==== Front PeerJ PeerJ PeerJ PeerJ 2167-8359 PeerJ Inc. San Diego, USA 15605 10.7717/peerj.15605 Microbiology Dentistry Pediatrics Saccharibacteria (TM7), but not other bacterial taxa, are associated with childhood caries regardless of age in a South China population http://orcid.org/0000-0001-5257-2922 You Yang 1 Yin Meixiang 12 Zheng Xiao 1 Liang Qiuying 1 Zhang Hui 1 Wu Bu-Ling 3bulingwu@smu.edu.cn Xu Wenan 1xu_wenan@smu.edu.cn 1 Department of Pediatric Dentistry, Shenzhen Stomatology Hospital (Pingshan), Southern Medical University, ShenZhen, GuangDong, China 2 Department of Stomatology, Shenzhen Samii Medical Center, ShenZhen, GuangDong, China 3 Department of Endodontics, Shenzhen Stomatology Hospital (Pingshan), Southern Medical University, ShenZhen, GuangDong, China Sistla Srinivas 26 6 2023 2023 11 e156057 4 2023 31 5 2023 © 2023 You et al. 2023 You et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited. Background Human microbiome dysbiosis is related to various human diseases, and identifying robust and consistent biomarkers that apply in different populations is a key challenge. This challenge arises when identifying key microbial markers of childhood caries. Methods We analyzed unstimulated saliva and supragingival plaque samples from children of different ages and sexes, performed 16S rRNA gene sequencing, and sought to identify whether consistent markers exist among subpopulations by using a multivariate linear regression model. Results We found that Acinetobacter and Clostridiales bacterial taxa were associated with caries in plaque and saliva, respectively, while Firmicutes and Clostridia were found in plaque isolated from children of different ages in preschool and school. These identified bacterial markers largely differ between different populations, leaving only Saccharibacteria as a significant caries-associated phylum in children. Saccharibacteria is a newly identified phylum, and our taxonomic assignment database could not be used to identify its specific genus. Conclusion Our data indicated that, in a South China population, oral microbial signatures for dental caries show age and sex differences, but Saccharibacteria might be a consistent signal and worth further investigation, considering the lack of research on this microbe. Saccharibacteria Childhood caries 16S rRNA Saliva Plaque Microbiota President Foundation of Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University2021A001 Oral Infectious Disease Mechanism Research and Clinical Translation Application Innovation team of Guangdong Province of China2021KCXTD033 Chinese Stomatological Association Dental caries prevention and treatment capacity improvement programCSA-ICP2022-03 This work was supported by grants from the President Foundation of Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University (grant no. 2021A001), the Oral Infectious Disease Mechanism Research and Clinical Translation Application Innovation team of Guangdong Province of China (grant no. 2021KCXTD033) and the Chinese Stomatological Association Dental caries prevention and treatment capacity improvement program (grant no. CSA-ICP2022-03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. ==== Body pmcIntroduction Childhood caries is the most common disease in children, affecting 0.5 billion children worldwide and leading to a heavy medical and economic burden (Global oral health status report: towards universal health coverage for oral health by 2030, WHO). Dental caries not only threaten the oral health of children but also affect the physical and mental health of children in severe cases (Jeffrey, 2016). According to the 4th National Oral Health Survey in mainland China (Wang, 2018), the prevalence of dental caries in children was 50.8%, 63.6% and 71.9% for 3-, 4- and 5-year-olds, respectively; the mean number of decayed, missing, or filled teeth (dmft) at 5 years old was 4.24, and the untreated rate was 96.0%. There is no doubt that the oral microbiota is the key pathogenic factor leading to dental caries, and many factors affect its development. The human oral cavity hosts a highly diverse bacterial community; however, only approximately half of oral microbes can be cultured (Paster et al., 2006). Furthermore, children’s transition from primary dentition to early mixed dentition begins when the children are 3 to 8 years old. The experience of caries in primary teeth can lead to a high risk of decay in permanent dentition (Zou et al., 2022). This critical period determines the oral health status in adulthood and is also a key period for the establishment of oral health concepts and behaviors. However, the flora was significantly different between individuals or in different regions of the same individual (Mark Welch, Ramírez-Puebla & Borisy, 2020), and different key bacteria have been observed in various studies, with the influencing factors behind the bacteria being of interest. Therefore, to study oral microbiota, it is necessary to understand their influencing factors. In microbiome research, population association analysis is usually used to obtain key bacteria, and then the causal relationship and interaction mechanism between them and diseases are explored. A balanced microbiota is the foundation of oral health, as dysbiosis causes tooth decay (Lamont, Koo & Hajishengallis, 2018). For the past few decades, acid-producing bacteria represented by Streptococcus mutans have been considered the main pathogenic bacteria involved in dental caries (Palmer et al., 2010), and most preventive measures and risk assessment methods against caries target Streptococcus mutans (Cugini et al., 2019; Zhang et al., 2021). In recent years, high-throughput sequencing has been used to study caries-associated microorganisms, and the sequencing results of the key oral bacteria for dental caries are often inconsistent. It has been found that S. mutans is not the dominant bacterium in plaque biofilm, as it is present at a low level in some caries patients and can also be detected in caries-free individuals (Dinis et al., 2022). Thus, other bacteria must be involved in the process of caries development. However, a growing body of evidence supports the view that S. mutans is not a single causative factor for caries (Kleinberg, 2002; Simón-Soro & Mira, 2015). In addition to S. mutans, other bacterial genera, including Lactobacillus, Actinomyces, and Veillonella, are thought to be involved in the occurrence and development of dental caries (Becker et al., 2002; Aas et al., 2008). Some bacteria, such as Veillonella, have been found to exhibit high relative abundance at all stages of dental caries (Aas et al., 2008), and they can be involved in acid production at high glucose levels (Bradshaw & Marsh, 1998). The relationship between bacterial flora and dental caries is complicated, and studies indicate that the population characteristics of key oral bacteria for dental caries remain unclear. Many studies have shown that dietary habits (Mahmoud et al., 2022), oral hygiene behaviors (Finlayson et al., 2019), genetics (Valles-Colomer et al., 2023), and the environment (Shaw et al., 2017) all affect children’s oral microbiota. Therefore, it is increasingly important to identify the key bacteria that can be used as targets for pathological analysis or targeted drug therapy to avoid the side effects of prolonged therapies with broad-spectrum drugs, both on the oral microbiota and at a systemic level (Mahendra et al., 2021). For children, the situation becomes more complicated due to tooth replacement. One possibility is that the key bacteria in children differ based on age, dentition and sex. The relationship between dental caries and bacterial flora may be a new target for dental caries risk assessment and intervention. Most previous studies involving dental caries in children have focused on the comparison of oral microbes between children with and without caries (Jiang et al., 2016; Kahharova et al., 2020; Qudeimat et al., 2021). However, there are few studies on the correlation between children with different caries severity. Dental plaque is the driving factor of caries, but the primary medium in the oral environment is saliva. It is often used as a sampling site for microbial detection because of the convenience of this analysis. Therefore, this experiment was designed to collect supragingival plaque and unstimulated saliva from children. In this cross-sectional study, we explored the relationship between caries severity and the oral microbiota. In total, 102 children with caries were included in this study, and high-throughput sequencing of supragingival plaque and unstimulated saliva samples from children with caries was used to analyze similarities and differences in key caries-related microbes according to the children’s age and sex. Materials and Methods Participants The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University (202201A). Study participants were recruited from the Department of Pediatric Dentistry in our hospital from March 2022 to September 2022 during their first visit. Patients who were scheduled to see a doctor at 9--11 am were selected for this study, and 127 children aged 3 to 8 years old were initially enrolled. After sample sequencing screening, donors with unqualified PCR and samples with less than 30,000 reads sequenced were excluded. Finally, 102 children remained. All parents or other legal guardians of the participating children signed the informed consent form. A telephone call was made the day before the first visit, and preliminary communication was made in accordance with the inclusion and exclusion criteria. Criteria Inclusion criteria were as follows: children aged 3–8 years old children with caries (ICDAS of any tooth surface ≥3) who have never been treated before children in the mixed dentition stage had no loss of deciduous teeth within 1 month children in the mixed dentition stage in whom the deciduous molars had not been replaced and without caries in the new permanent teeth lack of systemic diseases or congenital diseases no obvious active bacterial or viral infection informed consent was obtained from guardians. The exclusion criteria were as follows: use of antibiotics, probiotics, synbiotics or fluoride in the 3 months prior to the study developmental diseases of teeth extrinsic black tooth stain completion of orthodontic treatment within 6 months or undergoing orthodontic treatment mucosa or salivary system disease Sample collection According to the “Oral Microbiome: Methods and Protocols” (Hussein, 2021), on the day before the clinic visit, we communicated with participants’ guardians about their intention to participate by telephone. Participants were required to refrain from tooth brushing the night before and the morning on the day they visited and to fast and not drink within 2 h before arriving at the hospital. Supragingival plaque was assessed using a sterile dental excavator and sterile swabs to collect the plaque on the surface of teeth. The samples were placed into a 1.5 ml sterile microcentrifuge tube (FCT217; Beyotime, Jiangsu, China) containing 0.8 ml PBS (C0221A; Beyotime, Jiangsu, China). For the collection of unstimulated saliva, we allowed the patients to place a sterile cotton roll (Ruixue Medical Supplies, RX58, Shijiazhuang City, Hebei Province, China) under their tongue without moving, and then advised them to gently closed their mouths, lower their heads, rest, and avoid swallowing for 1 min; we then removed the cotton roll from the mouth with a dental tweezer and placed it into a 5 ml sterile microcentrifuge tube (FTUB020; Beyotime, Jiangsu, China). All the tubes were quickly placed in the freezer box of a foam incubator and stored in the refrigerator at −80 °C within 2 h until further processing. Clinical examination Oral examinations were performed by an experienced dentist. Community periodontal index (CPI) probes and mirrors under optimal light assisted by air water syringe conditions were available for all dental examinations. Oral examination of the patients was performed using the International Caries Detection and Assessment System (ICDAS) criteria (Ismail et al., 2007). We polished the teeth with a rubber cup first and then moistened and dried the teeth for 5 s. The visual examination was coded 0--6. 16S rRNA amplicon sequencing DNA was extracted using Mabio Bacterial DNA Extraction Mini Kits (Mabio Biotechnologies. Co., Ltd., Guangzhou, China) for the corresponding sample. The concentration and purity were measured using a NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) to test the concentration and purification of the final DNA. Primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the V3-V4 hypervariable regions of the bacterial 16S rRNA gene using the PCR instrument Bio-Rad S1000 (Bio-Rad Laboratory, Hercules, CA, USA). Data were collected as previously described in Chen et al. (2023). Following the manufacturer’s instructions, sequencing libraries were created using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA), and index codes were added. The Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific, MA, USA) was used to evaluate the library’s quality. On the Illumina Nova6000 platform, the library was finally sequenced, and 250 bp paired-end reads were produced. Statistical analysis After sequencing, 30,000 reads were set for data extraction, and the sample sequences were randomly selected to establish a uniform data volume to ensure the uniformity of the sample sequences. Bioinformatics analysis was conducted using QIIME2. DADA2 was used for sequence filtering, dereplication, sample inference, chimera identification, and merging of paired-end reads. All sequences were divided into amplicon sequence variants (ASVs) to reduce redundant computation during analysis. Microsoft Excel 2016 was used for two-person data entry, and then SPSS 25.0 software was used to analyze the demographic data and experimental data. The chi-square test was used to analyze count data, such as sex and caries prevalence. Measurement data, such as age, BMI, and mean number of caries, were expressed as the mean ± standard deviation, and a t-test was used for analysis. For sequencing data, a Kruskal‒Wallis nonparametric ANOVA calculated at the feature level was used to determine taxonomic group differences. For all statistical analyses, a p value of less than 0.05 or a q value of less than 0.25 indicated a significant difference. Results Study demographics and sequencing results The analyses included 102 children from 3 to 8 years of age that were caries-affected pediatric participants who lived in PingShan District, ShenZhen, with a mean ± SD age of 79.9 ± 21.47 months (Table 1). Each child contributed two samples from supragingival dental plaque and unstimulated saliva. Thus, a total of 204 samples were collected from these children. There were no significant differences in age, BMI, Hellman stage, number of decayed deciduous teeth or decayed deciduous teeth surface between the male and female groups. 10.7717/peerj.15605/table-1 Table 1 Demographic and clinical characteristics of the individuals. Total (n = 102) Male (n = 61, 59.8%) Female (n = 41, 40.2%) p-value* Age month (mean ± SD) 79.91 ± 21.47 80.75 ± 20.57 78.66 ± 22.95 0.562a BMI 15.62 ± 2.46 15.59 ± 2.36 15.66 ± 2.61 0.768a Age 0.711b Preschool 45 (44.1%) 26 19 Primary school 57 (55.9%) 35 22 Hellman stage 0.187b IIA 36 24 12 IIC 8 2 6 IIIA 55 33 22 IIIB 3 2 1 dt 0.688b 1--4 28 (27.45%) 15 13 5--8 43 (42.16%) 26 17 9--20 31 (30.39%) 20 11 ds 13.18 ± 10.44 13.85 ± 10.89 12.17 ± 9.79 0.534a Notes: Abbreviations: BMI, body mass index; dt, decayed deciduous teeth; ds, decayed deciduous teeth surface. a t-test. b Chi-Square. * p-value Comparison between sexes. A total of 24,274,860 high-quality 16S rRNA sequences were produced, with an average of 95,948 reads per sample. The number of ASVs was 10798. There were 1,849 ASVs in common between the two groups, 5,110 ASVs in the supragingival plaque group and 3,839 ASVs in the saliva group (Fig. S1). The rarefaction curves of all the samples trended flat at the end (Fig. S2). Sequences from these samples were classified into 28 phyla, 129 classes, 232 orders, 342 families, 467 genera, and 579 species. Microbiome profile in children with caries We assessed alpha diversity, which is indicative of species abundance within each group. There were significant differences between the plaque and saliva groups in terms of richness (observed OTUs, Kruskal‒Wallis, p < 0.001; Fig. 1A), alpha diversity (PD whole tree, Shannon index, Kruskal‒Wallis, p < 0.001; Fig. 1A), and beta diversity (Bray‒Curtis, PERMANOVA, p = 0.001; Fig. 1B). In saliva, the Shannon index values, which represent the richness and evenness of the distribution of the microbial community, were significantly lower than those in the plaque site (p < 0.001). Our findings suggest that the bacterial community in the plaque is more evenly distributed than that in saliva. 10.7717/peerj.15605/fig-1 Figure 1 Microbiota composition in the plaque (P) and saliva (S) groups. (A) Alpha diversity of the plaque and salivary microbiome among groups. Comparison of observed OTUs, PD whole tree and Shannon index; (B) A principle coordinate analysis (PCOA) plot was generated using ASV metrics based on beta diversity (Bray‒Curtis index) for different groups. PERMANOVA was used for these statistical comparisons between groups; (C and D) mean relative abundances of predominant phyla (C) and genera (D) in the two groups are displayed; (E) the linear discriminant analysis (LDA) effect size (LEfSe) method was performed to compare taxa between the plaque and saliva groups. The colored nodes from the inner to outer circles represent the most abundant taxa from the phylum to genus level. The children’s bacterial community was primarily composed of six phyla, namely Firmicutes, Proteobacteria, Actinobacteriota, Fusobacteriota, Bacteroidetes, and Saccharibacteria (formerly known as TM7) (Fig. 1C). Figure 1D depicts the top 10 prevalent bacterial genera. In both saliva and plaques groups, Streptococcus (P:13,51%; S:37.49%) was the most dominant genus, followed by Neisseria (P:14.47%; S:6.14%), Leptotrichia (P:9.88%; S:5.12%), Haemophilus (P:4.23%; S:7.30%), Fusobacterium (P:7.80%; S:2.63%), Corynebacterium (P:6.46%; S:2.45%), Actinomyces (P:6.46%; S:2.42%), Rothia (P:3.70%; S:2.37%), Veillonella (P:4.87%; S:1.43%), and Prevotella (P:4.23%; S:1.87%). Following this, we utilized LEfSe to identify the taxa that substantially varied across the samples for plaque and saliva, as depicted in Fig. 1E. As shown in supplementary, at the phylum level (Fig. S3A), Firmicutes was enriched in the saliva group, while Proteobacteria, Fusobacteria, and Actinobacteria were enriched in the plaque group. At the genus level (Fig. S3C), Streptococcus and Haemophilus were enriched in the saliva group, and Neisseria, Leptotrichia, Fusobacterium, Corynebacterium, Actinomyces, and Veillonella were rich in the plaque samples, and the differences were statistically significant (p < 0.05). The relationship between oral microbiota and dental caries in the overall population To investigate the correlation between dental caries and the microbiome in this study, we stipulated that the severity of caries was expressed in terms of the number of decayed teeth (dt), and then we plotted the correlation of dt with flora. In the plaque group, there was no significant difference in richness (observed OTUs, Kruskal‒Wallis, p = 0.657; Fig. 2A), alpha diversity (Shannon index, Kruskal‒Wallis, p = 0.624; Fig. 2A), or beta diversity (Bray‒Curtis, PERMANOVA, p = 0.055; Fig. 2B). In the saliva group, there was no significant difference in richness (observed OTUs, Kruskal‒Wallis, p = 0.785; Fig. 2C) or alpha diversity (Shannon index, Kruskal‒Wallis, p = 0.260; Fig. 2C), but there was a significant difference in beta diversity by executing PCoA in terms of Bray‒Curtis (PERMANOVA, p = 0.019; Fig. 2D). 10.7717/peerj.15605/fig-2 Figure 2 Scatter plot of bacterial diversity of plaque and saliva samples correlated with dt. (A and C) The observed and Shannon diversity of (A) plaque and (C) saliva samples correlated with dt using nonparametric Mann‒Whitney U tests; (B and D) For beta diversity, Bray‒Curtis distances were calculated, followed by PCoA. dt, decayed teeth. Then, we used MaAsLin2 to identify the specific bacteria that were most associated with dental caries. Table 2 shows all the microorganisms associated with dt. In both groups, Saccharibacteria were the most associated bacteria. In the plaque group, Acinetobacter was the only microbiome related to dt, while Actinomycetales and Clostridiales were related to dt in the saliva group. 10.7717/peerj.15605/table-2 Table 2 The bacteria correlated with dt in the plaque and saliva groups. Group Feature P S R 2 p q R 2 p q Both c__TM7.3 0.026 0.001 0.018 0.013 0.009 0.122 c__Clostridia 0.026 0.027 0.191 0.017 0.026 0.123 p__Saccharibacteria 0.026 0.001 0.012 0.013 0.009 0.078 P g__Acinetobacter −0.006 0.01 0.246 – – – S o__Actinomycetales – – – 0.039 0.019 0.185 o__Clostridiales – – – 0.017 0.026 0.185 c__Actinobacteria – – – 0.039 0.019 0.123 p__Actinobacteria – – – 0.039 0.019 0.087 Note: P, supringival plaque; S, unstimulated saliva. Community profiling at different ages associated with caries First, we distinguished plaque and saliva samples according to the participants’ age, so we obtained four subgroups: preschoolers’ plaque group, PP; schoolers’ plaque group, PS; preschoolers’ saliva group, SP; and schoolers’ saliva group, SS. The results in Figs. 3A and 3C show that, in the P and S groups, there were no significant differences in richness and evenness, but there was a significant difference in β diversity between different ages (Bray‒Curtis, PERMANOVA, p < 0.05, Figs. 3B and 3D). 10.7717/peerj.15605/fig-3 Figure 3 Diversity of dental plaque and saliva samples from children within different ages. (A and C) For alpha (within-sample) diversity, the observed OTUs and Shannon index were calculated according to (A) plaque samples and (C) saliva samples in both age groups. (Mann‒Whitney U tests were used for group comparisons); (B and D) For beta (between-sample) diversity, Bray‒Curtis distances were calculated, followed by PCoA. The plot shows the separation of (B) plaque samples and (D) saliva samples according to children’s age. In terms of the severity of caries, we plotted the correlation chart between different subgroups, as shown in Fig. 4. In the plaque group, only primary school children had a significant difference in beta diversity (Bray‒Curtis, PERMANOVA, p = 0.007; Fig. 4D). In the saliva group, the preschool children had a significant difference in beta diversity (Bray‒Curtis, PERMANOVA, p = 0.032; Fig. 4F). There were no significant differences in richness and evenness among the four groups. 10.7717/peerj.15605/fig-4 Figure 4 Scatter plot of bacterial diversity of plaque and saliva samples within different ages. The observed and Shannon diversity correlated with dt using nonparametric Mann‒Whitney U tests; For beta diversity, Bray‒Curtis distances were calculated, followed by PCoA. (A and B) Plaque Preschool; (C and D) Plaque Primary School; (E and F) Saliva Preschool; (G and H) Saliva Primary School. dt, decayed teeth. Community profiling in different sexes associated with caries We further analyzed differences in the diversity of bacterial flora in plaque and saliva between children of different sexes. All samples were categorized by sampling site and gender, and four subgroups were obtained: male plaque group, PM; female plaque group, PF; male saliva group, SM; and female saliva group, SF. The results in Figs. 5A and 5C show that in the P and S groups, there were no significant differences in α and β diversity between the different sexes (Fig. 5, p > 0.05). 10.7717/peerj.15605/fig-5 Figure 5 Bacterial diversity of dental plaque and saliva samples from children within females and males. (A and C) For alpha diversity (within-sample), the observed, Shannon diversity and richness measures were calculated according to (A) plaque samples and (C) salivary samples in both gender groups. Mann‒Whitney U tests were used for group comparisons; (B and D) For beta (between-sample) diversity, Bray‒Curtis distances were calculated, followed by PCoA. The plot shows the separation of (B) plaque samples and (D) saliva samples according to children’s gender. The core microbiome in preschoolers and schoolers is associated with decayed teeth Since there was no significant difference between females and males, we also intended to investigate the relationship between preschool and primary school children, so we used MaAsLin2 to identify the specific microbiome. In plaque, Saccharobacteria was the only phylum associated with caries severity in both subgroups, and Firmicutes was the key bacteria in the PP group; Clostridia, Bacilli and TM7.3 were the key bacteria in the PS group (Table 3). In the saliva group, no bacteria were the same in SP and SS; Gemellaceae, Micrococcaceae, and Lachnospiraceae were the key bacteria in the SP group; and Capnocytophaga and Aggregatibacter were the key bacteria in the SS group (Table 4). Differences between sexes are shown in the attached tables (Tables S1 and S2), and no bacteria were found in common between males and females. 10.7717/peerj.15605/table-3 Table 3 Plaque bacteria correlated with dt in the preschool and primary school groups. Group Feature PP PS R 2 p q R 2 p q Both p__Saccharibacteria 0.032 0.027 0.124 0.019 0.037 0.243 PP p__Firmicutes 0.040 0.003 0.031 – – – PS g__Paludibacter – – – −0.006 0.010 0.171 g__Porphyromonas – – – −0.015 0.012 0.171 g__Veillonella – – – 0.0420 0.009 0.171 g__Granulicatella_f__Carnobacteriaceae – – – −0.011 0.031 0.243 g__Moryella – – – 0.008 0.034 0.243 g__Actinobacillus – – – −0.012 0.032 0.243 f__Veillonellaceae – – – 0.044 0.006 0.111 f__Porphyromonadaceae – – – −0.014 0.015 0.177 f__Carnobacteriaceae – – – −0.011 0.031 0.248 o__Clostridiales – – – 0.048 0.003 0.073 c__Clostridia – – – 0.048 0.003 0.049 c__Bacilli – – – −0.035 0.047 0.189 c__TM7.3 – – – 0.019 0.037 0.189 Note: PP, Preschoolers’ supragingival plaque; PS, Schoolers’ supragingival plaque. 10.7717/peerj.15605/table-4 Table 4 The saliva bacteria correlated with dt in the preschool and primary school groups. Group Feature SP SS R 2 p q R 2 p q SP f__Gemellaceae −0.030 0.006 0.111 – – – f__Micrococcaceae 0.028 0.049 0.240 – – – f__Lachnospiraceae 0.011 0.032 0.240 – – – f__Cardiobacteriaceae 0.008 0.041 0.240 – – – o__Gemellales −0.030 0.006 0.062 – – – o__Actinomycetales 0.058 0.034 0.162 – – – o__Fusobacteriales 0.042 0.037 0.162 – – – o__Cardiobacteriales 0.008 0.041 0.162 – – – c__TM7.3 0.025 0.006 0.076 – – – c__Actinobacteria 0.058 0.034 0.120 – – – c__Bacilli −0.063 0.032 0.120 – – – c__Fusobacteriia 0.042 0.037 0.120 – – – p__Saccharibacteria 0.025 0.006 0.052 – – – p__Actinobacteria 0.058 0.034 0.111 – – – p__Fusobacteria 0.042 0.037 0.111 – – – SS g__Capnocytophaga – – – −0.012 0.025 0.228 g__Aggregatibacter – – – −0.020 0.021 0.228 c__Flavobacteriia – – – −0.034 0.046 0.226 c__BD1.5 – – – −0.002 0.025 0.226 p__GN02 – – – −0.002 0.025 0.218 p__SR1 – – – −0.004 0.048 0.218 Note: SP, Preschoolers’ unstimulated saliva; SS, Schoolers’ unstimulated saliva. According to the above results, we found that the age and severity of caries correlated significantly but that Saccharibacteria was the only type of bacteria associated with the severity of caries in both groups. The relative abundance of microbiome features was converted to units of log fold change, then we analyze the correlation between the abundances of Saccharibacteria and other microbial genera. We found that Saccharibacteria abundance was associated with the abundance of many species at the genus level. Among these, the abundance of Fusobacterium demonstrated the highest positive correlation (r = 0.586), while the abundance of Streptococcus showed the highest negative correlation (r = −0.593) with the levels of Saccharibacteria (Fig. S4). Discussion The present study revealed the similarity and differences in the caries-associated microbiota by different oral sampling sites and among subpopulations. The results of this experiment showed that different sampling sites and different ages affected the identification of key microorganisms in caries. This study found that Saccharibacteria and age group at different positions is the only consistent bacterial classification, suggesting that it may have value in future research. In addition, the relationship between oral flora and caries may have population heterogeneity, so it is necessary to consider the specific population characteristics in future research and transformation. Under normal physiological conditions, a dynamic balance is maintained between microorganisms and the host to jointly maintain the health of the host. However, the external environment or host factors can disrupt this balance, and some beneficial microorganisms become pathogenic, leading to dental caries, periodontal disease and other oral infectious diseases (Gao et al., 2018). A previous study suggested that the overall composition and proportion of microbes differ among the different regions of the oral cavity (Socransky & Manganiello, 1971; Gibbons & Houte, 1975). Unlike the microbiome of other parts of the body, the oral microbiome is considered to be highly diverse (Xiao, Fiscella & Gill, 2020). Although dental plaque, saliva, and buccal mucosa are in close contact, they have different microbial communities (Mark Welch, Ramírez-Puebla & Borisy, 2020). Hall et al. (2017) found significant differences in the microbial communities present in supragingival plaque patches, saliva, and tongue samples from healthy subjects, suggesting the presence of site-specific oral microbial communities. The Human Microbiome Project (HMP) compared microbial diversity across five major body regions in 242 healthy individuals, and the results showed that supragingival plaque had a higher bacterial alpha diversity than the oral mucosa (Human Microbiome Project Consortium, 2012), which is consistent with the results of the present study. Although saliva samples are easy to collect, distinctive taxonomic profiles between saliva and dental plaque have been observed in previous research (Cui et al., 2021; de Jesus et al., 2021). Our results showed that the microbial communities in supragingival plaque were significantly different in diversity from those in unstimulated saliva, and the microbial communities were more evenly distributed in supragingival plaque than in saliva. Plaques contain many microorganisms that closely adhere to the tooth surface and provide continuous nutrition for bacteria. The flow rate, viscosity, buffering capacity and remineralization capacity of saliva are important factors affecting caries (Kim et al., 2021), which regulate the progression and outcome of dental caries to some extent. By analyzing the correlation between the enriched groups and the number of carious teeth, we found that Saccharibacteria was the only bacterial group with an abundance that was most correlated with the severity of caries in both plaque and saliva, while other bacteria showed large differences, suggesting the heterogeneity of key bacteria across sites and the potential research value of Saccharibacteria. Saccharibacteria (formerly known as TM7) are widely present in a variety of habitats, and their physiological and ecological roles and pathogenic properties remain unknown due to the difficulty of obtaining them by traditional culture methods (McLean et al., 2020). In previous studies, Saccharibacteria were found only in peri-implantitis pockets of aggressive periodontitis patients, leading scientists to suspect that this group of bacteria could cause periodontal disease (Sousa et al., 2017). Baker, (2021) found that an increase in the levels of salivary immune markers (EGF, CSF2, IL13, etc.) has a similar trend with the increase in Saccharibacteria abundance. Genomic analysis revealed that a Saccharibacteria representative (RAAC3) lacks nucleotide, lipid and amino acid biosynthetic pathways, suggesting that Saccharibacteria may be auxotrophic and metabolically dependent on other organisms (Kantor et al., 2013). “Candidatus Saccharibacteria” was proposed as the new phylum name for Saccharibacteria based on genomic analysis, which suggested that these bacteria, which show reduced genomes, primarily consume sugar compounds (Bor et al., 2019). In 2015, He et al. (2015) first cultivated a TM7 phylotype (TM7x) from the human oral cavity. This microbe was cocultured with its bacterial host, Actinomyces odontolyticus subspecies actinosynbacter, XH001 (Bedree et al., 2018). The results of this study suggested the presence of a relationship between Saccharibacteria and dental caries, but still could not explain the role of Saccharibacteria in dental caries. However, we observed that the abundance of Saccharibacteria was most negatively correlated with Streptococcus abundance, suggesting that there may be some interaction between them that needs further investigation. A number of studies have confirmed that among Streptococcus spp, Streptococcus mutans and Streptococcus sobrinus are the main causative agents of human dental caries (Li, Wyllie & Jensen, 2021; Lemos et al., 2019; Philip, Suneja & Walsh, 2018). Saccharibacteria may be a protective factor in this population. A recent study showed that Saccharibacteria suppresses gingival inflammation and bone loss in mice and could protect mammalian hosts from inflammatory damage induced by bacteria in the host (Chipashvili et al., 2021). The latest research shows that environmentally-derived Saccharibacteria acquired an arginine deiminase system during their evolution and colonization in the human oral cavity; this system can metabolize arginine to provide energy (ATP) and protects Saccharibacteria and its host bacteria against the acidic environment in oral cavity (Tian et al., 2022). Acinetobacter was the key bacteria found in caries in the plaque group, while Actinomycetales and Clostridiales were the key bacteria found in caries in the saliva group. Clostridiales, a member of the “orange” complex in subgingival plaque, has traditionally been considered an important periodontal pathogen (Socransky et al., 1998). However, an increasing number of studies have noted its enrichment and predictive potential for ECC occurrence (Zhu et al., 2018; Chen et al., 2021). In the present study, the abundance of Clostridiales in saliva showed a significant positive correlation with dt, again highlighting its possible role in caries progression. A large number of studies have shown that the diversity of the oral bacterial community in children with caries is lower than that in children without caries, and caries-related differential flora, such as Streptococcus, Prevotella, Veillonella, Neisseria, and Rothia, have been found (de Jesus et al., 2021, 2020; Wang et al., 2019; Baker et al., 2021). This is similar in other inflammatory diseases in which the α diversity of the affected group is reduced (Chen et al., 2020). Previous studies have suggested that the predominance of S. mutans may reduce community diversity (Richards et al., 2017). Some scholars have speculated that S. mutans may compete with other oral commensal bacteria to disrupt the microbial balance through its strong capacity to produce acid, tolerance to acidic conditions and exopolysaccharide production (Zhang et al., 2022). In this study, due to the limitation of 16S sequencing at the species level, there was no in-depth analysis of which environments showed enrichment in S. mutans. However, it is certain that Streptococcus is the most abundant bacterium among different groups in the population of individuals with caries. We found that the key bacteria associated with the severity of caries varied with the age of the children. In this study, Saccharibacteria was found to be the only bacterial category in supragingival plaque that was associated with caries severity in children of all ages, suggesting that there may be age differences in caries prevention targets of the oral microbiota. Some scholars have shown that, among bacterial genera with high relative abundance, only TM7x is strongly correlated with the risk of caries (Kalpana et al., 2020; Baker et al., 2021). This finding is consistent with the results of this experiment. Conclusions In summary, our data indicated that, in a South China population, oral microbial signatures for dental caries show age and sex differences, but Saccharibacteria might be a consistent signal, which explains the inconsistency of results regarding the key bacteria reported in different studies. The identity of the key oral bacteria involved in caries remains unclear, and further population studies are needed to explore the potential influencing factors, which is in the beginning stages of research. However, this study is a cross-sectional survey, which can only reflect clues related to the etiology of dental caries and not the causal relationship. The relevant clues still need long-term and in-depth observation through cohort studies and omics studies, and the specific mechanism remains to be further explored. This study suggests possible target groups and locus heterogeneity and should, thus, be considered in future research and transformation according to the characteristics of the people in the corresponding design. Supplemental Information 10.7717/peerj.15605/supp-1 Supplemental Information 1 Venn diagram at ASVs level among plaque (P) and saliva (S) groups. Click here for additional data file. 10.7717/peerj.15605/supp-2 Supplemental Information 2 Rarefaction curve and rank abundance of the 204 samples, which are used to reflect the depth of sequencing. Click here for additional data file. 10.7717/peerj.15605/supp-3 Supplemental Information 3 Relative abundance of the 10 most predominant. (A) phyla, (B) class, and (C) genera among plaque (P) and saliva (S) groups. Click here for additional data file. 10.7717/peerj.15605/supp-4 Supplemental Information 4 Linear regression relationship between different bacterial genera and Saccharibacteria. Click here for additional data file. 10.7717/peerj.15605/supp-5 Supplemental Information 5 Plaque bacteria correlated with dt in the female and male groups. Click here for additional data file. 10.7717/peerj.15605/supp-6 Supplemental Information 6 The saliva bacteria correlated with dt in male group. Click here for additional data file. We would like to thank Guangdong MagiGene Technology Co., LTD for sample sequencing and AJE for English language editing. Additional Information and Declarations Competing Interests Author Contributions Human Ethics Data Availability The authors declare that they have no competing interests. Yang You conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft. Meixiang Yin analyzed the data, prepared figures and/or tables, and approved the final draft. Xiao Zheng performed the experiments, prepared figures and/or tables, and approved the final draft. Qiuying Liang performed the experiments, prepared figures and/or tables, and approved the final draft. Hui Zhang analyzed the data, prepared figures and/or tables, and approved the final draft. Bu-Ling Wu conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft. Wenan Xu conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft. The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The study was conducted in accordance with the Declaration of Helsinki, and approved by the Ethics Committee of Shenzhen Stomatology Hospital (Pingshan) of Southern Medical University (202201A). The following information was supplied regarding data availability: The 16S rRNA bacterial profiling data generated in this study are available at NCBI: PRJNA948117. ==== Refs References Aas et al. (2008) Aas JA Griffen AL Dardis SR Lee AM Olsen I Dewhirst FE Leys EJ Paster BJ Bacteria of dental caries in primary and permanent teeth in children and young adults Journal of Clinical Microbiology 2008 46 4 1407 1417 10.1128/JCM.01410-07 18216213 Baker (2021) Baker JL Complete genomes of Clade G6 Saccharibacteria suggest a divergent ecological niche and lifestyle mSphere 2021 6 4 e0053021 10.1128/mSphere.00530-21 34378983 Baker et al. (2021) Baker JL Morton JT Dinis M Alvarez R Tran NC Knight R Edlund A Deep metagenomics examines the oral microbiome during dental caries, revealing novel taxa and co-occurrences with host molecules Genome Research 2021 31 1 64 74 10.1101/gr.265645.120 33239396 Becker et al. (2002) Becker MR Paster BJ Leys EJ Moeschberger ML Kenyon SG Galvin JL Boches SK Dewhirst FE Griffen AL Molecular analysis of bacterial species associated with childhood caries Journal of Clinical Microbiology 2002 40 3 1001 1009 10.1128/JCM.40.3.1001-1009.2002 11880430 Bedree et al. (2018) Bedree JK Bor B Cen L Edlund A Lux R McLean JS Shi W He X Quorum sensing modulates the epibiotic-parasitic relationship between Actinomyces odontolyticus and its Saccharibacteria epibiont, a Nanosynbacter lyticus strain, TM7x Frontiers in Microbiology 2018 9 2049 10.3389/fmicb.2018.02049 30319555 Bor et al. (2019) Bor B Bedree JK Shi W McLean JS He X Saccharibacteria (TM7) in the human oral microbiome Journal of Dental Research 2019 98 5 500 509 10.1177/0022034519831671 30894042 Bradshaw & Marsh (1998) Bradshaw DJ Marsh PD Analysis of pH-driven disruption of oral microbial communities in vitro Caries Research 1998 32 6 456 462 10.1159/000016487 9745120 Chen et al. (2021) Chen Y Dou G Wang D Yang J Zhang Y Garnett JA Chen Y Wang Y Xia B Comparative microbial profiles of caries and black extrinsic tooth stain in primary dentition Caries Research 2021 55 4 310 321 10.1159/000517006 34247164 Chen et al. (2020) Chen X Li P Liu M Zheng H He Y Chen M-X Tang W Yue X Huang Y Zhuang L Wang Z Zhong M Ke G Hu H Feng Y Chen Y Yu Y Zhou H Huang L Gut dysbiosis induces the development of pre-eclampsia through bacterial translocation Gut 2020 69 3 513 522 10.1136/gutjnl-2019-319101 31900289 Chen et al. (2023) Chen J Xu J Sun Y Xue Y Zhao Y Yang D Li S Zhao X Gut microbiota dysbiosis ameliorates in LNK-deficient mouse models with obesity-induced insulin resistance improvement Journal of Clinical Medicine 2023 12 5 1767 10.3390/jcm12051767 36902554 Chipashvili et al. (2021) Chipashvili O Utter DR Bedree JK Ma Y Schulte F Mascarin G Alayyoubi Y Chouhan D Hardt M Bidlack F Hasturk H He X McLean JS Bor B Episymbiotic Saccharibacteria suppresses gingival inflammation and bone loss in mice through host bacterial modulation Cell Host & Microbe 2021 29 11 1649 1662.e7 10.1016/j.chom.2021.09.009 34637779 Cugini et al. (2019) Cugini C Shanmugam M Landge N Ramasubbu N The role of Exopolysaccharides in oral biofilms Journal of Dental Research 2019 98 7 739 745 10.1177/0022034519845001 31009580 Cui et al. (2021) Cui Y Wang Y Zhang Y Pang L Zhou Y Lin H Tao Y Oral mycobiome differences in various spatial niches with and without severe early childhood caries Frontiers in Pediatrics 2021 9 748656 10.3389/fped.2021.748656 34869106 de Jesus et al. (2021) de Jesus VC Khan MW Mittermuller B-A Duan K Hu P Schroth RJ Chelikani P Characterization of Supragingival plaque and oral swab microbiomes in children with severe early childhood caries Frontiers in Microbiology 2021 12 683685 10.3389/fmicb.2021.683685 34248903 de Jesus et al. (2020) de Jesus VC Shikder R Oryniak D Mann K Alamri A Mittermuller B Duan K Hu P Schroth RJ Chelikani P Sex-based diverse plaque microbiota in children with severe caries Journal of Dental Research 2020 99 6 703 712 10.1177/0022034520908595 32109360 Dinis et al. (2022) Dinis M Traynor W Agnello M Sim M-S He X Shi W Lux R Tran NC Tooth-specific Streptococcus mutans distribution and associated microbiome Microorganisms 2022 10 6 1129 10.3390/microorganisms10061129 35744648 Finlayson et al. (2019) Finlayson TL Cabudol M Liu JX Garza JR Gansky SA Ramos-Gomez F A qualitative study of the multi-level influences on oral hygiene practices for young children in an Early Head Start program BMC Oral Health 2019 19 1 166 10.1186/s12903-019-0857-7 31349826 Gao et al. (2018) Gao L Xu T Huang G Jiang S Gu Y Chen F Oral microbiomes: more and more importance in oral cavity and whole body Protein & Cell 2018 9 5 488 500 10.1007/s13238-018-0548-1 29736705 Gibbons & Houte (1975) Gibbons RJ Houte JV Bacterial adherence in oral microbial ecology Annual Review of Microbiology 1975 29 1 19 44 10.1146/annurev.mi.29.100175.000315 Hall et al. (2017) Hall MW Singh N Ng KF Lam DK Goldberg MB Tenenbaum HC Neufeld JD Beiko G Senadheera DB Inter-personal diversity and temporal dynamics of dental, tongue, and salivary microbiota in the healthy oral cavity NPJ Biofilms and Microbiomes 2017 3 1 2 10.1038/s41522-016-0011-0 28649403 He et al. (2015) He X McLean JS Edlund A Yooseph S Hall AP Liu S-Y Dorrestein PC Esquenazi E Hunter RC Cheng G Nelson KE Lux R Shi W Cultivation of a human-associated TM7 phylotype reveals a reduced genome and epibiotic parasitic lifestyle Proceedings of the National Academy of Sciences of the United States of America 2015 112 1 244 249 10.1073/pnas.1419038112 25535390 Hussein (2021) Hussein H Adami GR Oral Sampling Techniques The Oral Microbiome: Methods and Protocols. Methods in Molecular Biology 2021 New York, NY Springer US 17 29 Human Microbiome Project Consortium (2012) Human Microbiome Project Consortium Structure, function and diversity of the healthy human microbiome Nature 2012 486 7402 207 214 10.1038/nature11234 22699609 Ismail et al. (2007) Ismail AI Sohn W Tellez M Amaya A Sen A Hasson H Pitts NB The International Caries Detection and Assessment System (ICDAS): an integrated system for measuring dental caries Community Dentistry and Oral Epidemiology 2007 35 3 170 178 10.1111/j.1600-0528.2007.00347.x 17518963 Jeffrey (2016) Jeffrey AD McDonald and Avery’s Dentistry for the child and adolescent 2016 10th Edition Amsterdam Elsevier Health Jiang et al. (2016) Jiang S Gao X Jin L Lo ECM Salivary microbiome diversity in caries-free and caries-affected children International Journal of Molecular Sciences 2016 17 12 1978 10.3390/ijms17121978 27898021 Kahharova et al. (2020) Kahharova D Brandt BW Buijs MJ Peters M Jackson R Eckert G Katz B Keels MA Levy SM Fontana M Zaura E Maturation of the oral microbiome in caries-free toddlers: a longitudinal study Journal of Dental Research 2020 99 2 159 167 10.1177/0022034519889015 31771395 Kalpana et al. (2020) Kalpana B Prabhu P Bhat AH Senthilkumar A Arun RP Asokan S Gunthe SS Verma RS Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India Scientific Reports 2020 10 1 21248 10.1038/s41598-020-78057-z 33277566 Kantor et al. (2013) Kantor RS Wrighton KC Handley KM Sharon I Hug LA Castelle CJ Thomas BC Banfield JF Small genomes and sparse metabolisms of sediment-associated bacteria from four candidate phyla mBio 2013 4 5 e00708 00713 10.1128/mBio.00708-13 24149512 Kim et al. (2021) Kim J-H Kim M-A Chae YK Nam OH Salivary characteristics, individual casual parameters, and their relationships with the significant caries index among Korean children aged 12 years International Journal of Environmental Research and Public Health 2021 18 6 3118 10.3390/ijerph18063118 33803534 Kleinberg (2002) Kleinberg I A mixed-bacteria ecological approach to understanding the role of the oral bacteria in dental caries causation: an alternative to Streptococcus mutans and the specific-plaque hypothesis Critical Reviews in Oral Biology and Medicine: An Official Publication of the American Association of Oral Biologists 2002 13 2 108 125 10.1177/154411130201300202 12097354 Lamont, Koo & Hajishengallis (2018) Lamont RJ Koo H Hajishengallis G The oral microbiota: dynamic communities and host interactions Nature Reviews Microbiology 2018 16 12 745 759 10.1038/s41579-018-0089-x 30301974 Lemos et al. (2019) Lemos JA Palmer SR Zeng L Wen ZT Kajfasz JK Freires IA Abranches J Brady LJ The biology of Streptococcus mutans Microbiology Spectrum 2019 7 1 10.1128/microbiolspec.GPP3-0051-2018 Li, Wyllie & Jensen (2021) Li JW Wyllie RM Jensen PA A novel competence pathway in the oral pathogen Streptococcus sobrinus Journal of Dental Research 2021 100 5 542 548 10.1177/0022034520979150 33876976 Mahendra et al. (2021) Mahendra J Mahendra L Mugri MH Sayed ME Bhandi S Alshahrani RT Balaji TM Varadarajan S Tanneeru S Rao ANP Srinivasan S Reda R Testarelli L Patil S Role of periodontal bacteria, viruses, and placental mir155 in chronic periodontitis and Preeclampsia—A genetic microbiological study Current Issues in Molecular Biology 2021 43 2 831 844 10.3390/cimb43020060 34449559 Mahmoud et al. (2022) Mahmoud SA El Moshy S Rady D Radwan IA Abbass MMS Al Jawaldeh A The effect of unhealthy dietary habits on the incidence of dental caries and overweight/obesity among Egyptian school children (A cross-sectional study) Frontiers in Public Health 2022 10 953545 10.3389/fpubh.2022.953545 36052005 Mark Welch, Ramírez-Puebla & Borisy (2020) Mark Welch JL Ramírez-Puebla ST Borisy GG Oral microbiome geography: micron-scale habitat and niche Cell Host & Microbe 2020 28 2 160 168 10.1016/j.chom.2020.07.009 32791109 McLean et al. (2020) McLean JS Bor B Kerns KA Liu Q To TT Solden L Hendrickson EL Wrighton K Shi W He X Acquisition and adaptation of ultra-small parasitic reduced genome bacteria to mammalian hosts Cell Reports 2020 32 3 107939 10.1016/j.celrep.2020.107939 32698001 Palmer et al. (2010) Palmer CA Kent R Loo CY Hughes CV Stutius E Pradhan N Dahlan M Kanasi E Arevalo Vasquez SS Tanner ACR Diet and caries-associated bacteria in severe early childhood caries Journal of Dental Research 2010 89 11 1224 1229 10.1177/0022034510376543 20858780 Paster et al. (2006) Paster BJ Olsen I Aas JA Dewhirst FE The breadth of bacterial diversity in the human periodontal pocket and other oral sites Periodontology 2006 42 1 80 87 10.1111/j.1600-0757.2006.00174.x Philip, Suneja & Walsh (2018) Philip N Suneja B Walsh L Beyond Streptococcus mutans: clinical implications of the evolving dental caries aetiological paradigms and its associated microbiome British Dental Journal 2018 224 4 219 225 10.1038/sj.bdj.2018.81 29449651 Qudeimat et al. (2021) Qudeimat MA Alyahya A Karched M Behbehani J Salako NO Dental plaque microbiota profiles of children with caries-free and caries-active dentition Journal of Dentistry 2021 104 6 103539 10.1016/j.jdent.2020.103539 33248211 Richards et al. (2017) Richards VP Alvarez AJ Luce AR Bedenbaugh M Mitchell ML Burne RA Nascimento MM Microbiomes of site-specific dental plaques from children with different caries status Infection and Immunity 2017 85 8 e00106–17 10.1128/IAI.00106-17 28507066 Shaw et al. (2017) Shaw L Ribeiro ALR Levine AP Pontikos N Balloux F Segal AW Roberts AP Smith AM The human salivary microbiome is shaped by shared environment rather than genetics: evidence from a large family of closely related individuals mBio 2017 8 5 e01237–17 10.1128/mBio.01237-17 28900019 Simón-Soro & Mira (2015) Simón-Soro A Mira A Solving the etiology of dental caries Trends in Microbiology 2015 23 2 76 82 10.1016/j.tim.2014.10.010 25435135 Socransky et al. (1998) Socransky SS Haffajee AD Cugini MA Smith C Kent RL Microbial complexes in subgingival plaque Journal of Clinical Periodontology 1998 25 2 134 144 10.1111/j.1600-051X.1998.tb02419.x 9495612 Socransky & Manganiello (1971) Socransky SS Manganiello SD The oral microbiota of man from birth to senility Journal of Periodontology 1971 42 8 485 496 10.1902/jop.1971.42.8.485 4998039 Sousa et al. (2017) Sousa V Nibali L Spratt D Dopico J Mardas N Petrie A Donos N Peri-implant and periodontal microbiome diversity in aggressive periodontitis patients: a pilot study Clinical Oral Implants Research 2017 28 5 558 570 10.1111/clr.12834 27170047 Tian et al. (2022) Tian J Utter DR Cen L Dong P-T Shi W Bor B Qin M McLean JS He X Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment Proceedings of the National Academy of Sciences of the United States of America 2022 119 2 e2114909119 10.1073/pnas.2114909119 34992141 Valles-Colomer et al. (2023) Valles-Colomer M Blanco-Míguez A Manghi P Asnicar F Dubois L Golzato D Armanini F Cumbo F Huang KD Manara S Masetti G Pinto F Piperni E Punčochář M Ricci L Zolfo M Farrant O Goncalves A Selma-Royo M Binetti AG Becerra JE Han B Lusingu J Amuasi J Amoroso L Visconti A Steves CM Falchi M Filosi M Tett A Last A Xu Q Qin N Qin H May J Eibach D Corrias MV Ponzoni M Pasolli E Spector TD Domenici E Collado MC Segata N The person-to-person transmission landscape of the gut and oral microbiomes Nature 2023 614 7946 125 135 10.1038/s41586-022-05620-1 36653448 Wang (2018) Wang X The 4th national oral health survey in the mainland of China 2018 Beijing People’s Medical Publishing House Co. Ltd Wang et al. (2019) Wang Y Wang S Wu C Chen X Duan Z Xu Q Jiang W Xu L Wang T Su L Wang Y Chen Y Zhang J Huang Y Tong S Zhou C Deng S Qin N Oral microbiome alterations associated with early childhood caries highlight the importance of carbohydrate metabolic activities mSystems 2019 4 e00450–19 10.1128/mSystems.00450-19 31690590 Xiao, Fiscella & Gill (2020) Xiao J Fiscella KA Gill SR Oral microbiome: possible harbinger for children’s health International Journal of Oral Science 2020 12 1 12 10.1038/s41368-020-0082-x 32350240 Zhang et al. (2022) Zhang Y Fang J Yang J Gao X Dong L Zheng X Sun L Xia B Zhao N Ma Z Wang Y Streptococcus mutans-associated bacteria in dental plaque of severe early childhood caries Journal of Oral Microbiology 2022 14 1 2046309 10.1080/20002297.2022.2046309 35251525 Zhang et al. (2021) Zhang Q Ma Q Wang Y Wu H Zou J Molecular mechanisms of inhibiting glucosyltransferases for biofilm formation in Streptococcus mutans International Journal of Oral Science 2021 13 1 30 10.1038/s41368-021-00137-1 34588414 Zhu et al. (2018) Zhu C Yuan C Ao S Shi X Chen F Sun X Zheng S The predictive potentiality of salivary microbiome for the recurrence of early childhood caries Frontiers in Cellular and Infection Microbiology 2018 8 423 10.3389/fcimb.2018.00423 30619773 Zou et al. (2022) Zou J Du Q Ge L Wang J Wang X Li Y Song G Zhao W Chen X Jiang B Mei Y Huang Y Deng S Zhang H Li Y Zhou X Expert consensus on early childhood caries management International Journal of Oral Science 2022 14 1 35 10.1038/s41368-022-00186-0 35835750