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ABSTRACTThe oral microbiome plays an important role in protecting oral health. Here, we established a controlled mixed-species in vitro biofilm model and used it to assess the impact of glucose and lactate on the ability of Streptococcus mutans, an acidogenic and aciduric species, to compete with commensal oral bacteria. A chemically defined medium was developed that supported the growth of S. mutans and four common early colonizers of dental plaque: Streptococcus gordonii, Actinomyces oris, Neisseria subflava, and Veillonella parvula. Biofilms containing the early colonizers were developed in a continuous flow bioreactor, exposed to S. mutans, and incubated for up to 7 days. The abundance of bacteria was estimated by quantitative polymerase chain reaction (qPCR). At high glucose and high lactate, the pH in bulk fluid rapidly decreased to approximately 5.2, and S. mutans outgrew other species in biofilms. In low glucose and high lactate, the pH remained above 5.5, and V. parvula was the most abundant species in biofilms. By contrast, in low glucose and low lactate, the pH remained above 6.0 throughout the experiment, and the microbial community in biofilms was relatively balanced. Fluorescence in situ hybridization confirmed that all species were present in the biofilm and the majority of cells were viable using live/dead staining. These data demonstrate that carbon source concentration is critical for microbial homeostasis in model oral biofilms. Furthermore, we established an experimental system that can support the development of computational models to predict transitions to microbial dysbiosis based on metabolic interactions.IMPORTANCEWe developed a controlled (by removing host factor) dynamic system metabolically representative of early colonization of Streptococcus mutans not measurable in vivo. Hypotheses on factors influencing S. mutans colonization, such as community composition and inoculation sequence and the effect of metabolite concentrations, can be tested and used to predict the effect of interventions such as dietary modifications or the use of toothpaste or mouthwash on S. mutans colonization. The defined in vitro model (species and medium) can be simulated in an in silico model to explore more of the parameter space.