Entre as ß-lactamases, as enzimas CTX-M têm despertado atenção especial pela alta incidência e grande capacidade de propagação. Eventos como recombinação gênica, transferência plasmideal e multirresistência podem ser a razão da manutenção e da ampla disseminação dos genes blaCTX-M. Este é um trabalho retrospectivo que teve como objetivo caracterizar genes blaCTX-M presentes em Klebsiella spp. Foram estudadas 27 linhagens de Klebsiella pneumoniae e 8 linhagens de Klebsiella oxytoca, produtoras de ?-lactamase de espectro estendido, isoladas de pacientes hospitalizados no período de janeiro a junho de 2000. A detecção e identificação dos genes blaCTX-M, assim como dos elementos relacionados com a mobilização destes genes, foi realizada por PCR e seqüenciamento. A localização genética e a mobilidade dos genes blaCTX-M foram pesquisadas por análise plasmideal e hibridação e por conjugação. Os perfis de sensibilidade das linhagens estudadas e das linhagens transconjugantes foram comparados pela determinação da concentração inibitória mínima de antibióticos das classes das cefalosporinas, cefamicinas, aminoglicosídeos e quinolonas. Foram encontrados genes blaCTX-M em plasmídeos conjugativos em 13 (37%) linhagens estudadas: blaCTX-M-9 em 4 K. oxytoca, e blaCTX-M-2 em 9 K. pneumoniae. Os genes blaCTX-M-9 estavam associados ao elemento de inserção ISEcp1, enquanto os genes blaCTX-M-2 estavam associados a integrons de classe I contendo ISCR1. O genes blaCTX-M-2, carreado por plasmídeo, pode estar relacionado com disseminação horizontal entre vários clones de K. pneumoniae, enquanto o gene blaCTX-M-9 foi encontrado sendo carreado por um único clone de K. oxytoca. Este estudo determinou a incidência e a diversidade de enzimas CTX-M no período estudado, além de fornecer dados epidemiológicos que podem explicar a sua prevalência no mundo e contribuir para o entendimento e controle da disseminação deste tipo de resistência. / CTX-M enzymes, the world\'s most prevalent ß-lactamases disseminate very easily. Genetic recombination, plasmid transference and multiresistance could be responsible for the wide spread of blaCTM-X genes. This retrospective study aims to characterize blaCTX-M genes found in Klebsiella spp. The strains were isolated in hospital patients from January to June 2000 and consisted of 27 ESBL-producing Klebsiella pneumoniae and 8 ESBL-producing Klebsiella oxytoca. PCR and sequencing were used in the detection and identification of blaCTX-M genes and genetic elements associated with their mobilization. Determination of genetic localization and mobility of blaCTX-M genes was by plasmid analyses, hybridization and transfer assays. The minimal inhibitory concentrations (MICs) of cephalosporins, cefamicins, aminoglycosides and quinolone antimicrobials evaluated the antibiotic susceptibility profile of transconjugants and strains in the study. The blaCTX-M genes were found in 13 strains (37%): blaCTX-M-9 in 4 K. oxytoca and blaCTX-M-2 in 9 K. pneumoniae. The insertion sequence ISEcp1 was associated with blaCTX-M-9 and blaCTX-M-2 was found in a class I integron bearing ISCR1. Plasmid blaCTX-M-2 genes dissemination was due to horizontal transfer among many K. pneumoniae clones, while blaCTX-M-9 dissemination was associated with a particular clone of K. oxytoca. The study characterized incidence and diversity of CTX-M enzymes during the period studied. Moreover it showed epidemiological data, which may explain CTX-M prevalence worldwide and contribute for the understanding and control of the resistance spread.

SYNTHESIS AND BILOGICAL SIGNIFICANCE OF 1,2,4-OXADIAXOLIDIN-5-ONE Chimdi kalu, Austin Miller and Dr. Abbas G. Shilabin Department of Chemistry, East Tennessee State University, Johnson City, TN 37614. ABSRTACT Due to the challenge posed by microbial resistance to broad spectrum of antibiotics, there has been a great need to synthesize of a novel compound which has a different mechanism of action on microbial activity. 1,2,4-oxadiaxolidin-5-One constitute an important class of compound with tremendous potential as pharmaceutical and otherwise biologically relevance substance due to the fact that its five member ring is a configurationally stable building block. This unit is found in other compound like alkaloids, with vast medical application. This study describes the synthesis of 1,2,4-oxadiaxolidin-5-one in two-step procedure using nitroethane and benzaldehyde as starting materials to produce nitrone, which in turn undergoes 1,3- dipolar cycloaddition with phenyl isocyanate to give 1,2,4-oxadiaxolidin-5-one. The product was characterized using proton NMR and GC-MS. There is an ongoing investigation on the summary of some important inhibitory activity against class A β-lactamase by 1,2,4-oxadiaxolidin-5-one heterocyclic core structure to provide effective antimicrobial β-lactamase inhibitors, hence, solving the problem of microbial resistance to currently used antimicrobial drugs.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21958号 / 医博第4500号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 松原 和夫, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The general idea of this study was to develop candidate specific IgY antibodies for an oral therapy targeting the ESBL-producing gram negative bacteria. As the family of ESBLs constantly grows and there is lack of their clear classification in the literature, the specific aim was to build a proof of concept study based on the parental enzyme ß-lactamase TEM-1 to investigate different specific IgYs strategies to inhibit the growth of TEM-1 producing E.coli. This research included a bioinformatic analysis of the TEM-1 structure in the context of TEM-derivative ESBLs. Then, two IgY strategies were designed to target the ß-lactamase TEM-1-producing E.coli (BW25113 ΔbamBΔtolC) with IgYs: as a complement to antibiotics (IgYs against the enzyme TEM-1 used in combination with ampicillin) and as an alternative to antibiotics (IgYs against the bacteria TEM-1-producing E.coli without the addition of ampicillin). A good inhibitory effect of (a)TIgY, (a)p2IgY (in the presence of ampicillin) and eIgY, hIgY (without the ampicillin) on TEM-1-producing E.coli was observed in vitro. Moreover, they had the typical configuration of avian antibodies and were highly specific to their antigens. This study presents a model system to develop specific IgYs against a therapeutic target of interest. The activity of these IgYs complementary or alternatively to antibiotics should be further investigated in vivo in an animal infectious model. IgYs developed in this study might also be good candidates for further investigation as a broad-spectrum treatment against a variety of ESBL-producing E.coli. The aTIgY which was developed against the whole TEM-1 might also target its derivatives, as they have similar 3D structure with single amino acids mutations in the sequence. The ap2IgY was generated against catalytic and conservative residues, characteristic for the whole class A of ß-lactamases, thus it might target also the active site of ESBL-s from this class. The strategy used to generate eIgY and hIgY was efficient and IgYs could be generated directly against ESBL-producing bacteria.

Gene expression of porin and beta-lactamases genes during the beta-lactam antibiotic treatment and effect of inoculum size on Klebsiella pneumoniae clinical isolates ABSTRACT In recent years, Klebsiella pneumoniae has been increasingly reported to be one of the most important nosocomial pathogens, and it is usually resistant to many antibiotics. In this work, we focused on the expression of the AmpC group β- lactamase DHA-1 and its negative regulator AmpR, as well as the porins OmpK35 and OmpK36 and on effect of inoculum. We used well-characterized Klebsiella pneumoniae strains in this study. Plasmids obtained from these strains were also transformed into different wild-type Klebsiella pneumoniae strains, which were typed by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Gene expression analysis was performed by RT-PCR using specific primers and TaqMan probes. In most strains, expression was dependent on the presence of an inducer. The highly resistant strain showed a different expression pattern, but the expression of blaDHA-1 remained inducible by cefoxitin. Different regulation was also observed in the transformants. Based on our data, we suggest that the previously described regulatory pathway for AmpC is not generally suitable, and we propose that there are more...

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Antimicrobial susceptibility profiles were assembled for isolates of Moraxella catarrhalis collected from the Mountain Home Veteran's Affairs Medical Center (VAMC) clinical laboratory in Johnson City, Tennessee. The goal of the study was to identify isolates for genetic characterization using comparisons of susceptibility profiles. Isolates of Moraxella catarrhalis collected from July 1984 through 1994 were analyzed for β-lactamase production using a Cefinase disk assay. A multi-drug profile consisting of 11 β-lactam antibiotics was performed on the 41 M. catarrhalis isolates. Kirby Bauer disk assays were performed for 7 cephalosporin and 4 non-cephalosporin antibiotics. In summary, 2 observations implicate more complex resistance determinants than the 2 known forms of the BRO β-lactamase. First, there was overlap in the ranges of inhibition zones. Second, several isolates had antibiotic-specific deviations from typical profiles. These data suggest either more variation in the M. catarrhalis BRO β-lactamase than described or contributions to resistance from undescribed determinants.

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京都大学 / 新制・論文博士 / 博士(工学) / 乙第13581号 / 論工博第4212号 / 新制||工||1990(附属図書館) / (主査)教授 松原 誠二郎, 教授 中尾 佳亮, 教授 浦山 健治 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM