The Analysis On Microbial Community Structure In Dental Caries Of Children In Bonan From Gansu Province

Objective:Based on the early epidemiological investigation of oral cavity, selected children in Bonan from Gansu province as objects. By constructing 16SrRNA clone library and sequencing, analyzing the differences of microbial community structure between caries group and caries-free group, from different taxonomic levels. To explore the main oral cariogenic microorganisms of children in this area, providing data support for further research on cariogenic mechanism. And taking early preventive measures from the perspective of the etiology, to reduce the incidence of dental caries, and promote oral health status of children.Methods:According to the WHO sampling standard,20 subjects were selected from children in Bonan, and divided them into caries (BC) and without caries (BH) group. Unstimulated saliva collected and extracted bacterial genomic DNA. Obtained 16SrRNA fragments by PCR amplification and transformed them into E.coli DH5a. Screened positive clones and constructed 16SrRNA clone library, then sequencing. Excluding carrier and chimeras, MOTHUR software was used in OTU classification and proceed on diversity of microbial flora. RDP was used to classified sequences according to different classification levels, and then submitted to the NCBI database for similarity comparison. MEGA software was used to construct phylogenetic tree. And finally, PCA and cluster analysis were determined combining with SPSS software for statistical analysis.Results:(1) 960 qualified sequences (average read length was 850bp) were screened from 1000 sequences, BC group was 573 and BH group was 387. Total of 170 OTUs obtained by MOTHUR software, which belonging to the 5 phyla,12 classes,16 orders,23 families and 28 genera.(2) The results of diversity index showed that the capacity of this two library was good. Shannon index in BC group was higher than in BH, indicating a richer diversity of BC group. But there was no significant difference (p> 0.05) of Shannon index and Simpson index between the two libraries.(3) 5 bacterial phyla:Firmicute, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. In addition to Fusobacteria which detection rate was less than 1% in BC group, the detection rate of other 4 phyla were more than 1%. The chi-square test results of detection rate, showed statistically significant difference (p<0.05) only Firmicute and Proteobacteria between the two groups; Wilcoxon test for the abundance of 5 phyla showed that no statistical significance (p> 0.05) between two groups.(4) Only Streptococcus was detected in all samples, the dominant genera (detection rate> 1%) were also different between the two groups. There were 11 dominant genera were found in BC group, including Streptococcus, Gemella, Granulicatella, Haemophilus, Veillonella, Lactobacillus, Neisseria, Rothia, Aggregatibacter, Abiotrophia and Pseudomonas. And a total of 8 genera: Streptococcus, Gemella, Granulicatella, Haemophilus, Veillonella, Lactobacillus, Neisseria and Pseudomonas were detected in BH group.(5) The statistical analysis showed, only Rothia has statistical difference (p<0.05) on the abundance and detection rate between the two groups. PCA and cluster analysis showed that, there were some differences on microbial communities between the two groups, and the difference was larger in BC than in BH group.(6) In addition to the currently known microbial genera, there were some species still could not accurately classified. And the role of these species in the occurrence of dental caries or maintain the oral health status, needing a further exploration.Conclusion:There were some differences in microbial community structure in saliva of children in Bonan. Lactobacillus, Rothia, Aggregatibacter and Abiotrophia as dominate genera were main cariogenic bacteria, especially the role of Rothia. Streptococcus was detected in all samples and had the highest abundance. It was the largest proportion of the oral species. There were a lot of bacteria about dental caries required in-depth research in the complex oral environment, such as Rothia, Aggregatibacter and Abiotrophia.16SrRNA clone library was a more mature and comprehensive research method, which was useful in researching the diversity of oral microbial community structure.