RND-Type And MATE-Type Efflux Pumps Involved In Multidrug Resistance In Clinical Isolates Of Acinetobacter Baumannii

Objective:Acinetobacter baumannii is a nonfermentative,gram-negative bacillus that is causing increasing health problems in the nosocomial setting,particularly intensive care units.Multidrug-resistant Acinetobacter poses treatment challenge. Active efflux pumps confer clinically relevant resistance.This study focuses on the resistance mechanisms of Acinetobacter baumannii clinical islates involved with effiux pumps of AdeABC,AdeIJK and AbeM using molecular biology method.Methods:1,Strains,Susceptibility testing and Interference testing of CCCP:A total of 35 strains of Acinetobacter baumannii were isolated from 3 hospitals in Tianjin during 2006-2008,including 30 multidrug-resistant isolates and 5 antibiotic-susceptible isolates.Antibiotic susceptibility was tested by micro-dilution broth method and disk diffusion on Mueller-Hinton agar.The MICs of 8 antibiotics were determined by micro-dilution broth method with CCCP and without it.2,Detection of adeB,adeJ,abeM and expression of mRNA:The adeB,adeJ and abeM gene were detected by PCR amplification.The expression of mRNA of 10 multidrug-resistant isolates and 5 antibiotic-susceptible isolates were detected by RT-PCR,and analyzed by semiquantitative analysis of gel electrophoresis.3,Determination of the sequence of the adeRS:The controlling gene adeRS were detected by PCR amplification.DNA sequencing of 2 multidrug-resistant isolates and 2 antibiotic-susceptible isolates were performed with specific primers with automatic sequencer ABI PRISM 3730XL.Results:1,Antibiotic susceptibility and Effect of CCCP:30 multidrug-resistant isolates were all resistant to ciprofloxacin,ofloxacin,piperacillin,cefotaxime,ceftazidime, cefoperazone,ceftriaxone,cefepime,amikacin and gentamicin.The resistance rate of levofloxacin,cefoperazone/sulbactam and imipenem were 50%,26.67%, 10%.The MICs of 8 antibiotics in all the clinical isolates decreased at least 4-fold in the presence of CCCE The MICs of ofloxacin in 2 susceptible isolates decreased 2-fold,the MIC of ceflazidime in 1 susceptible isolates decreased 2-fold,the MICs of gentamicin in 2 susceptible isolates decreased 2-4 fold.2,Distribution of adeB,adeJ,abeM and expression of mRNA:All the isolates were positive for adeB,adeJ and abeM gene.The relative intensity of mRNA expression of adeB in 10 MDR strains were 1.211-9.169,the mRNA expression of adeB in 5 sensitive strains were not examined.The relative intensity of mRNA expression of adeJ in 10 MDR strains were 0.956-4.519(the average was 1.75), and those in 5 sensitive strains were 0.356-0.576(the average was 0.426).The mRNA expression of abeM were not examined both in 10 MDR strains and 5 sensitive strains.3,Sequence analysis of adeRS:The sequence of adeR gene in AbS2 and AbS5 were the same,and showed 97%identity with that of AY426969 published on GenBank. The sequence of adeR gene in AbM1 and AbM4 were the same,and showed 97% identity with that of AY426969.The predicted amino acid sequence of AdeR in AbM1 and AbM4 had substitutions:Ile14→Val,Val120→Ile,Ala136→Val, compared with those in AbS2 and AbS5.The sequence of adeS gene in AbS2 and AbS5 were the same,and showed 97%identity with that of AY426969.The sequence of arieS gene in AbM1 and AbM4 were the same,and showed 96% identity with that of AY426969.The predicted amino acid sequence of AdeS in AbM1 and AbM4 had substitutions:Gly186→Val,Asn213→Asp,Ile285→Leu, Asn268→His,Val331→Ile,Val348→Ile,Gly356→Asn,His357→Asn,and amino acid absence after the 357th,compared with those in AbS2 and AbS5.Conclusions:1,The MICs of one or more antibiotics(ciprofloxacin,ofloxacin,levofloxacin, chloramphenicol,ceftazidime,cefoperazone/sulbactam,amikacin and gentamicin) in MDR isolates decreased in the presence of CCCP.The effect of efflux pumps contributes to multidrug resistance.2,The difference of mRNA expression of adeB and adeJ in 10 MDR strains and 5 sensitive strains may relate to the multidrut-resistance.3,The substitutions and absence of the predicted amino acid sequence of AdeRS in AbM1 and AbM4 may contribute to the difference of mRNA expression of adeABC.The GenBank accession number of adeR and adeS in AbS5:FJ495118, FJ600366.The relationship between the gene variations and the structure and function of effiux protein needs further study.