Pore-forming toxins (PFTs) exert physiological effects by rearrangement of the host cell cytoskeleton. Staphylococcus aureus-secreted PFTs play an important role in bovine mastitis. In the study, we examined the effects of recombinant Panton–Valentine leukocidin (rPVL) on cytoskeleton rearrangement, and identified the signaling pathways involved in regulating the process in bovine mammary epithelial cells (BMECs) in vitro. Meanwhile, the underlying regulatory mechanism of baicalin for this process was investigated. The results showed that S. aureus induced cytoskeleton rearrangement in BMECs mainly through PVL. S. aureus and rPVL caused alterations in the cell morphology and layer integrity due to microfilament and microtubule rearrangement and focal contact inability. rPVL strongly induced the phosphorylation of cofilin at Ser3 mediating by the activation of the RhoA/ROCK/LIMK pathway, and resulted in the activation of loss of actin stress fibers, or the hyperphosphorylation of Tau at Ser396 inducing by the inhibition of the PI3K/AKT/GSK-3β pathways, and decreased the microtubule assembly. Baicalin significantly attenuated rPVL-stimulated cytoskeleton rearrangement in BMECs. Baicalin inhibited cofilin phosphorylation or Tau hyperphosphorylation via regulating the activation of RhoA/ROCK/LIMK and PI3K/AKT/GSK-3β signaling pathways. These findings provide new insights into the pathogenesis and potential treatment in S. aureus causing bovine mastitis.

Panton–Valentine leukocidin (PVL) is a pore-forming toxin secreted by Staphylococcus aureus (S. aureus) and a significant virulence factor that plays a crucial role in the pathogenesis of dairy mastitis. Previous studies by our research group demonstrated that baicalin inhibits the apoptosis and hyperphosphorylation of cytoskeletal proteins induced by recombinant Panton–Valentine leukocidin (rPVL) in bovine mammary epithelial cells (BMECs). However, the effects of baicalin on the proliferation of BMECs and the underlying mechanism remain unclear. Consequently, this study aimed to explore this underlying mechanism through an LC-MS/MS analysis performed in 4D data-independent acquisition (DIA) mode. Quantitative analysis identified 757 differentially expressed phosphoproteins, among which phosphorylation levels of proteins involved in BMEC proliferation and cell cycle regulation exhibited significant alterations (p < 0.05). rPVL inhibited BMEC proliferation in a dose-dependent manner and induced G0/G1 phase arrest and dephosphorylation of the cell-cycle-related proteins BCLAF1^S285, CDK7^T170, NF2^S518, and PKM2S37. Preintervention with baicalin significantly upregulated the expression and phosphorylation of these proteins and alleviated the G0/G1 phase arrest induced by rPVL in BMECs in vitro. The establishment of the mitotic state in BMECs due to the effect of baicalin appears to be closely related to the regulation of the phosphorylation of CDK7, PKM2, BCLAF1, and NF2. Moreover, in vivo analysis revealed that S. aureus ATCC49775 and rPVL induced dramatic structural destruction and pathological impairment of mammary gland tissues in mice and that these histopathological changes were ameliorated after baicalin intervention. Quantitative immunohistochemical analysis revealed that baicalin mitigated the rPVL-induced dephosphorylation of the aforementioned cell-cycle-related proteins and increased their phosphorylation. Both in vitro and in vivo experimental evidence demonstrated that baicalin effectively reversed rPVL-induced G0/G1 phase arrest in BMECs (p < 0.01) by significantly increasing the phosphorylation levels of cell cycle regulatory proteins (p < 0.05). Additionally, baicalin alleviates pathological damage to mammary gland tissues in mouse models. These data suggest that baicalin possesses antibacterial and antitoxin effects, indicating that it is an effective preventive agent against bovine mastitis.

Multidrug-resistant (MDR) Enterococcus faecium is an opportunistic pathogen. Its resistance and virulence genes can spread through the food chain, posing risks to public health. This study investigated the antimicrobial resistance and genomic characteristics of MDR E. faecium isolated from milk samples from cows with mastitis in Ningxia between 2019 and 2024. From 2019 to 2024, 1341 milk samples were collected in Yinchuan, Yinnan, and Yinbei. MDR E. faecium was identified using plate screening, mass spectrometry, broth microdilution, and hemolysis detection. Whole-genome sequencing enabled SNP, MLST, pan-genome, and COG analyses, focusing on ARGs and MGEs. MRPP, AMOVA and PCoA were applied to compare gene communities and identify driver genes. Ninety-one E. faecium strains were isolated. Resistance to florfenicol, ceftiofur, and chloramphenicol exceeded 60%, while resistance to vancomycin and linezolid showed an overall increasing trend over the study period. Phylogenetic clustering revealed two subtypes, three clades, and 10 novel STs. Spearman correlation analysis revealed strong positive correlations among the resistance genes optrA, cfr(A), and vanF. Antibiotic resistance, particularly MDR, increased over time, and strains carried diverse ARGs and MGEs. Overall, strengthened surveillance of mastitis-derived E. faecium is warranted to support the control of bovine mastitis and safeguard public health.