Assessment of three Resistance-Nodulation-Cell Division drug efflux transporters of Burkholderia cenocepacia in intrinsic antibiotic resistance

Abstract

BackgroundBurkholderia cenocepacia are opportunistic Gram-negative bacteria that can cause chronic pulmonary infections in patients with cystic fibrosis. These bacteria demonstrate a high-level of intrinsic antibiotic resistance to most clinically useful antibiotics complicating treatment. We previously identified 14 genes encoding putative Resistance-Nodulation-Cell Division (RND) efflux pumps in the genome of B. cenocepacia J2315, but the contribution of these pumps to the intrinsic drug resistance of this bacterium remains unclear.ResultsTo investigate the contribution of efflux pumps to intrinsic drug resistance of B. cenocepacia J2315, we deleted 3 operons encoding the putative RND transporters RND-1, RND-3, and RND-4 containing the genes BCAS0591-BCAS0593, BCAL1674-BCAL1676, and BCAL2822-BCAL2820. Each deletion included the genes encoding the RND transporter itself and those encoding predicted periplasmic proteins and outer membrane pores. In addition, the deletion of rnd-3 also included BCAL1672, encoding a putative TetR regulator. The B. cenocepacia rnd-3 and rnd-4 mutants demonstrated increased sensitivity to inhibitory compounds, suggesting an involvement of these proteins in drug resistance. Moreover, the rnd-3 and rnd-4 mutants demonstrated reduced accumulation of N-acyl homoserine lactones in the growth medium. In contrast, deletion of the rnd-1 operon had no detectable phenotypes under the conditions assayed.ConclusionTwo of the three inactivated RND efflux pumps in B. cenocepacia J2315 contribute to the high level of intrinsic resistance of this strain to some antibiotics and other inhibitory compounds. Furthermore, these efflux systems also mediate accumulation in the growth medium of quorum sensing molecules that have been shown to contribute to infection. A systematic study of RND efflux systems in B. cenocepacia is required to provide a full picture of intrinsic antibiotic resistance in this opportunistic bacterium.

RND-4 efflux transporter gene deletion in Burkholderia cenocepacia J2315: a proteomic analysis

“…Specially, we identified the tetracycline repressor protein, tetR (BCAL3258) and the metallo-β-lactamase superfamily protein (BCAL1818). These findings are in agreement with the well-established role of the RND efflux pumps protein family in resistance to antibiotics as reported in our previous paper [12]. Among the up-regulated proteins in mutant D4, we identified proteins involved in post-translational modification, protein turnover or chaperones.…”

“…The resistance of Gram-negative bacteria against tetracyclines is frequently triggered by drug recognition of the Tet repressor. This enables expression of the resistance protein TetA, which is responsible for active efflux of tetracycline [34].The downregulation of this gene is in agreement with the wellestablished antimicrobial susceptibility of the B. cenocepacia D4 mutant [12].…”

“…Moreover, these proteins are known to play a role in RND efflux pumps and resistance to antibiotics. In fact, we have demonstrated that the RND-4 mutant is more susceptible than the wild type J2315 strain when exposed to a variety of drugs including β-lactams such as aztreonam [12].…”

“…The bacterial strains used in this work were the wild type strain B. cenocepacia J2315 and the mutant D4 harbouring a deletion of the two ORF BCAL2822-BCAL2820 (RND-4) [12]. These strains were grown in Luria-Bertani (LB) medium by shaking up to an OD 550 = 0.5.…”

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

“…This alteration is not due to gross defects in the LPS of the mutant compared with the parental strain (data not shown) and its mechanism is not clear. B. cenocepacia also exhibits high resistance to aminoglycosides (Chen & Roberts, 2000;Hamad et al, 2010;Waters & Ratjen, 2006), which depends for the most part on efflux pumps (Buroni et al, 2009;Hamad et al, 2010). DsuhB Bc does not show any significant differences in resistance to kanamycin or gentamicin in comparison with the parental strain (Fig.…”