Microbial contamination and biofilm-forming pathogens in dental unit waterlines: A cross- sectional study of opportunistic bacteria and infection control implications in an urban setting of China

Background Dental unit waterlines (DUWLs) are susceptible to microbial contamination, posing significant infection risks. This study aimed to assess microbial contamination levels, identify biofilm-forming pathogens, and evaluate infection control practices in DUWLs across multi-tier medical institutions. Methods A cross-sectional study was conducted using stratified random sampling of 214 dental chair units (DCU) from 55 medical institutions (16 tertiary, 9 secondary/below-level, and 30 private) in Nanchang, China. DUWLs management data were collected via questionnaires. Water samples were collected from four sites (source water, handpiece, triple-syringe, and rinse water; total n = 2,782) at four time points: pre-operation (0 mL drainage), after 200 mL drainage, and at the end of morning and afternoon sessions. Bacterial counts were cultured on nutrient and Legionella agars, with compliance defined as ≤ 500 CFU/mL. Species identification was performed using MALDI-TOF MS and Sanger sequencing. Biofilm formation was quantified by crystal violet staining (OD₅₇₀ₙₘ). Risk factors were analyzed using generalized estimating equations (GEE). Results Severe DUWLs contamination was observed. Pre-operation compliance rates were 10.75% (handpieces), 17.76% (triple-syringes), and 29.91% (rinse water). Drainage of 200 mL significantly improved compliance (41.59–63.08%). Despite the highest compliance rates observed at the end of sessions (time point 4), 16.82% of handpiece and 19.16% of triple-syringe samples still exceeded the limit. Source water compliance was 46.73%. Tertiary hospitals demonstrated better water quality compared to secondary/private institutions. Multivariate GEE analysis indicated a higher contamination risk in handpieces from secondary/below-level (OR = 2.088, 95% CI: 1.253–3.481, P  = 0.005) and private institutions (OR = 1.861, 95% CI: 1.178–2.939, P  = 0.008) compared to tertiary hospitals. Regular disinfection was identified as a protective factor (OR = 4.767, 95% CI: 2.321–9.790, P  < 0.001).Among 2,123 isolates (60 species), dominant genera included Sphingomonas (447 strains), Bacillus (431), and Acidovorax (320). A total of 383 (18.0%) isolates were identified as strong biofilm formers. Eight opportunistic pathogens were ranked by biofilm-forming capacity (OD₅₇₀ₙₘ) as follows: Staphylococcus xylosus > Bacillus cereus > Staphylococcus warneri > Acidovorax mediterraneus > Pseudomonas otitidis > Acinetobacter baumannii > Pseudomonas aeruginosa > Staphylococcus hominis . Conclusions DUWLs contamination is severe, with biofilm colonization being a core concern. Hospital tier (tertiary vs. non-tertiary) and regular disinfection significantly affect water quality. This study is the first to systematically identify eight strong biofilm-forming opportunistic pathogens in DUWLs, providing a scientific basis for targeted disinfection and optimized waterline management. Urgent interventions—emphasizing effective disinfection and enhanced infection control in primary care—are warranted.