The Role Of Active Efflux Pumps In Mediating The Resistance To Carbapenems Among Acinetobacter Species

[Objective] To investigate the role of active efflux pumps in mediating the resistance to carbapenems among Acinetobacter species.[Method] Non-repetitive 95 isolates of Acinetobacter species were collected from 14 hospitals from 1999 to 2006. Amplified rDNA restriction analysis (ARDRA) was used to identify all isolates at a genomic level. Agar dilution method was used to determine the minimal inhibitory concentration (MIC) of meropenem, imipenem, cef-tazidime,cefepime,cefoperazone-sulbactam,piperacillin-tazobactam,ciprofloxacin,am-ikacin, minocycline and polymyxin, and then the strains were divided into different groups depending on the resistant phenotypes. Pulsed field gel electrophoresis(PFGE) was used to select the same genetic background . Efflux pumps genes including adeB, adeD and adeE were amplified by PCR and sequenced. The expression levels of these genes were measured using real-time fluorescence PCR. OXA-type carbapenemases and insertion element ISAba1 were amplified by PCR, while the expression levels of OXA-51-like gene were measured using real-time fluorescence PCR.[Results] 68 isolates of Acinetobacter baumannii and 4 isolates of Acinetobacter genospecies 3 were collected into this study according on ARDRA. The strains were divided into three groups depending on MICs, including resistant to carbapenems, susceptible to carbapenems while resistant to at least 5 other antibiotics, susceptible to all antibiotics. PFGE showed that clone A contained different resistant phenotypes. Aba 32R and 32S belonged to the same clone, so did 4AS6 and its mutants 4AS6-1L, 4AS6-2L and 4AS6-3L. The positive rates of adeB, adeD and adeM are 81.94%,0 %and 100%, respectively. Real-time PCR identified 2.79-fold increases in adeB expression and 4.04-fold increases in adeM expression in carbapenem-resistant strain 32R compared to carbapenem-susceptible one 32S. Among 4AS6 and its mutants, adeM gene expression increased in the mutants especially 4AS6-3L, however, the adeB gene could not be detected in all of the four. In clone A, the expression levels of both adeB and adeM genes were very low in all antibiotics-susceptible group. The expression level of adeB gene in the group carbapenem-resistant was higher than the group carbapenem-susceptible. The expression level of adeM gene was relative higher in clone P compared to other clones. If we ignored the genetic background, the expressions of adeB and adeM genes were the lowest in the all antibiotics-susceptible group and a little higher in multidrug-resistant groups, however, there were no statistic differences between the group carbapenem-resistant and carbapenem-susceptible. AdeD gene might be few and far between in Acinetobacter species because it could not be detected in all 72 strains. An insertion sequence ISAba1, was found to exist in 7 A.baumannii strains, upstream of bla_oxa-51-like. The expression levels of bla_OXA-51-like in the carbapenem-resistant or intermediate A.baumannii strains were higher than the carbapenem-susceptible A.baumannii strains. The presence of the insertion sequence ISAba1 upstream of the bla_OXA-51-like gene might provide a promoter that allowed the overproduction of carbapenemase, and this resulted in carbapenem resistance. The overexpression of OXA-51-like carbapenemase and efflux pumps (adeB and adeM) might contribute to more resistant to meropenem than imipenem.[Conclusions] Active efflux pumps play an important role in mediating the resistance to carbapenems among Acinetobacter species. The increases of efflux pump expression may reduce susceptibilities to mutiple antibiotics. We should integrate all kinds of resistant mechanisms including enzymes producing, outer membrane loss and efflux pumps overexpression together to know multidrug-resistant Acinetobacter species better. Effective infection control measure and antimicrobial intervention policy should be conducted in order to control outbreak of multidrug-resistant Acinetobacter species.