Functional Characterization of Brucella melitensis NorMI, an Efflux Pump Belonging to the Multidrug and Toxic Compound Extrusion Family

Abstract

Two putative proteins (NorMI and NorMII) similar to the multidrug efflux protein NorM of Vibrio parahaemolyticus are encoded by the Brucella melitensis 16 M genome. We show that a drug-hypersusceptible Escherichia coli strain overexpressing NorMI displays increased resistance to norfloxacin, ciprofloxacin, gentamicin, tetraphenylphosphonium ion, acriflavine, and berberine. This elevated resistance was proven to be mediated by an energy-dependent efflux mechanism. NorMI belongs to the multidrug and toxic compound extrusion family and is the first multidrug efflux protein identified in Brucella spp.

Clinically Relevant Chromosomally Encoded Multidrug Resistance Efflux Pumps in Bacteria

“…MDR pumps of the MATE family have been described for several gram-negative bacteria: V. parahaemolyticus (NorM) (132), B. thetaiotaomicron (BexA) (129), V. cholerae (VcmA, VcrM) (65,66), Brucella melitensis (NorMI) (21), N. gonorrhoeae (NorM) (189), H. influenzae (HmrM) (231), and P. aeruginosa (PmpM) (58). The substrate profile typically includes a fluoroquinolone (norfloxacin and ciprofloxacin), DNAintercalating dyes, and detergents.…”

In vitro selection of fluoroquinolone resistance in Brucella melitensis

“…NorMI and NorMII [26]. Only NorMI has been fully characterised [26]. Thus, we can hypothesise that NorMI and/or NorMII are overexpressed in our B. melitensis M a n u s c r i p t moxifloxacin-resistant mutants.…”

“…Two putative MATE family efflux pumps have been recently described in B. melitensis, i.e. NorMI and NorMII [26]. Only NorMI has been fully characterised [26].…”

“…NormI and NormII are currently the only efflux pumps that have been characterised as functionally active in B. melitensis 16M [26]. They belong to the MATE (multidrug and toxic extrusion) efflux pump family and have been shown to be specifically susceptible to PAN [26].…”

“…They belong to the MATE (multidrug and toxic extrusion) efflux pump family and have been shown to be specifically susceptible to PAN [26]. Whole NorMI and NorMII genes plus a 250 bp upstream M a n u s c r i p t DNA fragment were amplified and sequenced in B. melitensis 16M and two fluoroquinolone-resistant mutants using the primers indicated in Table 1 to check for the presence of mutations in the genes or in promoters.…”