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

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.