title: Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics.
abstract: Rapid bacterial species identification and antibiotic susceptibility testing in positive blood cultures have an important impact on the antibiotic treatment for patients. To identify extended-spectrum beta-lactamases (ESBL) directly in positive blood culture bottles, we developed a workflow of saponin extraction followed by a bottom-up proteomics approach using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The workflow was applied to positive blood cultures with Escherichia coli and Klebsiella pneumoniae collected prospectively in two academic hospitals over a 4-month period. Of 170 positive blood cultures, 22 (12.9%) contained ESBL-positive isolates based on standard susceptibility testing. Proteomic analysis identified CTX-M ESBLs in 95% of these isolates directly in positive blood cultures, whereas no false positives were found in the non-ESBL producing positive blood cultures. The results were confirmed by molecular characterisation of beta-lactamase genes. Based on this proof-of-concept study, we conclude that LC-MS/MS-based protein analysis can directly identify extended-spectrum beta lactamases in E. coli and K. pneumoniae positive blood cultures, and could be further developed for application in routine diagnostics.
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Fleurbaaij
authors.affiliation: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center, 3015 CN, Rotterdam, The Netherlands.
authors.forename: W
authors.initials: W
authors.lastname: Goessens
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: H C
authors.initials: HC
authors.lastname: van Leeuwen
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: M E M
authors.initials: MEM
authors.lastname: Kraakman
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: S T
authors.initials: ST
authors.lastname: Bernards
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333 ZA, Leiden, The Netherlands.
authors.forename: P J
authors.initials: PJ
authors.lastname: Hensbergen
authors.affiliation: Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC, Leiden, The Netherlands. E.J.Kuijper@lumc.nl.
authors.forename: E J
authors.initials: EJ
authors.lastname: Kuijper
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D015415:Biomarkers; D000071997:Blood Culture; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D004927:Escherichia coli Infections / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology; D006801:Humans; D007710:Klebsiella Infections / Q000175:diagnosis / Q000188:drug therapy / Q000382:microbiology; D007711:Klebsiella pneumoniae / Q000187:drug effects / Q000235:genetics; D008826:Microbial Sensitivity Tests; D011446:Prospective Studies; D040901:Proteomics / Q000379:methods; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D015415:Biomarkers; D001618:beta-Lactamases
keywords: Beta-lactamase; Blood cultures; ESBL; Mass spectrometry; Proteomics