Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively. The two are closely related and can also be mistaken for B. thailandensis, a nonpathogenic species. To improve their differential identification, we describe a hydrolysis probe-based real-time PCR method using the uneven distribution of type III secretion system genes among these three species.

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Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals. The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease. We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell density-dependent regulation in B. pseudomallei. A search of the B. pseudomallei genome led to identification of a quorum-sensing system involving the LuxI-LuxR homologs PmlI-PmlR. PmlI directed the synthesis of an N-acylhomoserine lactone identified as N-decanoylhomoserine lactone. A B. pseudomallei pmlI mutant was significantly less virulent than the parental strain in a murine model of infection by the intraperitoneal, subcutaneous, and intranasal routes. Inactivation of pmlI resulted in overproduction of MprA at the onset of the stationary phase. A wild-type phenotype was restored following complementation with pmlI or addition of cell-free culture supernatant. In contrast, there was no significant difference between the virulence of a B. pseudomallei mprA mutant and the virulence of the wild-type strain. These results suggest that the PmlI-PmlR quorum-sensing system of B. pseudomallei is essential for full virulence in a mouse model and downregulates the production of MprA at a high cell density.

Although B. mallei MICs are often lower, the overall results underline the importance of resistance in both species. The susceptibilities measured are consistent with the current recommendations for the treatment of B. pseudomallei and B. mallei infections.

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

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We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance.

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.