Study On The Epidemiology And Resistant Mechanism Of Carbapenem-resistant Acinetobacter Baumannii

Objective:(1) To understand the clinical distribution, antibiotic resistance situation and molecular epidemiological characteristics of carbapenem-resistant Acinetobacter baumannii (CRAB). In order to provide suggestions for rational use of antibiotics and experimental evidence for preventing and controlling of the prevalence of these multidrug resistant strains.(2) To investigate the relationship between insertion sequence ISAbal, carbapenemase enzyme, AdeABC efflux pump, and carbapenem resistance of Acinetobacter baumannii.Methods:(1) From April2012to March2013, a total of270non-duplicated Acinetabacter baumannii strains resistant to carbapenemases from10different general hospitals were collected. Antimicrobial susceptibility to18antibiotics was tested by Kirby-Bauer method and the data were analyzed by WHONET5.6software.(2) The repetitive extragenic palindromic PCR (REP-PCR) was used to analyze the homology of91CRAB isolates from different hospitals.(3) Modified Hodge test was carried out to screen carbapenemase enzyme of all the strains; the double-disk synergy test was used to detect metallo-p-lactamase (MBL); multiplex PCR was applied to detect carbapenemase genes, including OXA-51, OXA-23, OXA-58and OXA-24, and MBL genes, including IMP, VIM and SIM. ISAbal-OXA-23, ISAbal-OXA-51, Tn2006and Tn2008were detected by PCR amplification and the positive products were sequenced.(4) Carbapenem-resistant strains were acquired from multistep selection resistance test using meropenem in vitro. The quantitation test of sensitivity of strains before and after induction was determined by the E-test method and CCCP inhibition test was used to screen efflux pump. PCR and sequencing analyses were used to analyze the changes of the regulatory genes adeR and adeS of the AdeABC efflux pump system in these strains before and after induction, and real-time PCR was used for quantitation of expressions of adeA, adeB, adeR, adeS before and after induction.Results:(1) The270strains of CRAB were mainly from respiratory tracts (78.90%) and the second source was wound secretions (10.74%). The number of strains isolated from the intensive care unit was the highest that accounted for50.0%, followed by the neurosurgery department, the neurology department and the burn department, which accounted for7.78%,7.41%and7.41%respectively. In the18drugs tested, the resistance rates of14were higher than90.0%, piperacillin was the highest (100.0%) and cefoperazone/sulbactam was the lowest (48.9%).(2) The91clinical CRAB isolates were divided into7types named genotype A-G by REP-PCR. Among them, genotype A had79strains, genotype B had4strains, genotype C, D and F had2strains and the rest had only1strain each. Genotype A included2subtypes, named Al and A2which had46strains and33strains respectively. Strains with genotype A existed in every hospital included in this study.(3) Among the270CRAB strains156(57.8%) were positive in the modified Hodge test, but none harbored MBLs in the synergy test. All the strains carried OXA-51gene and267strains carried OXA-23gene, but only one strain carried OXA-58gene. OXA-24, IMP, VIM and SIM genes were all negative in these CRAB strains. The41carbapenem-sensitivity strains used as controls carried only OXA-51gene, and did not carry OXA-23, OXA-58or OXA-24genes. The insertion sequence IS Abal was detected in the upstream of OXA-23gene of all the267OXA-23positive strains, but not in the upstream of OXA-51gene. Of the91CRAB strains tested, Tn2006was positive in40isolates and Tn2008was all negative.(4) The MICs of meropenem were0.38μg/ml and0.25μg/ml in parental sensitive strain S25595and S7257respectively, and the MICs of meropenem after induction were both above32μg/ml. Compared with parental sensitive strains, the expression of adeA, adeB, adeR and adeS mRNA ranged from2.45to9.44times, but there were no gene mutations or insertion sequences in the regulatory gene adeS and adeR.Conclusions:(1) The multidrug resistance of CRAB was serious.(2) There was prevalence of the same clones in these clinical isolated CRAB strains in this area, and genotype A was the major prevalence type.(3) OXA-51and OXA-23were the mainly carbapenemases in Acinetobacter baumannii in this area. ISAbal-OXA-23might be an important mechanism in the resistance of Acinetobacter baumannii to carbapenems, which was transmitted mainly by the transposon of Tn2006.(4) High expression of the AdeABC efflux pump system in Acinetobacter baumannii was closely associated with meropenem resistance. The rise of adeA and adeB expression was not caused by gene mutations or insertion sequences in the regulatory gene adeS and adeR and there might be other mechanisms participated.