Materials & Methods
Dental plaque samples were collected from eleven patients with S-ECC. Three boys and six girls, aged from 2.53 to 5.33 years old, were diagnosed S-ECC with decayed, missing, and filled tooth surface (dmfs) scores were 14.11  ± 3.59 ( Table S1 ). Participants were required to avoid eating, drinking and brushing their teeth at 2 h before dental plaque sampling. Ethical approval of the study was granted by Peking University School of Stomatology (PKUSSIRB-201839140), and written informed consent was obtained from all study participants’ guardians.

Eleven plaque samples were collected with a sterile curette from the labial/buccal smooth surfaces or the decayed cavity of the affected teeth. The dental plaque was transferred to a 1.5 mL sterile centrifuge tube full of pre-reduced medium (0.15 g sodium mercaptoacetate, 0.9 mL fresh 1% calcium chloride, and 100 mL distilled water, filtered for removing bacteria) and taken to the laboratory and processed for culture within 3 min. Samples were dispersed by vortexing for 30 s and diluted tenfold consecutively from 10 −1 to 10 −8 .

All samples were grown in duplicate at 37 °C under anaerobic conditions (80% N 2 , 10% H 2 , and 10% CO 2 ) and normoxic conditions (air with 5% CO 2 ). To mimic the nutritional environment of the dental plaque, a novel culture medium was designed. The basal liquid medium consisted of brain-heart infusion broth (OXOID, Basingstoke, UK) 24.5 g/L, agar (Solarbio, Beijing, China) 15 g/L, dextrose (Solarbio) 5 g/L, corn starch (Solarbio) 1.5 g/L, casein acid hydrolysate (Macklin, Shanghai, China) 17.5 g/L, hemin (Solarbio) 5 mg/L, VitK (Solarbio) 1 mg/L, VitB12 (Macklin) 100 µg/L, L-glutamine (Macklin) 0.1 g/L, adenine (Macklin) 10 mg/L, guanine hydrochloride (Macklin) 300 µg/L, p-aminobenzoic acid (Macklin) 130 µg/L, nicotinamide adenine dinucleotide (Macklin) 2.5 mg/L, thiamine pyrophosphate (Macklin) 1 mg/L, ferric nitrate (Macklin) 200 µg/L, cysteine hydrochloride (Solarbio) 0.259 g/L, L-cysteine (Solarbio) 11 mg/L, and sheep blood (Pingrui Biotechnology company) 5%. SHI medium was prepared as described before ( Tian et al., 2010 ).

Samples were enriched aerobically and anaerobically using two liquid media SHI and NEMM designed by this study and subcultured in solid media after optimal dilution, whose single colonies numbers range from 100 to 500. The cultures were divided into four groups: (1) aNEMM (samples grown in NEMM anaerobically); (2) aSHI (samples grown in SHI medium anaerobically); (3) nNEMM (samples grown in NEMM under normoxic condition); and (4) nSHI (samples grown in SHI medium under normoxic condition). Consecutive tenfold dilutions of the plaque samples in two media were prepared by PBS and used to inoculate NEMM and SHI-agar plates in triplicate. Plates were incubated and photographed at 0, 1, 3, 6, 9, 15, 21 and 28 days. Meanwhile, 500 µL suspension cultures at these different time points were collected for 16S rRNA gene sequencing analysis and bacterial isolation. For clinical isolates, suspension cultures were gradient diluted from 10 −1 to 10 −8 , and then the diluted bacterial solutions were plated on the NEMM- and SHI-agar plates. The clinical isolates were picked up from the plates according to morphology and then identified by sequencing the full length of the 16S rRNA gene as described below.

Nine clinical dental plaque samples and their cultures in NEMM and SHI media under anaerobic and normoxic conditions at eight indicated time points (9 clinical samples + 144 SHI cultures +144 NEMM cultures) were respectively subjected to high through-put 16S rRNA gene sequencing at Biomarker Technologies (Biomarker Technologies, Beijing, China) where the total DNA was isolated, amplified and sequenced according to their standard procedures. In brief, microbial DNA was extracted from the bacterial suspensions according to the manufacturer’s protocols. DNA concentration was assessed by a Nanodrop (Thermo Scientific) and quality was determined by agarose gel electrophoresis. PCR was used to amplify the variable regions 4 and 5 (V4-V5) of bacterial 16S rRNA gene by using 515F (ACTCCTACGGGAGGCAGCA) and 907R (GGACTACHVGGGTWTCTAAT, H = A/C/T, V = A/C/G, W = A/T) PCR primers. The PCR procedure was set as previously published research ( Li et al., 2017 ). Purified amplicons were pooled in equimolar solutions and paired-end sequenced (2 × 300) on an Illumina MiSeq platform. After passing data quality check, the original high-throughput sequencing data were converted into FASTQ format files for bioinformatics analysis. The high-quality tag data were obtained after filtering and compared with sequences in the SILVA database ( http://www.arb-silva.de ).

The bioinformatics analysis was conducted using QIIME2 2020.6 ( Fig. S1 ). The alpha diversity indices of Chao1, ACE, Shannon, Simpson and phylogenetic diversity (PD whole tree) were calculated using Mothur software (version v.1.30) with coverage over 99%. Beta diversity analysis was performed by non-metric multi-dimensional scaling (NMDS) analysis based on the weighted unifrac and unweighted unifrac algorithms. Linear discriminant analysis (LDA) effect size (LEfSe) was conducted to define the biomarkers of these groups. The threshold on the logarithmic LDA score for the distinguishing features was set to 4.0. Microbial functions were predicted using PICRUSt2 (v1.0.0) software following the online protocol and aligned to the Clusters of Orthologous Groups of proteins (COG) database. The profiles of Kyoto Encyclopedia of Genes and Genomes (KEGG) levels were generated based on the KEGG database. Analysis of Variance (ANOVA) is mainly used to test whether the mean of multiple samples is equal, so as to judge the significant difference of the average of two or more samples. Level of statistical significance ( P value) was set as <0.05 for all comparisons. The feature sequences of Top 80 abundance ratio at OTU/ASV taxonomic level were selected, multiple sequence comparison was performed with QIIME command line and a taxa tree was constructed. Then the R package was used to combine the taxa tree and species taxonomic abundance data.

A total of 1,344 single colonies from NEMM-agar plates and 1,312 single colonies from SHI-agar plates were picked up and inoculated into NEMM and SHI liquid media, cultured in aerobically or anaerobically, respectively. DNA from these clinical isolates was extracted using hexadecyl trimethyl ammonium bromide (CTAB) methods ( Zheng, Gao & Deng, 2012 ). The full-length of 16S rRNA gene was amplified via PCR reaction. 27F (AGAGTTTGATCCTGGCTCAG) and 1492R (GGTTACCTTGTTACGACTT) served as forward and reverse primers, respectively. PCR reaction conditions were set as follows: predenatured at 94 °C for 5 min, 35 cycles, including denaturation at 94 °C for 30s, annealing at 54 °C for at 30s, elongation 72 °C for 90s, and extended at 72 °C for 10 min. The full-length of 16S rRNA gene amplicons were sequenced using the Sanger method. The sequences of 16S rRNA gene were blasted in either NCBI ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ) or EzBioCloud ( https://www.ezbiocloud.net/ ) to establish taxonomic relationships based on currently recognized prokaryotic reference species. Sequences with homology below 98.65% were defined as potential new strains ( Kim et al., 2014 ).

The t-test was performed on the species abundance data between the groups using Metastats software. Principal component analysis was used to analyze the composition of the microbiome. BugBase was used to evaluate microbial phenotype followed by the Mann–Whitney U test. The species leading to the composition difference between the two groups of samples were determined by P value or Q value (correcting P value). The level of statistical significance ( P value or Q value) was set as <0.05.