Matrix-assisted laser desorption/ionization-time of flight MS as a tool for clonal discrimination in Streptococcus dysgalactiae subsp. equisimilis

<b>Introduction.</b> <i>Streptococcus dysgalactiae</i> subsp. <i>equisimilis</i> (SDSE) is an emerging pathogen closely related to <i>Streptococcus pyogenes</i>, causing infections from mild to severe, including necrotizing fasciitis and toxic shock syndrome. Understanding bacterial diversity is crucial for monitoring the spread of antimicrobial-resistant and highly virulent strains. Clonal analysis by whole-genome sequencing (WGS) is costly, time-consuming and requires specialized bioinformatics expertise. PFGE-defined clones are often linked to human infections, but PFGE is also demanding and costly and results can vary between laboratories.<b>Aim.</b> This study explores the use of matrix-assisted laser desorption/ionization-time of flight MS (MALDI-TOF MS) to detect discriminatory biomarkers that discriminate SDSE strains exhibiting distinct PFGE clonal types.<b>Methodology.</b> MALDI-TOF MS spectra were generated from SDSE strains using a Microflex LT mass spectrometer (Bruker) and analysed with BioNumerics software v7.6. To validate the genetic relevance of PFGE pulsotypes, WGS and phylogenomic reconstruction were performed.<b>Results.</b> Unique MALDI-TOF MS biomarker peaks consistently differentiated SDSE strains corresponding to PFGE patterns A and B, providing robust molecular signatures for discriminating these clonal types. Phylogenomic analyses further supported this distinction by clustering PFGE A and B strains into two distinct main clades with an elevated accuracy of 95.2 (95% confidence interval: 76.2-99.9%).<b>Conclusion.</b> MALDI-TOF MS is effective not only for species identification but also for rapid and reliable assessment of SDSE clonal diversity. This approach has the potential to enable epidemiological tracking of specific clones, enhance understanding of SDSE in human infections and provide a practical tool for research and clinical surveillance.