AUTHOR=Kosai Kosuke , Kawamoto Yasuhide , Mitsumoto-Kaseida Fujiko , Kaku Norihito , Hasegawa Hiroo , Takazono Takahiro , Izumikawa Koichi , Mukae Hiroshi , Yanagihara Katsunori 

TITLE=Direct detection of Gram-negative bacilli and extended-spectrum β-lactamase producers in positive blood culture specimens using MALDI-TOF MS

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=Volume 15 - 2025

YEAR=2025

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1701423

DOI=10.3389/fcimb.2025.1701423

ISSN=2235-2988

ABSTRACT=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.