Molecular Investigations of PenA-mediated β-lactam Resistance in Burkholderia pseudomallei

Burkholderia pseudomallei is the etiological agent of melioidosis. Because of the bacterium’s intrinsic resistance and propensity to establish latent infections, melioidosis therapy is complicated and prolonged. Newer generation β-lactams, specifically ceftazidime, are used for acute phase therapy, but resistance to this cephalosporin has been observed. The chromosomally encoded penA gene encodes a putative twin arginine translocase (TAT)-secreted β-lactamase, and penA mutations have been implicated in ceftazidime resistance in clinical isolates. However, the role of PenA in resistance has not yet been systematically studied in isogenetic B. pseudomallei mutant backgrounds. We investigated the effects of penA deletion, point mutations, and up-regulation, as well as tat operon deletion and PenA TAT-signal sequence mutations. These experiments were made possible by employing a B. pseudomallei strain that is excluded from Select Agent regulations. Deletion of penA significantly (>4-fold) reduced the susceptibility to six of the nine β-lactams tested and ≥16-fold for ampicillin, amoxicillin, and carbenicillin. Overexpression of penA by single-copy, chromosomal expression of the gene under control of the inducible Ptac promoter, increased resistance levels for all β-lactams tested 2- to 10-fold. Recreation of the C69Y and P167S PenA amino acid substitutions previously observed in resistant clinical isolates increased resistance to ceftazidime by ≥85- and 5- to 8-fold, respectively. Similarly, a S72F substitution resulted in a 4-fold increase in resistance to amoxicillin and clavulanic acid. Susceptibility assays with PenA TAT-signal sequence and ΔtatABC mutants, as well as Western blot analysis, confirmed that PenA is a TAT secreted enzyme and not periplasmic but associated with the spheroplastic cell fraction. Lastly, we determined that two LysR-family regulators encoded by genes adjacent to penA do not play a role in transcriptional regulation of penA expression

An improved selective culture medium enhances the isolation of Burkholderia pseudomallei from contaminated specimens.

Burkholderia pseudomallei is a Gram-negative environmental bacterium found in tropical climates that causes melioidosis. Culture remains the diagnostic gold standard, but isolation of B. pseudomallei from heavily contaminated sites, such as fecal specimens, can be difficult. We recently reported that B. pseudomallei is capable of infecting the gastrointestinal tract of mice and suggested that the same may be true in humans. Thus, there is a strong need for new culture techniques to allow for efficient detection of B. pseudomallei in fecal and other specimens. We found that the addition of norfloxacin, ampicillin, and polymyxin B to Ashdown's medium (NAP-A) resulted in increased specificity without affecting the growth of 25 B. pseudomallei strains. Furthermore, recovery of B. pseudomallei from human clinical specimens was not affected by the three additional antibiotics. Therefore, we conclude that NAP-A medium provides a new tool for more sensitive isolation of B. pseudomallei from heavily contaminated sites

Versatile Dual-Technology System for Markerless Allele Replacement in Burkholderia pseudomallei▿ †

Burkholderia pseudomallei is the etiologic agent of melioidosis, a rare but serious tropical disease. In the United States, genetic research with this select agent bacterium is strictly regulated. Although several select agent compliant methods have been developed for allelic replacement, all of them suffer from some drawbacks, such as a need for specific host backgrounds or use of minimal media. Here we describe a versatile select agent compliant allele replacement system for B. pseudomallei based on a mobilizable vector, pEXKm5, which contains (i) a multiple cloning site within a lacZα gene for facile cloning of recombinant DNA fragments, (ii) a constitutively expressed gusA indicator gene for visual detection of merodiploid formation and resolution, and (iii) elements required for resolution of merodiploids using either I-SceI homing endonuclease-stimulated recombination or sacB-based counterselection. The homing endonuclease-based allele replacement system is completed by pBADSce, which contains an araC-PBAD-I-sceI expression cassette for arabinose-inducible I-SceI expression and a temperature-sensitive pRO1600 replicon for facile plasmid curing. Complementing these systems is the improved Δasd Escherichia coli mobilizer strain RHO3. This strain is susceptible to commonly used antibiotics and allows nutritional counterselection on rich media because of its diaminopimelic acid auxotrophy. The versatility of the I-SceI- and sacB-based methods afforded by pEXKm5 in conjunction with E. coli RHO3 was demonstrated by isolation of diverse deletion mutants in several clinical, environmental, and laboratory B. pseudomallei strains. Finally, sacB-based counterselection was employed to isolate a defined chromosomal fabD(Ts) allele that causes synthesis of a temperature-sensitive FabD, an essential fatty acid biosynthesis enzyme

Proteomic analysis of colony morphology variants of Burkholderia pseudomallei defines a role for the arginine deiminase system in bacterial survival

Colony morphology variation of Burkholderia pseudomallei is a notable feature of a proportion of primary clinical cultures from patients with melioidosis. Here, we examined the hypothesis that colony morphology switching results in phenotypic changes associated with enhanced survival under adverse conditions. We generated isogenic colony morphology types II and III from B. pseudomallei strain 153 type I, and compared their protein expression profiles using 2D gel electrophoresis. Numerous proteins were differentially expressed, the most prominent of which were flagellin, arginine deiminase (AD) and carbamate kinase (CK), which were over-expressed in isogenic types II and III compared with parental type I. AD and CK (encoded by arcA and arcC) are components of the arginine deiminase system (ADS) which facilitates acid tolerance. Reverse transcriptase PCR of arcA and arcC mRNA expression confirmed the proteomic results. Transcripts of parental type I strain 153 arcA and arcC were increased in the presence of arginine, in a low oxygen concentration and in acid. Comparison of wild type with arcA and arcC defective mutants demonstrated that the B. pseudomallei ADS was associated with survival in acid, but did not appear to play a role in intracellular survival or replication within the mouse macrophage cell line J774A.1. These data provide novel insights into proteomic alterations that occur during the complex process of morphotype switching, and lend support to the idea that this is associated with a fitness advantage in vivo