Development and validation of a triplex quantitative real-time PCR assay to detect efflux pump-mediated antibiotic resistance in Burkholderia pseudomallei

摘要

Aim: To develop a probe-based triplex quantitative real-time PCR assay to simultaneously detect the upregulation of the efflux pumps AmrAB–OprA, BpeAB–OprB and BpeEF–OprC in Burkholderia pseudomallei strains exhibiting increased minimum inhibitory concentrations toward meropenem, doxycycline or trimethoprim-sulfamethoxazole. Methods: The triplex assay was developed and subsequently tested on RNA isolated from eight clinical and eight laboratory-generated B. pseudomallei mutants harboring efflux pump regulator mutations. Results: The triplex assay accurately detected efflux pump upregulation in all clinical and laboratory mutants, which corresponded with decreased antibiotic susceptibility or antibiotic resistance. Conclusion: Rapid detection of antibiotic resistance provides clinicians with a tool to identify potential treatment failure in near real time, enabling informed alteration of treatment during an infection and improved patient outcomes.