Study On The Resistant Genes Of Disinfectant And Antimicrobial Drug,and Combined Antibacterial Activity In Acinetobacter Baumannii

Objective:To investigate the resistance profile on commonly used antimicrobial agents and disinfectant resistant in 47 clinical isolates of Acinetobacter baumannii in a hospital,detection of antimicrobial agents and disinfection resistance genes to explore the relationship between drug resistance and disinfectant resistance.Analysis of homology and epidemiological characteristics of clinical isolates of A.baumannii with high minimum inhibitory concentration(MIC)level of disinfectant.In vitro antibacterial activity of chlorhexidine combined with antibacterial agents against multidrug resistant(MDR)and extensively drug resistant(XDR)A.baumannii,to provide laboratory data support for control and treatment of A.baumannii infection.Methods:Agar dilution and microdilution were used to determine the MIC of 47 A.baumannii clinical isolates to 21 kinds of antibiotics and 6 kinds of disinfectant,respectively.The antimicrobial and disinfectant resistance genes,adeB,adeG,adeJ,adeT1,adeT2,amvA,abeD,abeM,qacE,qacE?1,aceI,abuO,fabI,OXA-23,OXA-24,OXA-51 and OXA-58 were analyzed through polymerase chain reaction(PCR).The expression profile of efflux pump genes adeB,adeG,adeJ,abeD,abeM,aceI and triclosan target gene fabI were detected by quanitative real time PCR(qRT-PCR).In addition,pulsed-field gel electrophoresis(PFGE)and multilocus sequence typing(MLST)were used to analyze the epidemiology characteristics of clinical isolates of A.baumannii with high MIC level of disinfectant.The antibacterial activity of chlorhexidine acetate combined with levofloxacin,ciprofloxacin,imipenem,meropenem,minocycline and doxycycline against MDR and XDR clinical isolates of A.baumannii was tested in vitro by checkboard test.Results:1.The results of drug susceptibility showed that there were 6(12.77%)MDR isolates and 24(51.06%)XDR isolates.Resistance rates to carbapenems and third-generation cephalosporin were more than 60% and all were susceptible to polymyxin and tigecycline.The results of disinfectant susceptibility showed that MICs is within a range of 8-128 ?g/ml to chlorhexidine acetate,4-32 ?g/ml to benzalkonium bromide,7.5-22.50% to ethanol,47-376 mM to hydrogen peroxide,2-256 ?g/ml to triclosan,and 160-640 ?g/ml to sodiumhypochlorite.A.baumannii clinical isolates showed reduced susceptibility to chlorhexidine acetate,benzalkonium bromide,ethanol,hydrogen peroxide and triclosan,especially for chlorhexidine acetate and triclosan.2.The resistant genes OXA-23,OXA-24,OXA-51 and OXA-58 in A.baumannii were found with a rate of 63.83%,0%,93.62% and 0%,respectively.The rates of disinfectant resistance genes adeB,adeG,adeJ,abeD,abeM,amvA,adeT1,adeT2,fabI,aceI,qacE and qacE?1 ratio were above 68.09%,abuO ratio was 2.13%.Resistant genes adeJ,aceI,abeD and fabI existed in each isolate.Triclosan resistant strains showed a relative increase in adeB and adeJ expression,whereas chlorhexidine resistant strains showed increased abeM and fabI gene expression.The expression of adeB,adeJ,adeG and fabI was increased approximately 3-fold with sublethal concentrations of triclosan induction.Only four times adeB expression was induced by sublethal concentrations of chlorhexidine acetate.The GlyI95 Ser mutation of FabI was detected in only one triclosan-resistant strain.3.ST92,ST195,ST184,ST618 and 4 new STs,ST1277,ST1278,ST1279 and ST1280 were identified in 14 clinical isolates.ST92 was the major ST type.ST92 and ST195 belonged to clone complex(CC)92 which were the major clone complex in the hospital.Two distinct PFGE patterns were identified,of which 2 isolates belonged to pulsotype A,3 isolates to pulsotype B,and the other 9 isolates to different pulsotypes.Meanwhile,CC92 was corresponded to the pulsotype B.4.Checkboard test results showed that more than 50% strains exhibited ?0.5synergistic index FICI on chlorhexidine combined with minocycline,doxycycline,ciprofloxacin and meropenem which shows synergy effect.And more than 66.67% strains exhibited 0.5< FICI ?1 on chlorhexidine combined with levofloxacin and imipenem which shows additivity effect.Conclusion:1.A.baumannii clinical isolates showed the seriously drug resistant situations,and tigecycline and polymyxin was still susceptibility.A.baumannii showed reduced susceptibility to triclosan,chlorhexidine acetate,benzalkonium bromide,hydrogen peroxide and ethanol.2.The emergence of A.baumannii MDR and XDR isolates was related to the existence of OXA-23,OXA-51 and efflux pump genes adeB,adeG and adeJ.Reduced susceptibility in A.baumannii to disinfectant was related to the efflux pump.3.Epidemiological analysis showed that CC92 was the major clone complex of high level disinfectant resistant A.baumannii.4.Chlorhexidine acetate combined with a variety of antibacterial agents show the synergistic or additive antibacterial activity.