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Express yourself - Quantitative real-time PCR assays for rapid chromosomal antimicrobial resistance detection in Pseudomonas aeruginosa572.38 kB
Preprint VersionCC BY-NC-ND V4.0, Open Access
Preprint
Express yourself: Quantitative real-time PCR assays for rapid chromosomal antimicrobial resistance detection in Pseudomonas aeruginosa
medRxiv, Vol.21 March 2022
Cold Spring Harbor Laboratory Press
2022
Abstract
The rise of antimicrobial-resistant (AMR) bacteria is a global health emergency. One critical facet in tackling this epidemic is more rapid AMR diagnosis in serious multi-drug 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, 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 confirm 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 when compared with chronic obstructive pulmonary disease sputa, despite harbouring fluoroquinolone- and aminoglycoside-resistant strains, indicating that these loci are not contributing 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 (6.7-15x), 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.
Details
- Title
- Express yourself: Quantitative real-time PCR assays for rapid chromosomal antimicrobial resistance detection in Pseudomonas aeruginosa
- Authors
- Danielle Madden (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacyOlusola Olagoke (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacyTimothy Baird (Author) - Sunshine Coast University Hospital (Australia)Jane Neill (Author) - Sunshine Coast University Hospital (Australia)Kay A Ramsay (Author) - The University of QueenslandTamieka Fraser (Author) - University of the Sunshine Coast, Queensland, School of Science and Engineering - LegacyScott C Bell (Author) - The University of QueenslandDerek Sarovich (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - LegacyErin Price (Author) - University of the Sunshine Coast, Queensland, GeneCology Research Centre - Legacy
- Publication details
- medRxiv, Vol.21 March 2022
- Publisher
- Cold Spring Harbor Laboratory Press
- Date published
- 2022
- DOI
- 10.1101/2022.02.03.22270419
- Copyright note
- The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
- Organisation Unit
- School of Science and Engineering - Legacy; University of the Sunshine Coast, Queensland; School of Science, Technology and Engineering; Centre for Bioinnovation; School of Health and Behavioural Sciences - Legacy
- Language
- English
- Record Identifier
- 99627440802621
- Output Type
- Preprint
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