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.
Highlights
Bacteria can adapt to a wide range of environmental conditions
Through a combination of bacteriological, proteomic and transcriptomic analyses, we demonstrated that doxycycline was associated with an overexpression of various efflux pumps that are expressed at different levels, depending on the step of the antimicrobial selection, thereby revealing a reversible multidrug resistance phenotype
E264DC16 and E264DC32 showed only a 2–3 fold increase in their minimal inhibitory concentrations (MICs) of cyclines and quinolones compared to the parental strain, while MICs of these antibiotics increased between 16–170 fold for E264DC64 and E264DC128
Summary
Bacteria can adapt to a wide range of environmental conditions. Antimicrobial compounds constitute environmental chemical stresses for bacterial cells and many pathogens have developed appropriate mechanisms conferring protection against this external attack. Each RND efflux pump is encoded by an operon and is a tripartite complex: an integral inner transporter (AmrB, BpeB and BpeF respectively), a periplasmic adaptator named Membrane Fusion Protein (AmrA, BpeA and BpeE respectively) and the outer membrane channel named Outer Membrane Factor (OprA, OprB and OprC respectively) [3,4,5,6,7,8,9]. These complexes can export drugs outside the bacteria [10]. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure
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