Cloning and sequencing of lsaE efflux pump gene from MDR Enterococci and its role in erythromycin resistance

Abstract

Enterococci are opportunistic members of intestinal microbiota with notable ability to transmit antimicrobial resistance genes. Among the different resistance mechanisms, multidrug efflux is evolving as a huge problem in conferring multidrug resistance to bacterial cells because these pumps extrude a broad range of antimicrobials. Therefore, the aim of this work was to evaluate role of efflux pumps in the development of multi-drug resistance in Enterococci through studying the antimicrobial resistance profiles of Enterococci isolates, phenotypically and genotypically investigating the role of active efflux pumps in development of resistance, in addition to characterizing the most common efflux pump genes. The study involved the recovery of 149 Enterococci isolates from specimens of patients suffering infections in some hospitals in Egypt. Antimicrobial resistance profiles of isolates showed that only 1.3% of the isolates were resistant to each of linezolid, daptomycin, and fosfomycin. The highest resistance was to ampicillin (60.4%) while 47 of the isolates (31.54%) were found to be multidrug-resistant. Efflux pumps have shown to have a significant role in erythromycin resistance in 11 isolates (23.4% of MDR isolates) as indicated by an 8 or more fold decrease in minimum inhibitory concentration in the presence of the efflux pump inhibitor, carbonyl cyanide m- chlorophenylhydrazone (CCCP). End point PCR was used to detect efflux pump genes lsaE, msrC, and mefA in the 11 isolates at which efflux pumps were found to play a significant role in resistance. Nine out of the 11 isolates (81.8%) were found to carry lsaE gene. This gene was inserted into pUC21 vector and cloned into DH5α E. coli resulting in successful transformation and expression of erythromycin resistance in this host. Finally, sequencing of the lsaE gene was carried out. To the best of our knowledge, this is the first report on the cloning of lsaE gene from MDR Enterococcus isolates.