In Vitro Antimicrobial and Immunomodulatory Activity of Trans-4-Hydroxycinnamic Acid Against Bovine Mastitis-Associated Bacteria

Background/Objectives: Bovine mastitis is a major economic and health challenge in the dairy industry, heavily dependent on antibiotics and increasingly complicated by antimicrobial resistance (AMR). This study evaluated the in vitro therapeutic efficacy of trans-4-hydroxycinnamic acid (4-HCA) as a novel alternative therapeutic agent on bovine mammary epithelial cells (bMECs). Specifically, the objectives were to assess the biofilm-forming capacity of mastitis-associated bacterial isolates, determine the antibacterial and bactericidal profile of 4-HCA, and investigate its immunomodulatory effects on infected bMECs using quantitative real-time PCR (qRT-PCR).

Methods

A panel of fourteen bovine mastitis-origin bacterial isolates was evaluated for biofilm production using both the tube method and the microtiter plate assay. The antibacterial activity of 4-HCA was determined via agar well diffusion, disc diffusion, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) assays. Cytotoxicity and safe working concentrations of 4-HCA on bMECs were established using the MTT cell-viability assay (3.125 to 800 µg/mL). Furthermore, an in vitro inflammatory model using bMECs infected separately with Staphylococcus aureus and Escherichia coli (1 × 106 CFU/mL) was treated with 4-HCA (50 and 100 µg/mL) or ciprofloxacin. Relative transcriptional expression profiles of pro-inflammatory cytokines (IL-1, IL-6, and TNF- α) were quantified via qRT-PCR and analyzed using the comparative 2-ΔΔCT method.