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Objective:(1) To understand the clinical distribution, antibiotics resistance characteristics and pan-drug resistance status of carbapenem-resistant A. baumannii (CR-AB) in our hospital.(2) To investigate the antibiotic resistant mechanisms and molecular epidemiological characteristics of clinical isolated pan-drug resistant P. aeruginosa (PDR-PA) in Hunan.Methods:(1) Non-duplicated clinical isolates of CR-AB in Xiangya hospital were collected from September1,2011to June30,2012. While non-duplicated clinical isolates of pan-drug resistant P. aeruginosa from15hospitals in Hunan area were collected from January1,2011to December31,2011. Identification of isolates was carried out by automatic microorganism analytical system Vitek-2or API. Antimicrobial susceptibility tests and screening of pan-drug resistant strains were performed by Kirby-Bauer method and the data were analyzed by WHONET5.6software.(2) Modified Hodge test was used for the screening of carbapenemase. EDTA-synergy test and combination disk diffusion were used for phenotypic screening of metallo-beta-lactamase (MBL).(3) PCR were performed in37strains of PDR-PA for the detecting of33inactivated enzyme genes (including the genes of the P-lactam,16SrRNA methylase and aminoglycoside modifying enzymes),13movable elements genes,3efflux pump genes and outer membrane protein genes. The amplified products were sequenced and BLAST analyses were performed.(4) MICs of antibiotic combined with efflux pump inhibitors (CCCP) against pan-drug resistant strains were determined by agar dilution method, and changes of MICs were observed. Real-Time PCR was used for detection of the expression of efflux pump gene MexA and outer membrane protein gene OprD2.(5) Homology of PDR-PA was analyzed by PFGE and cluster analysis method.Results:(1) Two hundred and five strains of CR-AB were mainly isolated from respiratory samples (accounted for79.0%) and ICU (55.6%) was the main department. Cefoperazone/sulbactam with a sensitive rate of52.2%was the most sensitive antibiotics tested, and the resistant rate to other antibiotics ranged from52.7%to100%. There were8PDR-AB strains.(2) Eight strains showed positive in modified Hodge test, and the positive rate was21.6%. Five strains showed positive in MBL screening test, and the positive rate was13.5%.(3) Totally37strains of PDR-PA were isolated from15hospitals. Their inactivated enzyme genes included TEM-1, armA, rmtB, VIM, IMP and CARB, with the positive rate of100.0%,22.0%,5.0%,19.0%,22.0%and37.8%, respectively. Five movable elements genes, tnpU, tnp513, tnpA/tn21, merA and Int I, were positive, with the positive rates of16.0%,81.0%,49.0%,100.0%and100.0%, respectively. All the positive genes were identified by BLAST analyze, and the sequence homology was all above98.0%compared with known genes in Genbank.(4) Among the37PDR-PA strains,34had a positive result in efflux pump inhibit tests, and the positive rate was91.9%. The relative expression levels of MexA gene in control and experimental group were0.70±0.13and1.95±0.48(P=0.018), respectively, while the relative expression levels of OprD2gene in control and experimental group were3.18±0.60and0.94±0.08(P=0.002), respectively.(5) Thirty-seven PDR-PA strains could be divided into21types, and type A (included5strains) was the main type. Subtype A1and E1appeared in two teaching hospitals at the same time.Conclusions:(1) CR-AB in our hospital presented with severe multi-drug resistance.(2) PDR-AB and PDR-PA were emerged in Hunan area.(3) Produce of inactivated enzymes, like IMP, VIM, TEM-1, CARB, armA and rmtB, high expression of efflux pump MexAB-OprM, and decreased expression of outer membrane protein OprD2were the main mechanisms that lead to the pan-drug resistance of PA in this area.(4) The movable elements, tnpU, tnp513, tnpA/tn21, merA and Int I played important roles in the dissemination of drug resistance.(5) Prevalence of PDR-PA in this area was sporadic, but there were clone disseminations between different hospitals or different departments within one hospital.
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