The Microflora Diversity And Profiles In Saliva From The Isolated Population With And Without Caries

Dental caries is one of the most common diseases which do harm to dental hard tissue. Both its occurrence and development are closely related with the microbial in dental ecosystem. As the repository and transfer media of dental microbial, saliva performs as a double-edged sword that can put both positive and negative effects on the occurrence of dental caries.As a part of dental external environment, on one hand, saliva plays an important part in dental caries prevention, on the other hand,it provides the bacteria a suitable ecological environment and sufficient nutrition for their attachmennt and development on another, and therefore increases the possibility of dental caries occurrence. Different oral microenvironment, including teeth, saliva, lip, tongue, buccal mucosa, palate mucosa etc., has its own different ecosystem particularity. The analysis on the microbial composition of dental plaque biofilm and mucosal tissue have been carried out both at home and abroad, but the whole picture of the microbial composition in saliva is not clear yet, whose importance in dental caries'prevention and therapy has not been ignored. Study on the oral microbial composition among a group of isolated people has greater comparability for the possible leading position of microorganism in infectious diseases because of their comparatively stable living environment, life style, diet and genetic background. And so far no research on this kind of study has been reported.Object:The present study used Human Oral Microbe Identification Microarray (HOMIM) to define microflora in dental plaque and saliva samples from a same group of minority children. To understand fully and comprehensively the microbial diversity in different parts of oral environment, and the correlation between the overall micro-ecology environment and the occurrence of dental caries.Furthermore this study compared and comprehensively analyzed microbial compositions in saliva and plaque after the high-throughout detection by Microarray technology. At the same time, through Real-time Q-PCR,this study has detected in the same samples for some commonly recognized cariogenic bacteria, including Streptococcus mutans,Streptococcus Sobrinus,Streptococcus oralis,Lactobacillus and Actinomyces naeslundii,so as to give a priliminary evalution microbial in Real-time Q-PCR used to determin the microbial profiles.Methods:Saliva sample were collected from 50 children from 6 to 8 years old including 20 caries free (dmfs=0) and 30 caries susceptive individuals (dmfs>6).16S rRNA genes were amplified by PCR with universal primers from bacterial DNA of saliva sample and labeled via incorporation of Cy3-dCTP in second nested PCR. After hybridization of labeled amplicons on The Human Microbe Identification Microarray (HOMIM),The microarray slides were scanned and full original data acquired from professional software were analyzed online to generate microbial profile maps, SYBR Green Real-time Q-PCR was used to detect Streptococcus mutans, Streptococcus Sobrinus, Streptococcus oralis,Lactobacillus and Actinomyces naeslundii.The detection rate and quantities of diverse bacteria in saliva were analyzed by cluster analyses, chi square test, Wilcoxon rank sum test et.alResults:DNA microarray analysis (1) Bacterial profile of children with 20 caries free and 30 caries susceptive individuals showed 94 bacterial species or clusters representing 6 bacterial phyla and 30 genus. The species/clusters detected in saliva with caries susceptive individuals (median is 33)was higher than that of the caries free (median is 25)(P<0.05).(2) Subjects with caries susceptive individuals were distinguished from caries-free by a significantly higher frequency or quantum of putative pathogens, such as Bacteroidetes[G-2J sp cloneAU126_ot274, Capnocytophaga granulosa cloneBB167_ot325, Capnocytophag sputigena_ot775,Fusobacterium periodontium_ot201,Fusobacterium nucleatum ss nucleatum and animalis_ot420₆98,Prevotella loescheii and sp clone GU027_ot472, Tannerella sp clone BU063_ot286, Cardiobacterium hominis_ot633, Campylobacter showae_ot763, Campylobacter Cluster I_ot580₇48₇63,Selenomonas infelix and sp clones EY047 and GT010 and IK004_ot126₄79₄81₆39_O54, Leptotrichia hofstadii_ot224,Leptotrichia buccalis and goodfellowii and Sneathia sanguinegens_ot563₈37₈45 (3)Species/clusters that were more prevalent in caries-free than in patients with caries susceptive individuals (P<0.05),included Haemophilus parainfluenzae ot718_W79, Gemella haemolysans_ot626_K63,Granulicatella elegans_ot596_AB29, Streptococcus intermedius_ot644,Streptococcus mitis bv2 and sp clone FP064_ot069₃98, Streptococcus oralis and sp clones C5MLM037and EK048_ot064₇07, Streptococcus sp clone FO042_ot067, Streptococcus infantis and sp clone FN042_ot065₆38, Rothia dentocariosa_ot587. (4)None of Streptococcus sobrinus,Lactobacillus and Actinomyces naeslundii has been found in all groups. Only one sample was found detectable level of Streptococcus mutans and Streptococcus salivarius respectively.(5) The microbial diversity with dental plaque (median 39) is higher than that in saliva (median 25) (P<0.05); for the same caries-free individuals,while the saliva microbial diversity was likly similar to dental plaque for those caries active individuals.(5)The results form comparing with dental plaque:â‘ The microbial diversity within dental plaque (median 39) is high than that in saliva (median 25) for the same caries-free individuals (P<0.05); while the saliva microbial diversity (median 33) was likely similar to dental plaque (median 30)for those same caries-active individuals of(P>0.05).â‘¡Both Fusobacterium periodontium_ot201_R20 and Fusobacterium nucleatum in saliva or plaque presented same prevalence that the two species showed higher detectable level in caries-active individuals than that in caries-free group (P<0.05).â‘¢Bacteroidetes [G-2] sp clone AU126_ot274_X57, Campylobacter showae_ot763_X35, Capnocytophaga sputigena_ot775_W46 were found the detection level in caries-free group were higher than that detected in caries-active group (P<0.05), while the saliva have a opposite level (P<0.05).â‘£Regarding the bacterial detection rate or quantum in dental plaque and saliva of same individuals in caries-active group:In addition to Streptococcus infantis and sp clone FN042_ot065₆38_Y74, Tannerella sp clone BU063_ot286_T83, Capnocytophaga sputigena_ot775_W46, Lachnospiraceae and Fusobacterium were found at higher levels in samples of saliva than in dental plaque(P<0.05).⑤Regarding the same caries-free group,the detection rate of 22 microbial species within dental plaque were higher than that of saliva,such as Capnocytophaga sputigena_ot775_W46, Campylobacter showae_ot763_X35, Streptococcus gordonii_ot622_F49,while there are only 5 species/clusters higher in saliva than in dental plaque,that are Streptococcus sp clone FO042_ot067,Gemella haemolysans_ot626,Granulicatella elegans_ot596,Streptococcus mitis bv2 and sp clone FP064_ot069 and Rothia dentocariosa_ot587 etal.â‘¥There are some bacteria at higher detection rate (greater than 80%).Saliva and dental plaque exhibited the highest proportions of Streptococcus intermedius_ot644,Leptotrichia buccalis and goodfellowii and Sneathia sanguinegens_ot563₈37₈45 in caries-active group,also likeCapnocytophaga granulosa and sp clone BB167_ot325,Neisseria Cluster ll_ot014₆09₆82₇64,Fusobacterium periodontium_ot201,Fusobacterium nucleatum ss nucleatum and animalis_ot420₆98 and Streptococcus Clusterâ…¡_ot071₇58 in caries-free group.Real-time Q-PCR analysis showed that (1) Streptococcus mutans, Streptococcus sobrinus Streptococcus sobrinus, Lactobacillus, Actinomyces naeslundii were detectable in all samples.(2)Streptococcus mutans, Streptococcus sobrinus, Actinomyces naeslundii detective frequencies in caries-active group were higher than in caries-free group, Streptococcus oralis and Lactobacillus hold an opposite detection level,but all the differences have no statistical significances((P>0.05)ConcIusion:The diversity of microbe within saliva derived from isolated population increased in caries-active status. The occurrence of dental caries is associated with some bacteria,such as Periodontal Fusobacterium, Fusobacterium nucleatum, Streptococcus mutans,Lactobacillus, Streptococcus sobrinus and some other known cariogenic bacterial may be not necessary for cariogenicity.The microbial composition of different micro-environment (just like saliva and dental plaque biofilm) from the same individual is diverse. The microbial diversity in dental plaque in caries-free state is much higher than that in saliva.However, the composition and distribution of bacteria in dental plaque may be similar to that in saliva when caries happenend.Oral micro-ecology is composed of many different micro-environment (including dental plaque and saliva).For the various interaction and complex distribution of microbial, there are still many bacteria need to be further explored,such as Bacteroidetes, Campylobacter showae, Capnocytophaga sputigena et al.Merely based on a certain bacterium level,saliva (or plaque) to evaluate caries susceptibility is not reliable.The overall microbial activity and unbalance may be the ultimate mechanism of dental caries.The study supports the ecology of the plaque.The minimum bacteria can be detectable after being amplification through Real-time fluorescence quantitative PCR,while failed to offer the overall objectiveness and comparability which could be achieved through DNA microarray.