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Folate pathway inhibitor resistance mechanisms in Burkholderia pseudomallei




Podnecky, Nicole L., author
Schweizer, Herbert P., advisor
Dow, Steven W., committee member
Slayden, Richard A., committee member
Stargell, Laurie A., committee member

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Antimicrobials are invaluable tools used to facilitate the treatment of infectious diseases. Their use has saved millions of lives since their introduction in the early 1900's. Unfortunately, due to the increased incidence and dispersal of antimicrobial resistance determinants, many of these drugs are no longer efficacious. This greatly limits the options available for treatment of serious bacterial infections, including melioidosis, which is caused by Burkholderia pseudomallei, a Gram-negative saprophyte. This organism is intrinsically resistant to many antimicrobials. Additionally, there have been reports of B. pseudomallei isolates resistant to several of the antimicrobials currently used for treatment, including the trimethoprim and sulfamethoxazole combination, co-trimoxazole. The overarching goal of this project was to identify and characterize mechanisms of trimethoprim and sulfamethoxazole resistance in clinical and environmental isolates, as well as in laboratory induced mutants. Prior to these studies, very little work has been done to identify and characterize the mechanisms by which B. pseudomallei strains are or could become resistant to folate-pathway inhibitors, specifically trimethoprim and sulfamethoxazole. During the initial phases of these studies, we determined the antimicrobial susceptibilities of a large collection of clinical and environmental isolates from Thailand and Australia (n = 65). A high frequency of naturally-occurring resistance to trimethoprim alone (40%) was observed. However these strains were susceptible to sulfamethoxazole and to the co-trimoxazole combination. Trimethoprim resistance in a subset of these strains was due to increased expression of an efflux pump belonging to the resistance nodulation and cellular division (RND) superfamily, BpeEF-OprC, in the presence of trimethoprim. This efflux pump had been previously shown to efflux trimethoprim, chloramphenicol and tetracyclines when expressed in surrogate bacterial strains. The molecular mechanism of increased BpeEF-OprC expression in these isolates remains unknown. Similarly, decreased susceptibility in laboratory mutants selected on trimethoprim were due to mutations leading to amino acid substitutions in BpeT, which caused overexpression of BpeEF-OprC, or FolA, the trimethoprim drug target. This is the first description of mutations to FolA conferring trimethoprim resistance in B. pseudomallei, though similar mutations have been observed in B. cenocepacia and Escherichia coli. A similar study to select for sulfamethoxazole resistance, instead suggested that B. pseudomallei may be able to tolerate high concentrations of the drug. Studies to characterize laboratory induced mutants selected on co-trimoxazole led to the identification of two novel resistance determinants. Mutations to BpeS, a newly named LysR-type regulator with high similarity to the cognate BpeEF-OprC efflux pump regulator, BpeT, cause increased BpeEF-OprC expression in these strains. Additionally mutations to Ptr1, an annotated pteridine reductase, partially contributed to the decreased co-trimoxazole susceptibility. However, it is unclear what function Ptr1 has in the folate synthesis pathway, as deletion of this gene also caused slight decreases in antimicrobial susceptibility. Finally, in a collection of co-trimoxazole resistant clinical isolates from Thailand, high-level expression of the BpeEF-OprC was found in the resistant isolates. A mutation to BpeS was also observed in two of the clinical isolates that had BpeT-independent BpeEF-OprC overexpression. Co-trimoxazole resistant isolates were each resistant to both trimethoprim and sulfamethoxazole individually. However, deletion of the bpeEF-oprC efflux pump structural genes in all isolates resistant to co-trimoxazole or isolates resistant to trimethoprim alone (except those with a mutant FolA) resulted in antimicrobial susceptibility to trimethoprim, co-trimoxazole and sulfamethoxazole. These data suggest that sulfamethoxazole is also a substrate of the BpeEF-OprC efflux pump and this RND pump is the major resistance determinant contributing to clinically relevant folate pathway inhibitor resistance in B. pseudomallei. To summarize, we have identified and described several resistance determinants in B. pseudomallei causing decreased susceptibilities to trimethoprim, sulfamethoxazole and/or co-trimoxazole; these include drug target and metabolic pathway modifications and overexpression of the BpeEF-OprC efflux pump. Further characterization of these mechanisms and the development of specific detection assays could allow for rapid determination of antimicrobial resistance and provide useful information for the development of novel antimicrobials against B. pseudomallei.


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