Milk metabolomic signatures associated with naturally occurring Staphylococcus chromogenes subclinical mastitis in early-lactation dairy cows

Background and Aim: Subclinical mastitis remains an important constraint on dairy production because it can impair udder health and milk quality without overt clinical signs. Staphylococcus chromogenes is a frequently isolated opportunistic pathogen associated with persistent subclinical intramammary infection; however, its pathogen-specific effects on the milk metabolome remain poorly characterized. This study aimed to characterize milk metabolomic alterations associated with naturally occurring S. chromogenes intramammary infection in early-lactation dairy cows and explore the underlying metabolic responses. Materials and Methods: A quarter-based comparative observational study was conducted on smallholder dairy farms in Khon Kaen, Thailand. Fifteen milk samples from nine early-lactation cows were analyzed, comprising eight S. chromogenes-infected and seven control quarters. Infection status was established using clinical assessment, the California Mastitis Test, somatic cell count (SCC), bacteriological culture, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and 16S rRNA gene sequencing. Milk metabolites were profiled using dual-polarity ultra-high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight tandem mass spectrometry. Multivariate discrimination was evaluated using orthogonal partial least squares-discriminant analysis (OPLS-DA), followed by univariate testing and metabolic pathway analysis. Results: SCC was significantly higher in infected than in control quarters (p < 0.05). Untargeted profiling detected 1,577 metabolites across positive and negative-ionization modes. OPLS-DA differentiated infected from control quarters, with R²X = 97.62%, R²Y = 96.89%, Q² = 55.28%, and permutation test p = 0.03. Five candidate discriminating metabolites were identified: cytidine 5′-diphosphate, citric acid, (S)-malate, and phenylacetylglycine showed increased abundance, whereas acetylcarnitine showed decreased abundance in infected quarters. Pathway analysis implicated the tricarboxylic acid cycle, glyoxylate and dicarboxylate metabolism, and phenylalanine metabolism at nominal significance; however, none remained significant after false discovery rate (FDR) correction. Conclusion: Naturally occurring S. chromogenes subclinical mastitis was associated with exploratory alterations in the milk metabolome, particularly involving energy, amino acid, and fatty-acid metabolism. The pathogen-specific design and naturally occurring infection model provide preliminary insight into host-pathogen metabolic interactions. However, given the small, partially paired sample and the absence of FDR-supported pathway associations, with only one of the five candidate metabolites surviving FDR correction, these findings should be considered hypothesis-generating. Larger independent cohorts with targeted quantitative validation are required before the identified metabolites can be considered diagnostic biomarkers.