Comprehensive identification of contagious, environmental, and emerging microorganisms associated with bovine mastitis in Northern Minas Gerais, Brazil, using MALDI-TOF mass spectrometry.

Background and Aim: Bovine mastitis remains one of the most economically significant diseases in dairy herds, driven by diverse etiological agents that vary in prevalence across regions and production systems. Rapid and reliable identification of mastitis-causing microorganisms is essential for targeted treatment, improved herd management, and enhanced biosecurity. This study aimed to identify and characterize the microorganisms associated with clinical and subclinical mastitis in dairy cows from northern Minas Gerais (Brazil) using Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), with special emphasis on uncommon and emerging bacterial species of potential public health concern. Materials and Methods: Milk samples (n = 321 isolates) were collected from cows diagnosed with clinical or subclinical mastitis between 2022 and 2024 across 15 farms. Bacteria were cultured on 5% sheep blood agar and identified by MALDI-TOF MS according to Bruker scoring criteria. Gram classification and contagious versus environmental categorization were performed. Descriptive statistics, chi-square analysis (p < 0.05), and Bray-Curtis similarity with Unweighted Pair Group Mathematical Average clustering were applied to determine distribution patterns and microbial diversity. Results: MALDI-TOF MS achieved an identification rate of 88%, predominantly at the species-level (99.38%). Gram-positive bacteria were significantly more frequent than Gram-negative bacteria (78%; χ² = 168.52; p < 0.000001). Most pathogens were classified as contagious (65%), followed by environmental agents (23%) (χ² = 64.40; p < 0.000001). The most prevalent organisms were Staphylococcus aureus (30.2%), Staphylococcus chromogenes (22.1%), and Sthaphylococcus epidermidis (4.9%). A combined frequency of 7.48% represented uncommon microorganisms, including Burkholderia cepacia, Arthrobacter koreensis, Ralstonia pickettii, Kosakonia radicincitans, Rothia terrae, and Paenibacillus azoreducens, some of which may pose emerging risks to bovine health and public health. Cluster analysis revealed two major microbial groups with distinct ecological and pathogenic profiles, highlighting the complexity of mastitis epidemiology in the region. Conclusion: This study provides an updated and region-specific overview of the mastitis microbiome in northern Minas Gerais, demonstrating the predominance of S. aureus and non-aureus staphylococci, alongside diverse environmental and rare pathogens. MALDI-TOF MS proved to be a powerful diagnostic tool for rapid species-level identification, supporting more precise mastitis control strategies. The detection of emerging or uncommon microorganisms underscores the need for sustained surveillance, improved biosecurity, and further research, including genomic characterization and antimicrobial resistance monitoring. These findings contribute to advancing dairy herd health, guiding targeted interventions, and informing One Health perspectives.