| Objective: The purpose of this study was to explore the species diversity,bacterial community structure,composition,function and differences of saliva microbes in caries-free and caries-high children with autism spectrum disorders by the third high-throughput sequencing platform combined with the metagenomics technique.The potential microorganisms related to caries in children with ASD were preliminarily identified,and the oral caries diagnosis model of children with ASD with high accuracy was established based on the information of salivary flora,providing a new strategy for the prevention and diagnosis of caries in special population with ASD.Methods: In this study,42 salivary samples were collected from ASD children in Qingdao Yilin School.According to the number of decay missing filled surfaces of teeth(dmfs),the samples were divided into two groups:caries-high group(CH group,dmfs ≥ 8;dmft≥6,n = 20)and caries-free group(CF group,dmfs = 0;n = 22).The metagenomic DNA genome was extracted from the 42 samples.and amplified by polymerase chain reaction(PCR).The full-length 16 Sr RNA gene was amplified and sequenced by Pacbio SMRT sequencing technique.T test was used to evaluate α diversity index and Good coverage.The β diversity between the two groups was evaluated by permutation multiple variance analysis and similarity analysis based on Unweighted Unifrac and Binary Jaccard Unweighted distances.Generate bar charts of different classification levels to describe the overall composition and abundance of microorganisms.Wilcoxon rank-sum test with P value was used to compare the characteristics of salivary microbes between the two groups of children with ASD.p<0.05 was considered to be statistically significant.Random forest algorithm and tenfold cross validation were used to select microbial markers and establish models.The performance of the caries model was evaluated by receiver operating characteristic curve(ROC curve).All analyses are in the R language "p ROC" package.Results:(1)Through comparative analysis of salivary microbial species diversity,we found that the diversity of salivary microbial species(Chao1index、ACE index、Shannon and Simpson)in CH group was higher than that in CF group,but not statisticantly different between the two groups(p > 0.05).(2)Through comparative analysis of salivary microbial community structure,we found that the difference in microbial structure between the CF and CH group was statistically significant(p < 0.05).The microbiome of the CF group is more variable(p <0.05).while the microbial community structure of the CH group is relatively conservative.(3)Through comparative analysis of salivary microbial species composition,we found that the salivary microbial compositions of CF and CH groups were similar from six taxonomical levels(i.e.,phylum,class,order,family,genus,and species)and there were no disease-specific taxa(only exists in the caries samples,not in healthy samples).Furthermore,the relative abundances of certain taxa were significantly different between CH and CF groups and moreover,at species level,we identified four caries-enriched taxa(p < 0.05),including Streptococcus mutans,Prevotella oris,Dialister invisus and Fusobacterium nucleatum,while three health-enriched taxa(p< 0.05),namely,Bergeyella spp.,Prevotella nanceiensis,and Staphylococcus aureus.(4)We built up a series of caries-diagnosis models based on salivary organismal compositions at six taxonomical levels individual,and the ROC curve model showed that species-based model performed best to distinguish caries samples from the healthy ones among ASD children.(5)Based on the predicted salivary microbial functional genes,lipid transport and metabolism were significantly lower in the CH group,in contrast with the CF group(p <0.05).Conclusion:(1)There were significant differences in microbial diversity,community structure and predicted metabolic pathway of salivary samples between the oral healthy and caries children with ASD.(2)The prediction model of dental caries established at species level is more valuable than the ones at the other levels,suggesting that salivary species could diagnosis caries status of ASD children with relatively high accuracy.(3)The seven species identified here can be served as markers/indicators for diagnosing and predicting dental caries in ASD children.The study characterized the caries-specific community structure and biomarkers of the oral microbiota in ASD patients,which might help develop novel strategies to prevention and diagnosis of dental caries in ASD children population. |
