Rapid species- and subspecies-specific level classification and identification of Lactobacillus casei group members using MALDI Biotyper combined with ClinProTools

Using common taxonomic methods such as 16S rDNA sequencing and physiological and biochemical analysis to identify members of the Lactobacillus casei group (LCG) is time-consuming, expensive and inaccurate. In this study, we applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to rapid discriminate LCG strains by creating an analytical in-house database (IHDB) and to develop a classification model for subspecies-level differentiation based on MS biomarkers using ClinProTools bioinformatics software (Bruker Daltonics, Billerica, MA). Genotypic methods (housekeeping gene sequencing and species-specific PCR) were also established to validate the MALDI-TOF MS platform. A total of 48 LCG reference strains were correctly identified at the species level (mean score: 2.45 ± 0.1) by using MALDI-TOF MS with an IHDB and had high score values, which was in accordance with results from mutL gene sequencing and specific PCR-based methods. However, one strain that was identified as L. casei had a relatively low score value (2.02 ± 0.02), lower sequence similarities (mutL: 90.4%), and failed to amplify a species-specific amplicon; it may therefore represent an undescribed novel species. In addition, after implementation of the classification model (based on 2 biomarker peaks: m/z 4,930 and 5,303), L. paracasei strains could be clearly and easily differentiated to the subspecies level. Afterward, 7 LCG-related isolates from different probiotic samples were analyzed and accurately identified. Our data demonstrate the high-resolution performance of MALDI-TOF MS for fast and accurate demarcation of LCG strains when used with an IHDB coupled to ClinProTools; this methodology can serve as an alternative for quality control of probiotic products.