Evaluation of Microbial Contamination and Practical Effectiveness of Chemical Disinfection in Dental Unit Waterlines Supplied with Reverse Osmosis Water: A Sequential and Non-Randomized Study

Abstract

Despite the recommendation of purified water, dental unit waterlines (DUWLs) readily form biofilm and suffer persistent microbial contamination, threatening both patients and dental staff. This study aimed to evaluate the microbial contamination and practical effectiveness of chemical disinfection in reverse osmosis (RO) water-supplied DUWLs. A single-center, sequential longitudinal study involving 15 dental chairs and 60 terminal outlets was conducted. Two disinfection strategies were sequentially evaluated: shock disinfection with a chlorine-based agent and continuous disinfection using slightly acidic electrolyzed water (SAEW). Heterotrophic plate counts (HPCs) were quantified via R2A culture, and microbial communities were characterized by metagenomic next-generation sequencing (mNGS) on 12 water samples (four terminal outlets and two source-water points, both pre- and post-disinfection). Water quality assessment revealed severe microbial contamination at the terminal outlets, with 73.33% of samples exceeding the threshold (500 CFU/mL) and a median HPC level of 805 CFU/mL. Notably, mNGS identified nucleic acid sequences of Legionella pneumophila, Pseudomonas aeruginosa, and Acanthamoeba in four pre-disinfection terminal-outlet samples. Chlorine-based shock disinfection achieved 100% compliance immediately after treatment; however, by week 5, the percentage above the threshold value had risen to 73.33%, with a median HPC level of 600 CFU/mL. The continuous SAEW disinfection protocol sustained effective microbial control over 55 weeks, with an overall percentage above threshold value of only 3.33%. mNGS analysis further confirmed a reduction in microbial relative abundance following one year of continuous SAEW disinfection. In conclusion, purified RO water alone could not guarantee DUWL microbiological safety, and this study provided practical evidence to support decision-making for DUWL maintenance.