title: Label-free SRM-based relative quantification of antibiotic resistance mechanisms in Pseudomonas aeruginosa clinical isolates.
abstract: 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 β-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 with 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.
authors.affiliation: Genomic Research Laboratory, Service of Infectious Diseases, Geneva University Hospitals Geneva, Switzerland.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Charretier
authors.affiliation: Department of Microbiology and Molecular Medicine, University of Geneva Geneva, Switzerland.
authors.forename: Thilo
authors.initials: T
authors.lastname: Köhler
authors.affiliation: Institute for Analytical Sciences, Joint Research Unit 5280 CNRS/Lyon 1 University Villeurbanne, France ; Technology Research Department, BioMérieux SA Marcy l'Etoile, France.
authors.forename: Tiphaine
authors.initials: T
authors.lastname: Cecchini
authors.affiliation: UMR1092 INSERM, Limoges University Limoges, France ; MD3, BioMérieux SA Marcy l'Etoile, France.
authors.forename: Chloé
authors.initials: C
authors.lastname: Bardet
authors.affiliation: Laboratory of Bacteriology, Department of Genetic and Laboratory Medicine, Geneva University Hospitals Geneva, Switzerland.
authors.forename: Abdessalam
authors.initials: A
authors.lastname: Cherkaoui
authors.affiliation: Laboratoire de Bactériologie, EA4266, Université de Franche-Comté Besançon, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Llanes
authors.affiliation: Laboratoire de Microbiologie, CHU Dinant-Godinne UCL Namur Yvoir, Belgique.
authors.forename: Pierre
authors.initials: P
authors.lastname: Bogaerts
authors.affiliation: Microbiology Unit, BioMérieux SA La Balme Les Grottes, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Chatellier
authors.affiliation: Technology Research Department, BioMérieux SA Marcy l'Etoile, France.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Charrier
authors.affiliation: Genomic Research Laboratory, Service of Infectious Diseases, Geneva University Hospitals Geneva, Switzerland ; Laboratory of Bacteriology, Department of Genetic and Laboratory Medicine, Geneva University Hospitals Geneva, Switzerland.
authors.forename: Jacques
authors.initials: J
authors.lastname: Schrenzel
keywords: AmpC cephalosporinase; OprD porin; Pseudomonas aeruginosa; RT-qPCR; SRM; carbapenem resistance; cephalosporin resistance; multidrug efflux system