Orientador: Maria Luiza Moretti Branchini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-07-27T12:44:09Z (GMT). No. of bitstreams: 1 Dantas_SoniaReginaPerezEvangelista_M.pdf: 4480348 bytes, checksum: e1174ec0dfc72f10164651708943e887 (MD5) Previous issue date: 2000 / Resumo: No Brasil, a carência de leitos hospitalares públicos especializados acarreta super lotação dos pronto-socorros e permanência de pacientes em observação por longos períodos, resultando em complicações como colonização por bactérias multi droga resistentes (MDR) e aquisição de infecções hospitalares. METODOLOGIA: No período de março de 1996 a junho de 1998 foi realizado um estudo tipo caso-controle nos pacientes assistidos na unidade de observação do pronto-socorro (UOPS) do Hospital das Clínicas (HC) - UNICAMP, para determinação de fatores de risco associados à colonização por bactérias MDR e pelo Acinetobacter baumannii MDR As cepas de A.baumannii MDR foram ana1iAAnas por dos métodos de tipagem molecular do DNA plasmidial e do DNA genômico por eletroforese em campo pulsátil (pFGE). Para o estudo da colonização por bactérias MDR foram realizadas coletas semanais de secreções de orofaringe, nasofaringe e endotraqueal dos pacientes assistidos na UOPS. O teste de Qui-quadrado, ou teste Exato de Fisher foram aplicados para a comparação entre proporções. Para variáveis contínuas ou ordenáveis entre três ou mais grupos, utilizou-se o teste de Kruskal-Wallis e o teste de Pearson para correlação linear. O critério de seleção de variáveis adotado foi o "stepwise" e o nível de significância adotado em todos os testes foi de 5%. RESULTADOS: Foram coletadas 481 amostras de secreções respiratórias em 232 pacientes e a taxa de colonização por MDR Joi de 25,43% (59 casos e 173 controles). Foram isolados 179 microrganismos e destes, 78 (16,21%) eram bactérias MDR Os isolados MDR foram o Acinetobacter baumannií (n=55), Staphylococcus aureus (n=21) e Pseudomonas aeruginosa (n=2). O tempo de permanência no PS foi de 13,9 dias para os casos e 9,8 dias para os controles (p=0,998). O tempo médio para diagnóstico da colonização foi de 9,9 dias (variação 3 a 25 dias). A doença neurológica representou 68% dos casos e 51 % dos controles (p=0,024). Na análise multivariada dos fatores de risco para colonização foram significativos a idade (p=0,05), uso de ventilação mecânica (p=0,0001) / Abstract: The absence of adequate number of beds in public hospitals for severely ill patients in Brazil leads to an excessive number of patients housing in the emergency rooms (ER). The prolonged permanence in these units resulting in patients colonization and hospital infections due to multi drug resistant (MDR) bacteria. Between March 1996 to June 1998 a case-control study was performed in patients housing in the ER of Hospital das Clínicas (HC)-UNICAMP. We studied the risk factors associated with colonization of nasal, oropharyngeal and tracheal secretions by MDR bacteria and MDR Acinetobacter baumannii. A molecular typing ana1ysis using plasmid DNA and pulsed-field electrophoresis (PFGE) were performed in MDR A. baumannii strains. For statistic ana1ysis, the Chi-square and Fisher tests was applied in the comparison of proportions, for continuous variables the Kruskal- Wallis test and for linear correlation the Pearson. The level of 5% was adopted in all tests. In our study 481 samples of respiratory secretions from 232 patients were collected. The colonization by MDR bacteria was 25,4% (59 cases and 173 controls). 179 microorganisms were isolated and 78 (16.2%) were MDR bacteria, inc1uding A. baumannii (55 isolates), Staphylococcus aureus (21 isolates) and Pseudomonas aeruginosa (2 isolates). The time of stay in the ER was 13.9 days for cases and 9.8 days for controls (p=0.998). The median length of stay prior the MDR isolation was 9.9 days (range 3 to 25 days). Neurological disorders represented 68% of cases and 51% of controls patients housing in the ER (p=O.024). Multivariated ana1ysisshowed as significant risk factors for 11DR colonization: age, use of mechanical ventilation, use of nasal gastric tubes and length of thorax drains. Mortality rates were significant1y higher in cases (27.1 %) than controls (12.75). Thirty strains of MDR A. baumannii isolated from patients in the ER were typed and 13 plasmid profiles were present among the isolates. Profile A was present in 9 strains and the other profiles were distributed in two or in individual strains. Genomic DNA typing revealed two endemic patterns (profile A-16 strains; profile B-13 strains) and one strain had its individual profile. Twenty MDR A. baumannii isolates from patients housing in different wards of the hospital were also typed by PFGE, as control strains. Among the controls strains, 8 profiles were identified inc1uding the two endemic profiles from ER. The hospital infections (lU) rate was 27.26 infections/1,000 patient-days and 59.3% were case patients (p=0.001). Pneumonia related to mechanical ventilation was 64.2 pneumonia / 1,000 ventilator-days, urinary tract infection was 8.4 UTI/1,000 indwelling urinary catheter-days and blood stream infections related to central venous catheter was 8.2 infections/1,0000 catheter-days. In conclusion, the lack of specialized beds in HC-UNICAMP for severely ill patients assisted in the ER resulted in prolonged permanence of these patients in the ER. In our study, 66% of the patients with prolonged permanence in the ER were patients with neurological disorders. Elderly, use of mechanical ventilation, nasal gastric tubes and thorax drains were independent1y variables for MDR colonization. Interestingly, the patients with RI resulted in significant1y higher rate of RI than patients hospitalized in the intensive care units of our hospital. The general conditions of the ER certainly contributed to the high prevalence of A baumannii MDR colonization and the predominance of only two MDR c10nes in this unit is still a subject of investigation. The detection of isolates with identical PFGE patterns in different units of the hospital suggests intra-hospital spread of MDR A. baumannii / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

La connaissance du métabolisme demeure incomplète mais sa compréhension reste un but majeur tant pour la recherche fondamentale qu’appliquée. Environ 40% des gènes des organismes procaryotes n’ont pas de fonction précise proposée. En conséquence, de nombreuses voies métaboliques sont incomplètes et d’autres restent à découvrir. Ces lacunes rendent très difficile la modélisation et la compréhension du métabolisme d’une cellule. Le laboratoire développe une nouvelle stratégie pour mettre au jour des voies métaboliques inconnues. Celle-ci est basée sur la bactérie modèle du laboratoire, Acinetobacter baylyi ADP1 (AP1). Elle exploite la banque complète de mutants de délétion de cet organisme (2600 mutants) ainsi que les avancées récentes du laboratoire dans le domaine de la métabolomique par chromatographie liquide couplée à la spectrométrie de masse à très haute résolution (LC/MS).Cette étude est liée à l’observation que lors d’un changement de source de carbone (succinate vs quinate) ADP1 produit de nombreux métabolites d’identité inconnue, non reliés à notre connaissance de son métabolisme. Ces métabolites orphelins (MO) participent à des voies métaboliques nouvelles et représentent des points d’entrée dans la partie obscure du métabolisme bactérien.Ce projet comporte deux volets. Il s’agit d’une part de procéder à l’élucidation structurale des MO et d’autre part d’identifier l'ensemble des gènes impliqués dans leur synthèse.Dans le cadre de ce travail de thèse, nous avons établi l’identité d’un premier MO : l’acide 3-(3-aminopropylamine)- 4-hydroxybenzoïque. Ce composé est un bien métabolite nouveau, jamais référencé auparavant. Dans un premier temps, des expériences de LC/MS impliquant notamment des fragmentations séquentielles MSn et des échanges isotopiques H/D ont permis de déterminer sa composition élémentaire, son nombre de protons échangeables et ont suggéré une structure aromatique de la molécule. Ensuite, son identification a nécessité sa purification à l’échelle de la centaine de µg. Puis, une analyse RMN complète (1H, 13C, COSY, 1H-13C HSQC, 1H-13C HMBC, et 1H-15N HMBC) a permis de préciser sa structure.Par ailleurs, pour identifier les gènes impliqués dans la synthèse des MO, nous avons procédé à l’analyse métabolomique de l’ensemble des mutants de la collection d’ADP1. Pour cela, nous avons développé des protocoles à haut-débit pour la préparation des métabolomes (100 métabolomes par jour), l’acquisition des données LC/MS (8 minutes par acquisition) et enfin leur analyse automatisée. Nous regardons, pour chaque mutant, quels sont les MO présents et absents. L’absence d’un MO chez un mutant donné indique que le gène excisé est impliqué dans sa synthèse. L’analyse systématique de la banque doit permettre in fine de dresser la liste de tous les gènes impliqués dans la synthèse de chaque MO. Les résultats du criblage, toujours en cours, permettront ainsi de commencer à reconstituer ces voies métaboliques nouvelles. / Our knowledge of metabolism remains incomplete and its understanding is a major goal for both basic and applied research. About 40% of prokaryotic genes have no specific function proposed. Many metabolic pathways are therefore incomplete and others remain to be discovered. These shortcomings (ou gaps ??) make the modelling and the understanding of cell metabolism very difficult. The laboratory is developing a new strategy to uncover unknown metabolic pathways. This is based on the model bacterium Acinetobacter baylyi ADP1 (ADP1). It exploits the complete library of deletion mutants of this organism (2600 mutants) as well as the recent advances of the laboratory in the field of metabolomics by liquid chromatography coupled with very high resolution mass spectrometry (LC / MS).This study is related to the observation that during a change of carbon source (from succinate to quinate), ADP1 produces many metabolites of unknown identity, unrelated to our knowledge of its metabolism. These orphan metabolites (MO) participate in novel metabolic pathways and represent entry points into the dark part of bacterial metabolism.This project has two objectives. The first is to elucidate of the structure of MOs, and the second is to identify all the genes involved in their synthesis.As part of this thesis, we established the identity of a first MO: 3- (3-aminopropylamine) - 4-hydroxybenzoic acid. This compound is a new metabolite, never referenced before. Firstly, LC / MS experiments involving sequential fragmentation MSn and isotopic H / D exchanges allowed us to determine its elementary composition, its number of exchangeable protons and suggested an aromatic structure of the molecule. Then, its identification required its purification on the scale of the hundred μg. Then, a complete NMR analysis (1H, 13C, COZY, 1H-13C HSQC, 1H-13C HMBC, and 1H-15N HMBC) allowed us to specify the structure of the compound.Furthermore, to identify genes involved in the synthesis of MOs, we proceeded to the metabolomic analysis of all the strains of the ADP1 mutant collection. For this, we developed high-throughput protocols for the preparation of metabolomes (100 metabolomes per day), for the acquisition of LC / MS data (8 minutes per acquisition) and for their automated analysis. For every mutant, we are looking which Mos are present and which are absent. The absence of an MO in a given mutant indicates that the excised gene is involved in its synthesis. The systematic analysis of the bank should allow to list all the genes involved in the synthesis of each MO. The results of the screening, still in progress, will thus make it possible to begin to reconstitute these new metabolic pathways.

Acinetobacter baylyi ADP1 (ADP1), une bactérie du sol aux capacités de dégradation des molécules aromatiques remarquables est utilisée au laboratoire comme organisme modèle pour l’élucidation de nouvelles fonctions enzymatiques et voies métaboliques. Tirant profit de deux qualités d’ADP1 (compétence naturelle et recombinaison homologue efficace), une collection complète de mutants knock-out a été obtenue. L’analyse de cette ressource génomique a fait apparaître des phénotypes non prédits par l’annotation aboutissant notamment à l’initiation de l’étude du métabolisme du soufre chez ADP1 avec deux axes : (1) la biosynthèse de la L-méthionine (L-Met) et plus spécialement l’étude des familles d’enzymes non homologues MetX et MetA catalysant l’acylation de l’homosérine dans le monde vivant, première étape de cette voie ; (2) le recyclage de la L-méthionine et les voies d’assimilation des molécules soufrées. La thèse s’inscrit dans ces deux thématiques. Tout d’abord, un vaste projet combinant criblage expérimental et analyse structurale des sites actifs de MetA et MetX avait permis précédemment d’identifier les résidus déterminant l’usage de l’acyl-CoA et de proposer des règles précises de prédiction de fonction pour ces deux familles finalement isofonctionnelles. Cette étude avait révélé entre autres que 10% des MetX n’étaient pas impliqués dans la biosynthèse de L-Met. Nous avons donc entrepris la caractérisation de ces paralogues à l’activité inédite L-sérine O-succinyltransférase (SST) et finalement impliquées dans la biosynthèse de la L-cystéine (L-Cys). Jusqu'alors, l'acétylation par les L-sérine O-acétyltransférases (SAT) était le seul moyen connu d'activer la L-sérine. La détermination des paramètres cinétiques des SST ainsi que la caractérisation par LCMS in vitro puis la détection in vivo d’O-succinyl-L-sérine (OSS) dans les métabolomes de la levure Schizosaccharomyces pombe et la bactérie Xanthomonas campestris ont permis de démontrer la fonction de ces paralogues. Pour compléter la démonstration, la caractérisation des cystéine synthases (CysK) respectives a montré qu’elles effectuent en effet la sulfhydrylation de l’OSS pour former de la L-Cys. Notre étude a ainsi également révélé que la voie décrite de la biosynthèse de la L-cystéine chez les levures jusqu’alors extrapolée à partir de la levure modèle Saccharomyces cerevisae (voie de transsulfuration réverse) était en réalité une exception et que la grande majorité des levures synthétisent la L-Cys à partir de la L-sérine via ce nouveau métabolite, l’OSS. Cette thèse, dans un deuxième temps, a initié l’exploration expérimentale des voies d’assimilation du soufre chez ADP1 chez qui notamment aucune voie de recyclage de la L-Met n’était prédite alors qu’elle y est source de soufre. Combinant des approches complémentaires (génétique inverse par phénotypage de la collection complète de mutants sur diverses sources de soufre, transcriptomique sur L-Met et L-Cys versus sulfate, criblage des enzymes à PLP d’ADP1 potentiellement impliquées, étude biochimique des candidats et construction de mutants d’ADP1), une image assez précise des voies d’assimilation de diverses sources de soufre se dessine. Par exemple, les voies d’assimilations de la L-Met et du DMSP semblent aboutir à la production de sulfite via la synthèse de méthanesulfonate sous le contrôle du régulateur transcriptionnel CBL. De plus, le KMBA et le méthanethiol sont probablement des intermédiaires de la voie de recyclage de la L-Met, alors que le DMSO et le DMSO2 semblent être des intermédiaires cataboliques uniquement pour le DMSP. / Acinetobacter baylyi ADP1 (ADP1), a soil bacterium with remarkable aromatic molecule degradation capacities is used in the laboratory as a model organism for the elucidation of new enzymatic functions and metabolic pathways. Taking advantage of two qualities of ADP1 (natural competence and effective homologous recombination), a complete collection of knock-out mutants has been obtained. Analysis of this genomic resource revealed unpredicted phenotypes by the annotation, leading in particular to the initiation of the study of sulphur metabolism in ADP1 with two axes: (1) the biosynthesis of L-methionine (L-Met) and more specifically the study of the families of non-homologous MetX and MetA enzymes catalysing the acylation of homoserine, the first step of this pathway; (2) the recycling of L-methionine. The thesis falls within these two themes. First, a vast project combining experimental screening and structural analysis of the active sites of MetA and MetX had previously made it possible to identify the residues determining the use of acyl-CoA and to propose precise function prediction rules for these two finally isofunctional families. Among other things, this study revealed that 10% of MetX were not involved in L-Met biosynthesis. We have therefore undertaken the characterization of these paralogues with novel activity L-serine O-succinyltransferase (SST) and finally involved in the biosynthesis of L-cysteine (L-Cys). Until then, acetylation by L-serine O-acetyltransferases (SAT) was the only known way to activate L-serine. The determination of kinetic parameters of SST as well as in vitro LCMS characterization and in vivo detection of O-succinyl-L-serine (OSS) in the metabolomes of Schizosaccharomyces pombe yeast and Xanthomonas campestris bacterium demonstrated the function of these paralogues. To complete the demonstration, the characterization of the respective cysteine synthases (CysK) showed that they indeed perform sulfhydrylation of OSS to form L-Cys. Our study also revealed that the described pathway of L-cysteine biosynthesis in yeasts hitherto extrapolated from the model yeast Saccharomyces cerevisae (reverse transsulfuration pathway) was actually an exception and that the vast majority of yeasts synthesize L-Cys from L-serine via this new metabolite, OSS. This thesis, in a second stage, initiated the experimental exploration of sulphur assimilation pathways in ADP1, in which no L-Met recycling pathway was predicted even though it is a source of sulphur. Combining complementary approaches (reverse genetics by phenotyping the complete collection of mutants on various sulphur sources, transcriptomics on L-Met and L-Cys versus sulphate, screening of ADP1 PLP enzymes, biochemical study of ADP1 candidates and mutants), a fairly accurate picture of the assimilation pathways of various sulphur sources is emerging. For example, the assimilation pathways of L-Met and DMSP appear to lead to the production of sulphite via methanesulphonate synthesis under the control of the transcriptional regulator CBL. In addition, KMBA and methanethiol are probably intermediates in the L-Met recycling pathway, whereas DMSO and DMSO2 appear to be catabolic intermediates only for DMSP.

La recrudescence de maladies infectieuses et le nombre grandissant de bactéries résistantes aux antibiotiques sont des problèmes majeurs de santé publique. Ainsi, le développement d’une nouvelle catégorie d’agents antimicrobiens est de plus en plus pressant. Nous nous sommes tournés vers les bactériophages afin d’en connaître davantage sur les moyens qu’ils utilisent pour tuer les bactéries. Cependant, jusqu’à maintenant il existe très peu de connaissances sur leur activité bactéricide. Nous avons donc caractérisé le mode d’action de la protéine de lyse P1 du phage AP205 à l’aide d’approches in vitro et in vivo. Ce phage a pour hôte Acinetobacter. La protéine P1 cible un élément essentiel à la survie bactérienne, la paroi de peptidoglycane. En fait, nous avons découvert qu’elle inhibe plusieurs enzymes impliquées au niveau de cette voie synthèse. Nous espérons que les connaissances acquises sur cette protéine antibiotique contribueront au développement d’une nouvelle classe d’agents antimicrobiens. / The constant increase of infectious diseases and bacterial resistance to antibiotics are major problems of public health. Thus, development of a new class of antimicrobial agents is very urgent. We have decided to explore bacteriophages to learn more about the way they kill bacteria. Therefore, until now, the knowledge about this bactericidal activity is limited. We have characterized the inhibition mechanism of P1 lysis protein from phage AP205 using in vitro and in vivo approaches. The host of this phage is Acinetobacter. The P1 lysis protein targets an essential element for the bacterial survival, the bacterial cell wall. Actually, we hope that knowledge about this protein will contribute to the development of a new class of antibiotics.

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Acinetobacter baumannii (Ab) é um dos principais agentes de infecção hospitalar, principalmente em Unidades de Terapia Intensiva (UTI). A principal característica da bactéria é a sua resistência intrínseca a diversos antimicrobianos, o que favorece sua persistência no ambiente hospitalar, causando infecções de difícil controle e tratamento. Nos esquemas terapêuticos, os antibióticos carbapenêmicos são os fármacos de escolha, porém nos últimos anos a resistência a estas drogas tem aumentado drasticamente em função da emergência e disseminação de cepas produtora de carbapenemases [i.e., oxacilinases (OXA) e metalo-beta-lactamases (MβL)]. O objetivo do presente trabalho foi avaliar a produção de enzimas carbapenemases do tipo OXA e MβLs, em 36 amostras multirresistentes de A. baumannii, previamente triadas, provenientes de 8 hospitais brasileiros, durante 2004/2008. A caracterização fenotípica e genotípica dos isolados foi realizada por meio da determinação de CIM, hidrólise enzimática e PCR para pesquisa dos genes blaOXA e blaMβL, assim como seqüências de inserção (ISAba-1, ISAba-3) responsáveis pela mobilização dos determinantes de resistência do tipo blaOXA. Finalmente a análise da diversidade genética foi realizada por ERIC-PCR com análise de clusters por coeficiente de Dice. Todos os 36 isolados apresentaram 100% de resistência para imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidima, ciprofloxacina e iperacilina/tazobactam, enquanto que os antibióticos com maior atividade in vitro foram a ampicilina/sulbactam (61,2%) > tobramicina (61,1%) > gentamicina (47,3%) > amicacina (28%). Em todos os isolados foi confirmada a presença intrínseca dos genes blaOXA-51 e ISAba-1, não apresentando colinearidade entre eles, enquanto que 41,6% dos isolados carregaram a combinação dos genes ISAba-1/blaOXA-23 (Genbank accession FJ628170), um isolado (2,7%) carregou a combinação de genes ISAba3/blaOXA-58/ISAba3 (Genbank accession FJ492877) e dois isolados (5,5%) clonalmente relacionados, carregaram o gene blaOXA-72 (Genbank accession FJ969387). Surpreendentemente, em um centro hospitalar foi documentada a presença de um surto de infecção por Ab produtor de OXA-23. Finalmente a tipagem epidemiológica dos isolados revelou a presença de 13 clusters, sendo que 8 diferentes clusters carregavam o gene blaOXA-23. Em resumo, nossos resultados confirmam a disseminação de cepas produtoras de OXA-23 associadas com ISAba-1 no Brasil, assim como a presença intrínseca do gene blaOXA-51 em Ab. Por outro lado, este é o primeiro relato de isolados carbapenem resistentes carregando os genes blaOXA-58 e blaOXA-72 em hospitais brasileiros, os quais aparentemente surgiram em 2004 e 2008, respectivamente. / Acinetobacter baumannii (Ab) is a leading cause of hospital infection, mainly in intensive care units. The main characteristic of the bacterium is its intrinsic resistance to diverse antimicrobial agents, which contribute to persistence in hospital environments causing infections of difficult control and treatment. In therapeutic schedules, carbapenems are choice antibiotics, however in recent years the resistance to these drugs has increased drastically in function of the emergency and dissemination of carbapenemase-producing isolates [i.e., oxacilinases (OXA) and metalo-beta-lactamases (MβLs)]. The aim of this work was to evaluate the production of OXA- and MβL-like carbapenemases, in 36 isolates previously screened as multidrug-resistant (MDR) A. baumannii, recovered from 8 Brazilian hospitals, during 2004/2008. Phenotypic and genotypic characterization of MDR Ab was carried out using CIM determination, enzymatic hydrolysis and PCR for screening of the blaOXA- and blaMβL-like genes, and insertion sequences (ISAba-1, ISAba-3) responsible for mobilization of blamOXA-like gene cassettes. Analysis of genetic relationship was carried out by ERIC-PCR with analysis of clusters for Dice`s coefficient. All of the 36 isolates showed 100% resistance to imipenem (CIM90 > 64 µg/mL), meropenem, ceftazidime, ciprofloxacin and piperacillin/tazobactam, whereas antibiotics exhibiting a best in vitro activity were ampicillin/sulbactam (61.2%) > tobramycin (61.1%) > gentamicin (47.3%) > amikacin (28%). Presence of blaOXA-51 and ISAba-1 genes was confirmed in all isolates, not presenting collinearity between them, whereas 41.6% isolates carried the ISAba-1/blaOXA-23 gene array (Genbank accession FJ628170). One Ab isolate harbored the ISAba-3/blaOXA-58/ISAba-3 gene array (2.7%) (Genbank accession FJ492877) and 5.5% of Ab isolates harbored the blaOXA-72 gene (Genbank accession FJ969387). Surprisingly, an outbreak of infection with MDR Ab producing OXA-23 enzyme was documented. Finally the ERIC-PCR typing revealed the presence of 13 clusters, of which 8 different clusters carried the blaOXA-23 gene. In summary, our results confirm the dissemination of OXA-23-producing Ab isolates associated with the ISAba-1 gene, in Brazil, as well as the intrinsic presence of the blaOXA-51 gene cassette. On the other hand, this is the first report of carbapenem-resistant Ab isolates harboring genes blaOXA-58 and blaOXA-72 recovered in Brazilian hospitals, which most likely emerged in 2004 and 2008, respectively.

by Leung Chi-man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 106-117). / Abstracts in English and Chinese. / ABSTRACT (English) --- p.i-ii / ABSTRACT (Chinese) --- p.iii / ACKNOWLEDGMENT --- p.iv / LIST OF CONTENTS --- p.v-viii / LIST OF TABLES --- p.ix-x / LIST OF FIGURES --- p.xi / ABBREVIATIONS --- p.xii / TERMS --- p.xiii-xiv / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1 --- History and taxonomic background of Acinetobacter --- p.1 -3 / Chapter 1.2 --- "Microbiology, ecology and habitats of Acinetobacter species" --- p.4 / Chapter 1.2.1 --- Isolation of Acinetobacter --- p.4 / Chapter 1.2.2 --- Clinical importance of Acinetobacter species --- p.5 / Chapter 1.2.3 --- Acinetobacter 226}0ؤ An endemic nosocomial pathogens of particular importance in Hong Kong --- p.6-7 / Chapter 1.2.4 --- Lack of knowledge of Acinetobacter genomic DNA groups --- p.7 / Chapter 1.2.5 --- Human carriage of Acinetobacter species --- p.8-9 / Chapter 1.2.6 --- Species from environment --- p.9-10 / Chapter 1.3 --- Identification of Acinetobacter --- p.10-11 / Chapter 1.3.1 --- DNA-DNA hybridization --- p.11 / Chapter 1.3.2 --- Phenotypic identification by conventional tests --- p.11 / Chapter 1.3.3 --- Genotypic identification by Amplified Ribosomal DNA Restriction Analysis (ARDRA) --- p.12-13 / Chapter 1.3.4 --- Other PCR methods --- p.13 / Chapter 1.3.5 --- Genotypic identification by tDNA fingerprinting --- p.14 / Chapter 1.4 --- Biotyping --- p.14-15 / Chapter 1.5 --- Background of this research project --- p.15 / Chapter 1.5.1 --- Distribution of different species of Acinetobacter --- p.15-16 / Chapter 1.5.2 --- Consideration of taxonomical problems --- p.16 / Chapter 1.5.3 --- Characterization of Acinetobacter isolates --- p.17 / Chapter 1.6 --- Research objectives --- p.18 / Chapter CHAPTER 2 --- MATERIALS AND METHODS / Chapter 2.1 --- Materials --- p.19 / Chapter 2.1.1 --- Reference strains --- p.19-20 / Chapter 2.1.2 --- Antimicrobial agents and chemicals --- p.20-21 / Chapter 2.1.3 --- "Carbohydrates, enzymes and other materials" --- p.22 / Chapter 2.1.4 --- Commercial media and media prepared manually --- p.23-26 / Chapter 2.1.5 --- Reagents --- p.27 / Chapter 2.1.6 --- Instruments and Software used in this study --- p.28 / Chapter 2.2 --- Methods --- p.28 / Chapter 2.2.1 --- Routine laboratory collection --- p.28 / Chapter 2.2.2 --- Blood culture collection --- p.29 / Chapter 2.2.3 --- Human carriage site collection --- p.29-31 / Chapter 2.2.4 --- Surveillance screening of clinical specimens --- p.31-32 / Chapter 2.2.5 --- Environmental samples 226}0´ؤؤ vegetable --- p.32 / Chapter 2.2.6 --- Environmental samples 一 soil --- p.32-34 / Chapter 2.3 --- General bacteriological techniques for genus identification --- p.34-37 / Chapter 2.4 --- Molecular techniques used for the delineation of genomic DNA groups --- p.37 / Chapter 2.4.1 --- Amplified ribosomal restriction DNA analysis (ARDRA) --- p.37-39 / Chapter 2.4.2 --- Characterization of acinetobacters by tRNA spacer (tDNA) fingerprinting analysis --- p.40-42 / Chapter 2.5 --- Biotyping of Acinetobacter spp --- p.42 / Chapter 2.6 --- Minimum Inhibitory Concentration (MIC) --- p.43-44 / Chapter CHAPTER 3 --- DISTRIBUTION OF ACINETOBACTER SPECIES / Chapter 3.1 --- Results --- p.45 / Chapter 3.1.1 --- Isolation of acinetobacters from surveillance screening of clinical specimens --- p.45-49 / Chapter 3.1.2 --- Isolation of acinetobacters from routine laboratory specimens --- p.49-50 / Chapter 3.1.3 --- Distribution of acinetobacter genomic DNA groups in all clinical specimens --- p.50-51 / Chapter 3.1.4 --- Isolation of acinetobacters from blood culture --- p.51 -52 / Chapter 3.1.5 --- Isolation of acinetobacters from human carriage sites --- p.53-55 / Chapter 3.1.6 --- Isolation of acinetobacters from environmental samples --- p.56-59 / Chapter 3.2 --- Discussion --- p.60 / Chapter 3.2.1 --- Prevalence of Acinetobacter species in clinical specimens --- p.60-61 / Chapter 3.2.2 --- Distribution of acinetobacter genomic DNA groups in clinical specimens --- p.61 -63 / Chapter 3.2.3 --- Distribution of different genomic DNA groups of Acinetobacter on carriage sites --- p.63-65 / Chapter 3.2.4 --- Distribution of different genomic DNA groups of Acinetobacter in environmental samples --- p.65-66 / Chapter CHAPTER 4 --- AN ASSESSMENT OF TDNA FINGERPRINTING IN THE IDENTIFICATION OF ACINETOBACTER SPECIES / Chapter 4.1 --- Results --- p.67 / Chapter 4.1.1 --- Complexity of tDNA fingerprint patterns --- p.67 / Chapter 4.1.2 --- Assessment of tDNA fingerprinting --- p.67-69 / Chapter 4.1.3 --- Construction of fingerprints database with the reference Acinetobacter strains --- p.70 / Chapter 4.1.4 --- Delineation of different genomic DNA groups in the fingerprints database --- p.71 / Chapter 4.1.5 --- Cluster analysis of tDNA fingerprints of Acinetobacter isolates classified by ARDRA --- p.71-73 / Chapter 4.2 --- Discussion --- p.74-75 / Chapter 4.3 --- Conclusion --- p.75-76 / Chapter CHAPTER 5 --- "BIOTYPING OF ISOLATES FROM CLINICAL SPECIMENS, CARRIAGE SITES AND ENVIRONMENTAL SAMPLES" / Chapter 5.1 --- Results --- p.77 / Chapter 5.1.1 --- Biotypes of A. baumannii --- p.77 / Chapter 5.1.2 --- Biotypes of genomic DNA group --- p.3 78 / Chapter 5.1.3 --- Biotypes of genomic DNA group 13TU --- p.78-79 / Chapter 5.2 --- Discussion --- p.79-80 / Chapter CHAPTER 6 --- ANTIMICROBIAL SUSCEPTIBILITIES OF ACINETOBACTER SPECIES / Chapter 6.1 --- Results --- p.81 / Chapter 6.1.1 --- Bacterial strains --- p.81 / Chapter 6.1.2 --- Susceptibilities of Acinetobacter genomic DNA groups --- p.82-86 / Chapter 6.1.3 --- Distribution of resistance patterns in Acinetobacter species --- p.87-90 / Chapter 6.2 --- Discussion --- p.91 / Chapter 6.2.1 --- Antimicrobial susceptibilities of different genomic DNA groups of Acinetobacter from different sources --- p.91-92 / Chapter 6.2.2 --- Emergence of P-Lactam resistance --- p.92 / Chapter 6.2.3 --- Activity of sulbactam --- p.93 / Chapter 6.2.4 --- Susceptibility of carbapenem --- p.93 / Chapter 6.2.5 --- Quinolones resistance --- p.94 / Chapter 6.2.6 --- Aminoglycoside resistance --- p.94-95 / Chapter 6.3 --- Conclusion --- p.95 / Chapter CHAPTER 7 --- GENERAL DISCUSSION / Chapter 7.1 --- Significance of delineation of genomic DNA groups of Acinetobacter --- p.96-98 / Chapter 7.2 --- Epidemiology and clinical implication of Acinetobacter species in Hong Kong --- p.99-104 / Chapter 7.3 --- Characterization of Acinetobacter --- p.104 / Chapter 7.4 --- Future work --- p.104-105 / REFERENCES --- p.106-117 / APPENDIX --- p.118-126

Acinetobacter baumannii, microorganisme responsable d’infections nosocomiales survenant le plus souvent par épidémies, pose un problème émergent de multi-résistance aux antibiotiques, notamment aux carbapénèmes. Cette résistance aux carbapénèmes est le plus souvent liée à l’acquisition et à l’expression de gènes codant pour des b-lactamases de classe D à activité de carbapénèmase (CHDLs). Le réservoir de ces gènes de résistance est inconnu.Nous avons montré que l’espèce bactérienne Acinetobacter radioresistens, espèce proche de A. baumannii, est le progéniteur du déterminant de résistance aux carbapénèmes le plus prévalent dans le monde chez A. baumannii : le gène blaOXA-23. Nous avons identifié l’espèce Acinetobacter lwoffii comme progéniteur d’un gène codant pour une nouvelle CHDL : OXA-134. Nous avons identifié les CHDLs naturelles des espèces Acinetobacter johnsonii, Acinetobacter calcoaceticus et Acinetobacter haemolyticus, dénommées respectivement OXA-211, OXA-213 et OXA-214. Nous avons ensuite étudié l’expression des gènes codant pour ces CHDLs naturelles en prenant l’exemple du gène naturel blaOXA-51/-66 de A. baumannii et nous avons démontré que ce gène était impliqué dans la réduction de sensibilité aux carbapénèmes chez A. baumannii, notamment lorsque la séquence d’insertion (IS) ISAba1 était présente en amont de ce gène. Nous avons ensuite identifié une nouvelle IS, ISAba9, à l’origine de la surexpression du gène blaOXA-51 naturel de A. baumannii. Enfin, nous avons identifié VIM-4, b-lactamase de classe B capable d’hydrolyser les carbapénèmes, dans une souche de Acinetobacter non-baumannii, Acinetobacter genomic species 16. / Carbapenem resistance in Acinetobacter baumannii is increasingly reported and leads to difficult-to-treat nosocomial infections. Carbapenem-hydrolyzing class D b-lactamases (CHDLs) represent the main mechanism of resistance to carbapenems in Acinetobacter spp. The origin (reservoir) of those CHDL genes remains unknown. We identified Acinetobacter radioresistens as the reservoir of the blaOXA-23-like genes currently emerging as the sources of carbapenem resistance in A. baumannii worldwide. Acinetobacter lwoffii was found to be a reservoir of a novel type of CHDL-encoding gene: blaOXA-134. We described the naturally-occurring CHDLs from Acinetobacter johnsonii, Acinetobacter calcoaceticus and Acinetobacter haemolyticus, respectively named OXA-211, OXA-213 et OXA-214. We reported an in vivo selection of reduced susceptibility to carbapenems in A. baumannii. This reduced susceptibility to carbapenems was related to selection of the ISAba1-related overexpression of the naturrally-occurring blaOXA-51/-69 gene from A. baumannii. We identified the novel insertion sequence ISAba9 as being involved in blaOXA-51/-69 gene overexpression, thus contributing to carbapenem resistance in A. baumannii.We reported the first identification of the metallo-b-lactamase VIM-4 determinant in Acinetobacter spp.

Made available in DSpace on 2015-07-22T20:49:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-31. Added 1 bitstream(s) on 2015-08-11T03:26:21Z : No. of bitstreams: 1 Publico-189.pdf: 1023260 bytes, checksum: 261ef67ec48667616e34852178c2c07d (MD5) / Objetivo geral: Avaliar o perfil de sensibilidade aos antimicrobianos carbapenêmicos e ampicilina/sulbactam, e produção de carbapenemases entre isolados clínicos de Acinetobacter spp. provenientes de infecção de corrente sanguinea de pacientes hospitalizados nos centros médicos do Programa de Vigilância SCOPE Brasil, entre junho de 2007 e julho de 2009; Objetivos específicos: (i) avaliar perfil de sensibilidade aos antimicrobianos imipenem, meropenem e ampicilina/sulbactam pelo método de diluição em ágar; (ii) caracterizar a freqüência e tipos de metalo-- lactamases presentes nos isolados resistentes aos carbapenêmicos; (iii) Determinar a freqüência dos genes codificadores de oxacilinases entre os isolados resistentes aos carbapenêmicos.Material e Métodos: Foram avaliadas 206 isolados de Acinetobacter spp. isoladas do primeiro episódio de infecção de corrente sanguínea (ICS) de pacientes hospitalizados em 16 centros médicos de diferentes regiões do país, participantes do isolados foram submetidos a diluição em ágar para imipenem, meropenem e ampicilina/sulbactam para obter a CIM desses antimicrobianos. Os isolados resistentes a imipinem e/ou meropenem foram submetidos à PCR multiplex em tempo real para detecção dos genes codificadores de ML(blaIMP, blaVIM, blaSIM, blaSPM, blaGIM) e PCR multiplex para detecção de genes codificadores de oxacilinases(blaOXA23-like, blaOXA24-like, blaOXA51-like, blaOXA58-like).Os isolados carreadores de blaIMP foram submetidos ao PFGE. Resultados e Conclusões: As taxas de resistência foram 63% para imipenem (CIM50 64 μg/mL e CIM90 256 μg/mL) e meropenem (CIM50 32 μg/mL e CIM90 256 μg/mL) e 45% para ampicilina/sulbactam (CIM50 8 μg/mL e CIM90 64 μg/mL). Dos 130 isolados submetidos a pesquisa ML 24 (18%) apresentaram o gene blaIMP e para pesquisa de OXA 94 (74%) apresentaram o gene blaOXA23-like e 24(20%) somente o gene blaOXA51-like. As altas taxas de resistência aos carbapenêmicos variaram em relação a região geográfica brasileira. A detecção do gene blaIMP em apenas 24% dos isolados demonsntra não ser este o principal mecanismo de resistência aos carbapenêmicos nesse estudo. Entretanto foi observada disseminação do gene blaOXA23-like em todas as regiões brasileiras,caracterizando o principal mecanismo de resistência frente aos carbapenêmicos. Foi observada disseminação clonal em um hospital em Fortaleza e entre 3 hospitais em São Paulo nos isolados carreadores do gene blaIMP. / Main Objective: To evaluate the susceptibility profile to antibiotics carbapenems and ampicillin/sulbactam, and the carbapenemases production among bloodstream isolates of Acinetobacter spp. from hospitalized patients participating in the SCOPE Program, period of june 2007 to July 2009. Specific objectives: (i) to evaluate the susceptibility to imipenem, meropenem and ampicillin/sulbactam by agar dilution; (ii) to characterize the frequency and types of metalo-B-lactamases in the carbapenem resistant isolates; (iii) to determine the frequency of oxacillinases coding genes among the carbapenem resistant isolates. Material and Methods: 206 isolates from the first blood stream nosocomial infection from 16 medical centers from the 5 regions of the country were susceptibility tested by agar dilution for imipenem, meropenem and ampicillin/sulbactam. The carbapenem resistant isolates were submitted to Real time multiplex PCR for detection of MBL genes (blaIMP, blaVIM, blaSIM, blaSPM, blaGIM) and oxacillinases coding genes (blaOXA23-like, blaOXA24-like, blaOXA51-like, blaOXA58-like). The isolates carring blaIMP were typed by PFGE . Results and Conclusion: 63% of the isolates were resistant to carbapenems (imipenem CIM50 64 μg/mL e CIM90 256 μg/mL); meropenem (CIM50 32 μg/mL e CIM90 256 μg/mL) and 45% to ampicillin/sulbactam (CIM50 8 μg/mL e CIM90 64 μg/mL). 24 (18%) carbapenem resistant isolates showed blaIMP. 94 (74%) showed blaOXA23 and 26 (20%) showed only blaOXA51. The highest resistant rate to carbapenem was observed in the Northeast region and the lowest rate in the South region. Clonal hospital and inter-hospital dissemination was observed for IMP carring isolates but not among the different regions. / TEDE / BV UNIFESP: Teses e dissertações