| BackgroundCaries is defined as localized damage to the hard tooth tissue caused by acidic byproducts of microbial fermentation of free sugars,and research on the caries-related microbiota has always been a major topic of discussion.Both supragingival plaque and saliva are commonly sampled for analysis of microbial communities.And it is unclear whether saliva can replace supragingival plaque for microbial studies of caries.Both cross-sectional and longitudinal studies have shown that deciduous tooth caries is a risk factor for caries in the first permanent molar.However,there are few studies on whether the microbial community of the first permanent molar is affected by the caries status of the deciduous molar when the first permanent molar is not carious.High-throughput sequencing technology provides strong support for microbial research.The next-generation sequencing platforms have the disadvantage of short reading length,which has certain influence on the analysis of microbial composition and structure.The third-generation sequencing platforms such as PacBio Sequel have overcome this limitation of next-generation sequencing technology with regard to read length.It enables more accurate taxonomic resolution of microbial communities owing to sequencing of full-length 16S rRNA genes,which can better analyze the oral microbiota.ObjectiveThe purpose of this study was to evaluate whether saliva samples could replace supragingival plaque for microbial studies of caries;to compare the difference of microbial community in caries children and healthy children aged 7-9 years;to compare the microbial community on the supragingival plaque of healthy first permanent molar from children with different caries status aged 7-9 years.MethodsSamples were taken from the supragingival plaque and non-stimulated saliva of 30 children aged 7-9 years.Fifteen children had dental caries on deciduous molars(Caries/C group),and fifteen didn’t have any caries(Healthy/H group).And all children’s first permanent molars were free of caries.Three types of samples were collected from each subject,and all of the samples were divided into six subgroups,including CD subgroup(supragingival plaque and caries lesions from deciduous molars of C group),CP subgroup(supragingival plaque from first permanent molars of C group),CS subgroup(saliva of C group),HD subgroup(supragingival plaque from deciduous molars of H group),HP subgroup(supragingival plaque from first permanent molars of H group)and HS subgroup(saliva of H group).Microbial DNA was extracted and full-length 16S rRNA gene were amplified,and the full length of the 16S rRNA gene was amplified.DNA library was constructed,and bioinformatics analysis was performed after Sequencing by PacBio Sequel using single-molecule real-time sequencing technology.The specific processes were as follow.1.Quality control was carried out on the obtained original sequences to obtain high-quality sequences.2.The high-quality sequences were clustered into operational taxonomic unit(OTU)at 97%similarity.The corresponding taxonomic information for each OTU was obtained,allowing us to determine the taxonomic composition at the phylum,class,order,family,genus,and species levels.3.The microbial richness and evenness were evaluated by alpha diversity analysis.4.The linear discriminant analysis Effective Size(LEfSe)method was used to identify microbial biomarkers in every subgroup,and linear discriminant analysis(LDA)score threshold was set as 4.0.5.Partial Least Squares Discriminant Analysis(PLS-DA)was used to compare the differences and similarities of microbial community structure among subgroups.6.Co-occurrence analysis was used to demonstrate the interactions of the microbiota.Results1.541,831 high-quality sequences were obtained from 90 samples.98.39%of the sequences were distributed between 1401 and 1600 base pair(bp),and the average length was 1510 bp.2.The sequences were clustered into 2495 OTU,representing 11 phyla,19 classes,28 orders,52 families,96 genera,and 370 species of bacteria.3.The results of rarefaction and species accumulation curve analyses showed that the sequencing depth and sample size in this study adequately reflected the microbial diversity.4.The results of alpha diversity analysis showed that there was no statistically significant difference in microbial richness and evenness between children with caries and healthy children(p>0.05);In Caries group,the salivary microbial richness and evenness were lower than those of the supragingival plaque on the deciduous molars and the first permanent molars(p<0.05);In healthy group,salivary microbial richness and evenness were lower than those of supragingival plaque on the first permanent molars(p<0.05).5.At the species level,for the supragingival plaque on the deciduous molars,LEfSe analysis showed that Streptococcus mutans,Veillonella dispar,and Propionibacterium acidifaciens were more abundant in caries children than healthy children(p<0.05),and Selenomonas noxia and Corynebacterium matruchotii were more abundant in healthy children than caries children(p<0.05);For the supragingival plaque on the first permanent molars,Selenomonas noxia was more abundant in healthy children than caries children(p<0.05).In salivary microniche,according to the LDA score threshold(4.0)set in this study,there was no statistical difference in microbial abundance between the two subgroups(CS subgroup and HS subgroup)(p>0.05)6.The PLS-DA showed that the microbial community structure of children with caries was completely different from that of healthy children in microneche of supragingival plaque on the deciduous molars;In saliva,the microbiome structure of children with caries was slightly similar to that of healthy children;In children with or without caries,the microbial community structure of supragingival plaque on the deciduous molars was slightly similar to the first permanent molars.7.The co-occurrence analysis showed that there was a positive correlation between Streptococcus mutans and Propionibacterium acidifaciens.ConclusionWe concluded the following.First,non-stimulated saliva samples cannot replace the supragingival plaque samples.Second,Streptococcus mutans,Veillonella dispar,and Propionibacterium acidifaciens are highly associated with the existence of deciduous caries,whereas Selenomonas noxia and Corynebacterium matruchotii are highly associated with the health of deciduous teeth.Third,the microbial composition of the supragingival plaque on the healthy first permanent molars is different with the caries status of the deciduous molar. |
