| Oral diseases are a global public health problem,and the cost of treating them places a significant financial burden on families and health care systems.Caries is a common oral infectious disease in people of different ages.It is the local destruction of tooth hard tissue caused by acidic byproducts produced by caries-causing bacteria fermenting free sugars.Previous studies on caries mainly focused on children,primary and secondary school students and the elderly,but neglected college students.Therefore,this study took college students as the research object to explore the microecological characteristics of saliva microflora with caries,in order to understand the structure and function of the oral saliva microflora in college students and discover the characteristic species and the characteristic functional genes related to caries in college students,and aims to isolate the bacteria species related to caries and the bacteria species in the normal flora in saliva.This study will accumulate strains for the diagnosis and prevention of caries and study of oral probiotics.In this study,non-irritating saliva samples were collected from 60college students(30 caries-free and 30 caries).Metagenomic sequencing was used to analyze the microbial community structure and functional genes of saliva flora and analyze the differential metabolic pathways.The pure culture technique was used to isolate,identify and preserve strains from oral cavity.The diversity and novelty of culturable bacteria in saliva and the differences between caries-free and caries groups were analyzed.Two new Streptococcus strains(HT-12 and QT-23)were isolated and analyzed by means of polyphasic taxonomic research from four aspects:molecular phylogenetic analysis,morphological and cultural characteristics,physiological and biochemical characteristics and fatty acid composition,so as to determine their taxonomic status.In this study,a gene set of saliva microflora in caries and caries-free college students was established.After genes assembled,1,358,647 non-redundant genes were predicted and annotated into NCBI NT database.A total of 47 phyla,96 classes,195 orders,426 families,1,360 genera and4,223 species of bacteria were detected in caries and caries-free group.The predominant phyla,Bacteroidetes,Proteobacteria,Firmicutes,Fusobacteria,Actinobacteri and Spirochaetes were found in the saliva of college students.The dominant bacteria genera are Prevotella,Streptococcus,Neisseria,Haemophilus,Veillonella,Fusobacterium,Rothia,Capnocytophag,Schaalia and Campylobacter.Differences in the composition of salivary bacteria between caries and caries-free college students:at the phylum level,the content of Fusobacteria was higher in caries-free group.At the genus level,except for Fusobacterium,the dominant genus,which has a higher content in the caries-free group,the other different bacteria genera are all low-abundance bacteria.The contents of Parascardovia,Olsenella,Halocynthiibacter,Propionibacterium and Cryptobacterium in the caries group were significantly increased compared with those in the caries-free group.At the species level,except for Fusobacterium pseudoperiodonticum,which is a dominant bacterium with a higher abundance in the caries-free group,the rest are low abundance.The contents of Fusobacterium varium and Hungateiclostridium saccincola in the caries-free group were significantly higher than those in the caries-free group.Streptococcus phage SM1,Parascardovia denticolens,Olsenella sp.oral taxon 807,Streptococcus periodonticum,Lactobacillus vadinalls,Rothia amarae,Cryptobacterium curtum and Propionibacterium acidifaciens were more abundant in the caries group.The non-redundant genes were annotated to the KEGG database for comparison.Global and overview maps and carbohydrate metabolism were the most abundant functional genes in saliva flora.The differences of functional genes between caries and caries-free college students were mainly distributed in carbohydrate and amino acid metabolism pathways.The genes of histidine metabolism,butanoate metabolism and propanoate metabolism increased in the caries group.In this study,a total of 134 culturable bacteria strains from oral cavity were isolated and preserved.By 16S r RNA gene sequence alignment,they belonged to 11 bacteria genera,Rothia,Actinomyces,Schaalia,Neisseria,Streptococcus,Granulicatella,Staphylococcus,Gemella,Veillonella,Haemophilus and Fusobacterium.Two Streptococcus isoalates,HT-12 and QT-23 showed the 16S r RNA gene similarity 98.64%and 98.50%with their closest relatives,respectively,and were identified as potential new species of Streptococcus.The two potential novel Streptococcus isolates HT-12 and QT-23 were identified by the method of polyphasic taxonomy.The 16S r RNA gene similarities between strain HT-12 and S.parasanguinis ATCC 15912~T,S.rubneri LMG 27207~T and S.australis ATCC 700641~T were 98.64%,98.21%and 98.09%,respectively.The phylogenetic trees based on 16S r RNA genes showed that strain HT-12 and S.parasanguinis ATCC 15912~Tclustered in branch and formed stable cluster.Multilocus sequence analysis(MLSA)based on concatenated sequences of the gro EL-rpo B-sod A-rec A-phe S genes and phylogenetic analysis based on the whole genome further confirmed the close relationship between strain HT-12 and S.parasanguinis ATCC 15912~T.The values of digital DNA-DNA hybridisation(d DDH)between the genomes of strain HT-12 and three reference strains were 54.4%,25.9%and 27.3%,respectively.The Average Nucleotide Identity(ANI)values between the genomes of strain HT-12 and the three reference type strains ranged from 81.26%～93.88%.Strain QT-23 was closed with S.oralis subsp.oralis ATCC 35037~T,S.oralis subsp.dentisani CECT 7747~T and S.downii CECT 9732~Twith 16S r RNA gene similarities 98.50%,98.50%and 98.23%,respectively.Phylogenetic trees based on 16S r RNA gene showed that strain QT-23 formed a branch independently.MLSA based on concatenated sequences of the gro EL-rpo B-sod A-rec A-phe S genes and phylogenetic analysis based on the whole genome showed that strain QT-23 and S.infantis ATCC 700779~T clustered in the same branch.The d DDH values between strain QT-23 and three reference strains were 25.9%,25.7%and 25.5%,respectively.The ANI values between the genomes of strain QT-23 and the three reference type strains ranged from 82.09%～82.36%.Therefore,the comparison and phylogenetic analysis based on the comprehensive analysis of 16S r RNA genes,housekeeping genes,and genomic sequences suggest that strain HT-12 and QT-23 are likely to represent different new species of Streptococcus.Both strains were grew at 22℃～42℃,but did not grow below 15℃.Both strains could grow on medium containing 0%～2.5%Na Cl.After strain HT-12 and QT-23 were cultured on blood agars for 24 h,the grass green hemolysis ring(α-hemolytic ring)was formed around the colony,and theα-hemolytic ring was more obvious around the colony after continued culture for 48 h.Strain HT-12 and QT-23 were Gram-positive,strain HT-12 arranged in pairs or short chains,and strain QT-23 arranged in long chains.Under transmission electron microscope,the single spherical cell diameter of strain HT-12 and QT-23 was about 0.4～0.7μm and 0.5～0.7μm,respectively.There were no spores and flagella in both strains.Both strains were negative for catalase and oxidase,and could be distinguished from other related strains in some biochemical reactions.Therefore,strain HT-12 and QT-23 were identified as two new species of Streptococcus by gene comparison,molecular phylogenetic analysis and phenotypic analysis.The contents of Parascardovia,Olsenella,Halocynthiibacter,Propionibacterium and Cryptobacterium in the saliva bacteria of college students in the caries group were significantly increased compared with those in the caries-free group,and functional genes in the caries group were mainly distributed in histidine metabolism,butanoate metabolism and propanoate metabolism,which may be the cause of caries development in college students. |
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