| Dental caries is one of the most common oral diseases worldwide,and is a highly prevalent biofilm-dependent disease.Many researchers have identified Streptococcus mutans as a major bacterial pathogen in dental caries.In recent years,Candida albicans has been frequently found in caries,particularly in early childhood caries(ECC)with coexistence of S.mutans.Sucrose is considered the most cariogenic sugar,and is directly related to the prevalence of caries.Finding sugar substitutes has long been one of the prevention strategies for caries,while artificial sweeteners are often associated with health hazards.As a natural sweetener with high sweetness,stevioside has been proved to inhibit the biofilm formation and acid production of S.mutans,reduce the bacteria viability and enamel demineralization.However,these previous studies conclude mainly on a singlespecies biofilm model,and the experimental content and evaluation index are relatively simple,so its promotion and application need to be verified by further complex and in-depth models.In this study,firstly,by comparing the differences of S.mutans and C.albicans in saliva of children with severe early childhood caries(S-ECC)and without caries,the correlation of C.albicans to caries or S.mutans was verified.Secondly,the mixed biofilm model of S.mutans and C.albicans was constructed to explore the preventive effects of stevioside,and sucrose background was also added to evaluate whether the effects of stevioside was affected by sucrose.Finally,based on RNA sequencing,the effects of stevioside on virulence genes and molecular pathways of the mixed biofilm were evaluated,and the mechanism of stevioside on the mixed biofilm was detected.Experiment 1 Metagenomic analysis of S.mutans and C.albicans in S-ECC saliva【Objective】To investigate the difference of S.mutans and C.albicans between S-ECC and healthy children by metagenomics sequencing.【Methods】Children aged 3-6 years old were selected as subjects.They were divided into S-ECC group and Healthy group according to the WHO standard of caries detection.Saliva and clinical information were collected and metagenomic analysis was performed.【Results】1.42 children were included.The decay,miss and filled teeth index(dmft)and the decay,miss and filled surfaces index(dmfs)were 15.56±2.89 and 37.30±12.61 respectively in SECC group.The largest number of decayed teeth were the upper and lower second deciduous molar.2.Metagenomics results showed that the salivary microbiota of S-ECC group and Healthy group were highly overlapping at phylum and genus level,but there were statistical differences in the diversity of composition structure.3.The detection rate of S.mutans and C.albicans in saliva of S-ECC group and Healthy group was 100%,and the difference between the two groups was not statistically significant in the relative abundance of S.mutans and C.albicans(P > 0.05).However,there was a high positive correlation between S.mutans and C.albicans.(r = 0.840,P < 0.05).【Conclusion】There is no significant difference between S.mutans and C.albicans in S-ECC group and Healthy group,but there is a positive correlation between them.Experiment 2 Effects of stevioside on mixed growth and biofilm formation of S.mutans and C.albicans【Objective】To explore the effects of stevioside on the mixed biofilm growth,acid production,biofilm morphological structure and function of S.mutans and C.albicans.【Methods】1.S.mutans UA159 and C.albicans SC5314 were cultured.TSB medium containing 1%stevioside(1% Ste),stevioside with the same sweetness of 1% sucrose(Ste),1% xylitol(1%Xy),1% sucrose(1% S),1% sucrose +1% stevioside(1% S +1% Ste),and blank TSB medium without sugar added were configured.2.The effect of stevioside on the planktonic growth of mixed suspension was observed by measuring absorbance in planktonic state.The effect of stevioside on the dual-species biofilm formation was observed and quantified by crystal violet staining.The effect of stevioside on acid production was detected by p H value measurement.The morphology and structure of the mixed biofilm were observed by scanning electron microscopy(SEM).The viability of mixed biofilms was observed by live/dead bacteria/fungi staining and counting colony forming units(CFU),and the viability inhibition rate was calculated by Image J.The effects of stevioside on the synthesis of extracellular polysaccharide(EPS)in mixed biofilms were observed by specific staining of EPS and bacteria/fungi.【Results】1.Compared with 1% S and 1% S +1% Ste groups,1% Ste and Ste groups inhibited the mixed planktonic growth,mixed biofilm formation,acid production and the viability of S.mutans in mixed biofilm(P < 0.05),while there had no significant effect on the viability inhibition rate of mixed biofilm(P > 0.05).2.Compared with TSB group,1% Ste and Ste groups had no significant effects on mixed planktonic and biofilm growth,acid production,the viability inhibition rate of mixed biofilm,and S.mutans viability(P > 0.05).3.Compared with 1% Xy group,the formation of mixed biofilm in 1% Ste group was significantly decreased(P < 0.001),and the viability inhibition rate was significantly increased(P < 0.05),while planktonic growth,acid production and S.mutans viability had no significant effects(P > 0.05).4.The observation of SEM and CLSM showed that compared to 1% S,1% Ste and Ste group had fewer S.mutans and C.albicans,with most C.albicans yeast phase,and the smallest colonies.The stereoscopic structure was not obvious and relatively sparse.Live and dead cells overlapped basically,and the production of EPS in mixed biofilms was significantly inhibited(vs 1% S).【Conclusion】Compared with sucrose,stevioside inhibits the mixed growth and acid production of S.mutans and C.albicans,inhibits the three-dimensional structure and extracellular matrix formation of mixed biofilm,and inhibits the activity of mixed biofilm and the production of EPS.However,in the sucrose added background,the inhibitory effect of stevioside is significantly reduced.Experiment 3 Effects of stevioside on transcription of S.mutans and C.albicans mixed biofilm【Objective】Transcriptomic sequencing is used to investigate the effects of stevioside on caries gene expression in mixed biofilm.【Methods】TSB medium containing 1% sucrose,1% xylitol,1% stevioside and TSB medium without any sugar added were configured.S.mutans and C.albicans were inoculated in different medium to form mixed biofilm,and the RNA of the mixed biofilm was extracted for transcriptomic sequencing and analysis.【Results】1.Effects of stevioside on the transcriptome of S.mutans in mixed biofilm.Compared with sucrose group,scr A was significantly downregulated in the stevioside group which inhibited the transport of sucrose in S.mutans.In galactose metabolism,the upregulated genes were galE,galK,galR,galT,lacEF,lacG,lacAB,lacC and lacD.Glg operon(glg B-glg C-glg Dglg A-glg P),encoding enzymes intracellular polysaccharide(IPS)synthesis by S.mutans,were significantly upregulated.In pyruvate metabolism,the PDH pathway was up-regulated(pdh D,pdh A,pdh B,pdh C),while the LDH and PFL pathways were down-regulated(ldh and pfl A).In addition,Cia RH system(cia R and cia H)was down-regulated.2.Effects of stevioside on the transcriptome of C.albicans in mixed biofilm.Compared with sucrose group,HWP1,PGA10,PGA23,PGA33 and PGA54 were down-regulated in stevioside group,which inhibited C.albicans adhesion and colonization.SAP4,SAP5,and SAP6 were also downregulated in the stevioside group than in the sucrose group which belong to the secreted aspartyl proteinase(SAP)family associated with extracellular hydrolase.Furthermore,ATP16,encoding F1FO-ATP synthetase,was down-regulated which is associated with C.albicans virulence and pathogenicity.TPS1,TPS2,TPS3,HSP31,HSP60 were also down-regulated that inhibited the oxidative stress response of C.albicans.【Conclusion】1.Stevioside inhibits sucrose transport,but promotes other sugars transport and synthesis pathways to compensate the lack of sucrose in S.mutans.2.Stevioside inhibits the genes expression associated with adhesion,colonizing,virulence,and pathogenicity in C.albicans. |
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