Quantification of five putative periodontal pathogens in female patients with and without chronic periodontitis by real-time polymerase chain reaction

Chronic periodontitis is a highly prevalent endogenous polymicrobial disease. To better understand the etiology of the disease a quantitative approach is mandatory and real-time PCR is the molecular technique currently preferred to achieve this purpose. Taking into account that such a kind of study is still scarce, we aimed to evaluate the association between periodontal microbiota and chronic periodontitis. A total of 60 low-income age-matched female adults, 30 with chronic periodontitis and 30 without periodontal disease, were enrolled. DNA obtained from subgingival specimens was used for quantification of Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, and Prevotella intermedia by real-time PCR. A. actinomycetemcomitans, E. corrodens, and F. nucleatum were detected in all subjects, P. gingivalis was observed in 70.0% and 46.6% and P. intermedia in 90.0% and 80.0% of chronic periodontitis patients and periodontally healthy subjects, respectively. P. gingivalis mean count was significantly higher in patients with chronic periodontitis than in periodontally healthy individuals. Accurate detection and quantification of five putative periodontal pathogens was feasible using a simple and fast real-time PCR protocol. Although P. gingivalis and P. intermedia have been found more commonly in chronic periodontitis patients, no statistical difference was observed between periodontally diseased and healthy groups. Quantitative data indicated association between P. gingivalis and chronic periodontitis. However, because of its uneven distribution, it should not be solely taken as a marker of periodontal status.     

Changes in the incidence of periodontal pathogens during long-term monitoring and after application of antibacterial drugs

The incidence of potential periodontal pathogens (Aggregatibacter actinomycetemcomitans, formerly Actinobacillus actinomycetemcomitans, Tannerella forsythia, Porphyromonas gingivalis, Prevotella nigrescens, Prevotella intermedia and Capnocytophaga ochracea) was monitored in patients with chronic periodontitis and in healthy control subjects. Two types of studies were carried out in which the composition of the bacterial communities in different niches of the same oral cavity ecosystem was investigated. Fluctuation or at least pronounced quantitative changes in the incidence of individual species in time were documented in the long-term study as well as after the local administration of antibacterial drug Chlo-Site or Metronidazole. Even within two weeks, a turnover of the monitored bacteria in separate niches of the oral biotope can be detected. A relatively high incidence of the tested periopathogens in the clinically healthy teeth of patients implies that even the “healthy” niches in the periodontal biotope function as a dynamic reservoir of periopathogenic microorganisms. This should be kept in mind when a local application of antibacterial compounds is used in the therapy of periodontal disease.     

Investigation of the Effect of Type 2 Diabetes Mellitus on Subgingival Plaque Microbiota by High-Throughput 16S rDNA Pyrosequencing

Diabetes mellitus is a major risk factor for chronic periodontitis. We investigated the effects of type 2 diabetes on the subgingival plaque bacterial composition by applying culture-independent 16S rDNA sequencing to periodontal bacteria isolated from four groups of volunteers: non-diabetic subjects without periodontitis, non-diabetic subjects with periodontitis, type 2 diabetic patients without periodontitis, and type 2 diabetic patients with periodontitis. A total of 71,373 high-quality sequences were produced from the V1-V3 region of 16S rDNA genes by 454 pyrosequencing. Those 16S rDNA sequences were classified into 16 phyla, 27 classes, 48 orders, 85 families, 126 genera, and 1141 species-level OTUs. Comparing periodontally healthy samples with periodontitis samples identified 20 health-associated and 15 periodontitis-associated OTUs. In the subjects with healthy periodontium, the abundances of three genera (Prevotella, Pseudomonas, and Tannerella) and nine OTUs were significantly different between diabetic patients and their non-diabetic counterparts. In the subjects carrying periodontitis, the abundances of three phyla (Actinobacteria, Proteobacteria, and Bacteriodetes), two genera (Actinomyces and Aggregatibacter), and six OTUs were also significantly different between diabetics and non-diabetics. Our results show that type 2 diabetes mellitus could alter the bacterial composition in the subgingival plaque.     

Relationship of periodontal clinical parameters with bacterial composition in human dental plaque

More than 600 bacterial species have been identified in the oral cavity, but only a limited number of species show a strong association with periodontitis. The purpose of the present study was to provide a comprehensive outline of the microbiota in dental plaque related to periodontal status. Dental plaque from 90 subjects was sampled, and the subjects were clustered based on bacterial composition using the terminal restriction fragment length polymorphism of 16S rRNA genes. Here, we evaluated (1) periodontal clinical parameters between clusters; (2) the correlation of subgingival bacterial composition with supragingival bacterial composition; and (3) the association between bacterial interspecies in dental plaque using a graphical Gaussian model. Cluster 1 (C1) having high prevalence of pathogenic bacteria in subgingival plaque showed increasing values of the parameters. The values of the parameters in Cluster 2a (C2a) having high prevalence of non-pathogenic bacteria were markedly lower than those in C1. A cluster having low prevalence of non-pathogenic bacteria in supragingival plaque showed increasing values of the parameters. The bacterial patterns between subgingival plaque and supragingival plaque were significantly correlated. Chief pathogens, such as Porphyromonas gingivalis, formed a network with other pathogenic species in C1, whereas a network of non-pathogenic species, such as Rothia sp. and Lautropia sp., tended to compete with a network of pathogenic species in C2a. Periodontal status relates to non-pathogenic species as well as to pathogenic species, suggesting that the bacterial interspecies connection affects dental plaque virulence.     

Detection rates of periodontal bacteria and herpesviruses in different forms of periodontal disease

The aim was to investigate the detection rates of periodontal bacteria (Porphyromonas gingivalis, Tannerella forsythia, Prevotella intermedia, and Aggregatibacter actinomycetemcomitans) and herpesviruses (herpes simplex virus-1 [HSV-1], cytomegalovirus [CMV], and Epstein-Barr virus [EBV]) in different forms and severity of periodontal disease, and to compare them with those in periodontally healthy subjects. One hundred and twenty-nine patients participated in the study: 39 diagnosed with periodontal abscess (PA), 33 with necrotizing ulcerative periodontitis (NUP), 27 with chronic periodontitis (CP), and 30 participants with healthy periodontal tissue represented a healthy control group. All patients with periodontal disease (PA, NUP, and CP) were also divided into two groups according to the severity of their disease: moderate and severe periodontitis. The subgingival samples were collected from the periodontitis active sites and the detection of microorganisms was performed by end-point polymerase chain reaction analyses. The results revealed significantly higher detection rates of P. gingivalis, T. forsythia, and P. intermedia in all three groups of patients with periodontitis than in healthy participants. The highest detection rate of A. actinomycetemcomitans was noticed in CP, which was significantly higher than that in PA, NUP, and healthy control. The occurrence of EBV was significantly higher in NUP than in CP and healthy participants. CMV was detected significantly more frequently in PA and NUP than in CP and healthy participants. Comparisons among healthy participants and patients with moderate and severe periodontitis showed significantly higher detection rates of EBV and CMV in patients with severe forms of periodontitis than in healthy participants and those with moderate periodontitis.     

Pyrosequencing Analysis of Oral Microbiota in Children with Severe Early Childhood Dental Caries

Severe early childhood caries are a prevalent public health problem among preschool children throughout the world. However, little is known about the microbiota found in association with severe early childhood caries. Our study aimed to explore the bacterial microbiota of dental plaques to study the etiology of severe early childhood caries through pyrosequencing analysis based on 16S rRNA gene V1–V3 hypervariable regions. Forty participants were enrolled in the study, and we obtained twenty samples of supragingival plaque from caries-free subjects and twenty samples from subjects with severe early childhood caries. A total of 175,918 reads met the quality control standards, and the bacteria found belonged to fourteen phyla and sixty-three genera. Our results show the overall structure and microbial composition of oral bacterial communities, and they suggest that these bacteria may present a core microbiome in the dental plaque microbiota. Three genera, Streptococcus, Granulicatella, and Actinomyces, were increased significantly in children with severe dental cavities. These data may facilitate improvements in the prevention and treatment of severe early childhood caries.     

Possible translocation of periodontal pathogens into the lymph nodes draining the oral cavity

Numerous publications have reported the presence of periodontopathogenic bacteria in peripheral and central vascular lesions. However, it is unclear how this bacterial translocation occurs. The objective of this study was to investigate whether periodontopathic bacteria are translocated to lymph nodes proximal to the oral cavity. Obtaining lymph node samples is not ethically feasible unless they are excised as part of the surgical management of patients with cancer. This study analyzed formalin-fixed and paraffin-embedded lymph nodes, histologically negative for cancer cell invasion, that were excised from 66 patients with histories of head and neck cancer. Real-time PCR was performed to amplify the 16S ribosomal DNA fragments from Porphyromonas gingivalis, Treponema denticola, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, and Prevotella intermedia. The relationship between bacterial detection and cancer severity, gender, and the use of anti-cancer therapy was examined by Fisher??s exact test. P. gingivalis, T. forsythia, and P. intermedia were present in 17%, 8%, and 8% of the samples of submandibular and submental lymph nodes, respectively. There were no significant relationships between bacterial detection and the cancer disease status, patient gender or use of anticancer therapy. According to these data, it appears that the translocation of periodontopathic bacteria may occur via lymphatic drainage, irrespective of the cancer disease status, gender or anticancer therapy.     

Periodontitis,periodontopathic bacteria and lactoferrin

Lactoferrin (LF) is a component of saliva and is suspected to be a defense factor against oral pathogens including Streptococcus mutans and Candida albicans. Periodontitis is a very common oral disease caused by periodontopathic bacteria. Antimicrobial activities and other biological effects of LF against representative periodontopathic bacteria, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia, have been widely studied. Association of polymorphisms in LF with incidence of aggressive periodontitis and the role of LF in the gingival crevicular fluid as a marker of periodontitis severity have also been reported. Periodontopathic bacteria reside as a biofilm in supragingival and subgingival plaque. Our recent study indicated that LF exhibits antibacterial activity against planktonic forms of P. gingivalis and P. intermedia at higher concentrations, and furthermore, LF effectively inhibits biofilm formation and reduces the established biofilm of these bacteria at physiological concentrations. A small-scale clinical study indicated that oral administration of bovine LF reduces P. gingivalis and P. intermedia in the subgingival plaque of chronic periodontitis patients. LF seems to be a biofilm inhibitor of periodontopathic bacteria in vitro and in vivo.     

Killing activity of LFchimera on periodontopathic bacteria and multispecies oral biofilm formation in vitro

Lactoferrin chimera (LFchimera), a heterodimeric peptide containing lactoferrampin (LFampin265–284) and a part of lactoferricin (LFcin17–30), possesses a broad spectrum of antimicrobial activity. However, there is no report on the inhibitory effects of LFchimera against multispecies oral biofilms. This study aimed to determine the effects of LFchimera in comparison to chlorhexidine digluconate (CHX) and minocycline hydrochloride (MH), on in vitro multispecies biofilms derived from subgingival plaque of periodontitis patients harboring Aggregatibacter actinomycetemcomitans. First the effects of LFchimera against planktonic and an 1-day old biofilm of the periodontopathic bacteria, A. actinomycetemcomitans ATCC 43718 were established. Then, the effects on biofilm formation and bacterial viability in the multispecies biofilm were determined by crystal violet staining and LIVE/DEAD BacLight Bacterial Viability kit, respectively. The results revealed that a significant reduction (P?<?0.05) in biofilm formation occurred after 15 min exposure to 20 µM of LFchimera or CHX compared to control. In contrast, MH at concentration up to 100 µM did not inhibit biofilm formation. The ratio of live/dead bacteria in biofilm was also significantly lower after 15 min exposure to 20 µM of LFchimera compared to control and 20–50 µM of CHX and MH. Altogether, the results obtained indicate that LFchimera is able to inhibit in vitro subgingival biofilm formation and reduce viability of multispecies bacteria in biofilm better than CHX and MH.     

Quantitative Proteomics Reveal Distinct Protein Regulations Caused by Aggregatibacter actinomycetemcomitans within Subgingival Biofilms

Periodontitis is an infectious disease that causes the inflammatory destruction of the tooth-supporting (periodontal) tissues, caused by polymicrobial biofilm communities growing on the tooth surface. Aggressive periodontitis is strongly associated with the presence of Aggregatibacter actinomycetemcomitans in the subgingival biofilms. Nevertheless, whether and how A. actinomycetemcomitans orchestrates molecular changes within the biofilm is unclear. The aim of this work was to decipher the interactions between A. actinomycetemcomitans and other bacterial species in a multi-species biofilm using proteomic analysis. An in vitro 10-species “subgingival” biofilm model, or its derivative that included additionally A. actinomycetemcomitans, were anaerobically cultivated on hydroxyapatite discs for 64 h. When present, A. actinomycetemcomitans formed dense intra-species clumps within the biofilm mass, and did not affect the numbers of the other species in the biofilm. Liquid chromatography-tandem mass spectrometry was used to identify the proteomic content of the biofilm lysate. A total of 3225 and 3352 proteins were identified in the biofilm, in presence or absence of A. actinomycetemcomitans, respectively. Label-free quantitative proteomics revealed that 483 out of the 728 quantified bacterial proteins (excluding those of A. actinomycetemcomitans) were accordingly regulated. Interestingly, all quantified proteins from Prevotella intermedia were up-regulated, and most quantified proteins from Campylobacter rectus, Streptococcus anginosus, and Porphyromonas gingivalis were down-regulated in presence of A. actinomycetemcomitans. Enrichment of Gene Ontology pathway analysis showed that the regulated groups of proteins were responsible primarily for changes in the metabolic rate, the ferric iron-binding, and the 5S RNA binding capacities, on the universal biofilm level. While the presence of A. actinomycetemcomitans did not affect the numeric composition or absolute protein numbers of the other biofilm species, it caused qualitative changes in their overall protein expression profile. These molecular shifts within the biofilm warrant further investigation on their potential impact on its virulence properties, and association with periodontal pathogenesis.     

Effect of Microsphaeropsis ochracea on Production of Sclerotia-borne and Airborne Conidia of Botrytis squamosa

Onion leaf blight, caused by Botrytis squamosa (Walker), is a destructive disease of onion. Conidia produced on overwintered sclerotia are the main source of initial inoculum, and those produced on lesions are responsible for secondary inoculum build-up. The biological control agent Microsphaeropsis ochracea (Carisse &; Bernier) was evaluated for its ability to control sclerotia-borne inoculum, to colonize onion leaves and reduce the production of conidia under field conditions. Colonisation by M. ochracea of onion leaves at different growth stages was monitored and its effect on B. squamosa sporulation on necrotic leaves was evaluated. Onion plots were treated with either Dithane® or with M. ochracea at 7–10-day intervals and according to inoculum production index (IPI). The concentration of airborne conidia and the number of lesions per leaf, on 20 plants per plot, were evaluated throughout the cropping season. The number of conidia produced per sclerotium treated with M. ochracea, was reduced by 75.5%. In the field, M. ochracea colonised only senescent or necrotic leaves and reduced the production of conidia on these leaves by an average of 82% as compared with untreated leaves. Best disease control was obtained by Dithane®, followed by M. ochracea applied at 7–10-day intervals. For the three years of the study, there were no significant differences in airborne concentrations of conidia in plots treated at 7–10-day intervals with Dithane® or M. ochracea. Fall application of M. ochracea could be used as a sanitation practice to reduce initial inoculum or as a part of an IPM program during the season.     

Pyrosequencing Analysis of Oral Microbiota Shifting in Various Caries States in Childhood

Dental caries is one of the most prevalent childhood diseases worldwide, but little is known about the dynamic characteristics of oral microbiota in the development of dental caries. To investigate the shifting bacterial profiles in different caries states, 60 children (3–7-year-old) were enrolled in this study, including 30 caries-free subjects and 30 caries-active subjects. Supragingival plaques were collected from caries-active subjects on intact enamel, white spot lesions and carious dentin lesions. Plaques from caries-free subjects were used as a control. All samples were analyzed by 454 pyrosequencing based on 16S rRNA gene V1-V3 hypervariable regions. A total of 572,773 pyrosequencing reads passed the quality control and 25,444 unique phylotypes were identified, which represented 18 phyla and 145 genera. Reduced bacterial diversity in the cavitated dentin was observed as compared with the other groups. Thirteen genera (including Capnocytophaga, Fusobacterium, Porphyromonas, Abiotrophia, Comamonas, Tannerella, Eikenella, Paludibacter, Treponema, Actinobaculum, Stenotrophomonas, Aestuariimicrobium, and Peptococcus) were found to be associated with dental health, and the bacterial profiles differed considerably depending on caries status. Eight genera (including Cryptobacterium, Lactobacillus, Megasphaera, Olsenella, Scardovia, Shuttleworthia, Cryptobacterium, and Streptococcus) were increased significantly in cavitated dentin lesions, and Actinomyces and Corynebacterium were present at significant high levels in white spot lesions (P?<?0.05), while Flavobacterium, Neisseria, Bergeyella, and Derxia were enriched in the intact surfaces of caries individuals (P?<?0.05). Our results showed that oral bacteria were specific at different stages of caries progression, which contributes to informing the prevention and treatment of childhood dental caries.     

Quantitative Molecular Detection of Putative Periodontal Pathogens in Clinically Healthy and Periodontally Diseased Subjects

Periodontitis is a multi-microbial oral infection with high prevalence among adults. Putative oral pathogens are commonly found in periodontally diseased individuals. However, these organisms can be also detected in the oral cavity of healthy subjects. This leads to the hypothesis, that alterations in the proportion of these organisms relative to the total amount of oral microorganisms, namely their abundance, rather than their simple presence might be important in the transition from health to disease. Therefore, we developed a quantitative molecular method to determine the abundance of various oral microorganisms and the portion of bacterial and archaeal nucleic acid relative to the total nucleic acid extracted from individual samples. We applied quantitative real-time PCRs targeting single-copy genes of periodontal bacteria and 16S-rRNA genes of Bacteria and Archaea. Testing tongue scrapings of 88 matched pairs of periodontally diseased and healthy subjects revealed a significantly higher abundance of P. gingivalis and a higher total bacterial abundance in diseased subjects. In fully adjusted models the risk of being periodontally diseased was significantly higher in subjects with high P. gingivalis and total bacterial abundance. Interestingly, we found that moderate abundances of A. actinomycetemcomitans were associated with reduced risk for periodontal disease compared to subjects with low abundances, whereas for high abundances, this protective effect leveled off. Moderate archaeal abundances were health associated compared to subjects with low abundances. In conclusion, our methodological approach unraveled associations of the oral flora with periodontal disease, which would have gone undetected if only qualitative data had been determined.     

Microbial Diversity Similarities in Periodontal Pockets and Atheromatous Plaques of Cardiovascular Disease Patients

Background and Objective

The immune and infectious alterations occurring in periodontitis have been shown to alter the development and severity of cardiovascular disease. One of these relationships is the translocation of oral bacteria to atheroma plaques, thereby promoting plaque development. Thus, the aim of this study was to assess, by 16s cloning and sequencing, the microbial diversity of the subgingival environment and atheroma plaques of patients concomitantly suffering from periodontitis and obstructive coronary artery atherosclerosis (OCAA).

Methods

Subgingival biofilm and coronary balloons used in percutaneous transluminal coronary angioplasty were collected from 18 subjects presenting with generalized moderate to severe periodontitis and OCAA. DNA was extracted and the gene 16S was amplified, cloned and sequenced.

Results

Significant differences in microbial diversity were observed between both environments. While subgingival samples mostly contained the phylum Firmicutes, in coronary balloons, Proteobacteria (p<0.05) was predominant. In addition, the most commonly detected genera in coronary balloons were Acinetobacter, Alloprevotella, Pseudomonas, Enterobacter, Sphingomonas and Moraxella, while in subgingival samples Porphyromonas, Filifactor, Veillonella, Aggregatibacter and Treponema (p<0.05) were found. Interestingly, 17 identical phylotypes were found in atheroma and subgingival samples, indicating possible bacterial translocation between periodontal pockets and coronary arteries.

Conclusion

Periodontal pockets and atheromatous plaques of cardiovascular disease patients can present similarities in the microbial diversity.     

Detection of periodontopathogenic bacteria in pregnant women by traditional anaerobic culture method and by a commercial molecular genetic method

To culture facultative and strict anaerobic bacteria is a well-established method for analyzing subgingival plaque samples. Micro-IDent^® and micro-IDent^® Plus (HAIN Lifescience GmbH, Nehren, Germany) tests are two commercially available rapid PCR-based methods for the identification and quantification of putative periodontopathogen bacteria. In this study, we compared these commercial PCR-based hybridization methods with conventional anaerobic culture technique. A total of 36 subgingival plaque samples were collected from periodontal pockets of pregnant women with chronic localized periodontitis. Aliquots of these samples were evaluated with species-specific probes provided by micro-IDent^® and micro-IDent^® Plus tests simultaneously, and from the same samples anaerobic and capnophylic bacteria were cultured on selective media. The overall agreement between both methods was excellent for Eubacterium nodatum, Tannerella forsythia and Porphyromonas gingivalis (97–92%), fair for Capnocytophaga sp, Eikenella corrodens, Actinobacillus actinomycetemcomitans, and Prevotella intermedia (91–89%) and poor for Fusobacterium nucleatum, Parvimonas micra (Micromonas micros), and Campylobacter rectus (86–78%). Discrepancies in the results may be explained by inability of culture method to distinguish between closely related taxa (e.i P. intermedia/Prevotella. nigrescens), and problems of keeping periodontopathogen bacteria viable, which is required for successful detection by standard culture method. Nucleic acid-based methods may replace cultivation method as frequently used methods in microbiological diagnosis of progressive periodontitis, thus micro-IDent^® andmicro-IDent^® Plus tests can be recommended where culture of periodontopathogenic bacteria is not performed in routine microbiology laboratories to analyze subgingival plaque samples.     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

Higher Prevalence of Epstein–Barr Virus DNA in Deeper Periodontal Pockets of Chronic Periodontitis in Japanese Patients

Periodontitis, a complex chronic inflammatory disease caused by subgingival infection, is among the most prevalent microbial diseases in humans. Although traditional microbiological research on periodontitis has focused on putative bacteria such as Porphyromonas gingivalis, the herpes virus is proposed to be involved in the pathogenesis of periodontitis because bacterial etiology alone does not adequately explain various clinical aspects. In this study, we established for the first time, more Epstein–Barr virus (EBV) DNA is found deeper in periodontal pockets of chronic periodontitis in Japanese patients. Subgingival samples were collected from 85 patients with chronic periodontitis having two periodontal sites with probing depths (PD) of ≤3 mm (shallow) or ≥5 mm (deep) and were subjected to a nested polymerase chain reaction. EBV DNA was more frequently detected in patients with deeper PD sites (66%) than in those with shallow PD sites (48%) or healthy controls (45%). Coexistence of EBV DNA and P. gingivalis was significantly higher in patients with deeper PD sites (40%) than in those with shallow PD sites (14%) or healthy controls (13%). Although no difference in clinical index for periodontitis, the odds ratio of EBV DNA in patients with deeper PD sites was 2.36, which was 2.07-fold higher than that in those with shallow PD sites. Interestingly, the odds of acquiring chronic periodontitis (PD ≥5 mm) were higher in the presence of both EBV DNA and P. gingivalis compared with either EBV DNA or P. gingivalis only. In addition, we also observed that EBV-encoded small RNA (EBER) in positive cells of human gingival tissues. These results would suggest that EBV DNA may serve as a pathogenic factor leading to chronic periodontitis among Japanese patients.     

Oral microbiota of children in a school-based dental clinic

ObjectivesDental caries disproportionately affects disadvantaged subjects. This study hypothesized that there were greater caries extent and higher levels of caries-associated and anaerobic subgingival bacterial species in oral samples of Hispanic and immigrant children compared with non-Hispanic and US born children.MethodsChildren from a school-based dental clinic serving a community with a large Hispanic component were examined, and the extent of caries was recorded. Microbial samples were taken from teeth and the tongues of children. Samples were analyzed using DNA probes to 18 oral bacterial species.ResultsSeventy five children were examined. Extent of caries increased with child age in immigrant, but not in US born or Hispanic children. There were no differences in the microbiota based on ethnicity or whether the child was born in US or not. There was a higher species detection frequency from teeth than tongue samples. Levels of Streptococcus mutans and other Streptococcus spp increased with caries extent. Prevotella intermedia, Tannerella forsythia and Selenomonas spp were detected at low levels in these children.ConclusionsWe conclude that, while there was a high rate of dental caries in disadvantaged school children, there were no differences in the caries-associated microbiota, including S. mutans, based on ethnicity or immigration status. Furthermore, while anaerobic subgingival, periodontal pathogens were also detected in children, there was no difference in species detection based on ethnicity or immigration status. Increased levels of streptococci, including S. mutans, however, were detected with high caries levels. This suggested that while it is beneficial to target preventive and treatment programs to disadvantaged populations, there is likely no additional benefit to focus on subgroups within a population already at high risk for dental disease.     

Distinct and complex bacterial profiles in human periodontitis and health revealed by 16S pyrosequencing

Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1 393 579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health- and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.     

Bacteriology of Most Frequent Oral Anaerobic Infections

The anaerobic infections most frequently found in the oral cavity are gingivoperiodontal diseases and pulpal and periapical infections. Gingivitis and adult periodontitis are the most frequent forms. In adult periodontitis the subgingival microbiota are complex and there is a prevalence of Porphyromonas gingivalis, Prevotella intermedia,Prevotella nigrescens , Actinobacillus actinomycetemcomitans, Bacteroides forsythus,Peptostreptococcus micros , Campylobacter rectus, and species of Fusobacterium, Eikenella andTreponema .The microflora associated with peri-implant infections are similar to the microflora found in periodontal diseases, particularly in partially edentulous patients. Implant placement is therefore not recommended in patients presenting with uncontrolled periodontal disease.Likewise, there is a similarity between the genera identified in periodontal pockets and infected root canals, and in periapical infections. However, some species are more prevalent than others in both infections. The following were predominantly observed inside the root canals: Prevotella intermedia, Prevotella nigrescens,Peptostreptococcus anaerobius , Peptostreptococcus micros, Eubacterium lentum, Eubacterium alactolyticum and Porphyromonas endodontalis, with strong associations among some species.Pericoronaritis is another infection associated with anaerobic Gram-negative bacilli and treponeme. There are a great number of methods for microbiological diagnosis, and treatment of some oral infections depends on close interaction between the microbiologist and the dentist.