Dental caries remains a global public health challenge, characterized by high prevalence and increasing severity, particularly for the low income families (Islam et al., 2007, Mathur and Dhillon, 2018). It is a microbially induced, chronic oral infectious disease, which eventually leads to localized destruction of teeth hard tissues through complex interactions between oral bacteria and dietary carbohydrates over time (Mathur and Dhillon, 2018, Opal et al., 2015, Selwitz et al., 2007). Therefore, it is of great importance to explore the pathogenesis microbiology and mechanism of caries and then find new avenues for caries prevention. Among this, Streptococcus mutans is considered as one of the main etiological agents of dental caries and also the key component of dental plaque, due to its virulence properties associated with cariogenicity such as acidogenicity, acid tolerance, adhesion and synthesis of intracellular and extracellular polysaccharides (Chen and Lin, 2022, Gabe et al., 2020, Krzysciak et al., 2014, Lemos et al., 2019). Moreover, human oral system is a complex micro-ecological environment. Most pathogenic microbes in oral cavity prefer to grow in the form of biofilms, which are orientated aggregations of microorganisms that tend to attach to each other or colonize teeth surface and are enmeshed in extracellular polymeric substance (EPS) excreted by themselves (Hibbing et al., 2010, Huang et al., 2011, Jiang et al., 2017). With the advancement of microbial technology, more and more microbial members potentially associated with dental caries have been discovered and attracted attention (Huang et al., 2014, Hurley et al., 2019, Kameda et al., 2020, Prabhu Matondkar et al., 2020).

Our preliminary studies focused on profiling of oral microbiota in caries-affected individuals based on cross-sectionally and longitudinally designed studies (Sun et al., 2018, Teng et al., 2015, Yang et al., 2016, Yang et al., 2012, Zhang et al., 2020, Zhang et al., 2021). Initially, in cross-sectional studies, we explored difference of the organismal lineage of oral microbiota in both caries-active and healthy individuals by pyrosequencing. Results demonstrated relatively elevated abundance of Prevotella species in severe early childhood caries (S-ECC) or caries-active adults (Sun et al., 2018, Yang et al., 2012). Moreover, to observe the temporal variation of the oral microbiota during ECC onset or transition, oral microbiota of 50 4-year-old children were tracked for up to 2 years. We found that only eight Prevotella species (P. denticola, P. pallens, P. verovalis, P. salivae, P. histicola, P. DO039 and P. maculosa, P. loescheii) could construct a caries risk assessment model with an accuracy of 74 % (Teng et al., 2015). Furthermore, based on the relative abundance of Prevotella denticola and S. mutans, a caries risk assessment model was proposed with an accuracy of 72 % in predicting caries sensitivity (Teng et al., 2015, Zhang et al., 2020). In addition, other research groups also reported the close association between Prevotella and caries by characterizing the oral microbiota in childhood caries or dynamic shift of microbial composition during disease transition (Grier et al., 2021, Hurley et al., 2019, Johansson et al., 2016, Kressirer et al., 2018, Wang et al., 2017, Xu et al., 2018).

Prevotella is a gram-negative obligate anaerobic coccus or short bacillus, inhabiting various body sites including oral cavity, gastrointestinal tract and vagina (Tett et al., 2021). Some studies have reported that Prevotella spp. have a strong correlation with the formation of dental plaque. In the early and middle stages of biofilm formation, Prevotella spp. could promote the adhesion or aggregation of microbes, forming highly structured and organized polymicrobial biofilms (Nadkarni et al., 2004, Simon-Soro et al., 2014, Takahashi and Yamada, 2000). However, researchers also reported that Prevotella lacked acidogenicity properties associated with cariogenicity (Takahashi & Nyvad, 2011). Till now, the study on how P. denticola involved and its corresponding mechanism on cariogenicity characteristics in the development of caries has not been reported yet. Here, we hypothesized that interactions between P. denticola and S. mutans may promote the establishment of hypervirulent biofilms on teeth surface and eventually influence the occurrence and development of caries. In this study, based on single-species biofilms of either P. denticola or S. mutans, and dual-species biofilms of both bacteria, we compared the virulence properties associated with cariogenicity such as carbohydrate metabolism and acidogenicity, synthesis of extracellular polysaccharides, biomass and architecture of biofilms and degree of enamel demineralization, to investigate the effect of symbiotic relationship between P. denticola and S. mutans on the virulence of plaque biofilms.