A pilot study on MALDI-TOF MS-based discrimination of Enterococcus faecalis and Lactococcus spp. isolated from bovine milk samples.

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is a rapid, cost-effective, and highly accurate technique for identifying microorganisms that are difficult to classify using traditional methods. Beyond microbial identification, MALDI-TOF MS has also potential for predicting antimicrobial resistance (AMR). Lactococcus and Enterococcus species are clinically important pathogens whose relevance is often underrecognized due to challenges in their identification. In this pilot study, we aimed primarily to assess the diagnostic accuracy of MALDI-TOF MS combined with Principal Component Analysis (PCA) for species-level identification of Lactococcus spp. and Enterococcus faecalis strains isolated from mastitic bovine milk, and secondarily to explore whether preliminary associations with antimicrobial resistance (AMR) could be observed in peptide and protein spectra. MALDI-TOF MS analysis was based on conserved bacterial structures (16 S rRNA), leading to the identification of E. faecalis, L. garvieae, L. lactis, and L. raffinolactis. Antimicrobial susceptibility testing using seven antibiotic discs revealed that the strains generally exhibited multidrug resistance. Within the Lactococcus genus, which includes closely related species, PCA analysis showed the formation of distinct clusters for three species, enabling their differentiation. However, PCA scatter plots and the Composite Correlation Index (CCI) visual matrix revealed no definitive differences associated with AMR. Due to the limited sample size and restricted AMR strain distribution, the results should be interpreted as preliminary and exploratory. Further studies with larger and more diverse isolate collections are necessary to validate and expand these findings.