Milk metabolomic signatures associated with naturally occurring Staphylococcus chromogenes subclinical mastitis in...

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.

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.

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.

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.