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Updated: Nov 26, 2025
Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS
Published on: July 11, 2016
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based identification of
Kym Antonation1, Dobryan Tracz1, Ashley Dreger1
1National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB.
Background:
The use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) systems for bacterial identification has rapidly become a front line tool for diagnostic laboratories, superseding classical microbiological methods that previously triggered the identification of higher risk pathogens. Unknown Risk Group 3 isolates have been misidentified as less pathogenic species due to spectral library availability, content and quality. Consequently, exposure to higher risk pathogens has been reported within Canadian laboratory staff following the implementation of MALDI-TOF MS. This overview aims to communicate the potential risk to laboratory staff of inaccurate identification of security-sensitive biological agents (SSBA) bacteria and to provide suggestions to mitigate.
Methods:
Cultures were manipulated in a Biosafety Level 3 laboratory, prepared for MALDI-TOF MS analysis via full chemical extraction and analysed on a Bruker Microflex LT instrument. Data were analyzed with Biotyper software; comparing raw spectra against MS profiles in three libraries: Bruker Taxonomy; Bruker Security-Restricted; and National Microbiology Laboratory (NML) SSBA libraries. Four years of Bruker MALDI-TOF MS data acquired in-house were reviewed.
Results:
In general, the Bruker MS spectral libraries were less successful in identifying the SSBA bacteria. More successful was the NML library. For example, using a high score cut-off (greater than 2.0), the Bruker SR library was unable to identify 52.8% of our Risk Group 3 agents and near neighbours to the species-level with confidence, whereas the custom NML library was unable to identify only 20.3% of the samples.
Conclusion:
The last four years of data demonstrated both the importance of library selection and the limitations of the various spectral libraries. Enhanced standard operating procedures are advised to reduce laboratory exposure to SSBAs when using MALDI-TOF MS as a front line identification tool.
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