Metagenomics Analysis Of Plaque Microbiomes From Root Caries And Healthy In Elderly

Caries is one of chronic bacterial infectious diseases with irreversible destruction progress. As the name implies, root caries (RC) occurs at the root cementum or dentine, and is caused by a supragingival microbial biofilm. The microbiological nature of dental plaque associated with root caries differs from that of crown caries, although it is still technically regarded as a kind of supragingival plaque infections. By the end of2012, the number of elderly people (over60years-old) in China has reached194million, which will overpass243million by2020, and exceed300million by2025. Considering the increasing population of elderly people, the incidence of root caries is rising. It is of great importance to investigate the etiological factor, risk assessment and prognosis on root caries.The etiology of root caries is influenced by four factors, including bacteria, substrate, host and time, among which bacteria microbial factors play a crucial role. However, oral cavity is one of the most complex microbial communities, thorough understanding of the effect of microbiological factors remains elusive because of our limited knowledge about their complex ecosystem. Knowledge of the relationship between oral bacteriology and RC is limited and confused, so the microbial etiology of root caries is still controversial. By using454GS FLX Titanium sequencing technology and real-time quantitative PCR (qPCR) technology, we analyzed the plaque and saliva microbiomes from root caries and healthy in order to explore the root surface caries microbial etiology of bacteriology in three parts.PART Ⅰ-Metagenomics analysis of plaque microbiomes from root caries and healthy in elderlyExp.1Comparing analysis the bacterial profiles of supragingival plaque between root caries and healthyAims:Root caries is one of the most common oral diseases in the elderly with limited knowledge. We used454pyrosequencing and qPCR methods to analysis the bacterial profiles of root caries and healthy plaque in the elderly to find out the microbiology factor on root caries.Methods:Supragingival plaques from Patient_cases, Patient_controls and Healthy_controls were sampled. Bacterial genomic DNA was isolated. Amplicons from V3-V5regions of the16S rRNA were sequenced by454GS FLX Titanium. Differences community profiles in health and disease were compared by BLAST against HOMD database.Results:Healthy-controls and Patient-cases showed no significant differences in terms of alpha diversity (Chao1and Shannon). As for beta diversity Healthy-controls and Patient-cases was distinguished from each other, appearing more variable community membership and structure in root caries microbiome but relatively conservative ones in health microbiome. PCoA (principle coordinate analysis) results represented that Patient-controls was at an intermediate stage between Healthy-controls and Patient-cases, but more inclined to the former. Differences between health-and caries-associated bacterial communities were observed at phyla, genera and species levels. Core supragingival microbiome associated with health and root caries were also defined.Conclusions:Propionibacterium acidifaciens, Streptococcus mutans, Olsenella profusa, Prevotella multisaccharivorax and Lactobacillus crispatus were showed more associated with root caries, whereas Delftia acidovorans, Bacteroidetes[G-2] sp., Lachnospiraceae[G-3] sp. and Prevotella intermedia were associated with health. The core supragingival microbiome in health and caries were defined;7caries-associated species representing little in health and having been defined as candidate cariogenic core microbiome (CCCM) required more attention. Exp.2Detection the total bacterial load of supragingival plaque between root caries and healthy and verification the method of454sequencingAims:To detect the total bacterial load of supragingival plaque in three groups (Healthy_controls, Patient_controls and Patient_cases) and the relative amounts of three selected bacteria. To verify the method of454sequencing, we compared the results from qPCR and454GS FLX Titanium.Methods:Supragingival plaques from Patient_cases, Patient_controls and Healthy_controls were sampled. Bacterial genomic DNA was isolated. The total bacterial load and the amount of DNA level of Streptococcus mutans, Bifidobacterium spp. and Lactobacillus spp. were detected by quantitative real-time PCR (qPCR). Results:Patient-controls was significant higher in terms of the total bacterial load than Patient_cases (p<0.05). Healthy-controls showed higher biomass than Patient_cases with no statistical significance (p>0.05). Similar results between qPCR and454sequencing were observed, showing that the454sequencing technology is verified reliable, stable and repeatable.Conclusions:The total bacterial load in health status (Healthy_controls and Patient_controls) showed higher DNA level than in disease status (Patient_cases). The results of454sequencing technology are reliable andrepeatable. PART Ⅱ-Analysis the bacterial profiles of plaque microbiomes from root caries and crown cariesAims:Dental caries are classified into root caries (RC) and crown caries (CC). Compare the bacterial profiles of plaque microbiomes from root caries and crown caries in order to explore the bacterial factor for dental caries.Methods:Supragingival plaques from RC_cases, CC_cases, Patient_controls and Healthy_controls were sampled. Bacterial genomic DNA was isolated. Amplicons from V3-V5regions of the16S rRNA were sequenced by454GS FLX Titanium. Differences community profiles in the four groups were compared by BLAST against HOMD database.Results:RC-cases showed higher alpha diversity (species, Chaol, Shannon and Simpson) than CC_cases with no statistical significance (p>0.05). As for unweighted UniFrac beta diversity, Healthy-controls was distinguished from other three groups, appearing more relatively conservative (p<0.05). RC_cases showed higher weighted UniFrac beta diversity than CC_cases and Healthy_controls (p<0.05). PCoA (principle coordinate analysis) results represented aggregative trend between RC-cases and CC-cases, but detrending with Healthy_controls. Similar bacterial communities between RC-cases and CC-cases were observed at different phylogenetic levels, and shared&unshared OTU curves, core microbiome associated with RC and CC were defined. A series of caries-associated and health-associated species were also found by comparing the bacterial communities between CC_cases and Healthy_controls. Conclusions:Bacterial communities and core microbiome of supragingival plaque between RC_cases and CC_cases were similar, implicating the bacterial factor for RC and CC may be similar. In addition, Propionibacterium acidifaciens, Streptococcus mutans, Olsenella profusa, Lactobacillus salivarius and Oribacterium sp. were more closely associated with caries, whereas Bacteroidetes [G-2J, Fusobacterium sp., Capnocytophaga leadbetteri, Prevotella intermedia and Prevotella sp. were associated with health. PART III-Quantification analysis five cariogenic bacteria in saliva from root caries and healthy in elderlyAims:Root caries is a significant oral public problem among humans due to improvement in health care, longer life expectancy, and increasing demand to maintain oral health. This study aimed to analyse the bacterial composition of five putative-cariogenic bacteria, namely Streptococcus mutans (S. mutans), Streptococcus sobrinus (S. sobrinus), Bifidobacterium spp.(Bif spp.), Lactobacillus spp.(Lac spp.) and Actinomyces naeslundii (A. naeslundii), in the saliva of the elderly and assess their association with RC.Methods:A total of43saliva samples were collected and divided into two groups:the root caries group (RCG, n=21) and the caries-free group (CFG, n=22). Bacterial DNA was isolated from the saliva samples. Quantitative real-time PCR (qPCR) was performed to determine the presence and amounts of the five microorganisms with specific primers. Spearman correlation test was conducted to evaluate the relationship between the numbers of root decayed filled surfaces (RDFS), root caries index (RCI) and salivary levels of the bacteria.Results:The prevalence of S. Sobrinus is significantly higher in RCG than in CFG (p <0.05); co-positive for S. mutans&Bif spp., S. sobrinus&Bif spp. and S. mutans&S. sobrinus&Bif spp. were manifested significantly higher in RCG than in CFG (p<0.05). The salivary levels of S. mutans, S. sobrinus, Bif spp. and Lac spp. were significantly higher in RCG than in CFG (p<0.05). RDFS and RCI (RDFS/RCI) were positively associated with salivary levels of S. mutans, S. sobrinus and Bif spp.(r=0.658/0.635, r=0.465/0.420and r=0.407/0.406, respectively). Conclusions:Co-positive from mutli-bacteria suggesting that co-positive with multi-bacteria could be valuable in risk assessment for RC. S. mutans, S. sobrinus and Bif spp. in saliva appear to be associated with RC in the elderly and may act as biomarkers for the risk assessment of RC.