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.
Highlights
Infections caused by antibiotic resistant Gram-negative bacteria are an increasing problem worldwide
We used negative blood culture bottles spiked with different amounts of liquid broth culture of E. coli BL21(DE3) pLysS cells to mimic different bacterial densities in positive blood cultures
In this study we developed a novel proteomic workflow for the direct identification of extended-spectrum beta-lactamases (ESBL) in positive blood culture bottles
Summary
Infections caused by antibiotic resistant Gram-negative bacteria are an increasing problem worldwide. In the Netherlands, resistance towards third generation cephalosporins through extended-spectrum beta-lactamases (ESBLs) is the most frequently found antibiotic resistance of medical importance [1]. Unrecognised, infections with ESBL-producing bacteria pose a serious threat, as they are associated with high morbidity and mortality rates [2]. Extended-spectrum beta-lactamases are a group of betalactamases which can hydrolyze third generation cephalosporins. The detection of these ESBL-enzymes is currently provided indirectly by the results of standard susceptibility testing of cultured bacteria, followed by a phenotypic confirmation assay or a genetic test. The inherent limitations of these instruments such as the limited dynamic range and resolution limit the general applicability to accurately detect the presence of ESBL, in identifying the nature of the underlying enzyme
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