Comparative Genomic Study Of Steptococcus Oralis And In Vivo Periodontal Pathogenic Analysis Of Fusobacerium Nucleatum

ObjectivesOral cavity is one of the five major bacterial libraries of human body,where more than 700 kinds of bacteria reside.Oral microorganisms are closely related to oral health and general health.Streptococcus oralis and Fusobacterium nucleatum are common microorganisms in human oral cavity,and both of them contain multiple subspecies,different subspecies have different effects on human health.Streptococcus oralis belongs to oral normal floral and is one of early colonized bacteria in human cavity.S.oralis can contribute to infective endocarditis.S.oralis consists of three subspecies(S.oralis subsp.oralis,S.oralis subsp.dentisani,S.oralis subsp.tigurinus).Different subspecies have different gene funtions.Comparation of gene functions of different strains and subspeices is incomplete.Our study analyzes and compares genomes of Streptococcus oralis strains.The similarities and differences of gene functions and virulence genes were discussed to provide references for the treatment and prevention of Streptococcus oralis infections.Fusobacterium nucleatum is a dominant periodontal bacterium,which is related to occurrence and severity of periodontitis.F.nucleatum can adhere to the early colonized bacteria and other bacteria.F.nucleatum also plays a crucial part in systemic health.Current studies have found that F.nucleatum is closely related with rectal cancer,adverse pregnancy outcomes,diabetes and other systemic disease.F.nucleatum consists four subspecies(F.nucleatum subsp.nucleatum,F.nucleatum subsp.animalis,F.nucleatum subsp.vincentii,F.nucleatum subsp.polymorphum).Detection rate of four subspecies are different in male and female.At present,studies on the pathogenicity of different subspecies in periodontitis are not complete in vivo.This experiment established in vivo models of periodontitis and infected with different subspecies of F.nucleatum to compare the pathogenicity of different subspecies and provide a reference for further studies on the pathogenicity of periodontitis among different subspecies.MethodsSmples were collected from human cavity with different oral conditions.The bacteria were cultured and isolated under different oxygen and nutrition conditions.Strains of S.oralis and F.nucleatum were aligned by 16S rRNA sequencing.Whole-genome DNA was extracted from three strains of streptococcus(SO,SOT,SOD).Genome-wide DNA was sequenced and assembled.The gene composition and function of three strains were predicted.The situation of gene island,CRISPR and prophage in three strains was predicted.KEGG database,COG database,VFDB pathogenic gene database and ARDB resistance gene database were used to predict gene function,virulence factors and resistance genes.Comparative genomic analysis was performed between the three strains and S.oralis representative strain Uo5.Three strains and Uo5 were compared by collinearity,single nucleotide polymorphism(SNP)and core-gene analysis,phylogenetic tree to analyze the genomic revolution.The in vivo periodontitis model was constructed against SD mouse.The mice were divided into 7 groups(6 mice each):contrast group(transient ligation with PBS injection),ligation group(ligation with PBS injection),FNN group(ligation with F.nucleatum subsp.nucleatum ATCC25586),FNA group,FNP group,FN51 190group,and FN49256 group(ligation with four newly cultured strains of F.nucleatum respectively).In the experimental group,PBS or bacteria were injected into gingival groove every two days for a total of 3 times.Normal diet for 2 weeks,death,seperated the maxillary bone.Micro-ct was used to measure the distance between enamel cementum junction and alveolar bone crest.The data were analyzed using GraphPadPrism 6.0 software for one-way anova,and the statistical results were expressed as mean ± standard deviation(SD).P<0.05 was statistically significant.Hematoxylin and eosin(HE)staining was used to observe degree of gingival epithelial inflammation.Results1.Oral bacteria isolation:1743 strains of bacteria were isolated and cultured from human oral samples.16S rRNA alignment revealed that the bacteria belonged to 48 genus and 129 species.Including three strains of S.oralis and four strains of F.nucleatum.2.Comparative genome results of Streptococcus oralis:whole-genome sequence alignment,gene function comparison and phylogenetic tree results showed that we isolated two strains of Streptococcus oralis(SOT,SOD)and one strain of Streptococcus infantis(SO).SOT and SOD were closely related in genetic evolution.SOT and SOD have same antibiotic resistance genes as well.Compared with the representative strain of Streptococcus oralis(Uo5),the consistency of SOD in SNP and core-gene was higher,while the consistency collinearity of SOT in was higher.There were significant genomic differences between strains SOT,SOD and SO.The virulence related genes and capsular related genes in three genomes was different.The consititution and location of CPS related genes were unlike in three strains.3.In vivo periodontal experiment results of F.nucleatum:Compared to contrast group,the alveolar bone height decreased in ligation group and bacteria injection group(P<0.05).Alveolar bone height of FN51190 is lower than ligation group.The alveolar bone height degrement between other bacterial injection groups and ligation group was not significant.The bacterial injection groups had obvious inflammation in the gingival epithelium in HE-stain experiments.Conclusions1.The species of oral bacteria are abundant,individual difference is reflected in strain level.There is uncertainty in analyzing the evolutionary relationship by single technical identification.For example,the similarity of 16S rRNA sequence between some strains of Streptococcus oralis and Streptococcus infantis is high.Using 16S rRNA is difficult to distinguish these two species.Specific genes are essential to distinguish them2.Streptococcus oralis strains obtained in the human cavity contains a variety of different pathogenic genes.Genes related to adhesion,colonization and immune escape are predicted in all strains,indicating that Streptococcus oralis has pathogenic potential.3.Fusobacterium nucleatum subspecies has destructive effects on periodontal tissue,especially in stimulating the inflammatory response.But the inflammatory reaction may progress slowly.