Dental spittoon biofilms as reservoirs of antimicrobial resistance: a longitudinal multi-omics study

ABSTRACT Dental chair spittoons are chronically exposed to saliva, aerosols, intermittent water flow, and chemical disinfectants, yet their biofilm ecology and antimicrobial resistance (AMR) dynamics remain poorly defined. We applied longitudinal 16S rRNA gene sequencing, shotgun metagenomics, and culture-based antimicrobial susceptibility testing to biofilms collected across four dental departments at three time points. Community analyses revealed significant temporal succession and department-specific structuring, indicating the establishment of stable, ecologically differentiated biofilm systems. Null-model analysis (Raup-Crick) indicated that community assembly remained predominantly stochastic, although later sampling periods showed modest evidence of increasing ecological filtering. Shotgun metagenomics identified metabolically versatile communities enriched in disinfectant-tolerant environmental taxa, with resistomes dominated by β-lactamases and aminoglycoside-modifying enzymes. Clinically associated plasmid replicons, including IncFII and Col440I, were detected in metagenomically analyzed samples. Culture-based testing of 162 isolates confirmed that 21.7% expressed phenotypic resistance to at least one antimicrobial agent, including multidrug- and carbapenem-resistant representatives of Pseudomonas and Acinetobacter . Together, these findings position dental spittoons as structured aquatic biofilm ecosystems that maintain viable antimicrobial-resistant populations and clinically relevant plasmid replicons under recurrent disturbance, highlighting their ecological role within the broader built-water resistome. IMPORTANCE Biofilms in healthcare environments can act as reservoirs of antimicrobial resistance, yet some potential niches remain poorly studied. Dental chair spittoons are continuously exposed to oral fluids, aerosols, water flow, disinfectants, and residual antimicrobial compounds, creating conditions that may favor biofilm formation and microbial selection. Despite this unique combination of ecological pressures, the microbial communities inhabiting these systems have received little attention. Using a longitudinal multi-omics approach combined with culture-based phenotypic testing, we show that spittoon biofilms harbor diverse microbial communities enriched in antimicrobial resistance determinants, including multidrug-resistant and carbapenem-resistant bacteria. These findings suggest that dental spittoons may represent previously overlooked reservoirs of antimicrobial resistance within clinical environments.