Mechanism Of Quinolone-resistant Acinetobacter Baumannii

【OBJECTIVE】To determine the quinolone resistance phenotype and to investigate the gene homology in the clinical isolates of Acinetobacte baumannii; To study the relations between resistance phenotype of Acinetobacter baumannii and gene mutations of gyrA and parC on the chromosomes;To study the expression of efflux pump gene in the clinical isolates of Acinetobacte baumannii.【METHODS】(1) Micro-broth dilution was used to determine the minimum inhibitory concentration(MIC) of Levofloxacin and Moxifloxacin of 90 Acinetobacter baumannii isolated from the clinical specimens. Gene homology was analyzed by repetitive extragenic palindrome.(2) The gyrA and parC was amplified by use of polymerase chain reaction(PCR). The PCR products were analysed by DHPLC and DNA sequencing.(3) Agar dilution was used to determine the influence of efflux pump inhibitor(CCCP) of quinolone in the Acinetobacter baumannii ; Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was used to amplify the adeB.Difference in the expression of adeB between the drug-resistant strains and drug-susceptible strains was analyzed by the relative gray value measured by Bandscan software.【RESULTS】(1) The resistances to Levofloxacin or Moxifloxacin were 63.3% and 60.0% in the 90 Acinetobacter baumannii respectively .There were nine genotypes in the clinical isolates including typeA(3,3.33%), typeB(40,44.44%), typeC(22,24.44%), typed(5,5.56%),typeE(7,7.78%),typeF(2,2.22%),typeG(2,2.22%),typeH(7,7.78%) , typeI(2,2.22%). The number of typeB was the most in all types.(2) Ninty strains had the expected sizes of 343bp of gyrA and 327bp of parC. Three out of nine genotypes, which were drug-resistant, were all found abnormal elution peaks (two or three peaks) by denaturing high-performance liquid chromatography DHPLC. DNA sequencing for the three drug-resistant genotypes demonstrated mutations in gyrA gene resulted in amino acid substitutions of Ser83→Leu and in parC resulted in Ser80→Leu. And there were 3 synonymous mutations in parC gene.All the drug-susceptible ones showed single peak, and the results of sequencing had no mutation.(3) After using CCCP, the MIC vaules of 71 strains declined in varying degrees. Sixteen strains were positive in efflux pump, incluing 14 drug-resistant strains and 2 drug-susceptible strains. RT-PCR showed that 84 out of 90 expressed adeB genes,with 63(63/65,96.7%)in drug-resistant strains and 21(21/25,84.0%) in drug-susceptible strains.【CONCLUSION】(1) At present, the resistance to four-genetaion quinolone has been close to three- generation, and we should pay more attention to it.(2) From December 2007 to April 2008 in our hospital there was the prevalence of a certain genotype of Acinetobacter baumannii (3) DHPLC, with its higher sensitivity,economy and convenience,had shown a wide application prospects in detecting bacteria gene mutation .(4) Resistance to quinolone-resistant Acinetobacter baumannii strains was related with high-frequency mutations in the gyrA and parC genes. The 3 synonymous mutations in parC genes suggested parC had mutations more easily.(5) Efflux pump inhibitor can enhance the antibacterial activity of quinolone. Active efflux has been widespread in the clinical isolates of Acinetobacter baumannii.(6) The effect of active efflux system is one of the major mechanisms of Acinetobacter baumannii.The expression enhancement of adeB gene plays an important role in inducing quinolone-resistant.