Explorative Study On Synergistic Cariogenicity Relationship Between Prevotella Denticola And Streptococcus Mutans

1.Background and Objective:Caries is one of the most common oral diseases with high prevalence and increasing severity and it is a global public health problem.Caries is a microbially induced,chronic oral infectious disease,which eventually leads to localized destruction of tooth hard tissues through complex interactions of various factors.Therefore,it is very important to explore the pathogeny microbiology and mechanism of caries.Most pathogenic microbes in oral cavity prefer to grow in the form of biofilms.Different species in biofilms interact with each other through signaling molecules or direct contact,which would affect their physiological characteristics and cariogenic potential.Among this,Streptococcus mutans is considered one of the main etiological agents of dental caries and also the key component of dental plaque,due to its virulence properties associated with cariogenicity such as acidogenicity,acid tolerance,adhesion and synthesis of extracellular polysaccharides.However,the occurrence of caries is the result of the imbalance of dental plaque microecology,and the role of each species in the process of caries could not be ignored.Our preliminary studies on profiling of oral microbiota found relatively elevated abundance of Prevotella denticola along with S.mutans in severe early childhood caries,and revealed the strong correlation between P.denticola and caries.Till now,the mechanism of the involvement of P.denticola in cariogenicity and its relationship with S.mutans in plaque biofilms remain unclear.Based on single-species of P.denticola/S.mutans and dual-species of above two taxa,we examined carbohydrate metabolism and acid productivity,biofilm formation,enamel demineralization ability and the expression of genes associated with cariogenic virulence in this study,aiming to investigate the cariogenic potential of P.denticola and the effect of symbiotic relationship between P.denticola and S.mutans on the virulence of plaque biofilms,which might improve the etiological microbiology of caries,and provide the research basis for the effective prevention and treatment of caries.2.Methods:2.1 Detection of bacterial glycometabolism and acid production abilityTo investigate the ability of ferment sugar and acid production for P.denticola/S.mutans and dual-species of above two taxa,the above bacterial suspensions were cultured under the same conditions and each sample were collected at a fixed point according to the time gradient.The p H value of the medium was detected by p H meter,and the metabolite profiling of grow medium supernatant was evaluated by high performance liquid chromatography（HPLC）.The content of glucose,sucrose,metabolite lactate and p H value in the supernatant were calculated with culture time.2.2 Detection of bacterial biofilm formation abilityP.denticola,S.mutans and mixed suspensions of above two taxa with the same concentration were incubated into 96-well microtiter plates placed or 24-well plates,respectively.The plates were cultured at 37℃under anaerobic conditions to form biofilms.Crystal violet（CV）assays were employed to quantify biofilm biomass.Moreover,after a series of treatments,such as fixing,dehydration and gold spraying,the morphology of the biofilms were observed by scanning electron microscopy.In addition,after staining with fluorescent dye,the distribution of each bacterial community and extracellular matrix in the biofilm was further observed by Confocal Laser Scanning Microscopy.The three-dimensional structure of the biofilm was constructed and the thickness of the biofilm formed by each bacterial species was calculated.2.3 Detection of bacterial demineralization abilityExtracted human third molars were collected and the teeth with complete crown surface,no caries,hidden cracks and no restorations were selected.After a series of treatments,uniform enamel slabs were selected for the study.The enamel slabs were co-cultured with P.denticola,S.mutans and mixed suspensions of above two taxa respectively under the same conditions to build a caries model in vitro.The hardness of enamel surface was measured by a Vickers microhardness tester and the microstructure of enamel surface was observed under scanning electron microscope.2.4 Expression analysis of genes associated with cariogenicity in S.mutansThe effects of P.denticola on gtfs related to carbohydrate metabolism and gbp B,which was closely associated with bacterial adhesion,genes expression in S.mutans were evaluated by q RT-PCR.S.mutans single-species biofilm and dual-species biofilm were obtained.Then total RNA in each biofilm were extracted respectively and reverse transcription and PCR amplification were performed.Finally,the results were analyzed by 2^-ΔΔCtmethod.3.Results:3.1 Both glucose and sucrose in the mediums,for P.denticola/S.mutans/dual-species,were gradually consumed in a time-dependent manner.The result of metabolizing sugars was an increase of lactate and fructose content.Sequentially,the p H value was also gradually decreased.Compared to S.mutans,glucose and sucrose were utilized and lactate was accumulated at a slower rate in P.denticola（P<0.05）.Compared with single-species,the glucose and sucrose were consumed at a slightly higher rate when the two bacteria were co-cultured.Nevertheless,there was no significant difference between S.mutans single-species and dual-species（P>0.05）.And the content of lactate accumulated by dual-species was more（P<0.05）.3.2 We found that both S.mutans and P.denticola had the ability to form biofilms on the glass coverslips.The results of crystal violet staining showed compared to S.mutans,the biofilm biomass formed by P.denticola was lesser,reflected in lower OD value（P<0.05）.However,compared with single-species biofilms,the biomass of dual-species biofilms increased significantly（P<0.05）.The morphology of the biofilms was observed by electron microscope with similar findings.We found that the biofilms formed by P.denticola were relatively thin,and the biofilms harbored few tiny colonies and sparse extracellular matrix,unlike in clusters of S.mutans.The structure of the dual-species biofilms was more complicated with dense microcolonies and abundant extracellular matrix.Furthermore,data showed that average thickness of dual-species biofilms was significantly increased than any single-species biofilms（P<0.05）.3.3 The microhardness and microstructure of enamel surface induced by P.denticola、S.mutans single-species biofilms and dual-species biofilms all changed obviously,which indicated that both P.denticola and S.mutans had cariogenic potential.Compared with P.denticola,the changes of the microhardness and microstructure of enamel surface induced by S.mutans were more obvious（P<0.05）.When the two bacteria were co-cultured,the enamel surface hardness decreased more obviously（P<0.05）,and the enamel demineralization was more serious.3.4 In the dual-species biofilms,relative expression levels of genes related to carbohydrate metabolism,gtf B,gtf C and gtf D in S.mutans were upregulated by 34%,39%and 40%,respectively.The m RNA level of the virulence gene gbp B,which was closely associated with adhesion,was significantly increased by 1.68-fold,which manifested that synergistic relationship between P.denticola and S.mutans enhanced the virulence of plaque biofilms.4.Conclusion:In this study,we found that P.denticola could attach to the enamel surface to form biofilm.Meanwhile,P.denticola could ferment glucose and sucrose,produce acid in vitro and demineralize the enamel,which indicated that P.denticola had cariogenic potential as such.Furthermore,in the symbiotic environment of the two bacteria,P.denticola could promote S.mutans metabolize sugars and enhance the adhesion between bacterial communities,which could promote the formation of biofilm.The presence of P.denticola could accelerate the demineralization of enamel.The study revealed synergistic relationship between P.denticola and S.mutans enhanced the virulence of plaque biofilms in vitro,which would further improve the etiologic microbiology of caries and provide new ideas for the effective prevention and treatment of dental caries.