Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR

Danielle E. Madden; Kate L. McCarthy; Scott C. Bell; Olusola Olagoke; Timothy Baird; Jane Neill; Kay A. Ramsay; Timothy J. Kidd; Adam G. Stewart; Shradha Subedi; Keat Choong; Tamieka A. Fraser; Derek S. Sarovich; Erin P. Price

doi:10.1099/jmm.0.001593

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Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR

Journal article

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Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR

Danielle E. Madden, Kate L. McCarthy, Scott C. Bell, Olusola Olagoke, Timothy Baird, Jane Neill, Kay A. Ramsay, Timothy J. Kidd, Adam G. Stewart, Shradha Subedi, …

Journal of Medical Microbiology, Vol.71, pp.1-10

2022

DOI: https://doi.org/10.1099/jmm.0.001593

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Abstract

antibiotic

AMR

ciprofloxacin

fluroquinolone

Pseudomonas aeruginosa

real-time PCR

SYBR-MAMA

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa. Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I (gyrA248) and GyrA D87N, D87Y and D87H (gyrA259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa. Methods. Following assay validation, the gyrA248 and gyrA259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing. Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes. Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa. An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Details

Title: Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR
Authors: Danielle E. Madden (Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Kate L. McCarthy (Author) - The University of Queensland
Scott C. Bell (Author) - Prince Charles Hospital
Olusola Olagoke (Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Timothy Baird (Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Jane Neill (Author) - Sunshine Coast University Hospital
Kay A. Ramsay (Author) - The University of Queensland
Timothy J. Kidd (Author) - Royal Brisbane and Women's Hospital
Adam G. Stewart (Author) - Sunshine Coast University Hospital
Shradha Subedi (Author) - Sunshine Coast University Hospital
Keat Choong (Author) - Sunshine Coast University Hospital
Tamieka A. Fraser (Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Derek S. Sarovich (Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Erin P. Price (Corresponding Author) - University of the Sunshine Coast, Queensland, Centre for Bioinnovation
Publication details: Journal of Medical Microbiology, Vol.71, pp.1-10
Publisher: The Microbiology Society
Date published: 2022
DOI: 10.1099/jmm.0.001593
ISSN: 1473-5644
Copyright note: © 2022 The Authors. This is an open-access article distributed under the terms of the Creative Commons Attribution NonCommercial License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Grant note: This work was supported by Advance Queensland (awards AQIRF0362018 and AQRF13016-17RD2), an Australian Government Research Training Program Scholarship, the Wishlist Sunshine Coast Health Foundation (award 2019-14), and the National Health and Medical Research Council (award 455919). The funders had no role in study design, data acquisition, analysis, interpretation, writing or submission of the manuscript.
Organisation Unit: School of Science and Engineering - Legacy; School of Science, Technology and Engineering; Centre for Bioinnovation; School of Health and Behavioural Sciences - Legacy
Language: English
Record Identifier: 99716027502621
Output Type: Journal article

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