Biofilm formation by clinical Clostridium perfringens isolates and its suppression by thymol

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 (OD₅₇₀) with and without 1% glucose, and tested thymol (6.25-1600 µg/mL) in a microplate model for effects on planktonic growth (OD₅₉₀) 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.