Dental Caries And Periodontal Diseases Associated Bacterial Colonization And Bacterial Diversity In Individuals With Gastric Precancerous Lesions

Dental caries and periodontal diseases are chronic biofilmbased diseases that occur in more than 90%(caries) and 35%(periodontal disease) in the adult population in the US. Mutans streptococci are generally considered to be the principal etiological agents of dental caries. S. mutans and S. sobrinus are the most commonly found bacteria in human plaque and are known to be associated with the development of dental caries. Periodontal diseases are characterized by gingival inflammation, periodontal pocket and attachment loss, which lead to tooth loss. In the past decades, increased epidemiological studies have presented evidence of positive association between periodontal diseases and the development of systemic diseases, including cardiovascular diseases, preterm delivery of low birth weight, diabetes mellitus, respiratory diseases, gastric cancer, AIDS and osteoporosis. However, the etiological details remain unclear. Nevertheless, more recent investigations have provided various results suggesting that periodontal infection and subsequent direct oral-hematogenous spread of bacteria are implicated in the development of various systemic diseases. Herein, we focus on the evaluation of the associations between the colonization of cariogenic and periodontal pathogens and their socio-demographic and clinical characteristics in individuals with gastric pre-cancerous lesions. Furthermore, we also evaluated the oral microbial diversity of the same group of individuals.Part One- The socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesionsGastric cancer is the second most common malignancy worldwide. The etiology of gastric cancer is not clear. There are many risk factors associated with gastric cancer, including life style, dietary structure, environmental, genetic and mental factors. Helicobacte pylori (H. pylori) was one of the most investigated etiological bacteria that are closely linked to chronic gastritis, peptic ulcer, gastric cancer. A few studies have suggested that H. pylori exist in subgingival plaque and therefore these sites are considered reservoirs for H. pylori. Meanwhile, several epidemiologic studies have also suggested a positive association between self-reported tooth loss and the risk of gastric cancer. Tooth loss in older age is more likely to be caused by chronic periodontal disease, whereas in younger age is usually caused by dental caries. Although these studies suggested that tooth lossis associated with an increased risk of gastric cancer, there was no detailed caries and periodontal assessment completed in these studies. More comprehensive clinical and microbial measurements are needed to investigate their association. The present study evaluated the socio-demographic and clinical oral health characteristics in individuals with gastric precancerous lesions.Methods The population of this study was consisted of 98 individuals who were scheduled for an upper endoscopy procedure at the gastrointestinal clinics in the Veterans Affairs New York Harbor Healthcare System and Bellevue Hospital Center from March 30,2009 to April 27,2011. All individuals were more than 30 years old and had no prior history of esophageal cancer, gastric cancer, gastric varices, or portal gastropathy. Based on biopsy diagnostics, these individuals were divided into two groups:Cases were those individuals with gastric precancerous lesion (chronic atrophic gastritis, intestinal metaplasia, or dysplasia). Controls were those individuals with no lesions or only superficial gastritis. Evaluation of the dental caries and periodontal status was conducted at the time of dental examination.Results Compared with the individuals in the control group, the individuals with gastric precancerous lesions harbored worse periodontal condition. They had deeper pocket depth, loss of attachment and more percent of sites with Pocket depth (PD) >4mm, Loss of attachment (LOA)≥3mm and LOA≥5mm. The percent of bleeding sites in the case group was significant higher than in the control groups (p<0.05).Conclusion There is association between gastric precancer and the periodontal condition.Part Two- The colonization of cariogenic and periodontal diseases pathogens in individuals with gastric precancerouslesionsThere is strong evidence that chronic inflammation is largely responsible for the early stages of disease progression. Because periodontal infection can lead to chronic systemic inflammation, which is an important risk factor in the development of gastric cancer, we hypothesize that major periodontal pathogens are associated with an increased risk for gastric cancer. To test this hypothesis, we conduct an exploratory hospital-based case-control study. The colonization of four periodontal pathogens, Porphyromonas gingivalis (Pg); Tannerella forsythensis (Tf); Treponema denticola (Td) and Actinobacillus actinomycetemcomitans (Aa) which are etiologically linked with destructive periodontal diseases; S. mutans (Sm) and S. sobrinus (Sb) which are cariogenic pathogens were measured in both saliva and subgingival plaque samples using the real-time quantitative PCR methods with species-specific DNA primers. We analyzed the microbial data together with the socio-demographic risk factors and clinical oral health characteristics, to see if the association exists between oral infectious diseases and gastric cancer.Methods The population of this study was the same as part one. Non-stimulated whole saliva and plaque samples were collected from each subject at the time of dental examination. Bacterial genomic DNA of the saliva and plaque was isolated using the MasterPure DNA purification kit. Standardization of the template DNA for real-time qPCR was established first so that the final results can be compared. The species-specific PCR primers for the four periodontal pathogens were used in this study. All reactions were carried out in duplicate, and the final analysis was based on the mean of the two qPCR reactions.Results The DNA level of periodontal pathogens in saliva was significantly associated with that in plaque (p<0.01). Subjects detected positive for Pg, Td and Tf had deeper pocket depth and higher percent of sites with PD>4mm and bleeding sites (p<0.05). The DNA level of Pg, Td and Tf in subgingival plaque was also significantly correlated with the percent of sites with PD>4mm and bleeding sites. The detection rates of Pg and Td were higher in the subjects with gastric precancerous lesion than the subjects in the healthy control groups. The detection rates of Aa and Tf were lower in the subjects with gastric precancerous lesion than the subjects in the control groups. The DNA level of four tested periodontal pathogens in both saliva and plaque was higher in case group than in control group, eventhough the difference didn't reach the statistic level.Conclusion Pg, Td and Tf were significantly associated with the clinical symptom, especially the pocket depth and bleeding on probing. The detection rates of cariogenic and periodontal pathogens were different in case and control groups. Subjects with gastric precancerous lesion were more likely to test positive for Pg and Td and had higher DNA level of four periodontal pathogens compared with control groups.Part Three-Oral microbial diversity in individuals with gastric precancerous lesionsThe bacterial community in the oral cavity is one of the most complex mixtures of bacteria known. Recent developments of molecular biological techniques for microbial identification have identified more than 500 microbial species. The oral cavity as a home to the microbial community plays an important role in human health and disease. Oral cavity is the entrance of the digestive tract, which is often regarded as the'inner outside'. The stomach also belongs to the digestive system. Their bacteria can interfere with each other to some extent. Oral bacteria can be taken to stomach along with the intake food, while some gastric cancer patients have GRED which might affect some bacteria in the oral cavity. Based on the information above, we hypothesize that the oral microbial diversity differs in individuals with gastric cancer compared with individuals without gastric cancer. To test this hypothesis, we conducted an exploratory hospital-based case-control study. In the present study, polymerase chain reaction-based denaturing gradient gel electrophoresis technique (PCR-DGGE) was used for the analysis of the microbial populations obtained from both saliva and subgingival plaque samples from all subjects, including individuals with gastric precancerous lesions and healthy controls. We also compared the variation of the oral bacterial population between the two study groups and among the colonization sites; and to determine if periodontal bacterial colonization has effect on oral microbial diversity.Methods A total of 98 subjects were enrolled in this study as part one. Bacterial samples were taken from all 98 subjects at the time of dental examination performed. Bacterial genomic DNA of the plaque or saliva was isolated as part two. Nested PCR was performed with use of the GeneAmp PCR SystemTM 9700. PCR products and species-specific DGGE reference markers were directly loaded in each lane, and electrophoresis was performed at a constant 60 V at 58℃for 17 hrs in 1×TAE, pH 8.5 buffer. After electrophoresis, the gels were rinsed and stained for 12 min in water containing 0.5μg/mL ethidium bromide, followed by 6 min of de-staining in water. DGGE profile images were digitally captured and recorded using the AlphalmagerTM 3300 System. DGGE profiles were analyzed using the BioNumerics Software.Results Subjects with gastric precancerous lesions had more DGGE bands (35.6) in pooled plaque sample, compared with healthy population (33.2), while there was no significant difference in saliva sample. The similarity of diversity between saliva and pooled plaque of the study population was 78.0%, lower than that among 6 subgingival plaques, which was 80.7%. The similarity of diversity between saliva and pooled plaque of the subjects with gastric precancerous lesions was lower compared with the control group. However, the similarity among 6 subgingival plaques of the case group was higher than that of the control group. The more similar among the 6 subgingival plaques the subjects had, the more microbial diversity in their pooled plaque they would have. Subjects had at least two teeth with none-zero value of the periodontal pathogens had less microbial diversity in saliva but more in pooled subgingival plaque.Conclusion Subjects with gastric precancerous lesions had more microbial diversity in pooled subgingival plaque, compared with healthy population. The similarity of same kind of sample (among plaques) was higher than different ones (saliva vs. plaque). There was highly correlation between diversity in subgingival plaque and the similarity among the subgingival plaques. The population had more tested positive teeth with periodontal pathogens had a decreased diversity in saliva but increased diversity in pooled subgingival plaque.