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

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Made available in DSpace on 2015-12-06T22:54:31Z (GMT). No. of bitstreams: 0 Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O presente estudo teve como objetivo determinar as β)lactamases envolvidas na resistência às cefalosporinas de amplo espectro em uma coleção de Pseudomonas aeruginosa isoladas de hemoculturas de pacientes internados em um complexo hospitalar universitário na cidade de São Paulo durante o ano de 2005. No primeiro trabalho descrevemos a produção de CTX)M)2 em um isolado que apresentava o estranho fenótipo de sensibilidade à ceftazidima e resistência à cefepima. O gene que codificava esta enzima estava localizado no cromossomo bacteriano, em um contexto genético idêntico àquele encontrado em plasmídeos que carregam blaCTX)M)2, que encontram)se disseminados entre enterobactérias. No segundo trabalho, descrevemos as β)lactamases envolvidas na resistência à ceftazidima em 43 isolados de P. aeruginosa da referida coleção, assim como o contexto e suporte no qual estavam inseridos os genes que codificavam as enzimas identificadas. Além disso, realizamos a tipagem molecular dos isolados estudados e pesquisamos os prontuários médicos dos pacientes infectados pelas amostras estudadas. A hiperprodução de AmpC foi considerada a única β)lactamase relacionada à resistência a ceftazidima em quatro isolados (9,3 por cento). Nove isolados (20,9 por cento) apresentaram a produção de β)lactamase de espectro estendido (ESBL), sendo que sete (16,3 por cento) apresentavam a enzima GES)1 e dois isolados (4,6 por cento) apresentavam a enzima CTX)M)2. A atividade hidrolítica contra os carbapenens foi detectada em trinta isolados (69,7 por cento), nos quais as enzimas IMP)1, GES) 5 e SPM)1 foram identificadas em 1, 2 e 27 isolados, respectivamente. Nenhum isolado apresentou produção simultânea de metalo)β)lactamase e ESBL. Os genes blaSPM)1 e blaCTX)M)2 estavam associados às seqüências de inserção ISCR4 e ISCR1, respectivamente, enquanto que os genes blaGES)1, blaGES)5 e blaIMP)1 estavam localizados em integrons da classe 1. Todos os genes codificadores de β)lactamases identificados apresentavam localização cromossomal. A tipagem molecular dos isolados estudados evidenciou a existência de sete genótipos distintos; porém, isolados produtores de uma mesma enzima freqüentemente apresentavam relação clonal. A análise dos prontuários médicos revelou que metade dos pacientes que apresentaram infecção da corrente sanguínea pelos isolados de P. aeruginosa estudados receberam terapia inadequada. Este estudo permitiu identificar a diversidade de genes codificadores de β)lactamases de amplo espectro hidrolítico que foram adquiridos por isolados clínicos de P. aeruginosa. Estes genes foram inseridos no DNA cromossomal destes isolados e, posteriormente, disseminados por transmissão cruzada.. / The aim of this study was to identify the β-lactamases involved in broad-spectrum cephalosporin resistance in P. aeruginosa isolates recovered from blood culture of patients hospitalized at a Brazilian teaching hospital located in São Paulo, between January and December 2005. In the first manuscript we have described the production of CTX-M-2 causing ceftazidime susceptibility and cefepime resistance in a P. aeruginosa clinical isolate. The CTX-M-2-encoding gene was located at the bacterial chromosome, and its vicinities were identical to those observed in blaCTX-M-2-carrying plasmids from enterobacterial isolates. In the second manuscript we have evaluated 43 ceftazidime-resistant P. aeruginosa isolates from the referred collection. We have described the β-lactamases involved in ceftazdidme resistance, as well as the genetic context and support of β-lactamaseencoding genes and we have performed molecular typing of isolates. The medical records of patients infected with isolates studied were also analyzed. AmpC overproduction was found to be the only β-lactamase-mediated mechanism responsible for ceftazidime resistance in four isolates (9.3%). Nine isolates (20.9%) produced an extended-spectrum β-lactamase (ESBL), either GES-1 (n = 7, 16.3%) or CTX-M-2 (n = 2, 4.6%). Carbapenemase activity was detected in 30 (69,7%) isolates, in which two (4.6%) produced the ESBL GES-5, a single isolate (2.3%) produced the metallo-β-lactamase (MBL) IMP-1; and 27 isolates produced the MBL SPM-1 (62.8%). None of the isolates coproduced both ESBL and MBL. Insertion sequence elements ISCR4 and ISCR1 were associated with blaSPM-1 and blaCTX-M-2 genes, respectively, whereas the blaGES and blaIMP-1 genes were part of class 1 integron structures. All β- lactamase-encoding genes identified were chromosomally located. Molecular typing showed the existence of seven distinct genotypes, in which producers of the same β- lactamase often belonged to a single clone. Clinical data revealed that half of the patients infected with isolates studied have received inadequate therapy, suggesting that empirical treatment protocols should be updated in the hospital studied. In this study we have identified a variety of broad-spectrum β-lactamase-encoding genes that have been initially acquired by P. aeruginosa clinical isolates, inserted on its chromosomal DNA and then spread between patients by cross-transmition. / BV UNIFESP: Teses e dissertações

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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE / Vita. / Bibliography: leaves 211-223. / by Douglas Elliott Koshland. / Ph.D.

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La Tesi descriu la prevalença de les soques portadores de betalactamases d'espectre ampliat (BLEA), cefamicinases i carbapenemases, principals mecanismes de resistència als antibiòtics betalactàmics, no només en l'àmbit clínic [mostres clíniques i malalts atesos al Servei d'Urgències (portadors fecals)] sinó en altres ecosistemes com en els subsòls de granges d'animals (aus, porcs i conills), en aigües residuals humanes, en aliments, i en portadors fecals afectats per un brot alimentari. A més a més s'han caracteritzat aquests enzims per tal de determinar la freqüència de cadascun dels tipus de betalactamases presents: tipus TEM, SHV i CTX-M com a BLEA, CMY, DHA o ACC com a cefamicinases, o VIM, IMP i altres com a carbapenemases. La conclusió del treball mostra com aquests enzims es poden trobar en tots els ecosistemes avaluats i, per tant, com la selecció de soques portadores d'aquests no es dóna només en l'àmbit clínic sinó que roman en tot l'entorn humà. Per altra banda, arran d'aquest treball s'han descrit dues noves betalactamases, la CTX-M-14b, i la CTX-M-51, ambdues derivades de la CTX-M-9. El fet que les soques portadores d'aquests enzims s'hagin trobat en tots els àmbits és causa de greu preocupació. Cal, doncs, intentar evitar la selecció d'aquestes soques, així com esbrinar quin o quins són els mecanismes que han permès aquesta expansió. L'estudi epidemiològic dels mecanismes de resistència, és de gran interès, no només en l'àmbit clínic sinó també veterinari, perquè coneixent la prevalença i el tipus d'enzims implicats es pot actuar oportunament dissenyant polítiques d'antibiòtics correctes. / This thesis describes the prevalence of strains carrying extended spectrum betalactamase (ESBL), plasmid-mediated AmpC and carbapenemase. There are the main mechanism of betalactam resistance, not only in clinic environment [clinical samples and patients attending the emergency room (faecal carriers)] also in others environments such as the floor of animal farms (poultry, pig and rabbit), the urban sewage, food, and human faecal carriers in the context of food-borne disease outbreaks. We have characterized these enzymes to know the frequency of each type of betalactamase: TEM, SHV and CTX-M type as ESBL, CMY, DHA and ACC as plasmid-mediated AmpC, or VIM, IMP and others as carbapenemase. The work's conclusion show's that these enzymes can be found in all environments evaluated. Then the selection of strains carrying this kind of enzymes is due not only to clinic environments otherwise to all human environments. In the other hand, we have described two new betalactamase, CTX-M-14b and CTX-M-51, both deriving from CTX-M-9.The finding of strains carrying these enzymes in all the environments is of great concerning. We need not to select these strains, and to know what are the mechanisms of expansion of these enzymes. The epidemiologic study of the resistance mechanism is of great interest, not only in hospitals also in veterinarian environment, because knowing the prevalence and the type of enzymes, we can design specific antibiotic policy.

Thesis (Ph. D.)--Miami University, Dept. of Chemistry and Biochemistry, 2003. / Title from first page of PDF document. Document formatted into pages; contains xi, 150 p. : ill. Includes bibliographical references.