*2.2. Biofilm Development*

In order to determine the role of the core microbiota, we established three experimental biofilm groups: "Core", "*S. mutans*", and "Core + *S. mutans"*. The *S. mutans* group contained only *S. mutans*. The Core group contained *V. parvula*, *F. nucleatum*, *P. denticola*, and *L. wadei*, and the Core + *S. mutans* group contained all bacteria in the Core group and *S. mutans*.

We first adjusted all bacterial suspensions to a 2 × 108 colony-forming unit (CFU)/mL. Then, the suspension of four bacteria from the core microbiota with equal volume was mixed to generate the Core group suspension for the follow-up experiment. For the Core group, a 100 μL suspension was added onto a saliva-coated glass coverslip in a 24-well cell culture plate with 1900 μL of SHI media [19]; for the Core + *S. mutans* group, a 100 μL suspension of *S. mutans* together with a 100 μL suspension of core microbiota were added to the system with 1800 μL of SHI media; for the *S. mutans* group, a 100 μL suspension of *S. mutans* and 1900 μL of SHI media were added. By doing so, the amount of *S. mutans* equaled the total amount of core microorganisms at the beginning of the biofilm formation.

The biofilms were incubated anaerobically for 24 h, 48 h, or 72 h and 1 mL of medium in each well was renewed every 24 h. The biofilm discs were harvested at the end of the incubation time and washed out by dip-washing with phosphate-buffered saline (PBS) three times [20].

#### *2.3. Biofilm Acidogenicity and S. Mutans Counting*

For each group, pH in culture medium, used as an indicator of biofilm acidogenicity, was measured each time when the medium was changed. The medium was collected and transferred to polystyrene tubes for pH evaluation with an Orion Dual Star pH/ISE electrode (Thermo Scientific, Waltham, MA, USA) [18]. For *S. mutans* counting, the biofilms were transferred to microcentrifuge tubes containing 1 mL PBS. Serial dilutions were performed in PBS and the CFU/disc of *S. mutans* was determined by plating in triplicate on MSB plates as described in previous studies [21]. Three specimens were tested for each group.

#### *2.4. DNA Isolation and Quantitative Analysis of Biofilm Composition*

The bacterial composition of the Core + *S. mutans* group and Core group was further quantified via species-specific real-time quantitative polymerase chain reaction (qPCR) by the method described before [22]. According to the manufacturer's instructions, the TIANamp Bacterial DNA Kit (TIANGEN, Beijing, China) was used to isolate and purify the total DNA of the biofilm. We used enzymatic lysis buffer (20 mM Tris-HCl, pH 8.0; 2 mM sodium EDTA, and 1.2% Triton X-100) containing 30 mg·mL-1 lysozyme to lyse the bacteria at 37 ◦C for 1 h. Three independent replicates from each parameter were analyzed in triplicate using a NanoDrop ND-1000 (Thermo Scientific) and stored at −20 ◦C before use. For qPCR, 20 μL mixture containing 10 μL of SYBR® Premix Ex Taq (Takara, Wan Chai, Hong Kong), 1.5 μL of template, and 250 nM (each) of the forward and reverse primer were placed in each well. Primer sequences are given in Table 1. Real-time PCR was performed as follows: 95 ◦C for 3 min, followed by 40 cycles of 95 ◦C for 10 s, and 56 ◦C for 30 s. The mean CT value was converted into the copy number for the calculation of the percentage of each strain in the biofilm. Melting curve analysis was performed on all primer sets to ensure a single peak, which indicates primer specificity.


**Table 1.** Primers used in this study.
