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. 2017 Sep;36(9):1621-1628.
doi: 10.1007/s10096-017-2975-y. Epub 2017 Apr 10.

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

F Fleurbaaij  1 , W Goessens  2 , H C van Leeuwen  1 , M E M Kraakman  1 , S T Bernards  1 , P J Hensbergen  3 , E J Kuijper  4
Affiliations

Direct detection of extended-spectrum beta-lactamases (CTX-M) from blood cultures by LC-MS/MS bottom-up proteomics

F Fleurbaaij et al. Eur J Clin Microbiol Infect Dis. 2017 Sep.

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.

Keywords: Beta-lactamase; Blood cultures; ESBL; Mass spectrometry; Proteomics.

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Not applicable (as discussed with the institutional medical ethical committee).

Figures

Fig. 1
Fig. 1
Coverage of SHV-1 sequence. Identified peptides by LC-MS/MS analysis are highlighted when they matched to the sequence of the SHV-1 beta-lactamase. The glycine at Ambler position 238 (underlined) is specific for the SHV-1 sequence, while SHV-2 type extended-spectrum beta-lactamases have a serine in this position. This peptide was not observed in LC-MS/MS analysis making it not possible to distinguish between the beta-lactamase types
See this image and copyright information in PMC

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