Abstract
Necrotic enteritis (NE) caused by Clostridium perfringens imposes major health and economic losses in broiler production, especially with coccidiosis co-infection. Beyond toxin secretion, C. perfringens forms biofilms that enhance persistence and reduce treatment efficacy. Thymol, a phenol from thyme, has broad antimicrobial and antibiofilm activities, but its effects on C. perfringens biofilms are not well defined. We profiled 23 clinical isolates, genotyped them (all cpa⁺, most cpβ2⁺; eight netB⁺; tpeL detected only in LLY_TpeL17), quantified biofilm biomass by 0.1% crystal violet staining (OD(570)) with and without 1% glucose, and tested thymol (6.25-1600 µg/mL) in a microplate model for effects on planktonic growth (OD(590)) and biofilm formation. Biofilm capacity varied widely, and glucose increased baseline biomass. Thymol inhibited planktonic growth and biofilm biomass in a dose-dependent manner, with the minimum inhibitory concentration (MIC) values of approximately 100 µg/mL in glucose-deprived medium and 200 µg/mL in 1% glucose-supplemented medium, where biofilm formation was significantly reduced at sub-MIC concentrations (25-50 µg/mL) under both conditions. In mature 72-h biofilms from high-biofilm producers, a 3-h thymol exposure reduced metabolic activity (resazurin RFU) and biomass, with strong effects at 50-100 µg/mL and near-baseline signals at ≥200 µg/mL. These findings reveal substantial isolate-to-isolate variability and support thymol as a promising natural antimicrobial and antibiofilm agent for NE control in poultry.