Made available in DSpace on 2014-06-12T18:30:31Z (GMT). No. of bitstreams: 2 arquivo3565_1.pdf: 5205665 bytes, checksum: d3f5d67863a34c85659602984a49cb65 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Klebsiella pneumoniae é uma enterobactéria responsável por uma alta incidência de infecções hospitalares, causadas geralmente por linhagens portadoras de multirresistência e produtoras de -lactamases de amplo espectro (ESBL). Este trabalho teve como objetivo determinar os genes envolvidos na formação de beta-lactamases clássicas e ESBLs em isolados de K. pneumoniae de Recife-PE, Brasil, assim como investigar o efeito ultra-estrutural causado por diferentes concentrações de antibióticos beta-lactâmicos, em isolados de K. pneumoniae produtores de diferentes tipos de betalactamases. Foram analisados 52 isolados de K. pneumoniae quanto a presença dos genes blaSHVe blaTEM, originados da microbiota normal e de infecções na comunidade e hospitalares e para a investigação do gene blaCTX-M foram utilizados 19 isolados clínicos de K. pneumoniae que apresentaram resistência a cefalosporinas de terceira geração ou ao aztreonam. Os MICs dos isolados foram determinados frente aos antibióticos ceftazidima, cefotaxima e aztreonam. Dois isolados de infecção nosocomial de K. pneumoniae foram submetidos a diferentes Sub-MICs de ceftazidima, cefotaxima e aztreonam para a análise pela Microscopia Eletrônica de Transmissão e de Varredura (MET e MEV). O gene blaSHV foi detectado em 16 isolados hospitalares, 4 da comunidade e 9 da microbiota, enquanto o gene blaTEM foi detectado em 18 isolados hospitalares, 4 da microbiota e 2 da comunidade. Através do sequenciamento de DNA, o gene blaCTX-M2 foi detectado em três isolados resistentes tanto a ceftazidima quanto a cefotaxima. Adicionalmente, foi encontrada uma nova variante SHV em um dos isolados e a presença de duas variantes anteriormente relatadas a SHV-28 e a SHV-108. As análises dos 2 isolados de K. pneumoniae pela MET e MEV mostraram grandes alterações morfológicas e ultra-estruturais dos isolados, quando submetidos aos diferentes Sub-MICs de ceftazidima e cefotaxima. *Nossos resultados mostram que isolados de K. pneumoniae portadores de diferentes β-lactamases e resistentes a diferentes antimicrobianos podem sofrer alterações ultra-estruturais significativas quando submetidos a Sub-MICs da droga, o que poderia possibilitar uma melhor atuação do sistema imune de um paciente infectado com esta espécie bacteriana

TEM and SHV β-lactamases are the most prevalent β-lactamases among Gram-negative bacteria. The introduction and widespread use of expanded-spectrum antibiotics, particularly third generation cephalosporins, has led to the evolution of bacterial strains expressing extended spectrum β-lactamases (ESBLs). ESBLs emerge by genetic point mutation from non-extended spectrum precursors. It was found that multiple β-lactamase families within single isolates complicate the process of detecting the resistance status of isolate using non-quantitative DNA diagnostic methods. Preliminary phenotypic characterisation of probable β-lactamase enzyme family types present in 100 isolates from the Asia-Pacific and South African locales showed that single isolates frequently contained multiple β-lactamase families. SHV, TEM, AMPC and CTX-M β-lactamase families were detected in these isolates using PCR detection methods. Ninety-eight percent of all isolates tested contained as least one β-lactamase gene, with up to four to β-lactamase gene families found to co-exist in single isolates. Kinetic PCR methods for interrogating the polymorphic sites at blaSHV codons 238 & 240 and blaTEM codons 164, 238, 240 as well as promoter polymorphism were developed. A high proportion of blaSHV 238 and 240 mutant alleles was found to correlate with cefotaxime, ceftazidime and aztreonam resistance levels. In an attempt to understand the molecular basis for the co-existence of multiple blaSHV alleles within single isolates, the blaSHV promoter region was cloned from one ESBL expressing isolate. Experimental results showed that blaSHV can exist downstream of two different promoters within a single isolate. Both promoters have previously been reported, and differ by the presence or absence of IS26, which results in a change in the transcription initiation site. The blaSHV gene copy numbers in cis with the different promoters were measured, and it was found that the copy number of the IS26::blaSHV promoter was positively correlated with resistance levels. Cloning and analysis of PCR products showed that different blaSHV variants existed in cis with promoters in individual isolates. However, mutant genes were more abundant downstream of the IS26 promoter. There were no ESBL+ isolates without this promoter. It was concluded that blaSHV in cis with the IS26 promoter is located on an amplifiable replicon, and the presence of the IS26 insertion may facilitate the acquisition of an ESBL+ phenotype. To further confirm the role of IS26 in resistance acquisition, ESBL negative isolates were subjected to serial passage in vitro evolution experiments and fluctuation assays. Results confirm that the insertion of the IS26 element upstream of blaSHV is positively correlated with the ability to exhibit an ESBL phenotype, when such isolates also contain the critical G238S substitution. It was also found that IS26 can catalyse the duplication and mobilisation of blaSHV within an isolate. Fluctuation experiments have shown that the frequency at which such genomic events occur resulting in ESBL phenotypes is extremely low and requires many generations of selection under sub-lethal conditions. A survey of a geographically diverse set of isolates has shown that IS26-blaSHV was found in all of the bacterial populations surveyed. However, it does not appear to be exclusively associated with SHV-mediated ESBL production.

Extended spectrum β-lactamases or ESBLs, which are derived from non-ESBL precursors by point mutation of β-lactamase genes (bla), are spreading rapidly all over the world and have caused considerable problems in the treatment of infections caused by bacteria which harbour them. The mechanism of this resistance is not fully understood and a better understanding of these mechanisms might significantly impact on choosing proper diagnostic and treatment strategies. Previous work on SHV β-lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains which contain plasmid-borne blaSHV are able to mutate to phenotypically ESBL-positive strains and there was also evidence of an increase in blaSHV copy number. Therefore, it was hypothesised that although specific point mutation is essential for acquisition of ESBL activity, it is not yet enough, and blaSHV copy number amplification is also essential for an ESBL-positive phenotype, with homologous recombination being the likely mechanism of blaSHV copy number expansion. In this study, we investigated the mutation rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive status by using the MSS-maximum likelihood method. Our data showed that blaSHV mutation rate of a non-ESBL expressing isolate is lower than the mutation rate of the other single base changes on the chromosome, even with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive (≥16 µg/mL for cefotaxime) is very high. This is because only gene copy number increase is needed which is probably mediated by homologous recombination that typically takes place at a much higher frequencies than point mutations. Using a subinhibitory concentration of novobiocin, as a homologous recombination inhibitor, revealed that this is the case.