Abstract
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.
Highlights
Burkholderia pseudomallei, the etiological agent of melioidosis, is a saprophytic Gram negative bacterium endemic to many tropical and subtropical regions of the world much of the disease and its investigation has historically been confined to Northern Australia and regions of SE Asia, notably NE Thailand, Singapore, and Malaysia (Cheng and Currie, 2005; Wiersinga et al, 2006; Currie et al, 2008)
Up-regulation of penA by single-copy expression from the isopropyl β-d-1thiogalactopyranoside (IPTG)-inducible Ptac (Bp82.21) significantly increased the MIC for seven of the eight β-lactams tested with meropenem showing only slight change. (Amoxicillin could not be tested as the resistance level for the wildtype was already beyond detection.) Quantitative real time PCR experiments showed that in the Ptac-penA strain (Bp82.21) penA transcript levels were 36-fold higher when compared to transcript levels observed in the wild-type strain
While mutations in penA can significantly affect the utility of ceftazidime and amoxicillin + clavulanic acid, the enzyme has a lesser effect on the activity of carbapenems and novel experimental drugs such as BAL30072
Summary
Burkholderia pseudomallei, the etiological agent of melioidosis, is a saprophytic Gram negative bacterium endemic to many tropical and subtropical regions of the world much of the disease and its investigation has historically been confined to Northern Australia and regions of SE Asia, notably NE Thailand, Singapore, and Malaysia (Cheng and Currie, 2005; Wiersinga et al, 2006; Currie et al, 2008). Because of its large genome and diverse repertoire of metabolic functions B. pseudomallei can survive hostile conditions and is resilient to many antimicrobial agents, including antibiotics (Holden et al, 2004). This makes choosing effective therapeutic strategies difficult. Initial parenteral therapy involves ceftazidime or a carbapenem for a minimum of 10–14 days and longer (4–8 weeks) for deep-seated infection. This regimen may be supplemented with trimethoprim–sulfamethoxazole given orally for treatment of patients with neurologic, prostatic, bone, or joint melioidosis. Oral eradication therapy is trimethoprim–sulfamethoxazole with or without doxycycline for at least 3–6 months (Peacock et al, 2008)
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