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Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

Published on: July 11, 2016

Creation and validation of improved MALDI-TOF MS libraries for S. bovis-S. equinus-complex subspecies identification

Jonas Öberg¹, Agnes Engberg², Malin Inghammar³

¹Department of Clinical Sciences Lund, Section for Infection Medicine, Lund University, Sölvegatan 19, Lund, Sweden; Department of Infectious Diseases, Helsingborg Hospital, Bergendals gata 2, Helsingborg, Sweden.

Diagnostic Microbiology and Infectious Disease

|July 17, 2025

Abstract

Objectives:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) libraries have limited capabilities for identification of Streptococcus bovis/Streptococcus equinus-complex (SBSEC) subspecies. The objective was to develop and validate novel MALDI-TOF MS libraries adapted to diagnostic culturing and sampling conditions for improved identification of SBSEC subspecies.

Methods:

SBSEC isolates had previously been identified by whole genome sequencing, constituting the reference standard. Two libraries were constructed based on 53 isolates cultured on blood agar plates under aerobic conditions (SBSEC-CMRS-BAE) and on chocolate agar under anaerobic conditions (SBSECCMRS-CAN). The performances of the new libraries, the Bruker MALDI Biotyper (MBT) Compass Library and the previously developed SBSECCMRS library based on isolates cultured in brain heart infusion-broth, were validated with 119 unique isolates. Each isolate was sampled using the direct transfer, on-target extraction, and full extraction methods under the two different culture conditions - in total 714 different isolates and conditions.

Results:

With a cutoff identification score of ≥2.0, the MBT compass library identified subspecies in 463/714 (65 %) in total for all culturing conditions and methods, while the broth-based SBSECCMRS library identified 674/698 (97 %). The novel SBSEC-CMRS-BAE and SBSECCMRS-CAN libraries identified subspecies in 707/713 (99 %) and 684/706 (97 %), respectively. The SBSEC-CMRS-BAE library performed best, regardless of the culturing conditions used or the three sample preparation methods employed, with up to 100 % correct subspecies identifications.

Conclusions:

The novel SBSEC-CMRS-BAE library performed excellently and improved MALDI-TOF MS performance in identifying SBSEC subspecies. It can be implemented directly into routine clinical practice where the Bruker MALDI Biotyper is used.

Keywords:

MALDI-TOF MS Streptococcus bovis Streptococcus gallolyticus ssp. gallolyticus Streptococcus gallolyticus ssp. pasteurianus Streptococcus infantarius Streptococcus lutetiensis Subspecies identification

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