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Submitted by Natalia Vieira (natalia.vieira@famerp.br) on 2016-05-30T18:51:03Z No. of bitstreams: 1 julianatiemitakahashi_dissert.pdf: 1874616 bytes, checksum: 4b57c05bf0ee3402a9384302e26ebd23 (MD5) / Made available in DSpace on 2016-05-30T18:51:03Z (GMT). No. of bitstreams: 1 julianatiemitakahashi_dissert.pdf: 1874616 bytes, checksum: 4b57c05bf0ee3402a9384302e26ebd23 (MD5) Previous issue date: 2015-11-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Introduction: Bacterial resistance to antimicrobials is an important public health problem, which results in increased treatment period, increase in mortality and in health care costs. This phenomenon is considered a result of the indiscriminate use of antimicrobials in human and veterinary medicine, and along the food chain. In recent years, it has been observed the frequent isolation of resistant bacteria from animal production and food of animal origin. In this context, have been highlighted the resistance to beta-lactâmicos and to quinolones, the first, due to extended-spectrum beta-lactamase (ESBL) production and the second, resultant from the acquisition of qnr genes encoding proteins that interfere in quinolone action on DNA gyrase and topoisomerase IV. Several bacterial species with antimicrobial resistance have been isolated from farm animals. Objective: This study evaluated the antimicrobial susceptibility profile and the presence of resistance genes blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS and oqxAB in Enterobacteriaceae isolated from meat market in the municipality of São José do Rio Preto- SP, acquired between November 2010 and August 2012. Material and Methods: The antimicrobial susceptibility was determined by disk diffusion according to CLSI. Strains resistant to beta-lactams and quinolones were submitted to PCR using specific primers for detection and sequencing of these genes for identification. Results: A total of 75 isolates were resistant to beta-lactam and 125 to quinolones. Among these, 6.6% blaCTX-M, 6.6% blaTEM, 16% blaSHV, 21.6% had qnr variants and 13.6% oqxAB. In a strain of Aeromonas sobria was detected the coexistence of qnrA and qnrS genes; the qnrB gene was detected in five strains of E. coli, five K.pneumoniae, two Salmonella spp. and 12 Citrobacter spp. The qnrB19 gene was detected in two K. pneumoniae, duas E. coli and one Citrobacter braakii. qnrB2 was detected in three K. pneumoniae. The qnrS1 gene was detected in a strain of K. pneumoniae. The sequencing blaCTX-M revealed blaCTX-M-2, all of which were derived from chicken meat samples. Conclusion: These data demonstrate there nay be association between excessive use of antimicrobials in animal production and isolation of bacteria resistant to these antimicrobials. The presence of bacteria resistant to antimicrobials in meat intended for human consumption consists in a public health problem and can affect commercials relationships of the country, since it the Brazil is one of the world's largest meat exporters. / Introdução: A resistência bacteriana aos antimicrobianos é um importante problema de saúde pública, que prolonga o período de tratamento, aumenta as taxas de mortalidade e custos da assistência à saúde. Este fenômeno pode ter considerado consequência do uso indiscriminado de antimicrobianos na medicina humana e veterinária, e ao longo da cadeia produtiva de alimentos. Nos últimos anos tem sido observado o frequente isolamento de bactérias resistentes a partir de animais de produção e alimentos de origem animal. Neste contexto, têm-se destacado a resistência aos beta-lactâmicos, devido à produção de beta-lactamase de espectro estendido (ESBL) e às quinolonas pela aquisição de genes qnr que codificam proteínas que interferem na ação das quinolonas sobre a DNA-girase e a topoisomerase IV. Diversas espécies bacterianas apresentando resistência aos antimicrobianos têm sido isoladas a partir de animais de produção. Objetivo: Este estudo teve como objetivo isolar enterobactérias resistentes aos beta-lactâmicos e quinolonas e detectar os genes blaCTX-M, blaSHV, blaTEM, qnrA, qnrB, qnrS e oqxAB em Enterobacteriaceae resistentes isoladas de carnes resfriadas de frango, suína e bovina comercializadas no município de São José do Rio Preto-SP, adquiridas entre novembro de 2010 e agosto de 2012. Material e Métodos: A suscetibilidade aos antimicrobianos foi determinada por disco-difusão de acordo com as normas do CLSI. As cepas resistentes aos beta-lactâmicos e às quinolonas foram submetidas à PCR com primers específicos para a detecção destes genes e ao sequenciamento para a identificação dos mesmos. Resultados: Um total de 75 isolados apresentou resistência aos beta-lactâmicos e 125 às quinolonas. Entre estes, 6,6% blaCTX-M, 6,6% blaTEM, 16% blaSHV, 21,6% apresentaram variantes de qnr e 13,6% oqxAB. Em uma cepa de Aeromonas sobria foi detectada a coexistência dos genes qnrA e qnrS; o gene qnrB foi detectado em cinco cepas de E. coli, cinco K.pneumoniae, duas Salmonella spp. e 12 Citrobacter spp. O gene qnrB19 foi detectado em duas K. pneumoniae, duas E. coli e uma Citrobacter braakii. qnrB2 foi detectado em três K. pneumoniae. O gene qnrS1 foi detectado em uma cepa de K. pneumoniae. O sequenciamento de blaCTX-M revelou blaCTX-M-2, sendo que todas foram provenientes de amostras de carne de frango. Conclusão: Esses dados revelam que pode haver associação entre o excessivo uso de antimicrobianos em animais de produção e o isolamento de bactérias resistentes aos mesmos. A presença de bactérias resistentes aos antimicrobianos em carnes destinadas ao consumo humano consiste em um problema de saúde pública e pode afetar as relações comerciais do país, já que o Brasil é um dos maiores exportadores de carne do mundo.

Les antibiotiques, et plus particulièrement les β-lactamines, furent pendant longtemps considérés comme l’arme absolue pour le traitement des infections bactériennes, du fait de leur efficacité, leur bonne tolérance et de leur faible coût. Cependant, les bactéries ont développé des mécanismes de résistance, y compris vis-à-vis des β-lactamines possédant le spectre d’activité le plus large, les carbapénèmes. Ces derniers sont considérés comme les antibiotiques de derniers recours pour le traitement des infections sévères à bacilles à Gram négatif en milieu hospitalier. Chez les entérobactéries, cette résistance émergente aux carbapénèmes est principalement due à l’expression d’enzymes appelées les carbapénèmases capables d’inactiver les carbapénèmes. L’émergence exponentielle à l’échelle mondiale de ces entérobactéries productrices de carbapénèmases (EPC) constitue un problème majeur de santé publique. Actuellement, les carbapénémases les plus répandues dans le monde sont les KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo- β- lactamase) et OXA-48 (Oxacillinase). La première KPC a été identifiée en 1996 aux Etats- Unis, NDM-1 en 2009 en Suède chez une patiente en provenance d’Inde et OXA-48 en 2003 en Turquie. En France, les carbapénèmases de type OXA-48 sont largement majoritaires et représentent près de 70% des EPC. Ainsi, le retentissement de leur dissémination et le besoin de développer de nouveaux inhibiteurs capables d’inactiver les carbapénèmases se fait de plus en plus pressante.Cette thèse a pour objectif de mieux comprendre le mode d’ action de ces 3 carbapénèmases d’un point de vue moléculaire et biochimique, afin d’ identifier les éléments structuraux nécessaires à leur fonctionnement, mais aussi de poser les bases du développement de « pan-inhibiteurs » et de nouveaux outils de diagnostics. Pour cela, la caractérisation biochimique des carbapénèmases de type KPC, NDM et OXA-48, de leurs variants naturels et de mutants générés in vitro a été entreprise. Il a pu être mis en exergue l’implication de résidus spécifiques et d’éléments structuraux nécessaires à l’ hydrolyse des carbapénèmes. L ’ étude cristallographique et RMN ainsi que l’étude in silico (modélisation) de ces enzymes et de leurs mutants respectifs, nous a permis de mieux comprendre le mode d’ interaction enzyme- substrat. Nous avons ainsi pu comprendre les bases de la capacité impressionnante de ces carbapénèmases à s’adapter et à évoluer en fonction des pressions de sélections exercées.En collaborations avec différentes équipes de chimistes nous avons développés différentes séries de molécules « pan-inhibiteurs » capables d’inhiber les 3 classes de carbapénèmases. Ainsi nous avons montré un effet inhibiteur de certains composés de la famille des flavonoïdes dont la myrécétine, molécule la plus active. Nous avons également identifié une série de composés, les imidazolines, possédant un effet pan-inhibiteur avec des valeurs de CI50 sub-micromolaires et donc compatible avec une utilisation in vivo.Enfin, nous avons participé au développement (en collaboration avec le CEA) d’ un outil de diagnostic rapide basé sur l’immunochromatographie, permettant détection des EPC en moins de 15 minutes. / Antibiotics, and particularly ß- lactams, have long been considered the ultimate weapon for the treatment of bacterial infections, because of their effectiveness, good tolerance and low cost. However, bacteria have developed mechanisms of resistance, including against β- lactams with the broadest spectrum of activity, carbapenems. The latter are considered as last resort antibiotics for the treatment of severe infections due to Gram negative bacilli in hospital. This emerging resistance to carbapenems in Enterobacteriaceae is mainly due to the expression of enzymes called carbapenemases capable of inactivating carbapenems. The worldwide exponential spread of these carbapenemase-producing enterobacteria (CPE) represents a major public health issue. Currently, the most common carbapenemases in the world are KPC (Klebsiella pneumoniae carbapenemase), NDM-1 (New Delhi metallo-β-lactamase) and OXA-48 (Oxacillinase). The first KPC was identified in 1996 in the United States, NDM-1 in 2009 in Sweden in a patient from India and OXA-48 in 2003 in Turkey. In France, OXA-48- type are the most abundant carbapenemases with nearly 70% of the EPCs. Thus, the repercussion of their dissemination and the development of new inhibitors capable of inactivating carbapenemases is becoming more and more urgent.The aims of this thesis were to better understand the mode of action of the 3 main carbapenemases from a molecular and biochemical point of view, in order to identify the structural elements necessary for their functioning, but also to lay the basis for the development of "pan-inhibitors" and noveldiagnostic tools. For this purpose, the biochemical characterization of carbapenemases of the KPC, NDM and OXA-48 type, their natural variants and mutants generated in vitro has been undertaken.We could highlight the involvement of specific residues and structural elements necessary for the hydrolysis of carbapenems. The crystallographic and NMR study as well as the in silico (modeling) studies of these enzymes and their respective mutants, allowed us to better understand the enzyme-substrate interaction mode. We could thus understand the basis of the impressive capacity of these carbapenemases to adapt and therefore to evolve according to the selection pressures exerted.In collaboration with several chemists we participated in the development of different series of "pan-inhibitors" that were able to inhibit the 3 classes of carbapenemases. Thus we could show the inhibitory properties of some compounds of the family of flavonoids including myricetin, the most active molecule. We have also been able to identify a series of compounds, imidazolines, possessing a pan- inhibitory effect with sub-micromolar IC50 values and therefore compatible with in vivo use. Finally, we participated in the development (in collaboration with the CEA) of a rapid diagnostic tool based on theimmunochromatographic, allowing the detection of EPCs in less than 15 minutes.

This document only includes an excerpt of the corresponding thesis or dissertation. To request a digital scan of the full text, please contact the Ruth Lilly Medical Library's Interlibrary Loan Department (rlmlill@iu.edu).

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Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005. / "A dissertation in pharmaceutical sciences and chemistry." Advisor: Steven H. Neau and Thomas P. Johnston. Typescript. Vita. Description based on contents viewed Mar. 12, 2007; title from "catalog record" of the print edition. Includes bibliographical references (leaves 97-108). Online version of the print edition.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Dissemination of CTX-M type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) is a serious health issue in Hong Kong. However, research knowledge concerning its dissemination mechanism and plasmid characteristics over time in health care setting is still limited. This study was conducted to characterize ESBL-producing E. coli from blood culture isolates and the epidemiology of the plasmids harboring CTX-M-14 collected from a healthcare region in Hong Kong from two periods of time. A total of 167 ESBL-producing E. coli in blood culture specimens were retrieved from period 1 (1996 - 1999, n = 50) and period 2 (2007 - 2008, n = 117). Antimicrobial susceptibilities were defined by disk diffusion method according to CLSI. Phylogenetic groups and CTX-M enzymes were detected among all the ESBL-producers. Clonal relatedness of the hosts was analyzed by pulsed-field gel electrophoresis and multi-locus sequence typing. A subset of 65 CTX-M-14-producing isolates was undergone for further plasmid characterization. Conjugation, PCR-based replicon typing, S1-PFGE, southern-blot hybridization, and genetic environment PCRs were performed. Plasmid PCR-restriction fragment length polymorphism (pRFLP), F-allele replicon sequence typing and variable region PCRs were studied in 54 F-plasmids obtained. Results showed that over half of the ESBL-positive isolates were non-susceptible to ciprofloxacin, cotrimoxazole and gentamicin. A surprisingly high number of CTX-M-type ESBL was carried by 98.2% (164/167) of the isolates. CTX-M-9 group (89.8%, 150/167) and CTX-M-14 (103/109) were predominantly found among both periods. Overall, nearly half (41.3%, 69/167) of the isolates belonged to 5 major clones. Clonal types undetermined-ST68 (n = 18) and O102-ST405 (n = 15) were dominant in period 1 while clonal types O25b-ST131 (n = 30), O15-ST69 (n = 5) and O12-ST12 (n = 3) emerged in period 2. Among a subset of 65 CTX-M-14 plasmids, most of them were transferable (84.6%, 55/65) with high frequency, similar plasmid sizes and genetic environment ISEcp1-blaCTX-M-14-IS903 (90.8%, 59/65). Replicon types of the CTX-M-14 encoding plasmids were FII (n = 48) or FII ± FIA/FIB types (n = 6), I1-I (n = 3), B/O (n = 2), K (n = 1) and undetermined (n = 4). Subtyping of 54 IncF plasmids by replicon sequence typing, pRFLP and PCR for marker genes (yac, malB, eitA, eitC and parAB) showed that 79.6% (43/54) of the plasmid subset exhibited identical or highly similar results with the completely sequenced plasmid, pHK01 (E. coli isolated from urine sample of a patient in Hong Kong, 2004). These 43 plasmids were originated from both period 1 (n = 11) and period 2 (n = 32). These pHK01-like plasmids were found to have spread to the major clones (ST68, ST405 and ST131) and multiple singleton isolates of all four phylogenetic groups. In conclusion, this study demonstrated the widespread dissemination of pHK01-like CTX-M-14 encoding plasmids among isolates of diverse genetic lineages over a decade. The dissemination was probably due to both clonal expansion and horizontal gene transfer of pHK01-like IncF plasmid. / published_or_final_version / Microbiology / Master / Master of Philosophy

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy