The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet of tackling this epidemic is more rapid AMR diagnosis in serious multidrug-resistant pathogens like Pseudomonas aeruginosa. Here, we designed and then validated two multiplex quantitative real-time PCR (qPCR) assays to simultaneously detect differential expression of the resistance-nodulation-division efflux pumps MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM, the AmpC β-lactamase, and the porin OprD, which are commonly associated with chromosomally encoded AMR. Next, qPCRs were tested on 15 sputa from 11 participants with P. aeruginosa respiratory infections to determine AMR profiles in vivo. We confirmed multiplex qPCR testing feasibility directly on sputa, representing a key advancement in in vivo AMR diagnosis. Notably, comparison of sputa with their derived isolates grown in Luria-Bertani broth (±2.5% NaCl) or a 5-antibiotic cocktail showed marked expression differences, illustrating the difficulty in replicating in vivo expression profiles in vitro. Cystic fibrosis sputa showed significantly reduced mexE and mexY expression compared with chronic obstructive pulmonary disease sputa, despite harboring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci do not contribute to AMR in vivo. oprD was also significantly downregulated in cystic fibrosis sputa, even in the absence of contemporaneous carbapenem use, suggesting a common adaptive trait in chronic infections that may affect carbapenem efficacy. Sputum ampC expression was highest in participants receiving carbapenems (6.7 to 15×), some of whom were simultaneously receiving cephalosporins, the latter of which would be rendered ineffective by the upregulated ampC. Our qPCR assays provide valuable insights into the P. aeruginosa resistome, and their use on clinical specimens will permit timely treatment alterations that will improve patient outcomes and antimicrobial stewardship measures.
Keywords: AMR; Pseudomonas aeruginosa; ampC; antibiotic; antibiotic resistance; assay development; diagnostics; efflux pumps; gene expression; genomics; in vivo expression technology; qPCR.