Investigation On Drug Resistence And Resistant Mechanisms Of Acinetobacter Baumannii Isolated From Patients In Neurosurgery

Objective To investigate the variation of drug resistance and molecular epidemiology of A. baumannii clinical isolates in neurosurgery of Shanghai Renji hospital in the past three years. In order to achieve the purpose of drug treatment and providing evidence for nosocomial infection control. We use polymerase chain reaction（PCR）to detect the genes of β-lactamases and main structural genes and regulatory genes of ade ABC、ade IJK and ade FGH efflux pump system of the RND family, comparing the distribution and expression of these genes in the A. baumannii isolates, and also to learn more about the carbapenems resistance mechanisms of Acinetobacter baumannii. By detecting the transposon structure of blα_oxa-23 gene and sequencing, we investigate the structure characteristics and spread mechanism, so as to better explain the spread of blα_oxa-23 gene in drug-resistant A. baumannii and to control transmission.Methods 1、A total of 87 clinical isolates of A. baumannii were collected from July 2011 to June 3013 in neurosurgery of Shanghai Renji hospital. The minimal inhibitory concentrations（MIC）of 12 antimicrobial agents against 87 A. baumannii isolates were determined by agar dilution method, and to investigate the variation of drug resistance. 2 、 Genotype was identified by multilocus sequence typing technique. 3、Polymerase chain reaction（PCR）was used to detect the genes of β-lactamases and main structural genes and regulatory genes of ade ABC、ade IJK and ade FGH efflux pump system of the RND family, comparing the distribution of thesegenes in the imipenem-resistant group and imipenem-sensitive group, further detecting its main efflux gene expression levels include ade B、ade J and ade G by using of the real-time fluorescence quantitative polymerase chain reaction（Realtime-PCR,RT-PCR）. 4、56 blα_oxa-23-producing carbapenems- resistant Acinetobacter baumannii were obtained from 87 isolates. PCR approach was carried out to determine the transposon structure of blα_oxa-23 gene. Plasmid extraction and Southem hybridizations were used to clarify the blα_oxa-23 gene location on the plasmid. Using a whole-genome shotgun approachto determine the complete sequence of the plasmid and analysis the structure.Results 1 、 Eighty-seven A. baumannii isolates were commonly resistant to Cephalosporin, Fluoroquinolone and Aminoglycosides, the resistance rates of A.baumannii to antibiotic drugs increases year by year. In the resisitant rates,minocyline had the lowest rate, was 33.2 %, 74.7 % and 80.5 % for imipenem and meropenem,others were all over 70 %. The resistance rate to co-trimoxazole were 100 %respectively, all higher than other antimicrobial agents. Those carbapenems resistant Acinetobacter baumannii（ CRAB） also showed high resistant rates to other antimicrobial agents. 2、Six genotypes were identified among 87 A. baumannii stains:ST 208（45 strains）、ST 191（15 strains）、ST 540（8 strains）、ST 195（2 strains）、ST 381（2 strains） and ST 368（1 strains）. There has been clonal spread of A.baumannii stains among patients in hospital, and ST 208 is the mainly epidemiology type. The ST types have the homologous relevance except for ST 381. 3、In this research, we detect OXA-23、OXA-24、OXA-51、OXA-58、Amp C、IMP-1 and VIM-1/2type gene. All of the strains carried OXA-51, and OXA-24、OXA-58 、VIM-1 and VIM-2 were all negative, the detection rate of Amp C、OXA-23、IMP-1 were 93.1 %、93.1 %、83.9 %. In Ade RS-ABC efflux pump, the detection of ade B、ade S、ade R was96.6 % 、 96.6 % and 83.9 % respectively. In Ade J and Ade FGH efflux pump, the detection of the ade J、ade L、ade F、ade G、ade H were all over 90 %. With the statistical analysis, the distribution rate differences of Amp C、OXA-23 and efflux pump genesade B、ade G in imipenem-resistant A.baumannii（IRAB）group and imipenem-sensitive A.baumannii（ISAB）group were statistically significant. The ade B, ade J and ade G gene expression levels of IRAB group are generally higher 5.254、1.263 and 6.387 times than ISAB group, ade B、ade G were statistically significant in IRAB group and ISAB group.4、PCR study showed the blα_oxa-23 gene located on the Tn 2006 and Tn 2008 transposon in plasmid.Conclusion: The sensitivity of Acinetobacter baumannii to antibiotic drugs decreases year by year in neurosurgery of Renji hospital. Isolates were commonly resistant to these antimicrobial agents, only slightly lower to minocycline, followed by imipenem and meropenem. Neurosurgery doctrors should choose drugs according to the resistant situation in their departments and the antimicrobial susceptibility results when empiric therapy. There has been clonal spread of carbapenem resistant Acinetobacter baumannii among patients in neurosurgery, which are mainly ST 208.We should take effective measures to intervene in a timely manner. Producing β-lactamase like Amp-C and OXA-23, high expression of active efflux system Ade ABC 、 Ade FGH play an important role in mediating carbapenem resistance of Acinetobacter baumannii. The prevalence of blα_oxa-23-positive resistant isolates has been increased worldwide. The blα_oxa-23 gene located on the Tn 2006 and Tn 2008 transposon in plasmid, and can transfer through different types of plasmids.