution de tem-1 vers la resistance a l'inhibition
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Mabilat, Claude. "Contribution à la caractérisation moléculaire des bêta-lactamases de type TEM à spectre élargi et à l'analyse de leur support génétique chez les entérobactéries." Paris 11, 1992. http://www.theses.fr/1992PA114808.
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Reynaud, Alain. "Bêta-lactamase à biogenèse chromosomique de Klebsiella oxytoca D488 : paramètres d'activité enzymatique et séquence protéique." Paris 11, 1991. http://www.theses.fr/1991PA114807.
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Pedrosa, Zilmara Vieira. "Atividade do coriandrum sativum l. sobre cepas de escherichia coli produtoras de β-lactamases de espectro estendido." Universidade Federal da Paraíba, 2014. http://tede.biblioteca.ufpb.br:8080/handle/tede/6817.
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There are some strains of Escherichia coli that are pathogenic and often cause urinary tract infections, septicemia and meningitis in neonates. Some produce enzymes known as β-lactamases of extended spectrum (ESBL) that decrease the therapeutic options, thereby arousing interest in the discovery of new antibacterial products. Medicinal plants and their essential oils are especially rich in metabolites with antimicrobial properties as soon researches are developed in searches of these substances. Thus, the present study aimed to evaluate the effect of essential oil of Coriandrum sativum and its major phytochemicals against strains of E. coli ESBL. Was initially determined the sensitivity of the tested strains to conventional antibiotics, as well as screening of the antibacterial activity of the oils against these strains. Chosen C. sativum oil, its chemical composition was determined by gas chromatography coupled to mass spectrometer (GC/MS) and the antibacterial activity of both the oil as the major phytochemicals were evaluated by determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC), the microdilution technique, the kinetics of microbial death oil was also analyzed. It has also played a modulating effect over the conventional antibiotics. It was found that the tested strains are producing ESBL. Among the phytochemicals, linalool (39.78%) presented itself as a major component, followed by linalool oxide (27.33%). The oil and linalool inhibited all strains having MIC and MBC of 256 μg/mL, 512 μg/mL and 1024 μg/mL, between 512 and 1024 μg/mL, respectively. Linalool oxide was inactive at the maximum test concentration of 1024 μg/mL. This oil has antibacterial activity dependent on its concentration and time of exposure in the micro-organism. Both oil and linalool modulates the action of the antibiotics ciprofloxacin and cefoxitin, synergistic interaction of oil and ciprofloxacin being observed. The combination with linalool showed synergistic effects with ciprofloxacin and norfloxacin. The results suggest that the essential oil of C. sativum can suppress the growth of species E. coli ESBL and its major phytochemical despite having weak antibacterial activity, may modulate the action of some antibiotics.
Existem algumas cepas de Escherichia coli que são patogênicas e frequentemente causam infecções urinárias, septicemias e meningites em neonatos. Algumas produzem enzimas conhecidas como β-lactamases de espectro estendido (ESBL) que diminuem as opções terapêuticas, despertando assim o interesse pela descoberta de novos produtos antibacterianos. As plantas medicinais e principalmente seus óleos essenciais são ricos em metabólitos com propriedades antimicrobianas, logo pesquisas são desenvolvidas em buscas destas substâncias. Assim, o presente estudo teve como objetivo avaliar o efeito do óleo essencial de Coriandrum sativum e seus fitoconstituintes majoritários contra cepas de E. coli ESBL. Inicialmente foi determinado o perfil de sensibilidade das cepas ensaiadas a antibióticos convencionais, bem como a triagem da atividade antibacteriana dos óleos contra estas cepas. Escolhido o óleo de C. sativum, a sua composição química foi determinada por Cromatografia Gasosa acoplada a Espectrômetro de Massa (CG/EM) e a atividade antibacteriana tanto do óleo quanto dos fitoconstituintes majoritários foram avaliadas pela determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM), pela técnica de microdiluição, a cinética de morte microbiana do óleo também foi analisada. Verificou-se também o efeito modulador desempenhado sobre os antibióticos convencionais. Constatou-se que as cepas ensaiadas são produtoras de ESBL. Entre os fitoconstituintes, o linalol (39,78%) apresentou-se como principal componente, seguido pelo óxido de linalol (27,33%). O óleo e o linalol inibiram todas as cepas apresentando CIM e CBM de, 256 μg/mL, 512 μg/mL e 1024 μg/mL, entre 512 e 1024 μg/mL, respectivamente. O óxido de linalol não apresentou atividade na concentração máxima ensaiada de 1024 μg/mL. Este óleo possui atividade antibacteriana dependente de sua concentração e do tempo de exposição no micro-organismo. Tanto o óleo quanto o linalol modulou a ação dos antibióticos ciprofloxacina e cefoxitina, sendo observada interação sinérgica do óleo e a ciprofloxacina.Já a combinação com linalol, apresentou efeitos sinérgicos com a ciprofloxacina e norfloxacina. Os resultados sugerem que o óleo essencial de C. sativum pode suprimir o crescimento de espécies de E. coli ESBL e que seu fitoconstituinte majoritário apesar de ter atividade antibacteriana fraca, pode modular a ação de alguns antibióticos