Abstract
ABSTRACTThe trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Previous studies demonstrated that the B. pseudomallei BpeEF-OprC efflux pump confers widespread trimethoprim resistance in clinical and environmental isolates, but this is not accompanied by significant resistance to co-trimoxazole. Using the excluded select-agent strain B. pseudomallei Bp82, we now show that in vitro acquired trimethoprim versus co-trimoxazole resistance is mainly mediated by constitutive BpeEF-OprC expression due to bpeT mutations or by BpeEF-OprC overexpression due to bpeS mutations. Mutations in bpeT affect the carboxy-terminal effector-binding domain of the BpeT LysR-type activator protein. Trimethoprim resistance can also be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. BpeS is a transcriptional regulator that is 62% identical to BpeT. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and thus to co-trimoxazole resistance. The majority of laboratory-selected co-trimoxazole-resistant mutants often also contain mutations in folM, encoding a pterin reductase. Genetic analyses of these mutants established that both bpeS mutations and folM mutations contribute to co-trimoxazole resistance, although the exact role of folM remains to be determined. Mutations affecting bpeT, bpeS, and folM are common in co-trimoxazole-resistant clinical isolates, indicating that mutations affecting these genes are clinically significant. Co-trimoxazole resistance in B. pseudomallei is a complex phenomenon, which may explain why resistance to this drug is rare in this bacterium.
Highlights
The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections
We have previously shown by heterologous expression in Pseudomonas aeruginosa that B. pseudomallei BpeEF-OprC, an efflux pump of the resistance nodulation cell division (RND) family, extrudes chloramphenicol and TMP [25]
We confirmed that increased expression of the BpeEF-OprC efflux pump contributes to the increased MICs observed in strains Bp82.102 and Bp82.103 using reverse transcription-quantitative PCR (RT-qPCR)
Summary
The trimethoprim and sulfamethoxazole combination, co-trimoxazole, plays a vital role in the treatment of Burkholderia pseudomallei infections. Trimethoprim resistance can be mediated by dihydrofolate reductase (FolA) target mutations, but this occurs rarely unless BpeEF-OprC is absent. Mutations affecting the BpeS DNA-binding or carboxy-terminal effector-binding domains result in constitutive BpeEF-OprC overexpression, leading to trimethoprim and sulfamethoxazole efflux and to cotrimoxazole resistance. The oral drug of choice is co-trimoxazole, a combination of trimethoprim and sulfamethoxazole These antibiotics target two distinct enzymes, FolA (dihydrofolate reductase) and FolP (dihydropteroate synthase), in the bacterial tetrahydrofolate biosynthetic pathway. Leishmania and related trypanosomatid protozoans lack a de novo pteridine biosynthetic pathway and are thought to rely on salvage of both pterins and folates [16] In bacteria such as Burkholderia spp. that possess a de novo pteridine biosynthetic pathway and phenylalanine hydroxylase (PhhA), FolM likely provides the essential tetrahydromonapterine cofactor for PhhA [14]. B. pseudomallei chromosome 2 encodes a gene (BP1026B_II0040) that is annotated as ptr, forms an operon with folE (Fig. 2) [18], and is likely a FolM homolog
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