Antibiofilm potential of luteolin against multidrug-resistant Staphylococcus aureus isolated from dairy goats and farm environments

Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for mastitis in dairy goats, and capable of contaminating farm environments. Luteolin is a naturally derived flavonoid found in many plant types. To our best of knowledge, this study involved the initial investigation into the prevalence of S. aureus and screened the multidrug-resistant (MDR) S. aureus from raw milk samples and farm environments. Furthermore, we explored the antimicrobial and antibiofilm activities of luteolin against MDR S. aureus. Antibiofilm activity was evaluated via crystal violet staining and confocal laser scanning microscopy (CLSM). Bacterial morphology and biofilm microstructure were observed via scanning electron microscopy (SEM), and the antibiofilm mechanisms were further explored based on extracellular polymeric substance (EPS) production, extracellular DNA (eDNA) content, and quantitative reverse transcription PCR (qRT-PCR). In total, 28 and 43 S. aureus isolates were isolated from raw milk and environmental samples, respectively. Raw milk samples had the highest prevalence of S. aureus (58.33%), followed by sewage sludge (35.42%), soil (27.78%), excrement (19.44%), bulk tank (12.50%), milking parlor (11.11%), and feed (7.50%). Among the isolated strains, 40 isolates (56.34%) expressed the MDR phenotype. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of luteolin against MDR S. aureus were 8-32 μg/mL and 16-64 μg/mL, respectively. Compared to that in the untreated control isolate, the number of dead cells increased, while the auto-aggregation and cell surface hydrophobicity decreased. Moreover, the cell membrane dissolved with the increase in luteolin concentration. Luteolin down-regulated the transcription of seven biofilm related genes: icaA, icaD, icab, hld, hla, agrA and RNAIII. These results indicated that S. aureus coexisted in raw milk and goat farm environments, and also suggested the potential of luteolin as a promising antibiofilm agent against MDR S. aureus.