In Vitro Antimicrobial and Immunomodulatory Activity of Trans-4-Hydroxycinnamic Acid Against Bovine...

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. Results: Quantitative biofilm evaluations identified 19% to 22% of the bacterial isolates as strong biofilm producers and 25% to 31% as moderate producers, with prominent biofilm formation observed in Enterococcus faecalis, Raoultella planticola, Shigella sonnei, and Staphylococcus aureus. 4-HCA exhibited robust bactericidal efficacy with mean MIC values ranging from 0.625 to 1.25 mg/mL and MBC values from 2.5 to 10 mg/mL. The MTT assay confirmed 100% bMEC viability at the experimental treatment concentrations of 50 and 100 µg/mL. qRT-PCR analyses revealed that 4-HCA treatment significantly modulated (p < 0.05) pro-inflammatory cytokine transcript profiles. In the S. aureus model, treatment significantly downregulated TNF-alpha expression while differentially upregulating IL-1 and IL-6 mRNA levels, demonstrating cytokine-specific immunomodulation. Conversely, in the E. coli model, 4-HCA induced a concentration-dependent reduction across all evaluated pro-inflammatory cytokines (IL-1, IL-6, and TNF- α). Conclusions: Trans-4-hydroxycinnamic acid demonstrates significant in vitro antibacterial activity, biofilm disruption potential, and targeted immunomodulatory effects on bovine mammary epithelial cells. These findings establish a strong foundational basis for exploring 4-HCA as a promising alternative therapeutic agent for managing bovine mastitis and mitigating antimicrobial resistance in veterinary medicine.