Phenotypic and Genotypic Studies on Antibacterial Efflux Pumps in Pseudomonas aeruginosa

Aim: The present study aims to determine the efflux pump resistance by phenotypic and genotypic methods. Methods: A total of 40 isolates of Pseudomoas aeruginosa were studied for antibiotic susceptibility pattern, , Effect of efflux pump inhibitors (reserpine, carbonyl cyanide mchloropheylhydorazone [CCCP] and omperazole) on the MIC of antibacterial agents, ethidium bromide (EtBr) efflux assay , outer membrane proteins , polymerase chain reaction for the amplification of resistance genes. Results: Nearly most of the isolates were multiple resistant as they were resistant to most antimicrobial classes used in this study. CCCP was found to have the highest effect on decreasing the MIC of different antibiotics comparable with reserpine and omperazole. It was found that all tested Pseudomonas aeruginosa isolates extrude EtBr resulting in decrease in fluorescence over the time of assay. In Pseudomonas aeruginosa isolate No.3 and 14 only the control cells extrude EtBr resulting in significant loss in fluorescence. In presence of each of reserpine, CCCP and omperazole, an insignificant loss in fluorescence was observed, reflecting blockage of EtBr by these compounds at different levels. All the isolates produced high amount of outer membrane proteins with apparent molecular mass of 54 KDa and 50 KDa. These proteins may be designated as OprJ and OprM or OprN. MexA and MexB genes was detected and amplified in most of the tested isolates of Pseudomonas aeruginosa genomic and plasmid DNA. The amplicons were visualized at 500 bp and 1000 bp respectively. While OprM gene was amplified successfully in the twenty tested isolates and the amplicons were visualized at size of 900 bp.