Research On Difference Of Salivary Biochemical Indices And Microbiome Structure Associated With Early Childhood Caries

Background:Early childhood caries（ECC）is a common chronic infectious disease.According to the fourth oral epidemiological survey of China,the prevalence of dental caries among 3-,4-,and 5-year-old children is 50.8%,63.6%,and 71.9%.The damage to the teeth of ECC is irreversible and it can cause acute pain,tooth loss and even affect the quality of children’s life.Therefore,improving the early diagnosis and preventive measures of ECC has specific clinical significance.Saliva is the partial microenvironment for the occurrence of dental caries,and the salivary microbiome and biochemical components of saliva are closely related to the progress of dental caries.Therefore,this study aims to explore the significance of salivary biochemical indicators and microbial components in diagnosing and predicting dental caries.Objective:This study aims to evaluate the differences in saliva biochemical indicators（p H,total protein,the concentration of 12 electrolytes）,microbial diversity,and community structure between children aged 4-6 suffering from dental caries and healthy children.Meanwhile,to explore the correlation between saliva biochemical indicators and microbiome.Finally,establish diagnosis models for ECC and provide the theoretical basis for individualized prevention,treatment,and risk assessment of ECC.Methods:Unstimulated saliva samples were collected from 331 children aged 4 to 6,including166 healthy children（H group）and 165 children with ECC（C group）.The p H value was measured by the p H meter and the total protein was measured by the microplate reader.The concentrations of 12 electrolytes,including Na⁺,K⁺,Ca²⁺,Mg²⁺,NH₄⁺,F^-,Br^-,Cl^-,NO₃^-,NO2^-,SO4^2-,PO4^3-were determined by the ion chromatography,and these biochemical indices between the two groups were compared.At the same time,the saliva microbiome was analyzed by 16S r RNA gene sequencing to identify potential cariogenic markers and explore the correlation between microbiome and saliva biochemical indicators.Finally,we constructed diagnosis models of ECC based on the selected salivary biochemical components and microbiome.Results:（1）Through analyzing the salivary biochemical indicators of children,we found that the concentrations of K⁺,Cl^-,NH₄⁺,Na⁺,SO₄^2-,Ca²⁺,Mg²⁺,and Br^-were significantly higher in the saliva of children with ECC compared with healthy children,while the p H value and NO₃^-concentration was significantly reduced in the children suffering from caries（p（27）0.05）.Among them,the concentrations of Cl^-,Br^-,Mg²⁺,Ca²⁺,NH₄⁺were positively correlated with the number of decayed,missing,and filling teeth（dmft index）with significance,while p H value and NO₃^-concentration was negatively correlated with the dmft index in deciduous dentition（p（27）0.05）.（2）Through the analysis of indices of saliva microbiome richness（Chao1）and microbiome diversity（Shannon and Simpson）,it was found that the microbiome richness and diversity indices of the C groups were higher than that of the H group with significance（p（27）0.05）.（3）By comparing the structure of the microbial community,it was found that the disease state was the major factor explaining the variation of the saliva microbiome（p=0.001,F=3.22）,while gender didn’t affect the variation of the saliva microbiome（p>0.05）.The microbial community structure of the C group is relatively conservative,while the microbiome of the H group is more variable.（4）According to the analysis of the microbial composition,we found that at different levels,the microbial compositions were similar between C and H groups.There were no disease-specific bacteria（only in ECC children,not in healthy children）and sex-specific bacteria（only in one gender,not in the other gender）,but the relative abundance of genera was significantly different between C and H groups.Compared with the H group,the relative abundance of Prevotella,Mogibacterium,Atopobium was increased in the C group,while the relative abundance of Porphyromonas and Capnocytophaga was decreased in the C group（p（27）0.05）.（5）Individual biomarkers closely related to caries were selected from saliva biochemical indicators（p H value,total protein,and electrolytes）and microbiome,and correspondingly diagnostic models were established.The results showed the accuracy of the ECC diagnosis model based on single saliva biochemical indicators was 94.20%,and the performance of the model based on single saliva microbial indicators was medium,with an accuracy rate of 69.50%,while the accuracy rate of the model based on composite indicators（biochemical indicators and microbiome）was 89.30%.Therefore,the ECC diagnostic model based on biochemical indicators has excellent performance exceeding the single saliva microbial indicators and the composite one.Conclusions:（1）Our study found that markers from the biochemical indicators of saliva（including p H,total proteins,and inorganic ions）and microbiome were closely related to ECC.（2）There were correlations between specific saliva biochemical indicators and microbiome.（3）The ECC diagnostic model constructed based on saliva biochemical indicators has the better performance,emphasizing that the use of saliva biochemical indicators can achieve an accurate diagnosis of ECC.Through the in-depth analysis of the saliva microenvironment,our study can explore the progression of caries in depth.It paved the way for regular monitoring and evaluation of caries,and provide more effective and personalized treatment programs for children with ECC.