Rapid fluoroquinolone resistance detection inPseudomonas aeruginosausing mismatch amplification mutation assay-based real-time PCR

AbstractBackgroundAntimicrobial 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.ObjectivesWe 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 inP. aeruginosa.MethodsFollowing assay validation, thegyrA248 andgyrA259 SYBR-MAMAs were tested on 84 clinicalP. aeruginosaisolates from Queensland, Australia, 45 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing.ResultsOur two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (84%) of isolates with intermediate/full FQ resistance. Importantly, all FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100% concordance with SYBR-MAMA genotypes.ConclusionsOur GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR inP. 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.