OBJECTIVES: This study aimed to analyze the bacteria in dental caries and establish an optimized dental-ca-ries diagnosis model based on 16S ribosomal RNA (rRNA) data of oral flora. METHODS: We searched the public databa-ses of microbiomes including NCBI, MG-RAST, EMBL-EBI, and QIITA and collected data involved in the relevant research on human oral microbiomes worldwide. The samples in the caries dataset (1 703) were compared with healthy ones (20 540) by using the microbial search engine (MSE) to obtain the microbiome novelty score (MNS) and construct a caries diagnosis model based on this index. Nonparametric multivariate ANOVA was used to analyze and compare the impact of different host factors on the oral flora MNS, and the model was optimized by controlling related factors. Finally, the effect of the model was evaluated by receiver operating characteristic (ROC) curve analysis. RESULTS: 1) The oral microbiota distribution obviously differed among people with various oral-health statuses, and the species richness and species diversity index decreased. 2) ROC curve was used to evaluate the caries data set, and the area under ROC curve was AUC=0.67. 3) Among the five hosts' factors including caries status, country, age, decayed missing filled tooth (DMFT) indices, and sampling site displayed the strongest effect on MNS of samples (P=0.001). 4) The AUC of the model was 0.87, 0.74, 0.74, and 0.75 in high caries, medium caries, low caries samples in Chinese children, and mixed dental plaque samples after controlling host factors, respectively. CONCLUSIONS The model based on the analysis of 16S rRNA data of oral flora had good diagnostic efficiency.
Humans ; Child ; Bacteria/genetics* ; Dental Caries/microbiology* ; Dental Caries Susceptibility ; Microbiota/genetics* ; RNA, Ribosomal, 16S

Streptococcus mutans (S. mutans) is generally regarded as a major contributor to dental caries because of its ability to synthesize extracellular polysaccharides (EPS) that aid in the formation of plaque biofilm. The VicRKX system of S. mutans plays an important role in biofilm formation. The aim of this study was to investigate the effects of vicK gene on specific characteristics of EPS in S. mutans biofilm. We constructed single-species biofilms formed by different mutants of vicK gene. Production and distribution of EPS were detected through atomic force microscopy, scanning electron microscopy and confocal laser scanning microscopy. Microcosmic structures of EPS were analyzed by gel permeation chromatography and gas chromatography-mass spectrometry. Cariogenicity of the vicK mutant was assessed in a specific pathogen-free rat model. Transcriptional levels of cariogenicity-associated genes were confirmed by quantitative real-time polymerase chain reaction. The results showed that deletion of vicK gene suppressed biofilm formation as well as EPS production, and EPS were synthesized mostly around the cells. Molecular weight and monosaccharide components underwent evident alterations. Biofilms formed in vivo were sparse and contributed a decreased degree of caries. Moreover, expressional levels of genes related to EPS synthesis were down-regulated, except for gtfB. Our report demonstrates that vicK gene enhances biofilm formation and subsequent caries development. And this may due to its regulations on EPS metabolism, like synthesis or microcosmic features of EPS. This study suggests that vicK gene and EPS can be considered as promising targets to modulate dental caries.
Animals ; Biofilms ; Dental Caries ; Dental Plaque ; Rats ; Streptococcus mutans/genetics*

Streptococcus mutans is a well-known cause of dental caries, due to its acidogenicity, aciduricity, and ability to synthesize exopolysaccharides in dental plaques. Intriguingly, not all children who carry S. mutans manifest caries, even with similar characteristics in oral hygiene, diet, and other environmental factors. This phenomenon suggests that host susceptibility potentially plays a role in the development of dental caries; however, the association between host genetics, S. mutans, and dental caries remains unclear. Therefore, this study examined the influence of host gene-by-S. mutans interaction on dental caries. Genome-wide association analyses were conducted in 709 US children (<13 years old), using the dbGap database acquired from the center for oral health research in appalachia (COHRA) and the Iowa Head Start programmes (GEIRS). A generalized estimating equation was used to examine the gene-by-S. mutans interaction effects on the outcomes (decayed and missing/filled primary teeth due to caries). Sequentially, the COHRA and GEIRS data were used to identify potential interactions and replicate the findings. Three loci at the genes interleukin 32 (IL32), galactokinase 2 (GALK2), and CUGBP, Elav-like family member 4 (CELF4) were linked to S. mutans carriage, and there was a severity of caries at a suggestive significance level among COHRA children (P < 9 × 10), and at a nominal significance level among GEIRS children (P = 0.047-0.001). The genetic risk score that combined the three loci also significantly interacted with S. mutans (P < 0.000 1). Functional analyses indicated that the identified genes are involved in the host immune response, galactose carbohydrate metabolism, and food-rewarding system, which could potentially be used to identify children at high risk for caries and to develop personalized caries prevention strategies.
Adolescent ; Child ; DMF Index ; Dental Caries ; microbiology ; Dental Caries Susceptibility ; genetics ; Galactokinase ; Genome-Wide Association Study ; Humans ; Streptococcus mutans ; genetics ; isolation & purification ; Tooth, Deciduous

Shwachman-Diamond syndrome (SDS) is an autosomal recessive genetic disorder, consisting of exocrine pancreatic insufficiency, chronic neutropenia, neutrophil chemotaxis defects, metaphyseal dysostosis, short stature, dental caries, and multiple organ involvements. Although SDS is the second most common hereditary abnormality of exocrine pancreas following cystic fibrosis in the Western countries, it has rarely been reported in Asia. We diagnosed a case of SDS in a 42-month-old girl, and genetic analysis including the relatives of the patient confirmed the diagnosis for the first time in Korea. She had short stature, steatorrhea, dental caries, and recurrent prulent otitis media and pneumonias. Laboratory studies revealed cyclic neutropenia, and serum levels of trypsin, amylase, and lipase were decreased. Simple radiography revealed metaphyseal sclerotic changes at the distal femur. A CT scan demonstrated a fatty infiltration and atrophy of the pancreas. On direct sequencing analysis of Shwachman-Bodian-Diamond Syndrome gene exon 2 region, the patient was homozygous for the c.258+2T>C mutation and heterozygous for the c.183_184TA>CT mutation and c.201A>G single nucleotide polymorphism. Treatment with pancreatic enzyme replacement, multivitamin supplementation, and regular to high fat diet improved her weight gain and steatorrhea.
Child, Preschool ; Dental Caries/*genetics ; Dysostoses/*genetics ; Exocrine Pancreatic Insufficiency/*genetics ; Female ; Humans ; *Mutation ; Neutropenia/*genetics ; Pedigree ; Syndrome

OBJECTIVETo evaluate the genotypic diversity of Streptococcus sobrinus (Ss) between children suffering with severe early childhood caries (S-ECC) and caries-free children by arbitrarily primed-polymerase chain reaction (AP-PCR).

METHODSA total of 178 children aged from 42 to 54 months were recruited from 14 urban kindergartens. The S-ECC group contained 87 children with more than 5 decayed teeth, and the control group was composed of 91 caries-free children. Stimulated whole saliva was collected by chewing paraffin. All mutans streptococcus isolates were subcultured, biochemically characterised and identified by PCR as Streptococcus mutans (Sm) and Ss. Then the Ss isolates were genotyped by AP-PCR.

RESULTSThe frequency of Ss detection was 18% in S-ECC children, which was significantly higher than 3% in caries-free children (P < 0.01). Twenty-two distinct genotypes of Ss were identified from 53 clinical isolates. In S-ECC group, one to three genotypes of Ss were detected in each saliva sample. Only one genotype of Ss was detected in all the caries-free children. One genotype of Ss were shared by three S-ECC children. The genotypes of isolates in S-ECC group were relate to decayed-missing-filled teeth (r = 0.50, P < 0. 05).

CONCLUSIONSThe rate of Ss detection was significantly higher in S-ECC children than in caries-free children. Isolates of Ss displayed genetic polymorphism. The multi-genotypes of Ss was related to differences in caries susceptibility. Strains of Ss with same genotype were present in unrelated subjects.

Child, Preschool ; Dental Caries ; microbiology ; Female ; Genotype ; Humans ; Male ; Polymorphism, Genetic ; Streptococcus sobrinus ; genetics ; isolation & purification

OBJECTIVETo compare the production of organic acid of different genotype Streptococcus mutans (S. mutans) isolated from children with different caries experience.

METHODS66 strains of S.mutans isolated from dental plaques of children aged from 3 to 5 with different caries experience were chosen as test bacteria. The quantities of organic acid include formic acid, acetic acid and lactic acid which produced by different genotype of S. mutans, were measured by gas chromatograph.

RESULTSThere were significant difference in production of organic acid among the different genotypes of S. mutans isolated from children with different caries susceptibility, and so were the strains isolated from children within the same caries susceptibility (P < 0.05). The more genotypes the strain had, the more organic acid it produced (P < 0.05). Among all the organic acid, the quantity of lactic acid was much more than that of formic acid and acetic acid.

CONCLUSIONSThere were significant difference in the ability of the strains with different genotypes to produce organic acid, and the more genotypes it has, the more organic acid it produced.

Carboxylic Acids ; metabolism ; Child, Preschool ; Dental Caries ; microbiology ; Dental Plaque ; microbiology ; Genotype ; Humans ; Streptococcus mutans ; genetics ; metabolism

OBJECTIVETo clone Streptococcus salivarius (Ss) 57. I urease gene, which can express ureolytic activity in Escherichia coli (Ec) without adding extra nickel ions.

METHODSUrease gene was cloned by polymerase chain reaction in three separate parts. The three separate plasmids were digested by specific restriction enzymes and ligated together. The expression of the complete urease gene in Ec was detected by phenol red assay and pH analysis.

RESULTSUrease gene of Ss 57.I was eventually cloned and proved correct. Urease activity of the obtained clone was positive in Ec. Without adding extra NiCl(2), the recombinant Ec could hydrolyze urea to produce ammonia, resulting in the increase of pH value.

CONCLUSIONSThe clone of Ss urease gene obtained in this study could express ureolytic activity in Ec without adding extra nickel ions. The current clone can be used to construct ureolytic effector strain used in replacement therapy in caries prevention.

Cloning, Organism ; Dental Caries ; microbiology ; prevention & control ; Escherichia coli ; genetics ; Hydrogen-Ion Concentration ; Nickel ; chemistry ; Streptococcus ; genetics ; Urease ; genetics

Objective: To detect and analyze the characteristics of oral microbiota in species composition, function and metabolism among caries, periodontitis and oral healthy individuals, hunting for the microbiome-derived biomarkers with specificity and sensitivity to estimate the occurrence of these two diseases. Methods: Saliva samples were collected from 10 patients with high caries risk [decayed-missing-filled teeth (DMFT)≥6, HC group] in Department of Endodontics, 10 patients with periodontitis of grade Ⅱ A-Ⅲ C (PG group) in Department of Periodontology and 10 oral healthy individuals (HH group) from School of Stomatology, The Fourth Military Medical University during from March 2022 to June 2022. A baseline examination was conducted on all participants, including their oral conditions of caries and periodontal health. Metagenomic sequencing (Illumina PE150 platform) and liquid chromatography-mass spectrometry were used to detect microorganisms and their metabolites in the samples respectively. The sequencing data were analyzed to obtain the information of microbial taxonomic composition, functional genes and metabolites in each group of samples. The basic oral conditions and saliva samples of subjects in each group were evaluated and collected by the same professional endodontist. Results: There were no significant difference in baseline characteristics such as age and sex among the subjects in each group (P>0.05). DMFT in HC group (9.0±1.7) was significantly higher than that in HH group (0) and PG group (0) (F=243.00, P<0.001). Sequencing data analysis showed that the taxonomic compositions of salivary microbiota in each group were mainly Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria at the phylum level, and Streptococcus, Neisseria, Rothia, Prevotella at the genus level. Differential analysis showed that, compared with the HH group, HC group and PG group had significant differences in taxonomic composition (P<0.05), and the most significant among them was Prevotella. At the species level, Prevotella pallens was the most significant change in HC group, and Porphyromonas gingivalis in PG group. Metabolite analysis showed that there were significant differences in metabolites between HC group and PG group. The results showed that, compared with the HH group, the most significant metabolite change was 3-hydroxy-1, 5-diphenylpentan-1-one in HC group (P=0.001) and N1 acetylspermine in PG group (P=0.002) respectively. Compared with the PG group, the metabolite of HC group with the most significant difference is D-glucosamine 6-phosphate (P=0.006). The metabolism gene function analysis showed that, the enrichment of carbohydrate metabolism related genes was highest in HC group, followed with HH group, and it was lowest in PG group. In addition, compared with the HH group, the abundance of functional genes related to glucose metabolism, such as ABC transporter and phosphotransferase system, were significantly decreased in PG group (P<0.05), but significantly increased in HC group (P<0.05). Conclusions: There is a significant correlation between the alternation of carbohydrate metabolism of salivary microbiota with the occurrence of caries and periodontitis. In the future, Prevotella pallens and 3-hydroxy-1, 5-diphenylpentan-1-one may be the potential biomarkers of caries; while Porphyromonas gingivalis and N1 acetylspermine work in the predictions of periodontitis.
Humans ; Saliva/microbiology* ; Dental Caries Susceptibility ; Periodontitis/microbiology* ; Microbiota/genetics* ; Porphyromonas gingivalis/genetics* ; RNA, Ribosomal, 16S/genetics*

OBJECTIVETo study the genetic diversity of F-ATPase alpha subunit gene uncA derived from Streptococcus mutans (S. mutans) clinical isolates and to investigate the relationship between the genetic diversity of acidurance factor and S. mutans aciduric ability, also and the cariogenicity.

METHODSSixty-four S. mutans strains derived from 34 caries-active individuals and 30 caries-free individuals, including 18 strains displaying high acid tolerance and 20 strains displaying low acid tolerance. Gene uncA was amplified with specific primers from S. mutans genomic DNA, then the PCR products were analyzed by RFLP and sequenced.

RESULTSTwo genotypes A and B of PCR-RFLP were revealed when digested with Hph I. Mbo II also produced two different pattern C and D. The distributions of A and B genotype strains with different caries-sensitivity groups were different (P < 0.05), and the proportion of A genotype strains from caries-activity group was higher than that from caries-free one. The distributions of C and D genotype strains with different acidurance strains were different (P < 0.05), and the proportion of C genotype strains from high acid tolerance group was higher than that from low acid tolerance group. These amplified uncA genes from different group were sequenced and there existed variation of Hph I and Mbo II recognized sites.

CONCLUSIONSThis study indicates that uncA gene of S. mutans F-ATPase obviously displayed genetic diversity. The different Hph I-RFLP and Mbo II-RFLP genotypes could be related to the cariogenicity and acid tolerance of S. mutans strains.

Bacterial Proton-Translocating ATPases ; genetics ; Dental Caries ; microbiology ; Genes, Bacterial ; Genotype ; Humans ; Polymorphism, Restriction Fragment Length ; Streptococcus mutans ; enzymology ; genetics

OBJECTIVETo isolate and identify Streptococcus mutans (Sm) and Streptococcus sobrinus (Ss) in dental plaque of children with high dmft and no caries by selective medium, biochemical methods and arbitrarily primed-polymerase chain reaction (AP-PCR).

METHODSA total of 401 3-4-year-old children from seven kindergartens were recruited using cluster sampling and their dental caries status were examined. From 30% of children with the highest dmft score (dmft >/= 5), 20 children were chosen randomly as test group and 20 age and gender-matched caries-free children were selected as control. Plaque samples were collected from buccal surfaces of the molars and plated onto TYCSB plate. Sm and Ss were primarily identified by colony morphology and biochemical characteristics. Then chromosomal DNA of the strains was isolated and Sm or Ss were confirmed by AP-PCR.

RESULTSThe proportion positive for Sm and Ss in children with high dmft was 100% and 40% respectively while that in caries-free children was 75% and 5% by AP-PCR analysis. The differences were statistically significant between the two groups.

CONCLUSIONSThe proportions positive for Sm and Ss detected by AP-PCR method were significantly higher in children with high dmft than in caries-free children and it is a risk factor for high dmft in deciduous teeth harboring Sm and Ss.

Child, Preschool ; Dental Caries ; microbiology ; Dental Plaque ; microbiology ; Female ; Humans ; Male ; Streptococcus mutans ; genetics ; isolation & purification ; Streptococcus sobrinus ; genetics ; isolation & purification