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.

The ß-lactam antibiotics are the most important antibacterial agents in the treatment of infectious diseases. A severe problem in ß-lactam therapy is the emergence of ß-lactam resistant bacteria. Clinical ß-lactam resistance is most often due to the production of ß-lactamases. ß-lactamase genes reside either on plasmids or on the chromosome. The aim of this study was to acquire an understanding of organisation and regulation of chromosomal ß- lactamase genes in different Gram negative species and to elucidate the mechanisms for ampC hyperproduction in the in vivo situation. By DNA hybridization with an ampC probe from Escherichia coli K-12 it was shown that other Gram negative bacteria contained an artpC like chromosomal gene, suggesting a common evolutionary origin. Furthermore, the preceding frd operon that overlaps the ampC gene in E. coli K-12 was found to be much more conserved than the ampC gene in the bacterial species investigated. The ampC and frd opérons in Shigella sonnei and Citrobacter freundii were cloned and characterized by physical mapping. The respective maps were compared to the ampC and frd region in E. coli K-12. The physical map of Sh. sonnei was almost identical to the E. coli K-12 map, whereas in C. freundii only the frd region exhibited any considerable homology. Moreover, in C. freundii, the anpC and frd regions were separated by 1100 basepairs. It is suggested that this DNA is involved in the induction of ß-lactamase production in this organism. A hypothesis for the evolution of the anpC operon in enterobacteria is presented. By isolating and characterizing six ß-lactam resistant clinica], isolates of E. coli hyperproducing the dhrcmosomal ß-lactamase, genetic mechanisms for in vivo evolved resistance was aimed at. These isolates exhibited a 24-48 fold increase in ß-lactamase production. The ß-lactamase produced was found to be biochemically and immunologically identical to the ß-lactamase produced by E. coli K-12. The ampC control region of these six E. coli isolates was DNA-seqenced. The cause of ß-lactamase hyperproduction in five of the clinical E. coli isolates, identical in the DNA segment sequenced, was due to a strong novel ampC promoter displaced 5 bp upstream of the ampC promoter defined in E. coli K-12. The ß-lactamase hyperproduction in the sixth clinical isolate was shown to be caused by two mutations affecting both the promoter and the attenuator in the regulatory region defined by E. coli K- 12. The obtained changes were sufficient to explain the increase in ampC ß- 1 act ama se expression exhibited in these clinical E. coli isolates. Sequence analysis of the ampC control region in Sh. sonnei revealed that it was, with one exception, identical to the one found in the five clinical E. coli ß-lactamase hyperproducers. The only difference was in a position that creates the strong novel ampC promoter in the E. coli hyperproducers. By isolating spontaneous Sh. sonnei mutants with a 40-fold increase in ß-lactamase production carrying the same novel ampC promoter as the clinical E. coli isolates it was concluded that this DNA segment has been transferred in vivo frcm Shigella to E. coli across the species barrier. / <p>Diss. (sammanfattning) Umeå : Umeå universitet, 1983; Härtill 5 uppsatser</p> / digitalisering@umu

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.

Made available in DSpace on 2015-07-22T20:50:06Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-09-29 / Centro de Integração Empresa Escola (CIEE) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação de Apoio e Desenvolvimento ao Ensino, Pesquisa e Extensão Universitária no Acre (FUNDAPE) / Nesta dissertação são apresentados dois estudos envolvendo isolados clínicos de Acinetobacter baumannii que apresentaram mecanismos enzimáticos de resistência aos carbapenêmicos, a produção de carbapenemases do tipo OXA. O primeiro estudo avalia a relação genética de amostras de A. baumannii multirresistentes produtoras de OXA-23 provenientes de distintas cidades brasileiras. 91 amostras clínicas de A. baumannii foram isoladas em 17 centros médicos localizados nas cidades de São Paulo (SP), Rio de Janeiro (RJ), Belo Horizonte (MG), Porto Alegre (RS), Blumenau (SC), Curitiba (PR), São Luiz (MA) e Salvador (BA). Nesta coleção observamos altas taxas de resistência aos carbapenêmicos (91.2%) e presença do gene blaOXA-23-like em 83,5% dos isolados. O elemento de insersão ISAba1 à montante ao gene blaOXA-23 foi detectado em todos os 76 isolados de A. baumannii produtores de OXA-23. Nove grupos de genótipos foram observados entre os 76 isolados produtores de OXA-23. Nossos achados sugerem que o gene blaOXA-23 presente nestes isolados estava localizado DNA cromossomal. Três principais grupos (A, B e D) foram observados circulando em seis cidades das regiões sudeste e sul do Brasil, sendo que o genótipo predominante (A) apresentou relação clonal àquele primeiramente descrito na cidade de Curitiba, em 2003. Em contrapartida foi encontrado um único genótipo de A. baumannii produtor de OXA-23 na cidade de São Luís. Embora existam estudos brasileiros prévios reportando a disseminação de clones de A. baumannii pordutores de OXA-23 localmente, nenhum estudo brasileiro demonstrou antes, i) a análise comparativa dos genótipos originários de diferentes e distantes cidades brasileiras, ii) a provável localização do gene blaOXA-23 e iii) a presença do elemento ISAba1 à montante ao gene blaOXA-23,o que pode ter levado ao alto grau de resistência aos carbapenens observado. Desta maneira, o estudo demonstra a circulação de clones de A. baumannii produtores de OXA-23 no Brasil e enfatiza que medidas de controle de infecção são urgentemente necessárias para reduzir tanto a disseminação de isolados multirresistenes quanto o número de infecções causadas por este patógeno. O segundo estudo trata de um relato de caso de um paciente internado em um hospital da cidade de São Paulo, que apresentou uma infecção por A. baumannii produtor de OXA-72. O isolado em questão, A 30235, apresentou resistência a todos os antimicrobianos testados, com excessão à ampicilina/sulbactam tendo apresentado redução da sensibilidade a esta associação. Foi realizada com sucesso a transformação do plasmídeo presente no isolado A30235 na cepa de A. baumannii ATCC 19606. A confirmação da presença do gene blaOXA-72 no transformante evidenciou a localização plasmidial deste determinante de resistência. O plasmídio contendo o gene blaOXA-72 apresentou um peso molecular de aproximadamente 86 Kb. Neste estudo identificamos pela primeira vez no Brasil, um isolado clínico de A. baumannii produtor de OXA-72. A presença deste determinante de resistência codificado por um gene localizado em um elemento genético móvel demonstra a crescente diversidade de carbapenemase do tipo OXA no Brasil com potencial de disseminação. Medidas de controles adequadas deverão ser tomadas para evitar a disseminação de isolados produtores de OXA-72 entre os hospitais brasileiros, o que tem ocorrido com os isolados de A. baumannii produtor de OXA-23. / In this dissertation we present two studies involving carbapenem-resistant Acinetobacter baumannii clinical isolates due to the production of carbapenems modifying enzymes, the OXA-type carbapenemases. The first study aimed to determine the genetic relationship of multi-drug-resistant A. baumannii producing OXA-23 that was isolated in distinct Brazilian cities. A total of 91 A. baumannii clinical isolates were recovered from 17 medical centers located at eight cities, namely São Paulo (SP), Rio de Janeiro (RJ), Belo Horizonte (MG), Porto Alegre (RS), Blumenau (SC), Curitiba (PR), São Luiz (MA) and Salvador (BA). In this collection we observed high rates of carbapenems resistance (91.2%). Also, the blaOXA- 23-like gene was present in 83.5% of isolates. The insertion sequence ISAba1 was positioned upstream the blaOXA-23 gene in all OXA-23-producing A. baumannii identified. Nine clusters were observed among OXA-23 producers. Our fidings suggest that the blaOXA-23 gene was probably chromosomally-located in all isolates studied. Three clusters (A, B and D) were found in six cities from southeast and southern reagions of Brazil. In addition, the predominant cluster (A) was clonally related to that first described in Curitiba, in2003. In contrast, a single cluster of A. baumannii producing OXA-23 was found in São Luís city. Although there were previous reports regarding the spread of OXA-23-producing A. baumannii in Brazil the following features had not yet been assessed: i) the comparative analysis of OXA-23 producers genotypes originating in distinct and distant Brazilian cities, ii) the genetic location of the blaOXA-23 gene and iii) the presence of ISAba1 upstream blaOXA-23 probably resulting in high degree of carbapenem resistance. Thus, our study demonstrates that the clonal dissemination of OXA-23- producing A. baumannii had occurred in Brazil. These findings emphasize that infection control measures are urgently needed to reduce both the spread of multidrug resistantstrains and the number of infections caused by this pathogen. The second study refers to a case report involving a hospitalized patient that presented an wound infection due to OXA-72-producing A. baumannii. The referred clinical isolate, A 30235, was resistant to most antibiotics tested and showed reduced susctptibility to ampicillin/sulbactam. Successful transformation assays using A. baumannii ATCC 19606 as the recipient strain revealed the plasmid location of the blaOXA-72 gene. This plasmid showed molecular weight of about 86 Kb. We identified for the first time in Brazil, an A. baumannii clinical isolate producing OXA-72. The presence of this resistance determinant encoded by a gene located in a mobile genetic elemet, points out to an increasing diversity of OXA-type carbapenemase in Brazil with potential spread. Appropriate control measures should be taken to prevent the spread of OXA-72 producers among Brazilian hospitals, which it we have experienced with OXA-23- producing-A. baumannii in this country. / TEDE / BV UNIFESP: Teses e dissertações

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

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

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In dieser Arbeit wurden zwei Aspekte der Yersinia β-Laktamasen bearbeitet: (1) Charakterisierung der β-Laktamasen hinsichtlich β-Laktam-Antibiotikaresistenz, Sekretion und Thermostabilität. (2) Untersuchung der Sekretionsfähigkeit von verschiedenen thermostabilen β Laktamasen über das Yersinia T3SS. Im ersten Teil wurden β Laktamase-Deletionsmutanten im Y. enterocolitica Serotyp O:8 Stamm WA-314 hergestellt, um den Einfluss der chromosomalen β Laktamasen auf die in vitro-Resistenz zu untersuchen. Es konnte gezeigt werden, dass WA-314 konstitutiv BlaA produziert und BlaA somit – unter nicht-induzierbaren Bedingungen – der dominante Faktor in der in vitro-Resistenz gegenüber Penicillinen mit erweitertem Wirkungsspektrum (z.B. Ampicillin) und Cephalosporinen der 1. Generation (z.B. Cefazolin) ist. Weiterhin konnte gezeigt werden, dass die zweite chromosomale β Laktamase AmpC (BlaB) unter Zugabe von subinhibitorischen Konzentrationen von Imipenem stark induziert wird. Keine der β Laktamasen ist in der Lage, in vitro-Resistenz gegenüber Carbapenemen und Monobactamen zu vermitteln. Die Konstruktion und Bestimmung der in vitro Antibiotika-Empfindlichkeit der β Laktamase-Deletionsmutanten dient als Grundlage für nachfolgende Untersuchungen im Mausinfektionsmodell. Weiterhin wurden die Transporteigenschaften beider β Laktamasen untersucht. In Gram-negativen Bakterien sind reife β Laktamasen im Periplasma lokalisiert und müssen somit nach der Synthese im Cytosol über die Cytoplasmamembran transportiert werden. Bis auf drei Ausnahmen (β Laktamasen aus Mycobacterium smegmatis, M. tuberculosis und Stenotrophomonas maltophila) sind bisher nur Sec-abhängige β Laktamasen beschrieben worden. Mittels Fusionsproteinen bestehend aus β Laktamase-Signalpeptiden und GFP konnte in dieser Arbeit eindeutig gezeigt werden, dass es sich bei Yersinia BlaA um ein Tat-Substrat handelt, bei Yersinia AmpC hingegen um ein Sec-Substrat. Somit konnte im Rahmen dieser Arbeit zum ersten Mal eine Tat-abhängige β Laktamase bei einer Bakterienart aus der Familie der Enterobacteriaceae nachgewiesen werden. Außerdem konnte gezeigt werden, dass die β Laktamase BlaA nicht diffus im Periplasma, sondern auf bestimmte Bereiche im Periplasma lokalisiert verteilt ist. Allerdings konnte die Art der Lokalisierung bisher nicht genau spezifiziert werden. Die cytosolische Faltung und die Tat-abhängige Translokation von BlaA lassen vermuten, dass eine besondere Thermostabilität von BlaA vorliegt. Deshalb wurde das BlaA-Enzym hinsichtlich seiner Thermostabilität und temperaturabhängigen enzymatischen Aktivität untersucht. Im Vergleich zur E. coli β Laktamase TEM-1 und der hitzestabilen TEM-1-Variante MEGA zeigte BlaA eine erhöhte Thermostabilität und einen starken Anstieg der Aktivität in einem Temperaturbereich zwischen 30 °C und 45 °C. Im zweiten Teil dieser Arbeit wurde geprüft, ob die charakterisierten Yersinia β Laktamasen als Reporterkonstrukte zur Untersuchung des Typ III Sekretionssystems (T3SS) geeignet sind. Y. enterocolitica besitzt ein pYV Virulenzplasmid, auf dem der vollständige Satz der Gene für das Ysc-T3SS und die Effektor-Yops (Yersinia outer protein) lokalisiert sind. Injektion der Yops in eukaryotische Zielzellen ermöglicht das extrazelluläre Überleben der Yersinien im Wirtsorganismus. Bei YopE handelt es sich um ein gut charakterisiertes Effektor-Yop, dessen N Terminus fusioniert an den reifen Teil der β Laktamase TEM-1 bereits vielfach als Reporterkonstrukt eingesetzt wurde. Unter Verwendung des fluoreszierenden β Laktamase-Substrats CCF4-AM kann die Translokation von YopEi-TEM-1 in Zielzellen in Zellkultur-Experimenten und im Mausinfektionsmodell visualisiert werden. In dieser Arbeit sollte deshalb die T3SS-Sekretionsfähigkeit von YopE-β Laktamase-Fusionsproteinen in Abhängigkeit von der „Schmelztemperatur“ (temperaturabhängige Stabilität, TM) untersucht werden. Yop-Substrate werden im ungefalteten Zustand (YscN wirkt dabei vermutlich als ATP-abhängige „Unfoldase“) über das Ysc-„Injektisom“ transloziert. YopEi-TEM-1 wird effizient sekretiert und transloziert (TM (TEM-1) = 50,8 °C). YopE-Fusionsproteine mit thermostabilen TEM-1 Varianten, YopEi-RLT bzw. YopEi-MEGA (TM (RLT) = 60,4 °C; TM (MEGA) = 69,2 °C) werden hingegen nur schwach bzw. nicht sekretiert. Weiterhin konnte gezeigt werden, dass die Sec-abhängige β Laktamase AmpC als YopE-Fusionsprotein (YopEi-AmpC) effizient T3SS-abhängig sekretiert und transloziert werden kann; das native Tat-Substrat BlaA (YopEi-BlaA) kann jedoch weder sekretiert noch transloziert wird. Eine mögliche Erklärung wäre, dass die ATPase YscN nicht in der Lage ist, BlaA und die thermostabilen TEM-1-Varianten zu entfalten und über das T3SS zu sekretieren und zu translozieren. RLT und MEGA können hingegen mithilfe ihrer nativen Signalsequenz über das Sec-System (und somit im ungefalteten Zustand) transloziert werden. / In this work, two aspects concerning the Yersinia β lactamases have been processed: (1) Characterization of the β lactamases in regard of β lactam antibiotic resistance, secretion and thermostability. (2) Analysis of the secretability of different thermostable β lactamases via the Yersinia T3SS. In the first part of this work we described the β lactam sensitivity pattern of the highly mouse pathogenic Y. enterocolitica strain WA 314 by creating a set of β lactamase deletion mutants and their subsequent characterization in vitro. In accordance to previous studies on biovar 1B, serovar O:8 strains, WA 314 produces both enzymes BlaA and AmpC (BlaB) and latter one is inducible using subinhibitory concentrations of imipenem. BlaA is dominant in providing in vitro-resistance towards extended-spectrum β lactams (e.g. ampicillin) and 1st generation cephalosporins (e.g. cefazolin). None of the β lactamases is able to mediate in vitro-resistance towards carbapenems and monobactams. The construction of β lactamase deletion mutants and their in vitro-characterization is the basis for their subsequent analysis within a murine infection model. Furthermore, we investigated the export mechanism of both β lactamases. In Gram-negative bacteria β lactamases are localized in the periplasmic space to be able to cleave the amide bond of the β lactam ring resulting in inactivation of the antibiotic. Proteins are translocated across the inner membrane either via the general secretory pathway (Sec system) or via the twin arginine pathway (Tat system). To date, only three β lactamases have been described as Tat-dependently translocated namely BlaC from Mycobacterium tuberculosis, BlaS from M. smegmatis and L2 from the plant pathogen Stenotrophomonas maltophila. In silico studies and experimental approaches lead to the assumption that the majority of β lactamases is translocated in a Sec-dependent manner. Analysis of β lactamase signal sequence-GFP fusion proteins revealed that Yersinia AmpC is a Sec-substrate whereas Yersinia BlaA is a Tat-substrate. Thus, BlaA of Y. enterocolitica is the first reported β lactamase within the family of Enterobacteriaceae which is secreted by the Tat system. Interestingly, closely related blaA genes are widely distributed within all Y. enterocolitica biovars and several environmental Yersinia species. It is generally accepted that Tat-dependent substrates rapidly fold in the cytoplasma. To compare the BlaA protein stability with that of the Sec-dependent β-lactamases of the TEM-1 family we determined thermal unfolding transitions and temperature dependent enzymatic activities. In contrast to TEM-1 β lactamase, BlaA shows an entire different activity profile with steep increase of activity in the temperature range of 30 °C to 45 °C and steep decrease between 50 °C to 60 °C. In the second part, the Yersinia β lactamases AmpC and BlaA characterized in this work were tested for suitablility as reporter constructs for analyzing the Ysc-T3SS. Y. enterocolitica harbors a pYV virulence plasmid which encodes genes for components of the Ysc-T3SS secretion machinery and the effector Yops (Yersinia outer protein). Injection of Yops into the eukaryotic host cell enables the extracellular survival of Yersinia within the host organism. YopE is a well described effector Yop and YopE-β lactamase (TEM-1) fusion proteins have been successfully applied to analyze translocation of YopE in cell culture experiments and in the murine mouse infection model by using the fluorescent β lactamase substrate CCF4-AM. In this work, we aimed to analyze the T3SS-dependent secretability of YopE-β-lactamase fusion proteins in dependency on the melting temperature (temperature dependent stability, TM). It has been described that Yop substrates are translocated in an unfolded conformation via the Ysc-“injectisom” (YscN has been discussed as an ATP-dependent unfoldase). YopEi-TEM-1 is efficiently secreted and translocated (TM (TEM-1) = 50.8 °C). However, YopE fusion proteins with heat stable TEM-1 variants, YopEi-RLT and YopEi-MEGA (TM (RLT) = 60.4 °C; TM (MEGA) = 69.2 °C), were weakly secreted or secretion is completely abolished, respectively. Furthermore we could show that a YopE fusion protein with the Sec-dependent β lactamase AmpC (YopEi-AmpC) is efficiently secreted and translocated via the T3SS. In contrast, the Tat-dependent β lactamase BlaA (YopEi-BlaA) can neither be secreted nor translocated T3SS-dependently. A putative explanation for that observation would be that the ATPase YscN is unable to unfold BlaA and the heat stable TEM-1 variants and therefore secretion and translocation is prevented. Interestingly, native RLT and MEGA β lactamases can be translocated in an unfolded conformation via the Sec system.

Les DD-peptidases sont des enzymes impliquées dans la synthèse de la paroi bactérienne. Elles sont aussi appelées "Penicillin Binding Proteins" (PBPs) car elles forment des acyl-enzymes stables avec des antibiotiques de type beta-lactames comme la pénicilline, la stabilité de ces complexes étant à l'origine de l'effet antibiotique. Certaines bactéries sont résistantes aux b-lactames grâce à la production de beta-lactamases, capables d'hydrolyser ces antibiotiques jusqu'à 10E8 fois plus rapidement que les PBPs selon un mécanisme impliquant deux étapes, d'acylation puis de désacylation. Il est généralement accepté que les beta-lactamases ont évolué à partir d'une PBP ancestrale en intégrant au site actif un mécanisme catalytique efficace pour la réaction de déacylation. L'objectif de cette thèse s'inscrit dans la compréhension des mécanismes d'évolution de la catalyse enzymatique au niveau moléculaire. Nous tenterons de reproduire un mécanisme évolutif en créant in vitro une activité b-lactamase à partir d'une DD-peptidase. La protéine de départ pour ce travail est la PBP-A de Thermosynechococcus elongatus, appartenant à une nouvelle famille de PBPs homologue aux beta-lactamases de classe A. L'analyse biochimique de cette protéine suggère que c'est une DD-peptidase, et une approche rationnelle par substitution d'un résidu dans le site actif de PBP-A a permis d'augmenter la vitesse de désacylation de l'acyl-enzyme formé avec la pénicilline d'un facteur 90. L'analyse des structures 3-D de PBP-A sauvage et mutante laisse ouverte la question de savoir comment évoluer cette PBP en beta-lactamase. Ainsi, pour l'évolution dirigée de cette protéine, deux banques ont été construites par approches aléatoire et semi-rationnelle. Il a été possible de sélectionner par "phage display" des enzymes améliorées pour la réaction d'acylation. L'obtention de mutants améliorés pour l'acylation ou la désacylation constitue des premiers pas vers l'évolution de PBP-A en beta-lactamase.