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

Jessica R. Webb; Erin P. Price; Nawarat Somprasong; Herbert P. Schweizer; Robert W. Baird; Bart J. Currie; Derek S. Sarovich

doi:10.2217/fmb-2018-0155

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

Jessica R. Webb, Erin P. Price, Nawarat Somprasong, Herbert P. Schweizer, Robert W. Baird, Bart J. Currie, Derek S. Sarovich

Research output: Contribution to journal › Article › peer-review

7 Citations (Scopus)

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.

Original language	English
Pages (from-to)	1403-1418
Number of pages	16
Journal	Future Microbiology
Volume	13
Issue number	12
DOIs	https://doi.org/10.2217/fmb-2018-0155
Publication status	Published - Sept 2018
Externally published	Yes

Bibliographical note

Funding Information:
This study was funded by the Australian National Health and Medical Research Council (award no. 1098337). JR Webb gratefully acknowledges the support provided by the Australian Federation of Graduate Women (Barbara Hale Fellowship), which made this work possible. EP Price was supported by a University of the Sunshine Coast fellowship, and DS Sarovich was funded by an Advance Queensland fellowship (AQRF13016-17RD2). Work in the HP Schweizer laboratory was funded in part by National Institute of Allergy and Infectious Diseases of the NIH (award no. U54 AI065357). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Australian National Health and Medical Research Council. Additional funding to HP Schweizer was provided by University of Florida Preeminence start-up funds. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Publisher Copyright:
© 2018 2018 Future Medicine Ltd.

Keywords

antibiotic resistance
Burkholderia pseudomallei
efflux pump
gene expression
melioidosis
qPCR

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title = "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.",

keywords = "antibiotic resistance, Burkholderia pseudomallei, efflux pump, gene expression, melioidosis, qPCR",

author = "Webb, {Jessica R.} and Price, {Erin P.} and Nawarat Somprasong and Schweizer, {Herbert P.} and Baird, {Robert W.} and Currie, {Bart J.} and Sarovich, {Derek S.}",

note = "Funding Information: This study was funded by the Australian National Health and Medical Research Council (award no. 1098337). JR Webb gratefully acknowledges the support provided by the Australian Federation of Graduate Women (Barbara Hale Fellowship), which made this work possible. EP Price was supported by a University of the Sunshine Coast fellowship, and DS Sarovich was funded by an Advance Queensland fellowship (AQRF13016-17RD2). Work in the HP Schweizer laboratory was funded in part by National Institute of Allergy and Infectious Diseases of the NIH (award no. U54 AI065357). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Australian National Health and Medical Research Council. Additional funding to HP Schweizer was provided by University of Florida Preeminence start-up funds. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Publisher Copyright: {\textcopyright} 2018 2018 Future Medicine Ltd.",

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AU - Price, Erin P.

AU - Somprasong, Nawarat

AU - Schweizer, Herbert P.

AU - Baird, Robert W.

AU - Currie, Bart J.

AU - Sarovich, Derek S.

N1 - Funding Information: This study was funded by the Australian National Health and Medical Research Council (award no. 1098337). JR Webb gratefully acknowledges the support provided by the Australian Federation of Graduate Women (Barbara Hale Fellowship), which made this work possible. EP Price was supported by a University of the Sunshine Coast fellowship, and DS Sarovich was funded by an Advance Queensland fellowship (AQRF13016-17RD2). Work in the HP Schweizer laboratory was funded in part by National Institute of Allergy and Infectious Diseases of the NIH (award no. U54 AI065357). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the Australian National Health and Medical Research Council. Additional funding to HP Schweizer was provided by University of Florida Preeminence start-up funds. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Publisher Copyright: © 2018 2018 Future Medicine Ltd.

PY - 2018/9

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N2 - 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.

AB - 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.

KW - antibiotic resistance

KW - Burkholderia pseudomallei

KW - efflux pump

KW - gene expression

KW - melioidosis

KW - qPCR

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JF - Future Microbiology

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ER -

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

Abstract

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Keywords

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