Microbiome and Metabolome Analyses of Milk From Dairy Cows With Subclinical Streptococcus agalactiae Mastitis—Potential Biomarkers

Abstract

The microbial ecosystem in the udders of dairy cows directly influences the flavor and quality of milk. However, to our knowledge, no published research has analyzed the complex relationship between the udder microbiome and its associated metabolism in animals with subclinical mastitis. We identified the bacterial species and measured relative population numbers in the milk of cows with subclinical <i>Streptococcus agalactiae</i> mastitis (GBS) and compared this information to that from the milk of healthy cows. Metabolite profiles were determined to investigate correlations between the milk microbiota and metabolic factors in healthy vs. GBS dairy cows. Six milk samples from GBS cows and six from healthy cows were subjected to 16S rRNA gene sequencing to identify the microbial species using a MiSeq high-throughput sequencing apparatus. The metabolites present in the milk were identified by gas chromatography time-of-flight mass spectrometry. Both principal component analysis and orthogonal partial least squares discriminant analysis indicated that the metabolites were well-separated from each other in the milk samples from the two groups. GBS dramatically altered microbial diversity, and the GBS group had significantly fewer <i>Proteobacteria, Actinobacteria</i>, and <i>Acidobacteria</i> than the CON group, with greater relative abundance of <i>Firmicutes</i> (<i>p</i> < 0.01). Several bacterial genera, such as <i>Streptococcus</i>, were significantly more abundant in milk from the GBS group than in milk from the CON group, and there was a tendency for greater abundance of <i>Turicibacter</i> (<i>p</i> = 0.07) and <i>Enterococcus</i> spp. (<i>p</i> = 0.07) in the GBS group. The levels of five milk metabolites were significantly higher in the GBS group than in the CON group: phenylpyruvic acid, the homogentisic acid: 4-hydroxyphenylpyruvic acid ratio, the xanthine: guanine ratio, uridine and glycerol. Metabolic pathway analysis of the different metabolites revealed that the following were enriched in both groups: galactose metabolism; pentose and glucuronate interconversion; starch and sucrose metabolism; alanine, aspartate and glutamate metabolism; arginine biosynthesis; citrate cycle (TCA cycle); D-glutamine and D-glutamate metabolism; and the neomycin, kanamycin, and gentamicin biosynthesis pathways. Several typical metabolites were highly correlated with specific ruminal bacteria, such as <i>Streptococcaceae, Lachnospiraceae, Lactobacillaceae</i> and <i>Corynebacteriaceae</i>, demonstrating the functional correlations between the milk microbiome and associated metabolites. These findings revealed that the milk microbiota and metabolite profiles were significantly different between the two groups of cows, raising the question of whether the microbiota associated with the bovine mammary gland could be related to mammary gland health. There was also a relationship between milk quality and the presence of spoilage bacteria. Other bacterial taxa should be investigated, as related information may provide insights into how perturbations in milk metabolomics profiles relate to differences in milk synthesis between healthy cows and those with subclinical mastitis.