Study of Antibiotic Resistance by Efflux in Clinical Isolates of Pseudomonas aeruginosa

Abstract

Twenty three multidrug resistant (MDR) strains were selected from 104 clinical isolates of P. aeruginosa and screened for resistance to ceftazidim, ceftriaxone, ciprofloxacin, ofloxacin and ethidium bromide by determining MICs. The MICs of EtBr and antibiotics were also measured in presence of proton conductor, carbonyl cyanide m-chlorophenyl hydrazone (CCCP). The presence of proton gradient-dependent efflux mechanism was assessed using ethidium bromide accumulation assays. Drug accumulation studies for these antibiotics were performed to determine the drug specificity of efflux. PCR was used to identify the mexAB-oprM gene as a major factor in MDR intrinsic resistance of clinical isolates of P. aeruginosa. In absence of CCCP, the MICs of these antimicrobial agents were > or = 4 microg L(-1). CCCP reduced the MICs of them at least in 1 dilution. Ethidium bromide accumulation assays confirmed the presence of proton gradient-dependent efflux mechanism in clinical isolates of P. aeruginosa and results of accumulation assays of drugs demonstrate that, active efflux in this bacterium are due to broadly-specific multidrug efflux system(s). PCR products demonstrate the presence of mexAB-oprM operon in 4 strains from 23 clinical isolates. These results confirmed the presence of proton gradient-dependent efflux mechanism in all of the clinical isolates of P. aeruginosa and demonstrate that, efflux pumps in this bacterium are broadly-specific multidrug efflux systems. In this study we show that MexAB-OprM multidrug efflux system was expressed in only 17% of clinical isolates of P. aeruginosa. These results confirmed the presence of other multidrug efflux pumps in clinical isolates of P. aeruginosa.