Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates

• Main Authors: Yannick eCharretier, Thilo eKöhler, Tiphaine eCecchini, Chloé eBardet, Abdessalam eCherkaoui, Catherine eLlanes, Pierre eBogaerts, Sonia eChatellier, Jean-Philippe eCharrier, Jacques eSchrenzel
• Format: Article
• Language: English
• Published: Frontiers Media S.A. 2015-02-01
• Series: Frontiers in Microbiology
• Subjects: Pseudomonas aeruginosa; RT-qPCR; SRM; Carbapenem resistance; OprD porin; multidrug efflux system
• Online Access: http://journal.frontiersin.org/Journal/10.3389/fmicb.2015.00081/full

Both acquired and intrinsic mechanisms play a crucial role in Pseudomonas aeruginosa antibiotic resistance. Many clinically relevant resistance mechanisms result from changes in gene expression, namely multidrug efflux pump overproduction, AmpC beta-lactamase induction or derepression, and inactivation or repression of the carbapenem-specific porin OprD. Changes in gene expression are usually assessed using reverse-transcription quantitative real-time PCR (RT-qPCR) assays. Here, we evaluated label-free Selected Reaction Monitoring (SRM)-based mass spectrometry to directly quantify proteins involved in antibiotic resistance. We evaluated the label-free SRM using a defined set of P. aeruginosa isolates with known resistance mechanisms and compared it against RT-qPCR. Referring to efflux systems, we found a more robust relative quantification of antibiotic resistance mechanisms by SRM than RT-qPCR. The SRM-based approach was applied to a set of clinical P. aeruginosa isolates to detect antibiotic resistance proteins. This multiplexed SRM-based approach is a rapid and reliable method for the simultaneous detection and quantification of resistance mechanisms and we demonstrate its relevance for antibiotic resistance prediction.