Study on the mechanism of hinokitiol in inhibiting the biofilm activity of Staphylococcus epidermidis.

Mastitis in dairy cows is one of the most prevalent infectious diseases in the dairy industry, leading to reduced milk production, increased production costs, and substantial economic losses. Antibiotics are commonly used to prevent and treat mastitis; however, persistent biofilm formation by Staphylococcus epidermidis (S. epidermidis) has contributed to rising antibiotic resistance and poses significant challenges for complete eradication. Therefore, the development of effective therapeutic strategies is urgently needed. This study investigated the antibacterial and anti-biofilm activities of hinokitiol, both alone and in combination with Ceftiofur sodium, against biofilm-producing S. epidermidis. The checkerboard assay revealed a significant synergistic effect between hinokitiol and Ceftiofur sodium (FICI ≤ 0.5). Importantly, hinokitiol exhibited negligible cytotoxicity toward mammalian cells and low hemolytic activity against sheep erythrocytes at therapeutic concentrations. Mechanism studies demonstrated that hinokitiol disrupted bacterial membrane permeability, as evidenced by increased NPN uptake, PI staining, and significant intracellular protein leakage. Furthermore, hinokitiol induced the accumulation of intracellular reactive oxygen species (ROS), leading to oxidative stress-mediated cell death. Transcriptomic analysis and RT-qPCR validation showed that hinokitiol significantly down-regulated the expression of key genes involved in biofilm formation (icaA, fnbA, aap), quorum sensing (agrA, luxS), and global regulation (sarA, sigB), while up-regulating the repressor icaR. These findings suggest that hinokitiol exerts its antibacterial and anti-biofilm effects by damaging the cell membrane, inducing oxidative stress, and modulating multi-target regulatory pathways, making it a promising candidate for treating S. epidermidis infections. However, this study is limited by the absence of in vivo mastitis models and pharmacokinetic data. Future research should focus on validating these findings in animal models, optimizing administration routes (e.g., intramammary formulations), and assessing clinical efficacy in field conditions to facilitate the translation of hinokitiol into a viable therapeutic option for refractory S. epidermidis mastitis.