Interplay between Three RND Efflux Pumps in Doxycycline-Selected Strains of Burkholderia thailandensis

Abstract

BackgroundEfflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure.Methodology/Principal FindingsWe used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect.Conclusions/SignificanceThese results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.

Loss of Methyltransferase Function and Increased Efflux Activity Leads to Doxycycline Resistance in Burkholderia pseudomallei

“…In E. coli, PA␤N at higher concentrations can inhibit efflux and destabilize outer membrane permeability (45); however, this phenomenon probably does not occur in B. pseudomallei due to the highest MICs being observed at the lowest PA␤N concentration, at least in the presence of gentamicin. Previous work in B. thailandensis has shown that the addition of PA␤N can upregulate amrB expression, which could potentially account for the increases in gentamicin MICs observed in this study (36). Caution should thus be taken when using efflux pump inhibitors, as they do not potentiate all antibiotic classes and can have the unintended consequence of increasing antibiotic resistance.…”

“…However, a recent publication reported that the presence of PA␤N at 50 and 100 g/ml decreased DOX resistance in B. pseudomallei under biofilm conditions (41). Similarly, reversion to a DOX-susceptible phenotype in the presence of PA␤N has been described in B. thailandensis (36). PA␤N has also been shown to have a competitive mechanism of inhibition in A. baumannii (42), P. aeruginosa (43), and E. coli (44).…”

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

“…When either of these pumps was inactivated, a third pump, BpeAB-OprB, was hyperexpressed, indicating the interplay among the three RND pumps (627), similar to those observed in P. aeruginosa (628) and Salmonella (629). Interestingly, an antagonistic effect between PA␤N (at 50 and 200 g/ml) and aminoglycosides/␤-lactams was observed, and the speculation was that PA␤N might have induced the overproduction of the AmrAB-OprA and BpeAB-OprB pumps (627). It remains to be determined whether an induction process exists for explaining the above-mentioned observations that PA␤N at 40 g/ml did not alter antibiotic susceptibility in B. pseudomallei (614,617) and that PA␤N at 20 g/ml did not affect the efflux activity of SmeDEF toward ciprofloxacin and tetracycline in S. maltophilia (630).…”

“…Several more recent studies have also highlighted such OM-permeabilizing effects (974,975,1006,1007), although their conclusions need to be examined carefully, as discussed above (see Efflux Pump Inhibitors). In Burkholderia spp., PA␤N at a concentration of up to 200 g/ml was considered most effective as an EPI (627,1005). With this high level, its impact on OM permeability should be investigated.…”

“…With this high level, its impact on OM permeability should be investigated. Moreover, PA␤N at 200 g/ml induced the expression of AmrABOprA and BpeAB-OprB of B. thailandensis (627), and this requires further study regarding the interplay between RND pumps and PA␤N.…”