*3.3. Core Microbiota Enhanced the Enamel Demineralization In Vitro*

To evaluate the demineralization ability of different biofilms, we harvested the biofilm demineralized enamel blocks after 72 h of treatment and tested them using transverse microradiography (TMR), which is the gold standard for detecting demineralization. The Core + *S. mutans* biofilm had the highest demineralization degree, followed by the *S. mutans* group biofilms, and the Core group had the lowest demineralization degree (Figure 3a). The integrated mineral loss was 2895 (±345.25) Vol%μm for the Core + *S. mutans* group; 1831.82 (±315.50) Vol%μm for the *S. mutans* group and 1077.78 (±175.98) Vol%μm for the Core group (Figure 3b). The demineralized depth was 72.05 (±7.18) μm for the Core + *S. mutans* group; 60.62 (±9.70) μm for the *S. mutans* group, and 53.74 (±10.43) μm for the Core group (Figure 3c). All three groups of biofilms exhibited demineralization ability on enamel and the Core + *S. mutans* biofilm had both the deepest demineralization depth and

the most integrated mineral loss (*p* < 0.01). These results showed that the core microbiota promoted the enamel demineralization of *S. mutans* biofilm in vitro.

**Figure 3.** Enamel demineralization ability of the biofilms (n = 3). (**a**) Demineralization of the 72 h biofilm treated enamel slides. (**b**) Quantification of the integrated mineral loss. (**c**) Quantification of the demineralized depth. \*\* *p* < 0.01.

## *3.4. The Core Microbiota Increased Cariogenic Potential in a Rat Model*

To explore whether the core microbiota would induce dental caries in vivo, we conducted the caries model on 20-day-old Wistar rats. For a better colonization of our selected bacteria, antibiotics were given by oral administration to eliminate the host microbiota. The bacteria were given for the first continuous seven days and the Keyes 2000 diet was fed to the rats for the whole experiment. These rats were sacrificed for Keyes scoring three weeks after bacterial challenge. As shown in Figure 4, significantly more carious lesions including enamel (E), moderate dentinal (Dm), and proximal-surface carious lesions were observed in the Core + *S. mutans* group compared with the *S. mutans* group.

**Figure 4.** In vivo cariogenesis ability of different biofilms. (**a**) Representative hemisectioned molars in each group under stereoscopic microscopy. (**b**) Caries score by Keyes' method (n = 5). E = Enamel caries; Ds = slight dentinal caries; Dm = moderate dentinal caries; Dx = extensive dentinal caries. The severity decreased in the order of Core + *S. mutans*, *S. mutans*, Core and control groups. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.

#### **4. Discussion**

Dental caries including ECC, is a multifactorial disease [37,38]. Microbes are considered as one of the main pathogenic factors of ECC. A large amount of evidence proves that there might be a core microbiome of diseases in the oral cavity [39,40]. This also partially explains why some people are susceptible to caries while others are not. However, most of the literature have inferred the existence of core microbiome by sequencing and have found that tremendous species and genera were related to caries but failed to construct models to verify the casual relationship between dental caries and the core microbiota. A longitudinal study further narrowed the core microbiome and demonstrated that the core microbiota played an important role in ECC [13]. Referring to this literature, for the first time, we developed a core microbiota biofilm model containing four representative bacteria of ECC during its occurrence and development, which proved to promote the progress of dental caries both in vitro and in vivo.

Many studies have also shown that the four microorganisms we selected including *V. parvula, F. nucleatum, P. denticola,* and *L. wadei* are closely related to caries, especially ECC. In our study, we found that after 24 h, 48 h, and 72 h of culture, *V. parvula* accounted for more than half of the core microbial biofilm. Similarly, a large number of studies have proven that *Veillonella* content in ECC is higher than that in non-caries children, suggesting that it is closely related to ECC [13,41–44]. In another study, *Fusobacterium* was found to be significantly higher in ECC [45], in which *F. nucleatum* is closely associated with ECC, especially severe ECC [43,46]. Our study found that the amount of *F. nucleatum* in the biofilm at 72 h was about twice that at 24 h in the Core + *S. mutans* biofilm, which also seems to suggest that *F. nucleatum* is more related to severe ECC. Meanwhile, with the extension of the Core + *S. mutans* biofilm culture time, the content of *P. denticola* also increased. *P. denticola* was found to be one of the most prevalent species in the S-ECC, shown to be associated with dental caries. There is also speculation that the proportion of *P. denticola* would reduce after comprehensive restorative and preventive dental treatment [47,48]. Our results showed that when *S. mutans* was added, the content of *L. wadei* increased at the early stage of biofilm formation, which implied that *S. mutans* might promote the growth of *L. wadei. L. wadei* was found to be overrepresented in caries-active dental plaque compared to caries-free [49]. Moreover, a large number of *Fusobacterium*, *Prevotella*, and *Leptotrichia* were observed in a group of children with active caries [13,43,50]. In summary, these previous studies proved the rationality of our choice of four strains as core microbiota.

We found that the core microbiota had the effects of promoting the growth of *S. mutans*. There have been some controversies about the effect of *V. parvula* on *S. mutans*. Some

previous studies have shown that *V. parvula* might contribute to acid production and the growth of *S. mutans* [43,51,52]. However, it has also been found that *V. parvula* had little effect on the *S. mutans* growth when co-cultured with *S. mutans* and *Streptococcus gordonii* compared with *S. mutans* alone [53]. There might be a certain relationship between *V. parvula* and *S. mutans*, but the impact of *V. parvula* in the Core microbiota on *S. mutans* remains to be studied. Similarly, *F. nucleatum* is also one of the first Gram-negative species to establish plaque biofilms [54]. It is one of the important "bridge" organisms in the naturally formed dental plaque [55], and has a central role in the ecology of dental plaque [56]. *F. nucleatum* has also been studied with the ability of affecting the growth and survival of *S. mutans*. However, the effect of core microbiota on *S. mutans* has not been previously studied. Our results showed that when the core microbiota presented, the growth of *S. mutans* increased by about four times, suggesting that the core microbiota promoted the *S. mutans* growth in the biofilms. In this study, we did not validate the function of these four species individually, but considered them as one factor. This is a limitation of our study, and we will further investigate the function of these individual species in our future studies.

The microbial composition of mature tooth biofilm is quite stable, but the pH of the biofilm could have small fluctuations [57]. A lower pH value is favored by demineralization, while a higher pH value is good for remineralization [9]. The fine-tuned balance of the oral ecosystem is disrupted, allowing disease-promoting bacteria to over grow and cause dental caries [8,37,58]. Interestingly, in our experiments, we found that the core microbiota itself produced limited acid, but could promote acid production of *S. mutans*. The integrated mineral loss of the enamel block increased by about 60% when the core microbiota existed. However, the environment in the oral cavity is very complicated, and saliva washing and food intake may have an impact on dental plaque. In vitro experiments cannot fully reproduce the complexity of in situ situation [59]. We therefore established an animal model to determine the role of the core microbiota with the presence of acidogenic and aciduric bacteria *S. mutans* and sugar. We found that rats in the Core + *S. mutans* group had the most severe dental caries.

#### **5. Conclusions**

In conclusion, we obtained core microbiota in ECC from a previous clinical research and validated its cariogenesis effects in vivo and in vitro. Subsequent clinical verification is needed in future investigations. The core microbiota containing four representative strains of ECC could promote the growth of *S. mutans*, acid production, demineralization, and the development of caries in rat (graphical abstract). This also brings new ideas and challenges to the prevention and treatment of ECC. However, how core microorganisms interact with *S. mutans* and promote the development of dental caries remain to be further studied.

**Author Contributions:** Conceptualization, J.C., L.K. and X.P.; Methodology, J.C., L.K., X.P. and Y.C.; Software, J.C., L.K. and X.P.; Validation, M.L. and J.L.; Formal analysis, J.C., L.K. and X.P.; Investigation, J.C., L.K. and X.P.; Resources, B.R.; Data curation, B.R., M.L. and J.L.; Writing—original draft preparation, J.C., L.K. and X.P.; Writing—review and editing, X.Z. and L.C.; Visualization, J.C., L.K. and X.P.; Supervision, X.Z. and L.C.; Project administration, X.Z. and L.C.; Funding acquisition, X.Z. and L.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the National Natural Science Foundation of China [81870759 (LC) 81430011(XZ)], the Youth Grant of the Science and Technology Department of Sichuan Province, China [2017JQ0028 (LC)], and Innovative Research Team Program of Sichuan Province (L.C). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the West China School of Stomatology Institute Review Board (WCSHIRB) ethics committee on 25 May 2017, and the record number was WCHSIRB-D-2017-115.

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** Our thanks go to the State Key Laboratory of Oral Diseases, West China School of Stomatology of Sichuan University for their support throughout the research project.

**Conflicts of Interest:** The authors declare no conflict of interest.
