Molecular Cloning and Characterization of All RND‐Type Efflux Transporters in Vibrio cholerae Non‐O1

Abstract

Resistance Nodulation cell Division (RND) efflux transporters are thought to be involved in mediating multidrug resistance in Gram‐negative bacteria, including Vibrio cholerae non‐O1. There are six operons for putative RND‐type efflux transporters present in the chromosome of V. cholerae O1 including two operons, vexAB and vexCD, which had already been identified. All of the six operons were cloned from V. cholerae non‐O1, NCTC4716 by the PCR method, introduced, and expressed in cells of drug hypersusceptible Escherichia coli KAM33 (AacrAB, AydhE). Only vexEF conferred elevated minimum inhibitory concentrations (MICs) of some antimicrobial agents in the E. coli cells. However, VexEF did not confer increased MIC of any drug tested in tolC‐deficient E. coli KAM43 cells. On the other hand, when E. coli KAM43 was transformed with vexAB, vexCD or vexEF together with tolCVc of V. cholerae NCTC4716, we observed elevated MICs of various antimicrobial agents. Among them, E. coli KAM43 expressing both VexEF and TolCVc showed much higher MICs and much broader substrate specificity than the other two. We also observed ethidium efflux activity via VexEF‐TolCVc, and the activity required Na+. Thus, VexEF‐TolCVc is either a Na+‐activated or a Na+‐coupled transporter. To our knowledge, this is the first report on the requirement of Na+ for an RND‐type efflux transporter.

Vibrio cholerae RND Family Efflux Systems Are Required for Antimicrobial Resistance, Optimal Virulence Factor Production, and Colonization of the Infant Mouse Small Intestine

“…Supporting this notion is the recent report showing that expression of the vexGH and vexIJK RND efflux systems may be induced in vivo in humans (31). In addition, Rahman et al recently reported that V. cholerae vexEF can produce a functional multiple-drug efflux system when recombinantly expressed in E. coli (51). Thus, it appears likely that the VexEF, VexGH, and VexLM RND efflux systems are produced under yet unknown conditions.…”

“…Ongoing work in our laboratory is focused on determining whether VexEF functions in this respect. Lastly, vexGH, vexIJK, and vexLM did not produce functional efflux systems when expressed in E. coli (51). Our results showed that the VexIJK efflux system functions in SDS and Triton X-100 resistance in V. cholerae.…”

SmdAB, a Heterodimeric ABC-Type Multidrug Efflux Pump, in Serratia marcescens

“…E. coli KAM32 (⌬acrB ⌬ydhE hsd⌬5), which lacks the major multidrug efflux pumps AcrAB and YdhE, is hypersusceptible to many antimicrobial agents (7). E. coli KAM42 (⌬acrB ⌬ydhE hsd⌬5 ⌬tolC), a tolC-deficient strain derived from KAM32, was constructed as described previously (32). Cells were grown in Luria (L) broth (18) at 37°C under aerobic conditions.…”

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

“…The V. cholerae genome contains 6 RND transporters (VexAB, VexCD/BreAB, VexEF, VexGH, VexIJK, and VexLM), and most of them require TolC for drug efflux (340)(341)(342)(343)(344)(345). VexAB seems to play a predominant role in the efflux of antibiotics (benzylpenicillin, erythromycin, and polymyxin B) and detergents (cholate, SDS, and Triton X-100) (341), although among the 6 RND systems cloned from a non-O1 strain, VexEF conferred the strongest resistance to an E. coli host (343). VexCD (confusingly "renamed" BreAB) is involved in bile efflux (340) and is induced by bile (342).…”