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Burkholderia pseudomallei is the etiologic agent of melioidosis, a difficult-to-treat disease with diverse clinical manifestations. β-Lactam antibiotics such as ceftazidime are crucial to the success of melioidosis therapy. Ceftazidime-resistant clinical isolates have been described, and the most common mechanism is point mutations affecting expression or critical amino acid residues of the chromosomally encoded class A PenA β-lactamase. We previously showed that PenA was exported via the twin arginine translocase system and associated with the spheroplast fraction. We now show that PenA is a membrane-bound lipoprotein. The protein and accompanying β-lactamase activity are found in the membrane fraction and can be extracted with Triton X-114. Treatment with globomycin of B. pseudomallei cells expressing PenA results in accumulation of the prolipoprotein. Mass spectrometric analysis of extracted membrane proteins reveals a protein peak whose mass is consistent with a triacylated PenA protein. Mutation of a crucial lipobox cysteine at position 23 to a serine residue results in loss of β-lactamase activity and absence of detectable PenAC23S protein. A concomitant isoleucine-to-alanine change at position 20 in the signal peptide processing site in the PenAC23S mutant results in a nonlipidated protein (PenAI20A C23S) that is processed by signal peptidase I and exhibits β-lactamase activity. The resistance profile of a B. pseudomallei strain expressing this protein is indistinguishable from the profile of the isogenic strain expressing wild-type PenA. The data show that PenA membrane association is not required for resistance and must serve another purpose., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Identification of a novel class of anti-Burkholderia compounds is key in addressing antimicrobial resistance to current therapies as well as naturally occurring resistance. The FabI enoyl-ACP reductase in Burkholderia is an underexploited target that presents an opportunity for development of a new class of inhibitors. A library of substituted diphenyl ethers was used to identify FabI1-specific inhibitors for assessment in Burkholderia pseudomallei ex vivo and murine efficacy models. Active FabI1 inhibitors were identified in a two-stage format consisting of percent inhibition screening and MIC determination by the broth microdilution method. Each compound was evaluated against the B. pseudomallei 1026b (efflux-proficient) and Bp400 (efflux-compromised) strains. In vitro screening identified candidate substituted diphenyl ethers that exhibited MICs of less than 1 μg/ml, and enzyme kinetic assays were used to assess potency and specificity against the FabI1 enzyme. These compounds demonstrated activity in a Burkholderia ex vivo efficacy model, and two demonstrated efficacy in an acute B. pseudomallei mouse infection model. This work establishes substituted diphenyl ethers as a suitable platform for development of novel anti-Burkholderia compounds that can be used for treatment of melioidosis.

The bacterial fatty acid biosynthesis pathway is a validated target for the development of novel chemotherapeutics. However, since Burkholderia pseudomallei carries genes that encode both FabI and FabV enoyl-acyl carrier protein (ACP) reductase homologues, the enoyl-ACP reductase that is essential for in vivo growth needs to be defined so that the correct drug target can be chosen for development. Accordingly, ΔfabI1, ΔfabI2, and ΔfabV knockout strains were constructed and tested in a mouse model of infection. Mice infected with a ΔfabI1 strain did not show signs of morbidity, mortality, or dissemination after 30 days of infection compared to the wild-type and ΔfabI2 and ΔfabV mutant strains that had times to mortality of 60 to 84 h. Although signs of morbidity and mortality of ΔfabI2 and ΔfabV strains were not significantly different from those of the wild-type strain, a slight delay was observed. A FabI1-specific inhibitor was used to confirm that inhibition of FabI1 results in reduced bacterial burden and efficacy in an acute B. pseudomallei murine model of infection. This work establishes that FabI1 is required for growth of Burkholderia pseudomallei in vivo and is a potential molecular target for drug development.

Emerging resistance to current antibiotics raises the need for new microbial drug targets. We show that targeting branched-chain amino acid (BCAA) biosynthesis using sulfonylurea herbicides, which inhibit the BCAA biosynthetic enzyme acetohydroxyacid synthase (AHAS), can exert bacteriostatic effects on several pathogenic bacteria, including Burkholderia pseudomallei, Pseudomonas aeruginosa, and Acinetobacter baumannii. Our results suggest that targeting biosynthetic enzymes like AHAS, which are lacking in humans, could represent a promising antimicrobial drug strategy.

Class D beta-lactamase OXA-57 was identified in a range of isolates of Burkholderia pseudomallei and Burkholderia thailandensis. Comparative kinetic analyses of wild-type and mutant forms of B. pseudomallei OXA-57 are reported. Implications of these data for beta-lactam resistance and the proposed role of Ser-104 in beta-lactam hydrolysis are discussed.

Burkholderia pseudomallei, the causative agent of melioidosis, is inherently resistant to a variety of antibiotics including aminoglycosides, macrolides, polymyxins, and beta-lactam antibiotics. Despite resistance to many beta-lactams, ceftazidime and beta-lactamase inhibitor-beta-lactam combinations are commonly used for treatment of melioidosis. Here, we examine the enzyme kinetics of beta-lactamase isolated from mutants resistant to ceftazidime and clavulanic acid inhibition and describe specific mutations within conserved motifs of the beta-lactamase enzyme which account for these resistance patterns. Sequence analysis of regions flanking the B. pseudomallei penA gene revealed a putative regulator gene located downstream of penA. We have cloned and sequenced the penA gene from B. mallei and found it to be identical to penA from B. pseudomallei.

Pseudomonas pseudomallei, the causative agent of melioidosis, is generally susceptible to some of the newer extended-spectrum cephalosporins or to combinations of a beta-lactam and clavulanic acid, a beta-lactamase inhibitor. Resistance to these agents may, however, emerge during treatment. We report on alterations in the chromosomal beta-lactamase associated with the development of resistance. Three resistance patterns resulted from three different mechanisms in the strains investigated. Derepression of the chromosomal enzyme resulted in a general increase in the MICs of all of the beta-lactams tested. The second mechanism observed was an insensitivity to inhibition of the beta-lactamase by clavulanic acid. In this case, the level of susceptibility to beta-lactams as independent entities remained unchanged. The final "resistance" pattern occurred in a patient treated with ceftazidime and resulted in a beta-lactamase that was capable of hydrolyzing this antibiotic at detectable levels, but with reduced efficacy against other beta-lactams. The net result was a strain that was generally susceptible to all of the beta-lactams tested except ceftazidime. In all cases, the level of susceptibility to antibiotics other than beta-lactams remained unchanged. Such variability found within one genus over a relatively short time course suggests that treatment of infections caused by this organism should be carefully monitored to detect susceptibility alterations to the chosen therapy.