The Mechanisms Of Active Efflux Transporters And Reverse Transcription Test In Ciprofloxacin-resistant Acinetobacter Baumannii

Objective The study was designed to understand drug resistance situation of Acinetobacter baumannii to ciprofloxacin in Changsha for providing instruction of curing clinical infection of Acinetobacter baumannii and making use of antibiotic reasonably, and to research the relationship of active efflux mechanism and drug tolerance of ciprofloxacin; Besides, initially researching drug reverse mechanism of Acinetobacter baumannii to ciprofloxacin and seeking a good method to recover sensitivity of Acinetobacter baumannii to ciprofloxacin.Methods (1) The broth microdilution method was performed to determine the minimal inhibitory concentration (MIC) of CIP against 56 Acinetobacter baumannii strains. (2) We screened the resistant strains with signifificantly increased efflux of NaN₃, and compared the efflux of NaN₃ of sensitive group with that of CIP-resistant group in two conditions of applied NaN₃ or no NaN₃. (3) Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to measure mRNA levels of active efflux pump gene (adeABC). (4) PCR was used to amplificate self-promoter region gene of adeABC, then sequenced. (5) Reverse experiment was used to observe inhibitory action of three EPIs to efflux.Results (1) Among 56 strains, there were 48 strains presenting CIP resistance with MIC>4mg/l and 8 strains presenting susceptible to CIP with MIC<1mg/l. (2) Choosed the 6 strains with largest cumulation discrepancy of inner CIP density as resistant group,then compared to sensitive group: When no NaN₃, sensitive group's cumulant (138.01±0.251 ng/ml) was obviously higher than that of resistant group (62.15±0.743ng/ml)(P<0.01); After added NaN₃, the cumulant of resistant group manifestly was higher than before (114.78±0.415ng/ml), had no discrepancy with sensitive group (138.01±0.251ng/ml) when no NaN₃ (P>0.05), but had obvious discrepancy with resistant group (62.15±0.743ng/ml)(P<0.05); There had no obvious change when added NaN₃. (3) The gene expression of adeABC obviously increased in resistant group, compared with that in the sensitive strains. (4) Self-promoter region had no sense mutation. (5) After added three EPIs seperately, the MIC of sensitive group changed a little; the MIC of resistant group all decreased, and MIC of 6,6 and 2 strains decreased to 1/4 of the prior MIC.Conclusions (1) The rate of CIP-resistant to Acinetobacter baumannii in Changsha is 85.7%. (2) Our data also demonstrated that measurement of CIP efflux is a useful method for identification of drug-resisitant strains induced by the excessive expression of active efflux pump. (3) The excessive expression of active efflux pump genes was correlative with the CIP resistant in Acinetobacter baumannii. (4) Self-promoter region had no sense mutation. (5) MC-207110 and NaN₃ could inhibit efflux distinguishly; but reserpine had little inhibitory action.