A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa

Nontaporn Rattanachak; Sattaporn Weawsiangsang; Robert A Baldock; Theerasak Jaifoo; Touchkanin Jongjitvimol; Jirapas Jongjitwimol

doi:10.3390/mps6050096

A Novel and Quantitative Detection Assay (effluxR) for Identifying Efflux-Associated Resistance Genes Using Multiplex Digital PCR in Clinical Isolates of Pseudomonas aeruginosa

Methods Protoc. 2023 Oct 8;6(5):96. doi: 10.3390/mps6050096.

Authors

Nontaporn Rattanachak¹, Sattaporn Weawsiangsang¹, Robert A Baldock², Theerasak Jaifoo³, Touchkanin Jongjitvimol⁴, Jirapas Jongjitwimol^{5

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Affiliations

¹ Biomedical Sciences Program, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
² School of Pharmacy and Biomedical Sciences, Faculty of Science and Health, University of Portsmouth, Portsmouth PO1 2DT, UK.
³ Master of Science Program in Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
⁴ Biology Program, Faculty of Science and Technology, Pibulsongkram Rajabhat University, Phitsanulok 65000, Thailand.
⁵ Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok 65000, Thailand.
⁶ Centre of Excellence in Biomaterials, Faculty of Science, Naresuan University, Phitsanulok 65000, Thailand.

Abstract

The rise of multidrug resistance of Pseudomonas aeruginosa highlights an increased need for selective and precise antimicrobial treatment. Drug efflux pumps are one of the major mechanisms of antimicrobial resistance found in many bacteria, including P. aeruginosa. Detection of efflux genes using a polymerase chain reaction (PCR)-based system would enable resistance detection and aid clinical decision making. Therefore, we aimed to develop and optimize a novel method herein referred to as "effluxR detection assay" using multiplex digital PCR (mdPCR) for detection of mex efflux pump genes in P. aeruginosa strains. The annealing/extension temperatures and gDNA concentrations were optimized to amplify mexB, mexD, and mexY using the multiplex quantitative PCR (mqPCR) system. We established the optimal mqPCR conditions for the assay (Ta of 59 °C with gDNA concentrations at or above 0.5 ng/µL). Using these conditions, we were able to successfully detect the presence of these genes in a quantity-dependent manner. The limit of detection for mex genes using the effluxR detection assay with mdPCR was 0.001 ng/µL (7.04-34.81 copies/µL). Moreover, using blind sample testing, we show that effluxR detection assay had 100% sensitivity and specificity for detecting mex genes in P. aeruginosa. In conclusion, the effluxR detection assay, using mdPCR, is able to identify the presence of multiple mex genes in P. aeruginosa that may aid clinical laboratory decisions and further epidemiological studies.

Keywords: Pseudomonas aeruginosa; RND-type efflux pumps; antibiotic resistant genes; multiplex digital polymerase chain reaction (mdPCR); multiplex quantitative polymerase chain reaction (mqPCR).

Grants and funding

This research was supported by National Science, Research and Innovation Fund (NSRF), grant number R2564B019 (J.J.) and the Global and Frontier Research University Grant, grant number R2566C052 (J.J.). This research was also partly funded by Pibulsongkram Rajabhat University (Research Fund from the Research and Development Institute: RDI-2-65-44, and Fundamental Fund: fiscal year 2024) (T.J.). N.R. thankfully acknowledges funding from the Royal Thai Government, grant number 3/2563. R.A.B. gratefully acknowledges funding from the Royal Society, grant number RGS\R2\192126.