DNA-Microarray Analysis And The Phylogenetic Tree Establishment Of The Microflora In Dental Plaque Biofilms From The Children With And Without Caries

Dental caries is one of the most common diseases which is harmful to human oral health. The incidence of caries in our country is high, which makes the prediction and prevention of this disease particularly important. Nevertheless, we do not currently have a complete understanding of its etiology, and the effective biological interventions to prevent caries have not been developed. Up to now, caries has been understood initiating in the micro-ecology environment, the dental plaque attaching and colonizing on the tooth surface, in which the bacteria reside and have specific relations and interactive effect with each other. According to plaque ecology theory, the bacteria are normal flora in the oral cavity. Under normal situations the composition of bacteria inter-flora is in a dynamic equilibrium, while when the dynamic equilibrium has been disturbed due to the alteration of external environment, the oral cavity's ecology would be loss of balance, then the amounts or(and) virulence of potential pathogens will increased, and the dental plaque would change to be carigenic from non-cariogenic that may result in dental caries owing to the serious expression of the microbial abnormal properties. There are about 700 oral bacterial species detected in oral biofilms, of which about 50% has not yet been cultivated, and the current studies on oral microflora diversity are limited to a few of foregone carigenic bacteria, instead on the entire and integrity cariogenic mechanism of dental plaque biofilms.Object:The present study used Human Oral Microbe Identification Microarray (HOMIM) to define the bacterial species and phylotypes of microflora in dental plaque from the children with sever caries and the healthy controls, including the diversity alteration of microbial distribution and their quantitative composition in dental plaque biofilms. To understand the phylogenetic relationship between different bacteria, the phylogenetic tree was established based on the bacteria detected in the dental plaque by using HOMIM.Methods:Plaque samples from 20 children with sever caries and 20 healthy controls were pooled from the intact enamel of the first permanent tooth. Bacterial DNA was extracted from each pooled plaque sample, and from which 16S rRNA genes were amplified by PCR with 16S rRNA universal primers and were labeled via incorporation of Cy3-dCTP in a second nested PCR. Hybridization of labeled amplicons on HOMIM, The microarray slides were scanned and full original data acquired from professional software were analyzed online to generate microbial profile maps, further were assayed the detected frequency and quantities of diverse bacteria in dental plaque with cluster analyses, Chi-Square x2 Test, and Wilcoxon rank sum test et.al. By using the ClustalX and MEGA program, the 16S rRNA gene sequences of all oral bacteria detected in HOMIM were analyzed to establish the phylogenetic tree.Results:(1) Bacterial profile of 20 children with sever caries and 20 healthy controls showed 123 bacterial species or clusters representing 9 bacterial phyla and 43 genus. The species/clusters detected in the plaque with sever caries ranged from 45 to 79(median is 53.5), and the healthy controls is 37 to 69(median is 51). (2) Subjects with sever caries were distinguished from healthy controls by a significantly higher frequency or quantum of putative pathogens, such as Streptococcus australis_ot073, Streptococcus oralis/sp clones C5MLM037/EK048_ot064₇07, Selenomonas sp clones DS071_otl38, Granulicatella elegans_ot596, Neisseria gonorrhoeae/polysaccharea_ot621₇37, Haemophilus sp clone BJ095_ot036 and Haemophilus parainfluenzae_ot718 (P<0.05). Species/clusters that were more prevalent in healthy controls than in patients with sever caries included Solobacterium moorei_ot678, Capnocytophaga sp clone DS022_ot332, Capnocytophaga sp. oral strain S3_ot337, Capnocytophaga granulose_ot325 (P<0.05). (3) Streptococcus mutans was detectable in only 1 case in the caries-free group, and just 1 smple from caries-active group presented detectable Lactobacillus. Both Streptococcus sobrinus and Streptococcus salivarius were not detectable in the two groups. (4) The phylogenetic tree was established based on the so many 16S rRNA gene sequences of all oral bacteria detected in this project.Conclusion:The relationship between microorganisms and caries was explored based on the standpoint of biofilm micro-ecology. In the oral ecological environment, the "core microbiome" may be existed. Some bacteria likely play important roles in the incipience progress of caries, e.g. Selenomonas, Granulicatella elegans, Neisseria and Haemophilus. S. mutans, S. sobrinus and Lactobacillus are not necessary for cariogenicity of dental plaque. Several other species are associated with caries, while some are associated with health. The present findings support the ecological plaque hypothesis in caries disease, and caries occurrence may be due to the complicated effective alteration of bacteria compostion in biofilms. Further studies of the potential etiological roles of these diverse bacterial communities, including additional and novel species, are recommened. The phylogenetic tree establised in this project could provide as a promising method and effective tool for thoese researches on the diversity and consanguinity of oral microorganisms.