Abstract #T61

# T61
Effects of plant-derived compounds on Staphylococcus aureus infection of primary bovine mammary epithelial cells.
Ellen V. Valley*1, Devi Jaganathan1, Kumar Venkitanarayanan1, Gary W. Kazmer1, Lynn Kuo2, Yu Bo Wang2, Kristen E. Govoni1, 1Department of Animal Science, University of Connecticut, Storrs, CT, 2Department of Statistics, University of Connecticut, Storrs, CT.

Mastitis is an inflammation of the mammary gland which results in losses to the dairy industry due to discarded milk, poor milk quality and culling. Plant-derived antimicrobials (PDA) have inhibitory effects on Staphylococcus aureus, an economically important cause of mastitis, and have less potential to induce bacterial antimicrobial resistance. We hypothesized that (1) eugenol (EG) and trans-cinnamaldehyde (TC) would reduce S. aureus adhesion to and invasion of primary bovine mammary epithelial cells (MEC), and (2) EG and TC would alter the expression of virulence factors in S. aureus and immune response genes in MEC. Mammary epithelial cells were collected from a lactating dairy cow at slaughter and isolated by selective trypsinization. Four strains of S. aureus, Thorn 17, S35, M9175, and Thorn 15, were obtained from the Connecticut Veterinary Medical Diagnostic Laboratory. Before inoculation, MEC and S. aureus were incubated at 37°C for 12 and 5 h, respectively, with sub-inhibitory concentrations (SIC, concentrations not inhibiting bacterial growth) of EG (0.03%) and TC (0.006%). Mammary epithelial cells were challenged with S. aureus in mid log phase and incubated for 2 h at 37°C. To quantify adhesion, cells were washed with PBS to remove unadhered bacteria and lysed with Triton-X. Lysed cells were serially diluted and plated on mannitol salt agar, and colonies were counted after 48 h. To quantify invasion, cells were incubated for 1 h with gentamycin before washing, lysing, and plating as performed for the adhesion assay. Experiments included MEC and S. aureus as controls, MEC or S. aureus pre-treated with PDA, and MEC and S. aureus both pre-treated with PDA (9 treatments). Experiments were performed in duplicate and repeated 3 times. Data were analyzed using PROC MIXED in SAS. For all strains, adhesion was reduced 0.2 to 0.8 log10 cfu/mL (P ≤ 0.05) and invasion was reduced 1.7 to 3.8 log10 cfu/mL (P ≤ 0.05). Gene expression analysis is currently underway. In conclusion, SIC of EG and TC effectively reduced S. aureus infection of MEC.

Key Words: bovine, mastitis, plant-derived antimicrobial