Targeting the Nonmevalonate Pathway in Burkholderia cenocepacia Increases Susceptibility to Certain ß-Lactam Antibiotics.

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr, the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 (B. cenocepacia K56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2-C-methyl-d-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-d-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2dxr showed markedly increased susceptibility to the ß-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected ß-lactam antibiotics is a useful strategy to combat B. cenocepacia infections.

Effect of ß-Lactamase inhibitors on in vitro activity of ß-Lactam antibiotics against Burkholderia cepacia complex species.

BACKGROUND: Bacteria belonging to the Burkholderia cepacia complex (Bcc) are an important cause of chronic respiratory tract infections in cystic fibrosis patients. Intrinsic resistance to a wide range of antimicrobial agents, including a variety of ß-lactam antibiotics, is frequently observed in Bcc strains. Resistance to ß-lactams is most commonly mediated by efflux pumps, alterations in penicillin-binding proteins or the expression of ß-lactamases. ß-lactamase inhibitors are able to restore the in vitro activity of ß-lactam molecules against a variety of Gram-negative species, but the effect of these inhibitors on the activity of ß-lactam treatment against Bcc species is still poorly investigated. METHODS: In the present study, the susceptibility of a panel of Bcc strains was determined towards the ß-lactam antibiotics ceftazidime, meropenem, amoxicillin, cefoxitin, cefepime and aztreonam; alone or in combination with a ß-lactamase inhibitor (clavulanic acid, sulbactam, tazobactam and avibactam). Consequently, ß-lactamase activity was determined for active ß-lactam/ß-lactamase inhibitor combinations. RESULTS: Clavulanic acid had no effect on minimum inhibitory concentrations, but addition of sulbactam, tazobactam or avibactam to ceftazidime, amoxicillin, cefoxitin, cefepime or aztreonam leads to increased susceptibility (at least 4-fold MIC-decrease) in some Bcc strains. The effect of ß-lactamase inhibitors on ß-lactamase activity is both strain- and/or antibiotic-dependent, and other mechanisms of ß-lactam resistance (besides production of ß-lactamases) appear to be important. CONCLUSIONS: Considerable differences in susceptibility of Bcc strains to ß-lactam antibiotics were observed. Results obtained in the present study suggest that resistance of Bcc strains against ß-lactam antibiotics is mediated by both ß-lactamases and non-ß-lactamase-mediated resistance mechanisms.

Synthesis and bioactivity of ß-substituted fosmidomycin analogues targeting 1-deoxy-D-xylulose-5-phosphate reductoisomerase.

Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the ß-position of the hydroxamate analogue of 2. While direct addition of a ß-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.