Changes in the lipidome of water buffalo milk during intramammary infection by non-aureus Staphylococci

Abstract

This study aimed to determine the lipidome of water buffalo milk with intramammary infection (IMI) by non-aureus staphylococci (NAS), also defined as coagulase-negative staphylococci, using an untargeted lipidomic approach. Non-aureus Staphylococci are the most frequently isolated pathogens from dairy water buffalo milk during mastitis. A total of 17 milk samples from quarters affected by NAS-IMI were collected, and the lipidome was determined by liquid chromatography-quadrupole time-of-flight mass spectrometry. The results were compared with the lipidome determined on samples collected from 16 healthy quarters. The study identified 1934 different lipids, which were classified into 15 classes. The abundance of 72 lipids changed in NAS-IMI milk compared to healthy quarters. Significant changes occurred primarily in the class of free fatty acids. The results of this study provided first-time insight into the lipidome of dairy water buffalo milk. Moreover, the present findings provide evidence that NAS-IMI induces changes in water buffalo milk's lipidome.

Figure 1

Schematic of the experimental design. Milk was collected from healthy and NAS-IMI quarters of water buffaloes and processed for lipidomic analysis through a liquid chromatography quadrupole time-of-flight (LC-Q-TOF) mass spectrometer. The figure was created using the web-based tool BioRender (https://app.biorender.com) to generate Fig. 1.

Figure 2

TAG, FA and Cer are the most abundant lipid classes in healthy buffalo milk samples. (A) Relative abundance of the main lipid classes in healthy buffalo milk samples. Histograms of the most abundant lipid classes, namely (B) TAG, (C) FA, and (D) Cer.

Figure 3

TAG, FA and Cer are the most abundant lipid classes in NAS-IMI affected milk quarter. (A) Relative abundance of the main lipid classes in NAS-IMI buffalo milk samples. Histograms of the most abundant lipid classes, namely (B) TAG, (C) FA, and (D) Cer.

Figure 4

Lipidome discriminates between H and NAS-IMI milk. (A) Scree plot of the principal component analysis (PCA) representing the total variance (green line) and the single variance (blue line) explained for each component (PC index). (B) 2D plot of the PCA representing sample distribution based on PC1 and PC2 of lipidomic profiles of healthy (pink) and NAS-IMI (cyan) milk samples. (C) 2D plot of K-means clustering based on PC1 and PC1 from PCA. (D) Details of samples distribution among K-means clusters.

Figure 5

NAS-IMI impairs specific lipids in the milk of water buffalos. (A) Cross-validation performed by the tenfold method to estimate the predictive ability of the generated models. Asterisk on model no. 5 indicates the highest and most consistent model (highest Q2, where Q2 estimates the model's predictive ability). (B) Variable importance in projection (VIP) scores of indicated metabolites listed in model no. 5. Blue and carmine red squares indicate low and high lipid levels. (C) Volcano plot representing down—(blue dots), upregulated (red dots), and unchanged lipids (grey dots) in NAS-IMI compared to H milk. Dark blue and carmine red dots indicate differentially abundant fatty acids. Significant lipids were considered with |log2 fold-change|> 1 and − log10 adjusted p value > 1.3.

Figure 6

Lipid classes do not discriminate the lipidome of NAS-IMI and H water buffalo milk. (A) Scree plot of the principal component analysis (PCA) representing the total variance (green line) and the single variance (blue line) explained for each component (PC index). (B) 3D scatter plot of the PCA representing sample distribution based on PC1, PC2, and PC3 of lipidomic profiles of healthy (pink) and NAS-IMI (cyan) milk samples. (C) Volcano plot representing down-, upregulated (red dot) and unchanged lipid classes (grey dots) in NAS-IMI compared to H buffalos. Significant lipid classes were considered with |log2 fold-change|> 1 and − log10 adjusted p value > 1.3.