Burkholderia cenocepacia is a member of the Burkholderia cepacia complex (Bcc), a group of phenotypically similar Gram-negative bacteria  that are opportunistic pathogens and sometimes cause serious life-threatening infections in cystic fibrosis (CF) patients [2, 3]. Infection in CF patients may result in asymptomatic carriage, but often leads to a rapid decline of the lung function and in some cases to the "cepacia syndrome", characterized by necrotizing pneumonia and sepsis .
B. cenocepacia and other members of the Bcc demonstrate high-levels of intrinsic resistance to most clinically relevant antibiotics, complicating the treatment of the infection . Multi-drug resistance in CF isolates is defined as resistance to all of the agents in two of three classes of antibiotics, such as quinolones, aminoglycosides, and β-lactam agents, including monobactams and carbapenems . Multiple antibiotic resistances in Bcc bacteria have been attributed to reduced permeability of the bacterial outer membrane [7–9], expression of antibiotic modifying enzymes , and alteration of cellular targets . Information relating to the contribution that drug efflux systems play in the drug resistance of Bcc bacteria is limited, as only a few multi-drug efflux pumps have been described to date in some clinical isolates [12–14]. In contrast, the contribution of multidrug efflux systems to antibiotic resistance in clinical isolates of Pseudomonas aeruginosa, another CF pathogen, is well documented. Two P. aeruginosa efflux pumps, MexAB-OprM and MexXY-OprM, contribute to intrinsic multidrug resistance, while MexCD-OprJ and MexEF-OprN are responsible for the acquired antimicrobial resistance of different mutant strains .
RND transporters are important mediators of multi-drug resistance in Gram-negative bacteria . RND transporters form protein complexes that span both the cytoplasmic and outer membrane. The complex comprises a cytoplasmic membrane transporter protein, a periplasmic-exposed membrane adaptor protein, and an outer-membrane channel protein. The Escherichia coli AcrAB-TolC and the P. aeruginosa MexAB-OprM complexes are extremely well characterized and the three-dimensional structures of various components have been resolved [17–21].
Two RND type multi-drug efflux pumps, AmrAB-OprA and BpeAB-OprB, have been described in Burkholderia pseudomallei (the causative agent of melioidosis) and both confer resistance to aminoglycosides and macrolides [22, 23]. The contribution of BpeAB-OprB and AmrAB-OprA, to the intrinsic resistance of B. pseudomallei to gentamicin, streptomycin and erythromycin explains why aminoglycoside-β-lactam combinations, which are commonly used to treat suspected cases of community-acquired sepsis in any part of the world, are ineffective for the treatment of melioidosis . Furthermore, the transport of acyl homoserine lactones, involved in quorum-sensing systems of B. pseudomallei, also requires the BpeAB-OprB efflux pump . Thus, targeted inhibition of BpeAB-OprB could be therapeutically beneficial.
Several possible strategies can be considered to specifically block the activity of these drug efflux pumps, such as jamming the outer membrane channel, generating competition at the inner membrane pump, altering the pump assembling or collapsing the energy component of the mechanism . The activity of efflux inhibitors, such as diamine compounds, has been demonstrated in animal models of P. aeruginosa infections and two of them are in preclinical development .
In B. cenocepacia the significance of RND efflux systems has not been determined. However, a salicylate-regulated efflux pump that is conserved among members of the Bcc has been identified [27, 28]. We are focusing our research in the B. cenocepacia J2315 strain. This strain is a prototypic isolate belonging to an epidemic clone that has spread by cross infection to CF patients in Europe and North America . Previously, we identified 14 genes encoding putative RND efflux pumps in the genome of B. cenocepacia J2315 . After the completion of the whole genome sequence , two additional genes encoding RND pumps were discovered. Reverse transcriptase analyses showed that some of these genes are indeed transcribed at detectable levels. As a first step towards understanding the contribution of RND pumps to B. cenocepacia antibiotic resistance we deleted genes encoding putative efflux pumps, RND-1, RND-3, and RND-4, containing the genes BCAS0591-BCAS0593 (located on chromosome 3), BCAL1674-BCAL1676, and BCAL2822-BCAL2820 (located on chromosome 1), respectively. In a previous publication, the genes encoding the membrane transporter component of the efflux pump, BCAS0592, BCAL1675, and BCAL2821 were referred to as Orf1, Orf3, and Orf4, respectively . In this investigation we show that deletion of rnd-3 and rnd-4 genes is associated with increased sensitivity to certain antibiotics and reduced secretion of quorum sensing molecules.