ORIGINAL RESEARCH article
Front. Cell. Infect. Microbiol.
Sec. Bacteria and Host
This article is part of the Research TopicAdvances in Diagnostic Platforms for Rapid Detection of Multidrug-Resistant Bacterial InfectionsView all 3 articles
Direct detection of Gram-negative bacilli and extended-spectrum β-lactamase producers in positive blood culture specimens using MALDI-TOF MS
Provisionally accepted
Kosuke Kosai*
Yasuhide Kawamoto
Fujiko Mitsumoto-Kaseida
Norihito Kaku
Hiroo Hasegawa
Takahiro Takazono
Koichi IzumikawaHiroshi Mukae
Katsunori Yanagihara- Nagasaki University, Nagasaki, Japan
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Bloodstream infections caused by antimicrobial-resistant Gram-negative bacilli are often difficult to treat. Here, we investigated the performance of a workflow for direct detection of Gram-negative bacilli and extended-spectrum β-lactamase (ESBL) producers in positive blood culture (PBC) specimens using matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS). Samples were prepared from spiked and clinical PBC specimens using a sample preparation kit, and bacterial identification and cephalosporinase activity assays were performed using MALDI-TOF MS. The results were compared with those obtained using the conventional method, which was performed for colonies as routine microbiological testing. Of the 34 spiked PBC specimens prepared using Escherichia coli and Klebsiella pneumoniae isolates, all 27 ESBL producers tested positive and seven non-producers tested negative for cephalosporinase activity. Of the 111 clinical PBC specimens analyzed, 99 (89.2%) showed concordance with the conventional method results, with scores ≥ 2.00. Among the 54 Enterobacterales, consisting of E. coli, K. pneumoniae, Klebsiella oxytoca, and Proteus mirabilis isolates identified in the directly prepared samples, 14 were ESBL producers in the colonies. Of the 14 ESBL producers, 11 (78.6%) were correctly identified as cephalosporinase producers in the directly prepared samples using MALDI-TOF MS. The time required for direct identification (8.4 h) was significantly shorter than that required for conventional identification (15.0 h). Moreover, the time required for cephalosporinase activity detection in the directly prepared samples (9.2 h) was significantly shorter than that required for phenotypic ESBL detection in colonies (32.3 h). This study demonstrates the performance of the workflow for direct detection of Gram-negative bacilli and cephalosporinase activity in ESBL producers from PBC specimens using MALDI-TOF MS.
Keywords: Bloodstream infection, antimicrobial resistance, β-lactamase, Rapid detection, workflow
Received: 08 Sep 2025; Accepted: 28 Oct 2025.
Copyright: © 2025 Kosai, Kawamoto, Mitsumoto-Kaseida, Kaku, Hasegawa, Takazono, Izumikawa, Mukae and Yanagihara. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Kosuke Kosai, k-kosai@nagasaki-u.ac.jp
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