| [Objective] 1) To investigate the molecular epidemiology and independent risk factors of nosocomial infections caused by Stenotrophomonas maltophilia. 2) To amplify and position L1, L2 P-lactamases genes, and confirm transferable resistance by conjugation experiment. 3 ) To compare 4 antibiotics including imipenem, meropenem, cefotaxime and ceftazidime inductivity to two p-lactamases, and survey the regulation of LI, L2 P-lactamases. 4) To explore the effect of amino acid substitution at position Asp74 of LI enzyme on its substrate specificity. 5 ) To study the resistance mechanism of Stenotrophomonas maltophilia to trimethoprim-sulfamethoxazole.[Methods] 1 ) 30 isolates of Stenotrophomonas maltophilia causing nosocomial infections were collected and were identified with API 20NE test strips. Minimal inhibitory concentration(MICs) of 14 antimicrobial agents against 30 isolates were determined by broth microdilution method. Case-control study and multivariate logistic regression analysis wre used for statistics to verify risk factors of infections caused by Stenotrophomonas maltophilia. ERIC-PCR typing was used to investigate the epidemiological characteristics of isolates. 2 ) Plasmid DNA were isolated from Stenotrophomonas maltophilia to ensure the numbers and sizes of plasmids. The genes of L1, L2 P-lactamases were amplified by PCR using chromosome and different plasmids as templates respectively to investigate the strains producing two P-lactamases and location of L1, L2 genes. Conjugation experiment was performed to identify whether there were the transferability of resistance determinants in bacterial strains. 3 ) RT-PCR was used to determine the effect of different concentrations (0.25,1,4×MIC) of clinical common antimicrobial agents on induction of L1, L2 p-lactamases and the regulation of two p-lactamases expression. 4 ) Wild-type L1 gene was cloned by PCR. Based onwild-type LI gene, three site-directed mutation genes(G220C, G220A and G220T) were constructed by the overlap extension method of PCR. Wild-type and three mutant-type LI genes were subcloned into pET28a, transformed into E.coli BL21(DE3) cells and overexpressed to get wild-type and three mutant-type LI enzymes by replacing Asp74 with His, Asn or Tyr respectively. The amount and activity of LI p-lactamase were compared between wild-type and three mutant-type LI enzymes. 5 ) Class 1 integrase gene and sull gene were amplified using chromosomal DNA as templates from 30 isolates of Stenotrophomonas maltophilia, and subsequently were cloned and sequenced. The difference carrying class 1 integrase gene and sull gene was found between trimethoprim-sulfamethoxazole resistance and susceptibility strains.[Results] 1 ) 30 isolates of Stenotrophomonas maltophilia were high resistant to imipenem, meropenem, cefotaxime, aztreonam and amikacin, but showed certain susceptibility to cefoperazone/sulbactam, piperacillin/tazobactam,trimethoprim-sulfamethoxazole and ticarcillin /clavulanic acid, 96.7%, 76.7%, 73.3% and 60%% respectively. The independent risk factors leading to infections of Stenotrophomonas maltophilia were mechanical ventilation(OR=7.629) and over 60 days of length of stay(OR=4.466). ERIC-PCR typing revealed 26 different clones of strains in all isolates of Stenotrophomonas maltophilia. 2)12 strains were found to posses approximately 12kb plasmids in 13 isolates of Stenotrophomonas maltophilia. L2 gene was located in each of 30 isolates of Stenotrophomonas maltophilia, LI gene was in 27 isolates of Stenotrophomonas maltophilia by using chromosomal DNA as templates, and LI, L2 genes were also meanwhile in 12kb pJasmid by using different sizes of plasmids as templates. The amplicons were individually cloned into pMD18-T vector and were sequenced. The molecular evolutionary trees of LI and L2 genes were constructed by DNAStar biosoft among sequences sequenced and registered in GeneBank. Among 9 nucleotide sequences of LI gene, 8 sequences showed higher homology with Lie gene, and the lowest homology with Lie gene. Three nucleotidesequences of L2 gene exhibited higher homology with L2b genes, and lower homology with L2d gene. Conjugons could grow and generate in the media containing ampicillin, rifampicin and nalidixic acid, but could not grow in the media containing rifampicin, nalidixic acid and imipenem (meropenem, cefotaxime, ceftazidime). At the same time 12kb plasmid was isolated from conjugons. 3 ) Different concentrations of antibiotics (imipenem, meropenem, cefotaxime and ceftazidime) were mixed into the media of Stenotrophomonas maltophilia that could produce LI and L2 enzymes. Total RNA were isolated from the incubated isolations of Stenotrophomonas maltophilia, and RT-PCR was carried out by using specific primers. Electrophoresis strips of LI, L2 amplicons were not found in strains of blank control and those whose media had imipenem, meropenem or cefotaxime(4> |
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