Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Webb JR

PROVIDER: S-EPMC6190177 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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

Webb Jessica R JR Price Erin P EP Somprasong Nawarat N Schweizer Herbert P HP Baird Robert W RW Currie Bart J BJ Sarovich Derek S DS

Future microbiology 20180926

<h4>Aim</h4>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.<h4>Methods</h4>The triplex assay was developed and subsequently tested on RNA isolated from eight clinical and eight laboratory-generated B. pseudomallei mutants harb ...[more]

PMID: 30256166

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Project description:Bacterial efflux pumps are an important pathogenicity trait because they extrude a variety of xenobiotics. Our laboratory previously identified in silico Burkholderia collagen-like protein 8 (Bucl8) in the hazardous pathogens Burkholderia pseudomallei and Burkholderia mallei. We hypothesize that Bucl8, which contains two predicted tandem outer membrane efflux pump domains, is a component of a putative efflux pump. Unique to Bucl8, as compared to other outer membrane proteins, is the presence of an extended extracellular region containing a collagen-like (CL) domain and a non-collagenous C-terminus (Ct). Molecular modeling and circular dichroism spectroscopy with a recombinant protein, corresponding to this extracellular CL-Ct portion of Bucl8, demonstrated that it adopts a collagen triple helix, whereas functional assays screening for Bucl8 ligands identified binding to fibrinogen. Bioinformatic analysis of the bucl8 gene locus revealed it resembles a classical efflux-pump operon. The bucl8 gene is co-localized with downstream fusCDE genes encoding fusaric acid (FA) resistance, and with an upstream gene, designated as fusR, encoding a LysR-type transcriptional regulator. Using reverse transcriptase (RT)-qPCR, we defined the boundaries and transcriptional organization of the fusR-bucl8-fusCDE operon. We found exogenous FA induced bucl8 transcription over 80-fold in B. pseudomallei, while deletion of the entire bucl8 locus decreased the minimum inhibitory concentration of FA 4-fold in its isogenic mutant. We furthermore showed that the putative Bucl8-associated pump expressed in the heterologous Escherichia coli host confers FA resistance. On the contrary, the Bucl8-associated pump did not confer resistance to a panel of clinically-relevant antimicrobials in Burkholderia and E. coli. We finally demonstrated that deletion of the bucl8-locus drastically affects the growth of the mutant in L-broth. We determined that Bucl8 is a component of a novel tetrapartite efflux pump, which confers FA resistance, fibrinogen binding, and optimal growth.

Dataset Information

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

Publications

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

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