Supplementary Figure 1. Heatmap of the antibiotic resistance profile of 309 NAS isolates. The colorful blocks (red, orange, and green) indicate the antimicrobial susceptibility results, with R = resistant, I = intermediate, and S = sensitive. Blocks in shades of blue indicate the clinical status of the cow from which each isolate was obtained: dark blue = clinical mastitis; medium blue = high somatic cell count (SCC > 200,000 cells/mL); light blue = low somatic cell count (SCC < 200,000 cells/mL). Antibiotics: GEN = gentamicin (10 μg), OXA = oxacillin (1 μg), CEF = cefoxitin (30 μg), TET = tetracycline (30 μg), ERY = erythromycin (15 μg), CLI = clindamycin (2 μg), TOB = tobramycin (10 μg), CHL = chloramphenicol (30 μg), and PEN = penicillin (10 μg).

Non-aureus staphylococci and mammaliicocci (NASM) are frequently isolated from bovine milk in cases of mastitis and intramammary infection, as well as from extramammary environments. However, most studies do not identify NASM at the species level, limiting the understanding of their epidemiological relevance. This study aimed to provide a comprehensive characterization of NASM isolated from bovine milk across the five major dairy-producing regions of Brazil. Using MALDI-ToF MS, 309 NASM isolates obtained from cows with low and high somatic cell counts (SCC) and from clinical mastitis cases were identified at the species level. All isolates were evaluated for antimicrobial susceptibility and screened for the presence of methicillin resistance genes (mecA, mecC), disinfectant resistance genes (qacAB, qacC), and enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei). In addition, the association between NASM occurrence and herd management practices was assessed.Eighteen NASM species were identified, with Staphylococcus chromogenes (58.6%), S. simulans (8.4%), and S. haemolyticus (8.1%) being the most prevalent. Forty-one isolates (13.3%) were classified as multidrug-resistant, and 15 carried the mecA gene. The most prevalent enterotoxin gene was seb, detected in 15.2% of isolates. Management-related analyses indicated that S. chromogenes occurred widely across farms with adequate milking hygiene. In contrast, non-chromogenes species were more frequently associated with herds where hygienic practices were less consistent.This study provides the first large-scale, species-level evaluation of NASM across multiple Brazilian regions, integrating phenotypic, genotypic, and management-related data. The findings underscore the importance of NASM as common intramammary pathogens and highlight their potential roles in the persistence of mastitis, the dissemination of antimicrobial resistance, and challenges in dairy herd management.