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
[...] Read more.
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.
Full article
