Lee, Ching Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 90-103). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Table of Contents --- p.vi / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction / Chapter 1.1. --- Quinolone Antimicrobial Agents --- p.1 / Chapter 1.1.1. --- Development --- p.1 / Chapter 1.1.2. --- Mode of action --- p.3 / Chapter 1.1.3. --- Mechanisms of resistance to quinolones --- p.4 / Chapter 1.1.3.1. --- Target genes mutations --- p.4 / Chapter 1.1.3.2. --- Decreased intracellular quinolone accumulation --- p.5 / Chapter 1.1.3.3. --- Plasmid-mediated quinolone resistance --- p.6 / Chapter 1.2. --- Plasmid-mediated Quinolone Resistance Genes (qnr) --- p.8 / Chapter 1.2.1. --- Discovery of qnrA genes --- p.8 / Chapter 1.2.2. --- Discovery of qnrS genes --- p.9 / Chapter 1.2.3. --- Discovery of qnrB genes --- p.10 / Chapter 1.2.4. --- Discovery of qnrC genes --- p.11 / Chapter 1.2.5. --- Discovery of qnrD genes --- p.12 / Chapter 1.2.6. --- Origins of qnr genes --- p.12 / Chapter 1.2.7. --- Qnr proteins and mode of action --- p.14 / Chapter 1.2.8. --- Epidemiology and quinolones resistance activity of qnr genes --- p.16 / Chapter 1.2.9. --- Epidemiology of fluoroquinolone-resistant Enterobacteriaceae --- p.17 / Chapter 1.2.10 --- Multidrug-resistant in extended-spectrum-B-lactamase- and AmpC-producing Enterobacteriaceae --- p.19 / Chapter 1.3. --- Background of Study --- p.20 / Chapter 1.4. --- Objectives of Study --- p.21 / Chapter Chapter 2 --- Materials & Methods / Chapter 2.1. --- Study Design --- p.22 / Chapter 2.2. --- Antimicrobial Susceptibility Testing --- p.24 / Chapter 2.2.1 --- Bacterial isolates --- p.24 / Chapter 2.2.2. --- Screening for ESBL and AmpC production by disk diffusion test --- p.24 / Chapter 2.2.3. --- Determination of minimal inhibitory concentrations (MICs) --- p.25 / Chapter 2.3. --- "Detection of qnrA, qnrB and qnrS Genes by Multiplex PCR" --- p.27 / Chapter 2.3.1. --- Total DNA preparation --- p.27 / Chapter 2.3.2. --- "Multiplex PCR assay for qnrA, qnrB and qnrS genes detection" --- p.27 / Chapter 2.3.3. --- Agarose gel electrophoresis --- p.29 / Chapter 2.4. --- "Detection of TEM-, SHV-, CTX- and PMAmpC Type B-Lactamase Genes by PCR" --- p.30 / Chapter 2.5. --- PCR Assays for Further Genotypic Characterization Purpose --- p.32 / Chapter 2.5.1. --- PCR assay to amplify qnrB genes --- p.32 / Chapter 2.5.2. --- PCR assay to amplify qnrS genes --- p.33 / Chapter 2.5.3. --- "PCR assays for genotypic characterizations of the co-existed blaTEM, blaSHV, blaCTX-M and PMAmpC genes of all qnr-positive isolates" --- p.33 / Chapter 2.5.3.1. --- Genotypic characterizations of the co-existed bla-TEM and genes --- p.33 / Chapter 2.5.3.2. --- PCR assays to amplify the co-existed blaCTX_M genes --- p.33 / Chapter 2.5.3.3. --- PCR assay to amplify the co-existed PMAmpC genes --- p.34 / Chapter 2.5.4. --- Sequencing reaction --- p.36 / Chapter 2.5.4.1. --- Purification of PCR product and sequence determination --- p.36 / Chapter 2.5.4.2. --- Sequence analysis --- p.37 / Chapter 2.6. --- Collection of Clinical Data --- p.38 / Chapter 2.6.1. --- Demographics and clinical data --- p.38 / Chapter 2.6.2. --- Definitions --- p.38 / Chapter 2.6.3. --- Data analysis --- p.40 / Chapter Chapter 3 --- Results / Chapter 3.1. --- Bacterial Isolates --- p.41 / Chapter 3.2. --- "Demographics, Medical History, Clinical Features and Clinical Outcomes of Patients" --- p.42 / Chapter 3.3. --- Antimicrobial Susceptibility Testing --- p.44 / Chapter 3.4. --- Detection of qnr Genes --- p.48 / Chapter 3.4.1. --- "Detection of qnrA, qnrB and qnrS genes by multiplex PCR" --- p.48 / Chapter 3.5. --- Detection of ESBLs --- p.49 / Chapter 3.5.1. --- Detection of TEM- and SHV-type ESBLs --- p.49 / Chapter 3.5.2. --- Detection of CTX-M- type ESBLs --- p.51 / Chapter 3.6. --- Detection of PMAmpC Genes --- p.52 / Chapter 3.6.1. --- Detection of PMAmpC genes --- p.52 / Chapter 3.7. --- "The Distribution of qnr and bla Genes for TEM, SHV, CTX-M and PMAmpC" --- p.53 / Chapter 3.8. --- The Characteristics of qnr Isolates --- p.54 / Chapter 3.8.1. --- Genotypes of qnrB and qnrS --- p.54 / Chapter 3.8.2. --- Antimicrobial susceptibility of qnr isolates --- p.58 / Chapter 3.9. --- "The Associations of qnr Genes with Other Bacterial Resistance Genotypes, and the Clinical Characteristics and Outcomes of Patients" --- p.62 / Chapter 3.9.1. --- "Univariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics and outcomes of patients" --- p.62 / Chapter 3.9.2. --- "Multivariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics and outcomes of patients" --- p.65 / Chapter 3.9.2.1. --- "Multivariate analysis of the associations of qnr genes with other bacterial resistance genotypes, and the clinical characteristics of patients" --- p.65 / Chapter 3.9.2.2. --- "Multivariate analysis of the associations of mortality with qnr genes, bacterial resistance genotypes and other clinical characteristics of patients" --- p.66 / Chapter Chapter 4 --- Discussion / Chapter 4.1. --- Prevalences and Susceptibility of ESBL and PMAmpC in Bacteraemic Enterobacteriaceae Isolates --- p.67 / Chapter 4.2. --- Epidemiology of Plasmid-mediated Quinolone Resistance (qnr) Genes --- p.69 / Chapter 4.3. --- Genotypes of qnr-positive Isolates --- p.72 / Chapter 4.4. --- Antimicrobial Susceptibility of qnr-positive Isolates --- p.75 / Chapter 4.5. --- "The Associations of qnr Genes with Other Bacterial Resistance Genotypes, and the Clinical Characteristics of Patients" --- p.79 / Chapter 4.6. --- "The Associations of Mortality with qnr Genes, Bacterial Resistance Genotypes and Other Clinical Characteristics of Patients" --- p.80 / Chapter 4.7. --- Clinical Importance and Clinical Implications of qnr Genes --- p.82 / Chapter 4.8. --- Limitations of the Current Study --- p.85 / Chapter 4.9. --- Future Studies --- p.87 / Chapter 4.10. --- Conclusions --- p.89 / References --- p.90

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Em diversos estados do Brasil, foram relatadas epidemias de infecções causadas por micobactérias de crescimento rápido (MCR) desde o ano 2000. A maioria dos casos foi principalmente associada ao clone BRA100 de Mycobacterium massiliense, recentemente renomeada para Mycobacterium abscessus subsp. bolletii, isolado de pacientes submetidos a procedimentos invasivos nos quais os instrumentos médicos não foram adequadamente esterilizados e/ou desinfetados. Sendo as quinolonas uma opção no tratamento de infecções por MCR e sugerida para esquemas terapêuticos para esses surtos, foram avaliadas nesse trabalho as atividades in vitro de quatro gerações de quinolonas para cepas clinicas e de referência de MCR através da microdiluição em caldo. Também foram analisadas as sequências peptídicas das regiões determinantes da resistência a quinolonas (RDRQ) das subunidades A e B da DNA gyrase (GyrA e GyrB) após o seqüenciamento de DNA seguido pela tradução da sequência de aminoácidos. Cinquenta e quatro cepas de M. abscessus subsp bolletii, incluindo o clone BRA100, isoladas em diferentes estados do Brasil, e 19 cepas de referência de MCR foram caracterizadas. Todas as 54 cepas clínicas de M. abscessus subsp. bolletii foram resistentes a todas as gerações de quinolonas e mostraram o mesmo resíduo nas RDRQ, incluindo Ala-83 em GyrA, Arg-447 e Asp-464 em GyrB, descritos como sendo responsáveis por gerar um baixo nível de resistência a quinolonas em micobactérias. Porém, outras espécies de MCR apresentaram diferentes susceptibilidade e padrões de mutações contrários aos classicamente já definidos, sugerindo que outros mecanismos de resistência, diferentes de mutações em gyrA e gyrB também possam estar envolvidos na alta resistência a quinolonas. / Several outbreaks of infections caused by rapidly growing mycobacteria (RGM) have been reported in many Brazilian states since 2000. Most of the cases were mainly associated to Mycobacterium massiliense, recently renamed as Mycobacterium abscessus subsp. bolletii, BRA100 clone recovered from patients who had undergone invasive procedures, in which medical instruments have not been properly sterilized and / or disinfected. Since quinolones have represented an option for the treatment of general RGM infections and suggested for therapeutic schemes for these outbreaks, we evaluated the in vitro activities of four generations of quinolones for clinical and reference RGM by broth microdilution, and analysis of peptide sequences of the quinolone resistance determining regions (QRDR) of GyrA and GyrB after DNA sequencing followed by amino acid translation. Fifty four isolates of M. abscessus subsp bolletii, including clone BRA100, recovered in different states of Brazil, and 19 reference strains of RGM species were characterized. All 54 M. abscessus subsp. bolletii isolates were resistant to all generations of quinolones and showed the same amino acids in the QRDR including the Ala-83 in GyrA, Arg-447 and Asp-464 in GyrB, described as responsible for an intrinsic low level of resistance to quinolones in mycobacteria. But other RGM species presented distinct susceptibilities to this class of antimicrobials and patterns of mutations contrary to what has been traditionally defined, suggesting that other mechanisms of resistance, different from gyrA or gyrB mutations, may also be involved in resistance to high levels of quinolones.

Maintenance of a topological homeostasis by introduction and removal of the supercoils to relieve excessive strain on the DNA is a hallmark of topoisomerase function in the cell. The requirement of the topoisomerases during DNA transaction processes marks a ubiquitous presence of the enzymes in all the life forms. Different reactions carried out by the enzymes include relaxation of positive and negative supercoils required majorly during DNA replication and transcription, decatenation at the end of DNA replication to separate the daughter chromosomes and removal of lethal knots generated in the circular chromosome. In eubacteria, the enzymes introduce negative supercoils to facilitate easier strand separation for DNA transaction processes. However, in thermophiles, a different enzyme maintains the genome in a positively supercoiled form to protect from denaturation by excessive heat. These varied functions are carried out by different topoisomerases. Therefore, each organism maintains a minimum required set of the enzymes and the absence of a certain enzyme may be compensated for by topoisomerases with dual functions. For example, Mycobacterium tuberculosis and many other slow growing mycobacteria do not possess topoisomerase IV or its homologs. In these organisms, the DNA gyrase is suggested to carry out both negative supercoiling and decatenation reactions. Therefore, the mycobacterial DNA gyrase must be able to manage between both the functions in vivo. In contrast, Mycobacterium smegmatis and few other mycobacteria contain an additional type II topoisomerase which does not resemble any known type II enzyme but could catalyze relaxation and decatenation reactions. Importantly, the enzyme displays a unique ability to introduce limited positive supercoils and may have certain functions inside the cell which remains to be studied. Owing to the indispensability for bacterial survival topoisomerases present themselves as important drug targets. A large number of inhibitors have been found to inhibit the enzyme and thereby killing the bacterial. Among these, quinolones are successfully being used as broad spectrum antibacterial drugs. Although the commonly used quinolones inhibit many bacterial pathogens, a reduced susceptibility is exhibited by some of the pathogens e.g. Mycobacterium tuberculosis. To circumvent the lower efficacy of existing drugs, new and modified quinolones have been developed which are highly effective against mycobacteria. The difference in the susceptibility may be conferred by a difference in the chemical property of the drug and the interacting residues present in the enzyme. In the present thesis efforts have been made to understand the mechanism of the type II topoisomerases from mycobacteria and drug action on these enzymes. The thesis is divided into four chapters. In Chapter I of the thesis an introduction is provided on the topoisomerases, their classification and different reactions catalyzed by these enzymes. As the work in present thesis has been carried out with type II topoisomerases, introduction of type II enzymes, their structure and mechanisms is elaborated. DNA gyrase, its mechanism of reaction and in vitro and in vivo functions are explained in great detail. DNA gyrase and topoisomerase IV are targeted by a range of different inhibitors. These different classes of inhibitors and their mechanism of action are described. Finally, the mechanism of mycobacterial DNA gyrase with structural information and the current understanding of quinolone action on the enzyme are explained. The chapter ends with the objective of the study in the present thesis. In chapter II, the studies are aimed at understanding the molecular basis for decatenation carried out by mycobacterial DNA gyrase. Previous work from the laboratory showed that the enzyme can carry out decatenation more efficiently than its homolog from E. coli. It was shown that the mycobacterial enzyme binds two DNA molecules in trans in a length dependent manner. The ability to bind the second DNA is conferred upon the holoenzyme by ATPase subunit (GyrB) subunit which alone can bind DNA. Similar studies using topo IV from E. coli, the strongest known decatenase showed binding of two DNA molecules and the second DNA binding by ATPase (ParE) subunit. However, GyrB subunit from E. coli DNA gyrase, a weaker decatenase, does not bind second DNA molecule efficiently. The results provide a general mechanism for decatenation by type II enzymes in which efficient binding of second DNA is important. In Chapter III, studies have been carried out using topo NM, an atypical type II topoisomerase from Mycobacterium smegmatis. The enzyme has been characterized previously in the laboratory. In addition to efficient decatenation and relaxation, the enzyme exhibits a unique ability to introduce positive supercoils into the DNA. As demonstrated for the mycobacterial DNA gyrase and topo IV in the Chapter II, the ATPase subunit (Topo N) of topo NM, binds second DNA efficiently. The binding of both gate and transport segments increases with the length of the DNA. Binding of two DNA molecules by the holoenzyme appears to be a cumulative effect of DNA binding to individual subunits. In the absence of any inhibitor, the enzyme accumulates cleaved DNA products with shorter DNA but not with larger DNA. The cleavage of the shorter DNA is supported only in the presence of Mg2+ and Mn2+. Another important property of the enzyme is to introduce positive supercoils which appears to be due to its efficient utilization of ATP and a high rate of reaction. Chapter IV deals with the interaction of mycobacterial gyrase with fluoroquinolones (FQs). Although DNA gyrase is the sole target of the FQs in M. tuberculosis, the lower susceptibility to commonly used FQs have led to the studies to find out more effective quinolones. Previous studies from the laboratory showed a lower susceptibility of the mycobacterial gyrase to ciprofloxacin, but moxifloxacin could inhibit the enzyme efficiently. The better inhibition by moxifloxacin appears to be due to efficient trapping of the enzyme-DNA covalent complex. Both ciprofloxacin and moxifloxacin bind the DNA gyrase from mycobacteria, E. coli and E. coli topo IV, independent of DNA. The extent of binding also correlates with the inhibition potential of the drug against a given enzyme. A general model of quinolone enzyme interaction is provided wherein the quinolones are shown to interact with GyrA subunit or holoenzyme or the enzyme- DNA complex which would finally result in the trapping of the covalent complex.

[pt] A fibrose cística é uma doença genética causada por mutações no gene CFTR, que implica na alteração do transporte de íons pela membrana das células de diversos órgãos, aumentando a quantidade e viscosidade do muco, suor e secreções pancreáticas. A principal consequência são os danos pulmonares, caracterizados por frequentes infecções e insuficiência respiratória. Com isso, vários estudos vêm sendo desenvolvidos ao longo dos anos, com o intuito de descobrir fármacos que possam agir como moduladores do CFTR, melhorando a função pulmonar desses pacientes. O presente trabalho teve o objetivo de sintetizar derivados de 3-fenilindeno[1,2-d][1,2,3]triazol-8(3H)-ona, 3-fenil-3,4- di-hidro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-ona e 3-(1-fenil-1H-1,2,3-triazole-4- carbonil)quinolin-4(1H)-ona caracterizar a relação estrutura atividade desses compostos como potenciadores do CFTR. Para a síntese de 3-fenilindeno[1,2-d][1,2,3]triazol-8(3H)-ona inicialmente utilizou-se a 2 bromoacetofenona para, através do reagente de Gold produzido a partir do cloreto de cianurila, produzir a enaminona. A etapa chave de obtenção dos triazóis foi realizada através de uma reação de cicloadição entre as (E)-3-(dimetilamino)-1-(2-nitrofenil)prop-2-en-1-ona e aril azidas azido-benzeno, 1-azido-4-bromo-benzeno, 1-azido-4- nitrobenzeno e ácido 4-azido-benzóico. Foram obtidos quatro compostos com rendimentos entre 20 por cento e 50 por cento. Foram realizadas tentativas de ciclização intramolecular catalisada por paládio na presença de fosfinas, porém o produto esperado não foi obtido. Para a obtenção da 3-fenil-3,4-di-hidro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-ona obteve-se o intermediário (E)-3-(dimetilamino)-1-(2-nitrofenil)prop-2-en-1-ona, que através de uma reação de cicloadição com a azido-benzeno obteve-se o triazol com rendimento de 30 por cento. A proposta seria a redução do grupo nitro para amina, e posterior substituição da amina por azida, para fazer a arilação intramolecular do triazol via catálise fotoredox. Para a obtenção de 3-(1-fenil-1H-1,2,3-triazole-4-carbonil)quinolin4(1H)-ona foram utilizados anilina e dietil etoximetilenomalonato como precursores para sintetizar 4-oxo-1,4-di-hidroquinolino-3-carboxilato de etilo, que foi obtida com rendimento de 50 por cento. Realizou-se uma reação de adição de alcino seguida da eliminação de etanol, para obter a 3-(1-fenil-1H-1,2,3-triazole-4-carbonil)quinolin-4(1H)-ona com etiniltrimetilsilano e então realizar uma reação click com azidas. Todos os compostos obtidos foram caracterizados por RMN1H e RMN13C. / [en] Cystic fibrosis is a genetic disease caused by mutations in the CFTR gene, which involves changing the transport of ions across the membrane of various organ cells, increasing the amount and viscosity of the mucus, sweat and pancreatic secretions. The main consequence is lung damage, characterized by frequent infections and respiratory failure. Several studies have been developed over the years with the intention of discovering drugs that can act as modulators of CFTR, improving the lung function of these patients. The present work aimed to synthesize derivatives of 3-phenylindeno[1,2-d [1,2,3]triazol-8(3H)-one, 3-phenyl-3,4-dihydro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-one and 3-(1-phenyl-1H1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one to characterize the relationship structure of these compounds as enhancers of CFRT. For the synthesis of 3-phenylindeno[1,2-d][1,2,3]triazol-8(3H)-one, the 2-bromoacetophenone was initially used to produce the (E)-3-(dimethylamino) -1- (2-nitrophenyl) prop-2-en1-one through the Gold reagent obtained from the cyanuric chloride. The key step of obtaining the triazoles was accomplished by a cycloaddition reaction between the (E)-3-(dimethylamino)-1-(2-nitrophenyl) prop-2-en-1-one and the aryl azides azido-benzene, 1-azido-4-bromo-benzene, 1-azido-4-nitrobenzene and 4-azido benzoic acid. Four compounds in yields between 20 percent and 50 percent were obtained. Attempts were made for intramolecular cyclization catalyzed by palladium in the presence of phosphines, but the expected product was not obtained. To obtain 3- phenyl-3,4-dihydro-9H-[1,2,3]triazolo[4,5-b]quinolin-9-one, first It was obtained the intermediate (E)-3- (dimethylamino)-1-(2-nitrophenyl) prop-2-en-1-one through cycloaddition reaction with the azido-benzene gave the triazole in 30 percent yield. The proposal would be the reduction of the nitro group to amine, and subsequent replacement of the amine with azide, to make the intramolecular arylation of triazole via photoredox catalysis. To obtain 3-(1-phenyl-1H-1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one, aniline and diethyl ethoxymethylene malonate were used as precursors to synthesize ethyl 4-oxo-1,4-dihydroquinoline3-carboxylate, which was obtained in 50 percent yield. An addition reaction of alkyne followed by the elimination of ethanol was performed to obtain the 3-(1-phenyl1H-1,2,3-triazole-4-carbonyl)quinolin-4(1H)-one with ethynyltrimethylsilane and then carried out a click reaction with azides. All compounds obtained were characterized by 1H-NMR and 13C-NMR.

Orientador: Mara Corrêa Lelles Nogueira / Banca: Miyoko Jakabi / Banca: Márcia Cristina Bisinoti / Resumo: Bactérias resistentes aos antimicrobianos são um importante problema de saúde pública, pois causam infecções de difícil tratamento que resultam em maiores períodos de internação, aumento das taxas de mortalidade e dos custos da assistência à saúde. Entre as estratégias para a prevenção e controle deste problema, a detecção de bactérias resistentes no ambiente tem ganhado importância, e neste contexto, o ambiente aquático tem recebido atenção por ser um importante reservatório de genes de resistência, destacando-se os genes que codificam as -lactamases de espectro estendido (ESBLs) e de resistência às quinolonas. A produção de ESBLs é uma ameaça à terapia antimicrobiana, pois estas enzimas inativam as cefalosporinas de terceira geração, que constituem a principal opção para o tratamento de infecções graves por bactérias Gramnegativas. A resistência às quinolonas também é um problema porque estes antimicrobianos são alternativas para o tratamento de infecções por bactérias Gram-negativas produtoras de ESBLs. Este estudo teve como objetivos investigar no Sistema Municipal de Abastecimento de São José do Rio Preto-SP, a presença de coliformes resistentes às cefalosporinas de terceira geração e às quinolonas, e investigar nos isolados a presença de genes determinantes da produção de ESBLs do tipo TEM, SHV e CTX-M, e de resistência às quinolonas (qnrA, qnrB e qnrS). O total de 7899 amostras de água foi coletado entre julho a novembro de 2011. Estas foram submetidas à filtração em membrana utilizando o Ágar Endo para o isolamento bacteriano. Os testes de suscetibilidade aos antimicrobianos foram realizados de acordo com o CLSI e em adição foi realizado teste fenotípico para produção de ESBL. PCR com primers... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Bacteria resistant to antimicrobial cause infections which require longer periods of hospitalization, increase the mortality rates and also the expenses with health care. Among the strategies for the prevention and control of this problem, the detection of resistant bacteria in the environment has been more important and, in this context, the aquatic environment has been paid attention for being an important reservoir of resistance gene, especially the genes encoding the extended-spectrum β-lactamases (ESBLs) and quinolone resistance. The production of ESBLs is a threat to antimicrobial therapy because these enzymes inactivate third generation cephalosporins, which are the primary choice for treatment of severe infections caused by Gram-negative bacteria. The quinolone resistance is also a problem because they are alternatives for antimicrobial treatment of infections caused by Gram-negative bacteria producing ESBLs. This study aimed to investigate in Municipal System of Supply in São José do Rio Preto-SP, the presence of coliform bacteria resistant to third generation cephalosporins and to quinolones and also investigate in isolated bacteria the presence of determinant genes for ESBLs production (TEM, SHV and CTX-M) and of quinolones resistance (qnrA, qnrB and qnrS). All the 7899 water samples were collected from July to November 2011. They were subjected to membrane filtration using Endo Agar for bacterial isolation. The antimicrobial susceptibility testings were carried out according to CLSI and in addition was performed phenotypic test for ESBL production. PCR with specific primers were performed in order to detect genes blaSHV, blaTEM, blaCTX-M, qnrA, qnrB e qnrS. None of the 72 isolated coliforms presented this phenotypic test positive for ESBL production, but 22 isolated coliforms presented resistance to... (Complete abstract click electronic access below) / Mestre

Made available in DSpace on 2014-06-11T19:30:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-05-21Bitstream added on 2014-06-13T20:40:08Z : No. of bitstreams: 1 pereira_lo_me_sjrp.pdf: 659708 bytes, checksum: 0787226dfdc454edee30ed321bef35a6 (MD5) / Bactérias resistentes aos antimicrobianos são um importante problema de saúde pública, pois causam infecções de difícil tratamento que resultam em maiores períodos de internação, aumento das taxas de mortalidade e dos custos da assistência à saúde. Entre as estratégias para a prevenção e controle deste problema, a detecção de bactérias resistentes no ambiente tem ganhado importância, e neste contexto, o ambiente aquático tem recebido atenção por ser um importante reservatório de genes de resistência, destacando-se os genes que codificam as -lactamases de espectro estendido (ESBLs) e de resistência às quinolonas. A produção de ESBLs é uma ameaça à terapia antimicrobiana, pois estas enzimas inativam as cefalosporinas de terceira geração, que constituem a principal opção para o tratamento de infecções graves por bactérias Gramnegativas. A resistência às quinolonas também é um problema porque estes antimicrobianos são alternativas para o tratamento de infecções por bactérias Gram-negativas produtoras de ESBLs. Este estudo teve como objetivos investigar no Sistema Municipal de Abastecimento de São José do Rio Preto-SP, a presença de coliformes resistentes às cefalosporinas de terceira geração e às quinolonas, e investigar nos isolados a presença de genes determinantes da produção de ESBLs do tipo TEM, SHV e CTX-M, e de resistência às quinolonas (qnrA, qnrB e qnrS). O total de 7899 amostras de água foi coletado entre julho a novembro de 2011. Estas foram submetidas à filtração em membrana utilizando o Ágar Endo para o isolamento bacteriano. Os testes de suscetibilidade aos antimicrobianos foram realizados de acordo com o CLSI e em adição foi realizado teste fenotípico para produção de ESBL. PCR com primers... / Bacteria resistant to antimicrobial cause infections which require longer periods of hospitalization, increase the mortality rates and also the expenses with health care. Among the strategies for the prevention and control of this problem, the detection of resistant bacteria in the environment has been more important and, in this context, the aquatic environment has been paid attention for being an important reservoir of resistance gene, especially the genes encoding the extended-spectrum β-lactamases (ESBLs) and quinolone resistance. The production of ESBLs is a threat to antimicrobial therapy because these enzymes inactivate third generation cephalosporins, which are the primary choice for treatment of severe infections caused by Gram-negative bacteria. The quinolone resistance is also a problem because they are alternatives for antimicrobial treatment of infections caused by Gram-negative bacteria producing ESBLs. This study aimed to investigate in Municipal System of Supply in São José do Rio Preto-SP, the presence of coliform bacteria resistant to third generation cephalosporins and to quinolones and also investigate in isolated bacteria the presence of determinant genes for ESBLs production (TEM, SHV and CTX-M) and of quinolones resistance (qnrA, qnrB and qnrS). All the 7899 water samples were collected from July to November 2011. They were subjected to membrane filtration using Endo Agar for bacterial isolation. The antimicrobial susceptibility testings were carried out according to CLSI and in addition was performed phenotypic test for ESBL production. PCR with specific primers were performed in order to detect genes blaSHV, blaTEM, blaCTX-M, qnrA, qnrB e qnrS. None of the 72 isolated coliforms presented this phenotypic test positive for ESBL production, but 22 isolated coliforms presented resistance to... (Complete abstract click electronic access below)