Mechanisms of Resistance to Folate Pathway Inhibitors in Burkholderia pseudomallei : Deviation from the Norm

Abstract

The 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.

Raising the stakes: Loss of efflux-pump regulation decreases meropenem susceptibility in Burkholderia pseudomallei

“…Up-regulation of multi-drug efflux pumps can also contribute to antibiotic resistance in B. pseudomallei . There are three characterized pumps known to confer resistance (AmrAB-OprA, BpeEF-OprC or BpeAB-OprB) and up-regulation of one or more of these pumps in combination with mutations in methylation or metabolism pathways lead to doxycycline or TMP/SMX resistance, respectively (14, 19, 20).…”

Development and validation of a triplex qPCR assay to detect efflux pump-mediated antibiotic resistance inBurkholderia pseudomallei

“…229 RND efflux pump upregulation is common in B. pseudomallei strains that exhibit 230 decreased susceptibility towards clinically relevant antibiotics. This mechanism can 231 either by itself, or in concert with additional mutations, cause resistance towards DOX, 232 SXT, and MEM (17,22,23,24), three invaluable antibiotics in melioidosis treatment 233 regimens. Therefore, the main goal of this study was to design, optimize and validate a 234 triplex qPCR probe-based assay targeting the AmrAB-OprA, BpeAB-OprB, and BpeEF- 235 OprC RND efflux pumps in B. pseudomallei (40).…”

“…258 The mechanisms conferring DOX and SXT resistance, and decreased MEM sus-259 ceptibility, have recently been unraveled in B. pseudomallei. Importantly, all resistant 260 strains analyzed to date have upregulated efflux pump expression (17,22,23,24 folM/ptr1, are required for the shift towards high-level DOX and SXT resistance (23,24). 268 The two-step nature of these mechanisms means that the triplex qPCR assay alone 269 cannot be used to definitively determine if a strain is resistant to DOX or SXT.…”

“…369 The strain details for these eight cases, including regulatory mutations and MIC 370 data, are shown in Table 1. Additionally, an amrR knockout of the final isolate retrieved were created via site-directed mutagenesis and knockouts to investigate the mecha-400 nisms of SXT resistance in B. pseudomallei using previously described methods (24). 401 Bp82.280 ∆(amrAB-oprA) ∆bpeR was derived from a previously created strain (Bp82.27) 402 as previously described (27,56) (23).…”

“…The triplex assay accurately detected efflux pump upregulation between isogenic 23 pairs, which corresponded with decreased antibiotic susceptibility. We further verified 24 assay performance on eight laboratory-generated B. pseudomallei mutants encoding 25 efflux pump regulator mutations. Targeting antibiotic resistance in B. pseudomallei 26 using molecular genotyping provides clinicians with a rapid tool to identify potential 27 treatment failure in near real-time, enabling informed alteration of treatment during 28 an infection and improved patient outcomes.…”