EC Microbiology

Sports mouthguards are essential for preventing orofacial injuries but may act as reservoirs for microbial contamination when not properly disinfected. Ethylene-vinyl acetate (EVA), the material commonly used for mouthguards, facilitates bacterial adhesion and biofilm formation. This in vitro study evaluated the effectiveness of different chemical and mechanical disinfection methods for removing Escherichia coli biofilm from EVA surfaces. EVA plates (3 × 3 cm, 3 mm thick; Bio-Art, Brazil) were sterilized with ethylene oxide and immersed in Brain Heart Infusion (BHI) broth containing E. coli (10⁸ CFU/mL) at 37°C for 72h to promote biofilm formation. After incubation, 25 specimens were divided into five groups (n = 5): control (sterile saline), brushing (sterile toothbrush and saline), 0.12% chlorhexidine digluconate, 1% sodium hypochlorite, and effervescent tablet (Corega Tabs^®). Disinfection was performed for 5 min, followed by rinsing with sterile saline. Detached biofilms were vortexed, serially diluted, plated on BHI agar, and incubated at 37°C for 24h. Colony-forming units (CFU/mL) were counted and log-transformed. Data were analyzed using ANOVA and Tukey’s test (p < 0.05). All treatments significantly reduced E. coli biofilm compared with the control (p < 0.05). Mean log₁₀ CFU/mL values were: effervescent tablet 1.6 ± 0.4 (4.7-log reduction), chlorhexidine 2.3 ± 0.2 (4.0-log), sodium hypochlorite 2.8 ± 0.3 (3.5-log), brushing 3.5 ± 0.3 (2.8-log), and control 6.3 ± 0.4. Effervescent tablets and chlorhexidine achieved the greatest antimicrobial effect without significant difference (p > 0.05). All evaluated disinfection methods effectively reduced E. coli biofilm on EVA surfaces. Effervescent tablets and 0.12% chlorhexidine provided the best combination of efficacy and material safety, representing practical and reliable options for daily mouthguard hygiene and biofilm control.

 Keywords: EVA; Mouthguard; Biofilm; Disinfection; Escherichia coli

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