Subclinical Mastitis and Antibiotic Exposure Reshape the Milk Bacteriome, Resistome, and Metabolic Functional Profiles in Dairy Cows.

Abstract

Subclinical mastitis (SCM) reduces milk yield and promotes antibiotic use, contributing to antimicrobial resistance. However, information on milk microbiome dysbiosis and resistome alterations in dairy cows remains limited. We performed shotgun metagenomic sequencing of 30 milk samples from 20 cows classified as SCM (n = 10), antibiotic-treated (ANT; n = 10), and healthy milk (HM; n = 10). Bacteriome, resistome, and metabolic functions were analyzed using bioinformatic tools. Microbial alpha diversity was significantly higher in HM than SCM and ANT samples (p < 0.05), with the greatest richness observed in Local Zebu and Middle-aged (5-8 years) cows. However, beta diversity did not differ significantly among groups (PERMANOVA, p > 0.05). A total of 665 bacterial species belonging to 15 phyla were identified, with Pseudomonadota, Actinomycetota, and Bacillota comprising 90%-99% of total abundance. SCM milk was dominated by Actinoalloteichus sp. (40.55%) and Vibrio furnissii (37.01%), while Staphylococcus aureus was enriched (1.74%) compared with HM and ANT (< 0.01%). ANT milk showed increased abundance of Acinetobacter johnsonii (10.52%), whereas HM samples contained beneficial commensals, including Pseudomonas lundensis, Bacteroides thetaiotaomicron, and Lactococcus lactis. Similar taxonomic shifts were observed across breeds and age groups. In total, 194 antimicrobial resistance genes (ARGs) were detected, with SCM harboring the largest repertoire (n = 161). Nevertheless, macrolide-, aminoglycoside-, and β-lactam-resistance genes were significantly enriched in ANT samples (p < 0.01). Correlation analyses linked Escherichia coli and A. johnsonii with multiple ARGs, while Acinetobacter and Pseudomonas were associated with lipid metabolism, membrane transport, and xenobiotic biodegradation pathways. These findings reveal bacteriome disruption, ARG enrichment, and functional alterations in SCM and antibiotic-exposed milk.