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Macrolide resistance mechanisms in Enterobacteriaceae: Focus on azithromycinGomes, Cláudia, Martínez Puchol, Sandra, Palma, Noemí, Horna, Gertrudis, Ruiz-Roldán, Lidia, Pons, Maria J, Ruiz, Joaquim 27 October 2016 (has links)
From its introduction in 1952 onwards, the clinical use of macrolides has been steadily increasing,
both in human and veterinary medicine. Although initially designed to the treatment of Grampositive
microorganisms, this antimicrobial family has also been used to treat specific Gram-negative
bacteria. Some of them, as azithromycin, are considered in the armamentarium against
Enterobacteriaceae infections. However, the facility that this bacterial genus has to gain or
develop mechanisms of antibiotic resistance may compromise the future usefulness of these antibiotics
to fight against Enterobacteriaceae infections. The present review is focused on the mechanisms
of macrolide resistance, currently described in Enterobacteriaceae.
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Functional analysis of the two subunits of DNA methyltransferase EcoHK311. / CUHK electronic theses & dissertations collectionJanuary 2006 (has links)
All mC5-MTases are monomeric enzymes, except M. EcoHK31I and M. AquI which are MTases composed of two poly peptides. M.EcoHK31I is a mC5-MTase which recognizes the sequence 5-YGGCCR-3' and consists of polypeptide alpha and beta, with the latter gene encoded in an alternative reading frame of the former. All of the conserved motifs in mC5-MTases can be found in polypeptide alpha, except motif IX, which is located in polypeptide beta. Both polypeptides are required for in vitro methylation. / Methylation of cytosine residues in DNA occurs in diverse organisms from bacteria to humans. In higher eukaryotic organisms cytosine-C5 methyltransferase (mC5-MTase) is the only type of DNA MTase and it plays an important role in controlling a number of cellular processes including transcription genomic imprinting and DNA repair. In bacteria, there are three types of MTases, mC4-, mC5- and mAb-, classified according to the methylation site of the DNA. MTase and its cognate restriction endonuclease (ENase) form restriction-modification system. The role of MTase is to protect the host from its own ENase digestion while the ENase acts to degrade the invasion of foreign DNA. Sequence comparison of nearly 50 bacterial mC5-MTases has shown that these enzymes share an overall common protein architecture. Ten conserved motifs (I to X), each 10 to 20 amino acids in length, have been identified, five of which are highly conserved (I, IV, VI, VIII and X). In addition, all of these enzymes have a hypervariable region lying between motifs VIII and IX. It is called the target recognition domain (TRD), and is responsible for the specificity of DNA recognition and the choice of base to be methylated. / Since both of the polypeptides alpha and beta of M.EcoHK31I are sequenced and cloned into the expression vector separately, the role of DNA recognition and subunits interaction of individual polypeptides can be studied. By electromobility shift assay, we found that polypeptides alpha and beta complex recognize specific double strand oligos substrate. Polypeptide alpha-DNA formed aggregates and polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists the proper binding of polypeptide alpha to DNA substrate. Complex of polypeptide alpha and a polypeptide beta variant with N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive MTase. By surface plasmon resonance assay, the dissociation equilibrium constant (KD) of polypeptides alpha and beta complex was found to be 56.2nM and the KD for polypeptide alpha and DeltaN46-polypeptide beta complex was increased by about 95 folds, contributing by a drastic decrease in dissociate rate constant (kd) and an increase in association rate constant (ka). This indicated that the N-terminal region of polypeptide beta takes part in subunit interaction. / To pinpoint which amino acid residues located at the variable region of polypeptide alpha are important for DNA binding and subunits interaction, "charge-to-alanine scanning mutagenesis" were performed on 16 charge residues between Asp213 and Glu271 in the small domain. It was found that the five charge residues upstream of motif X are not required for activity. For other residues except K225, E240 and D245, the protein is active when the same charge is maintained. / Fung Wai To. / "March 2006." / Adviser: P. C. Shaw. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6376. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 180-201). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
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Prevalência de beta-lactamases de amplo espectro e metilases RNAr 16S em isolados clínicos de Pseudomonas aeruginosa multirresistentes recuperados em diferentes hospitais de São Paulo / Prevalence of Extended-spectrum beta-lactamase and 16S rRNA methylases in clinical isolates of multidrug-resistant Pseudomonas aeruginosa recovered from differents hospitals in São PauloSilva, Mariama Tomaz Nogueira da 07 December 2009 (has links)
Introdução e objetivos: A produção de beta-lactamases de espectro ampliado (ESBLs) tem sido restrita a espécies do gênero Klebsiella spp. e E. coli, sendo associada a altos índices de resistência, morbidade e mortalidade. Uma vez que os determinantes genéticos para ESBLs (genes blaESBL) são mediados por plasmídios, a sua disseminação para outras espécies de importância médica é considerada uma urgência epidemiológica. Os genes que codificam para ESBLs são mais comumente encontrados em membros da família Enterobacteriaceae, porém, plasmídeos, integrons, e sequências de inserção têm contribuído para o aumento da incidência de genes blaESBL entre outras bactérias Gramnegativas, incluindo Pseudomonas aeruginosa. Infelizmente, uma das maiores dificuldades associada à identificação precoce da produção de ESBL em agentes de infecção hospitalar como Pseudomonas aeruginosa tem sido a padronização de métodos fenotípicos, os quais são afetados por resultados falso-negativos, decorrentes de mecanismos intrínsecos que mascaram a presença destas enzimas (i.e., produção de beta-lactamase AmpC de origem cromossômica). O presente estudo teve como objetivo caracterizar feno e genotipicamente a produção de ESBLs em isolados clínicos de P. aeruginosa recuperados de diferentes hospitais do Estado de São Paulo durante o período de 2004-2008. Materiais e métodos: 35 amostras de P. aeruginosa provenientes de 4 diferentes centros médicos de São Paulo, com perfil de resitência às cefalosporinas de terceira geração, foram submetidas à triagem fenotípica para a produção de ESBL na presença e ausência de inibidores específicos (ácido clavulânico e cloxacilina). A confirmação genotípica foi realizada por PCR e sequenciamento, usando iniciadores para pesquisa dos genes blaCTX-M, blaTEM, blaSHV, blaOXA, blaPER e blaGES, e para o mapeamento de integron Classe 1. A diversidade genética das amostras foi realizada com auxílio do método de ERIC PCR e o índice de similaridade calculado empregando-se o coeficiente de Dice. Resultados e conclusão: Os métodos fenotípicos identificaram 3 cepas produtoras de ESBL, porém, a presença dos genes blaCTX-M e blaOXA-10 foi confirmada em 9 (33%) e 2 (7%) cepas de P. aeruginosa, respectivamente, recuperadas em 3 centros. O mapeamento e seqüenciamento do integron classe 1 encontrado revelou que duas cepas carregam 2 diferentes integrons classe 1 com genes blaCTX-M-2, aac6, e aadA6 de resistência para as cefalosporinas de terceira geração e para aminoglicosídeos. A transferência horizontal dos genes blaESBL não foi confirmada por transformação. Este estudo descreve que o aparecimento e disseminação de genes blaCTX-M em isolados clínicos de P. aeruginosa no Brasil teve sua origem a partir de 2005. Uma vez que ESBLs do tipo CTX-M têm sido amplamente descritas em Enterobactérias, a identificação destes genes em isolados de P. aeruginosa é alarmante e mostra que a mobilização horizontal do gene blaCTX-M entre diferentes gêneros e espécies é uma realidade no Brasil. Os genes que conferem resistência às metilases 16s RNAr não estavam relacionados a cepa de Pseudomonas aeruginosa produtores da enzima ESBL isoladas nas amostras de São Paulo. As cepas de Pseudomonas aeruginosa do presente estudo mostraram vários elementos de mobilização genéticos, como integrons e sequências de inserção, que podem estar participando na disseminação de resistência aos antibióticos beta- lactâmicos de amplo espectro. / Introduction and aim: The production of extended-spectrum beta-lactamases (ESBLs) has been restricted to Klebsiella spp. and Escherichia coli, being associated with high rates of resistance, morbidity and mortality. Since genetic determinants for ESBLs (blaESBL genes) are mediated by plasmids, the spread to other medical important species is considered an epidemiological urgency. These genes encoding ESBLs are commonly found in members of the Enterobacteriaceae however, plasmids, integrons, and insertion sequences have contributed to the increase in the incidence of blaESBL genes among other gram-negative bacteria, including Pseudomonas aeruginosa. Unfortunately, one of the biggest difficulties associated with the early identification of the production of ESBL in nosocomial agents as Pseudomonas aeruginosa has been the standardization of phenotypic methods, which are affected by false-negative results stemming from intrinsic mechanisms which can mask the presence of these enzymes (i.e., production of chromosomal beta-lactamase AmpC). The aim of this study is to characterize phenotypical and genotypical ESBL production in clinical isolates of P. aeruginosa recovered in different hospitals in São Paulo during the period of 2004 to 2008. Materials and methods: 35 P .aeruginosa isolates intermediately resistant or resistant to third generation cephalosporin were phenotypically analyzed for the presence of ESBL with and without inhibitors (clavulanic acid and cloxacilin).Genotipic confirmation with specific primers for blaCTX-M, blaTEM, blaSHV, blaOXA, blaPER and blaGES and the characterization of the genetic environment of blaCTX-M was performed by PCR and DNA sequencing.The random amplified polymorphism DNA technique with primer ERIC-2 was carried out for the isolates genotyping and the similarity index calculated with coefficient of Dice. Results and conclusion: the phenotypic methods identified 3 ESBL producing strains, however, the presence of genes blaCTX-M and blaOXA-10 has been confirmed in 9 (33%) and 2 (7%) strains of P. aeruginosa, respectively, from 3 medical centers. The mapping and sequencing of integron class 1 revealed that two strains harbored two different class 1 integrons with aadA6-like , aac6 gene and blaCTX-M-2-like genes conferring resistance to aminoglicosydes and third generation cephalosporins. The blaESBL horizontal transferation has not been confirmed by transformation. This study describes that the emergence and spread of genes blaCTX-M in clinical isolates of P. aeruginosa in Brazil had its origin from 2005. Since CTX-M type ESBLs have been widely described in Enterobacteriaceae, the identification of these genes in P. aeruginosa is alarming and shows that the blaCTX-M horizontal mobilization among different genus and species is a reality in Brazil. The genes that confer resistance to 16s RNAr methylases are not related to ESBL positive Pseudomonas aeruginosa strains in samples of São Paulo. The Pseudomonas aeruginosa strains of this study showed several genetic mobilization elements, such as integrons and insertion sequences, which may be participating in the spread of resistance to broad spectrum betalactams.
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The effect of putative vesicular stomatitis virus methyltransferase mutants on transcription and replicationTower, Dallas Lauren, January 2005 (has links)
Thesis (M.S.)--University of Florida, 2005. / Typescript. Title from title page of source document. Document formatted into pages; contains 57 pages. Includes Vita. Includes bibliographical references.
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Prevalência de beta-lactamases de amplo espectro e metilases RNAr 16S em isolados clínicos de Pseudomonas aeruginosa multirresistentes recuperados em diferentes hospitais de São Paulo / Prevalence of Extended-spectrum beta-lactamase and 16S rRNA methylases in clinical isolates of multidrug-resistant Pseudomonas aeruginosa recovered from differents hospitals in São PauloMariama Tomaz Nogueira da Silva 07 December 2009 (has links)
Introdução e objetivos: A produção de beta-lactamases de espectro ampliado (ESBLs) tem sido restrita a espécies do gênero Klebsiella spp. e E. coli, sendo associada a altos índices de resistência, morbidade e mortalidade. Uma vez que os determinantes genéticos para ESBLs (genes blaESBL) são mediados por plasmídios, a sua disseminação para outras espécies de importância médica é considerada uma urgência epidemiológica. Os genes que codificam para ESBLs são mais comumente encontrados em membros da família Enterobacteriaceae, porém, plasmídeos, integrons, e sequências de inserção têm contribuído para o aumento da incidência de genes blaESBL entre outras bactérias Gramnegativas, incluindo Pseudomonas aeruginosa. Infelizmente, uma das maiores dificuldades associada à identificação precoce da produção de ESBL em agentes de infecção hospitalar como Pseudomonas aeruginosa tem sido a padronização de métodos fenotípicos, os quais são afetados por resultados falso-negativos, decorrentes de mecanismos intrínsecos que mascaram a presença destas enzimas (i.e., produção de beta-lactamase AmpC de origem cromossômica). O presente estudo teve como objetivo caracterizar feno e genotipicamente a produção de ESBLs em isolados clínicos de P. aeruginosa recuperados de diferentes hospitais do Estado de São Paulo durante o período de 2004-2008. Materiais e métodos: 35 amostras de P. aeruginosa provenientes de 4 diferentes centros médicos de São Paulo, com perfil de resitência às cefalosporinas de terceira geração, foram submetidas à triagem fenotípica para a produção de ESBL na presença e ausência de inibidores específicos (ácido clavulânico e cloxacilina). A confirmação genotípica foi realizada por PCR e sequenciamento, usando iniciadores para pesquisa dos genes blaCTX-M, blaTEM, blaSHV, blaOXA, blaPER e blaGES, e para o mapeamento de integron Classe 1. A diversidade genética das amostras foi realizada com auxílio do método de ERIC PCR e o índice de similaridade calculado empregando-se o coeficiente de Dice. Resultados e conclusão: Os métodos fenotípicos identificaram 3 cepas produtoras de ESBL, porém, a presença dos genes blaCTX-M e blaOXA-10 foi confirmada em 9 (33%) e 2 (7%) cepas de P. aeruginosa, respectivamente, recuperadas em 3 centros. O mapeamento e seqüenciamento do integron classe 1 encontrado revelou que duas cepas carregam 2 diferentes integrons classe 1 com genes blaCTX-M-2, aac6, e aadA6 de resistência para as cefalosporinas de terceira geração e para aminoglicosídeos. A transferência horizontal dos genes blaESBL não foi confirmada por transformação. Este estudo descreve que o aparecimento e disseminação de genes blaCTX-M em isolados clínicos de P. aeruginosa no Brasil teve sua origem a partir de 2005. Uma vez que ESBLs do tipo CTX-M têm sido amplamente descritas em Enterobactérias, a identificação destes genes em isolados de P. aeruginosa é alarmante e mostra que a mobilização horizontal do gene blaCTX-M entre diferentes gêneros e espécies é uma realidade no Brasil. Os genes que conferem resistência às metilases 16s RNAr não estavam relacionados a cepa de Pseudomonas aeruginosa produtores da enzima ESBL isoladas nas amostras de São Paulo. As cepas de Pseudomonas aeruginosa do presente estudo mostraram vários elementos de mobilização genéticos, como integrons e sequências de inserção, que podem estar participando na disseminação de resistência aos antibióticos beta- lactâmicos de amplo espectro. / Introduction and aim: The production of extended-spectrum beta-lactamases (ESBLs) has been restricted to Klebsiella spp. and Escherichia coli, being associated with high rates of resistance, morbidity and mortality. Since genetic determinants for ESBLs (blaESBL genes) are mediated by plasmids, the spread to other medical important species is considered an epidemiological urgency. These genes encoding ESBLs are commonly found in members of the Enterobacteriaceae however, plasmids, integrons, and insertion sequences have contributed to the increase in the incidence of blaESBL genes among other gram-negative bacteria, including Pseudomonas aeruginosa. Unfortunately, one of the biggest difficulties associated with the early identification of the production of ESBL in nosocomial agents as Pseudomonas aeruginosa has been the standardization of phenotypic methods, which are affected by false-negative results stemming from intrinsic mechanisms which can mask the presence of these enzymes (i.e., production of chromosomal beta-lactamase AmpC). The aim of this study is to characterize phenotypical and genotypical ESBL production in clinical isolates of P. aeruginosa recovered in different hospitals in São Paulo during the period of 2004 to 2008. Materials and methods: 35 P .aeruginosa isolates intermediately resistant or resistant to third generation cephalosporin were phenotypically analyzed for the presence of ESBL with and without inhibitors (clavulanic acid and cloxacilin).Genotipic confirmation with specific primers for blaCTX-M, blaTEM, blaSHV, blaOXA, blaPER and blaGES and the characterization of the genetic environment of blaCTX-M was performed by PCR and DNA sequencing.The random amplified polymorphism DNA technique with primer ERIC-2 was carried out for the isolates genotyping and the similarity index calculated with coefficient of Dice. Results and conclusion: the phenotypic methods identified 3 ESBL producing strains, however, the presence of genes blaCTX-M and blaOXA-10 has been confirmed in 9 (33%) and 2 (7%) strains of P. aeruginosa, respectively, from 3 medical centers. The mapping and sequencing of integron class 1 revealed that two strains harbored two different class 1 integrons with aadA6-like , aac6 gene and blaCTX-M-2-like genes conferring resistance to aminoglicosydes and third generation cephalosporins. The blaESBL horizontal transferation has not been confirmed by transformation. This study describes that the emergence and spread of genes blaCTX-M in clinical isolates of P. aeruginosa in Brazil had its origin from 2005. Since CTX-M type ESBLs have been widely described in Enterobacteriaceae, the identification of these genes in P. aeruginosa is alarming and shows that the blaCTX-M horizontal mobilization among different genus and species is a reality in Brazil. The genes that confer resistance to 16s RNAr methylases are not related to ESBL positive Pseudomonas aeruginosa strains in samples of São Paulo. The Pseudomonas aeruginosa strains of this study showed several genetic mobilization elements, such as integrons and insertion sequences, which may be participating in the spread of resistance to broad spectrum betalactams.
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Identification and characterization of virulence factors of mycoplasmasLuo, Wenyi. January 2009 (has links) (PDF)
Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from PDF title page (viewed on July 1, 2010). Includes bibliographical references.
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