Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus.

Recent genomic data have revealed multiple interactions between Neanderthals and modern humans, but there is currently little genetic evidence regarding Neanderthal behaviour, diet, or disease. Here we describe the shotgun-sequencing of ancient DNA from five specimens of Neanderthal calcified dental plaque (calculus) and the characterization of regional differences in Neanderthal ecology. At Spy cave, Belgium, Neanderthal diet was heavily meat based and included woolly rhinoceros and wild sheep (mouflon), characteristic of a steppe environment. In contrast, no meat was detected in the diet of Neanderthals from El Sidrón cave, Spain, and dietary components of mushrooms, pine nuts, and moss reflected forest gathering. Differences in diet were also linked to an overall shift in the oral bacterial community (microbiota) and suggested that meat consumption contributed to substantial variation within Neanderthal microbiota. Evidence for self-medication was detected in an El Sidrón Neanderthal with a dental abscess and a chronic gastrointestinal pathogen (Enterocytozoon bieneusi). Metagenomic data from this individual also contained a nearly complete genome of the archaeal commensal Methanobrevibacter oralis (10.2× depth of coverage)-the oldest draft microbial genome generated to date, at around 48,000 years old. DNA preserved within dental calculus represents a notable source of information about the behaviour and health of ancient hominin specimens, as well as a unique system that is useful for the study of long-term microbial evolution.

Probiotic Lactobacillus rhamnosus GG prevents alveolar bone loss in a mouse model of experimental periodontitis.

AIM: This study investigated the role of Lactobacillus rhamnosus GG (LGG) on bone loss and local and systemic inflammation in an in vivo mouse model of experimental periodontitis (PD). MATERIALS AND METHODS: Experimental PD was induced in mice by oral inoculation with Porphyromonas gingivalis and Fusobacterium nucleatum over a period of 44 days. The probiotic LGG was administered via oral inoculation or oral gavage prior to, and during disease induction. The antimicrobial activity of LGG on the inoculum was also tested. Alveolar bone levels and gingival tissue changes were assessed using in vivo microcomputed tomography and histological analysis. Serum levels of mouse homologues for IL-8 were measured using multiplex assays. RESULTS: Pre-treatment with probiotics either via oral gavage or via oral inoculation significantly reduced bone loss (p < .0001) and gingival inflammation (p < .0001) when compared with PD group. Oral gavage treatment group had significantly less tartrate-resistant acid phosphatase positive cells (p < .02) then PD group. LGG showed no antimicrobial activity against P. gingivalis and F. nucleatum. CONCLUSIONS: Lactobacillus rhamnosus GG effectively suppresses bone loss in a mouse model of induced PD irrespective of the mode of administration.

Porphyromonas gingivalis peptidylarginine deiminase substrate specificity.

UNLABELLED: While a group of oral commensals have been implicated in the aetiology of chronic periodontitis; the asaccharolytic Gram negative anaerobe Porphyromonas gingivalis is most commonly reported to be associated with severe forms of the disease. Although a variety of human tissues can produce a number of peptidylarginine deiminase (PAD), enzymes that convert peptide bound arginine residues to citrulline, P. gingivalis is one of the few prokaryotes known to express PAD. Protein and peptide citrullination are important in the development of rheumatoid arthritis and in recent years a number of authors have suggested a possible link between periodontitis and rheumatoid arthritis (RA). Indeed, some have linked P. gingivalis directly to RA via the action of PAD. Accordingly, the prime purpose of this study was to further characterise PAD in P. gingivalis cells particular emphasis on substrate specificity, using arginine containing peptides and RA relevant proteins. METHODS: P. ginigvalis W50 was anaerobically cultured in BHI broth, cells harvested and resuspended in assay buffer. A colourimetric assay was developed to measure citrulline and employed to determine enzyme activity using the substrate BAEE. The assay was employed to investigate the effects of environmental pH and temperature on activity. Citrullination of BAEE by sonicated cells allowed the proportion of intracellular enzyme to be estimated. Enzyme specificity and substrate preference were investigated by using various arginine containing peptides, proteins and arginine analogues, as substrates and measuring the rate of citrullination. The influence of gingipains on citrullination was assessed by measuring the rates of citrullination of bovine serum albumin in the presence of protease inhibitors. RESULTS: Enzyme activity decreased by 13% following exposure of cells to 60 °C for 10 min. A comparison of intact and disrupted cells indicated that 90% of PAD activity is cell surface associated and the remainder cytoplasmic. Optimal pH for enzyme activity was between pH 7.5 and 8. All small arginine-containing peptides were citrullinated with reaction rates faster than that for free arginine with rates that varied with arginine residue position and number. Arginine analogues exhibited minimal effect and influence when tested as either substrates or competitive inhibitors. Cells were able to citrullinate yeast enolase, human vimentin and fibrin at varying rates. All proteins were modified at slower rates than those for peptide substrates. Inhibition of gingipains had no influence on the rate of protein citrullination. CONCLUSIONS: P. gingivalis PAD is a primarily cell surface associated, heat stable, enzyme that exhibits optimal activity under alkaline conditions similar to those present in the inflammatory environment. The enzyme displays high specificity for arginine residues in peptides and modified arginine in all positions and the gingipains did not influence the rate of protein citrullination. The ability of the enzyme to convert arginine residues in all proteins tested would indicate that its presence in inflamed tissue may promote autoimmune reactions by creation of altered host epitopes.

Ancient dental calculus reveals oral microbiome shifts associated with lifestyle and disease in Great Britain.

The prevalence of chronic, non-communicable diseases has risen sharply in recent decades, especially in industrialized countries. While several studies implicate the microbiome in this trend, few have examined the evolutionary history of industrialized microbiomes. Here we sampled 235 ancient dental calculus samples from individuals living in Great Britain (∼2200 BCE to 1853 CE), including 127 well-contextualized London adults. We reconstructed their microbial history spanning the transition to industrialization. After controlling for oral geography and technical biases, we identified multiple oral microbial communities that coexisted in Britain for millennia, including a community associated with Methanobrevibacter, an anaerobic Archaea not commonly prevalent in the oral microbiome of modern industrialized societies. Calculus analysis suggests that oral hygiene contributed to oral microbiome composition, while microbial functions reflected past differences in diet, specifically in dairy and carbohydrate consumption. In London samples, Methanobrevibacter-associated microbial communities are linked with skeletal markers of systemic diseases (for example, periostitis and joint pathologies), and their disappearance is consistent with temporal shifts, including the arrival of the Second Plague Pandemic. This suggests pre-industrialized microbiomes were more diverse than previously recognized, enhancing our understanding of chronic, non-communicable disease origins in industrialized populations.

A proteomic investigation of Fusobacterium nucleatum alkaline-induced biofilms.

BACKGROUND: The Gram negative anaerobe Fusobacterium nucleatum has been implicated in the aetiology of periodontal diseases. Although frequently isolated from healthy dental plaque, its numbers and proportion increase in plaque associated with disease. One of the significant physico-chemical changes in the diseased gingival sulcus is increased environmental pH. When grown under controlled conditions in our laboratory, F. nucleatum subspecies polymorphum formed mono-culture biofilms when cultured at pH 8.2. Biofilm formation is a survival strategy for bacteria, often associated with altered physiology and increased virulence. A proteomic approach was used to understand the phenotypic changes in F. nucleatum cells associated with alkaline induced biofilms. The proteomic based identification of significantly altered proteins was verified where possible using additional methods including quantitative real-time PCR (qRT-PCR), enzyme assay, acidic end-product analysis, intracellular polyglucose assay and Western blotting. RESULTS: Of 421 proteins detected on two-dimensional electrophoresis gels, spot densities of 54 proteins varied significantly (p < 0.05) in F. nucleatum cultured at pH 8.2 compared to growth at pH 7.4. Proteins that were differentially produced in biofilm cells were associated with the functional classes; metabolic enzymes, transport, stress response and hypothetical proteins. Our results suggest that biofilm cells were more metabolically efficient than planktonic cells as changes to amino acid and glucose metabolism generated additional energy needed for survival in a sub-optimal environment. The intracellular concentration of stress response proteins including heat shock protein GroEL and recombinational protein RecA increased markedly in the alkaline environment. A significant finding was the increased abundance of an adhesin, Fusobacterial outer membrane protein A (FomA). This surface protein is known for its capacity to bind to a vast number of bacterial species and human epithelial cells and its increased abundance was associated with biofilm formation. CONCLUSION: This investigation identified a number of proteins that were significantly altered by F. nucleatum in response to alkaline conditions similar to those reported in diseased periodontal pockets. The results provide insight into the adaptive mechanisms used by F. nucleatum biofilms in response to pH increase in the host environment.

Probiotic Lactobacillus Rhamnosus GG Protects Against P. Gingivalis And F. Nucleatum Gut Dysbiosis.

OBJECTIVES: This study investigated changes induced by Porphyromonas gingivalis and on gastrointestinal histology and gut microbiome in a mouse model of experimental periodontitis. The effect of probiotic Lactobacillus rhamnosus GG (LGG) in altering these changes was also investigated. METHODS: IThirty-six mice were allocated into six groups. Experimental alveolar bone loss was induced by oral inoculation with P. gingivalis and F. nucleatum. LGG was orally inoculated or orally gavaged. Gastrointestinal tissue changes were assessed using histological analysis and immunohistochemistry. Caecal microbiome was analysed by sequencing 16S rRNA genes of caecal content. RESULTS: Inoculation with P. gingivalis and F. nucleatum induced inflammation throughout gastrointestinal tract (p less than 0.05), increased expression of IL-6 in ileum (p = 0.052) and altered composition of caecal microbiome (p less than 0.05) in experimental mice compared to controls. Mice treated with LGG had reduced tissue inflammation in duodenum (p = 0.044) and lowered levels of IL-6 in ileum (p = 0.048) when compared with disease. LGG therapy influenced gut microbiome changes. CONCLUSION: P. gingivalis and F. nucleatum inoculation induced significant changes in intestinal inflammation and caecal microbiome. Oral gavage with LGG exerted a protective effect against intestinal inflammation and limited gut microbiome changes associated with P. gingivalis and F. nucleatum.

Probiotics and Periodontitis - A Literature Review.

OBJECTIVE: This review was designed to explore the use of probiotics in prevention or treatment of periodontitis. METHODS: A search was performed using MEDLINE and bibliographies from previous reviews in order to identify any randomised controlled animal and human probiotic interventions in periodontitis. RESULTS: Five studies using probiotics in animal models of periodontitis and eight clinical studies using probiotics in patients with chronic periodontitis were analysed. The analysis of the animal models showed reduction in periodontal pathogens and bleeding on probing when probiotics were used in adjunction to mechanical debridement and significant increase in alveolar bone levels and bone density in the probiotic groups when compared with placebo. Some of the results of the clinical studies indicated decreased clinical parameters (gingival inflammation, bleeding on probing, plaque index) and decreased pro-inflammatory markers levels in saliva or gingival crevicular fluid in treated periodontitis patients when compared with controls or placebo. Other results included decreased periodontal pocket depth and clinical attachment loss for scaling and root planing plus probiotic treatment versus scaling and root planing alone or placebo and also reduction in Porphyromonas gingivalis numbers and the total viable count and proportion of obligate anaerobic bacteria. CONCLUSIONS: Within the limitations of this review, the results are encouraging, supporting the notion that there is a place for probiotics in the treatment of periodontitis. Future independent studies are needed to investigate specific probiotic strains, doses, delivery methods, treatment schedules, mechanisms of action, safety and how to maintain the results of the probiotic interventions.

Genetic and environmental influences on dentofacial structures and oral health: studies of Australian twins and their families.

Our studies of the teeth and faces of Australian twins commenced at the School of Dentistry, The University of Adelaide in the early 1980s. There are now over 900 pairs of twins enrolled in our continuing investigations, together with 1200 relatives. There are 3 main cohorts of participants. The first cohort comprises around 300 pairs of teenage twins for whom various records have been collected, including dental casts, facial photographs, finger and palm prints and information on laterality, including handedness. The second cohort comprises around 300 pairs of twins who have been examined at 3 stages of dental development from approximately 4 years of age to about 14 years: at primary, mixed, and permanent dentition (excluding 3rd molars) stages. The most recent study of tooth emergence and oral health, for which we are currently recruiting twins, will provide a third cohort of around 500 twin pairs aged from around birth to 3 to 4 years of age. Our broad aim in these studies has been to improve our understanding of how genetic and environmental factors contribute to variation in dental and facial features, and to oral health. We have also used our data to investigate aspects of the determination of laterality, particularly the fascinating phenomenon of mirror imaging. We plan to maximize the use of the longitudinal data and DNA we have collected, and continue to collect, by performing genome-wide scans for putative genetic linkage peaks for a range of dental features, and then to test for association between a series of likely candidate genes and our phenotypes.

Growth pH and transient increases in amino acid availability influence polyglucose synthesis by Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum is a Gram-negative anaerobe that has been implicated in the aetiology of several diseases including periodontal diseases. Like other fusobacteria, it derives energy from the fermentation of amino acids and, in resting (non-growing) cells, this enables the organism to transport glucose and synthesise intracellular polyglucose (IP). The continued availability and fermentation of amino acids inhibits IP breakdown. We have grown F. nucleatum in continuous culture in a chemically defined medium under amino acid limitation and determined the fate of glucose during growth at steady state and during transient increases in the concentration (pulses) of serine and glutamate. When grown under steady state conditions, IP synthesis dramatically increased at culture pH values of 6.1 and 7.8 and appeared to be a result of cell stress. IP synthesis also increased when the culture was pulsed with serine or glutamate but was rapidly metabolised as the added amino acids were depleted. These results may help to explain the role of IP synthesis in response to environmental stress.

Porphyromonas gingivalis peptidylarginine deiminase, a key contributor in the pathogenesis of experimental periodontal disease and experimental arthritis.

OBJECTIVES: To investigate the suggested role of Porphyromonas gingivalis peptidylarginine deiminase (PAD) in the relationship between the aetiology of periodontal disease and experimentally induced arthritis and the possible association between these two conditions. METHODS: A genetically modified PAD-deficient strain of P. gingivalis W50 was produced. The effect of this strain, compared to the wild type, in an established murine model for experimental periodontitis and experimental arthritis was assessed. Experimental periodontitis was induced following oral inoculation with the PAD-deficient and wild type strains of P. gingivalis. Experimental arthritis was induced via the collagen antibody induction process and was monitored by assessment of paw swelling and micro-CT analysis of the radio-carpal joints. Experimental periodontitis was monitored by micro CT scans of the mandible and histological assessment of the periodontal tissues around the mandibular molars. Serum levels of anti-citrullinated protein antibodies (ACPA) and P. gingivalis were assessed by ELISA. RESULTS: The development of experimental periodontitis was significantly reduced in the presence of the PAD-deficient P. gingivalis strain. When experimental arthritis was induced in the presence of the PAD-deficient strain there was less paw swelling, less erosive bone damage to the joints and reduced serum ACPA levels when compared to the wild type P. gingivalis inoculated group. CONCLUSION: This study has demonstrated that a PAD-deficient strain of P. gingivalis was associated with significantly reduced periodontal inflammation. In addition the extent of experimental arthritis was significantly reduced in animals exposed to prior induction of periodontal disease through oral inoculation of the PAD-deficient strain versus the wild type. This adds further evidence to the potential role for P. gingivalis and its PAD in the pathogenesis of periodontitis and exacerbation of arthritis. Further studies are now needed to elucidate the mechanisms which drive these processes.

Qualitative comparison of sonic or laser energisation of 4% sodium hypochlorite on an Enterococcus faecalis biofilm grown in vitro.

The effectiveness of sonic activation, laser activation and syringe irrigation of 4% sodium hypochlorite in removing an Enterococcus faecalis biofilm was compared. Biofilms were grown in extracted human single rooted teeth using a flow cell apparatus. After 4 weeks' growth, teeth were subjected to each treatment using 4% sodium hypochlorite and radicular dentinal surfaces of the root canals were analysed by scanning electron microscopy. Results showed that sonic activation and syringe irrigation with sodium hypochlorite showed reduced numbers of bacterial cells on the radicular dentine but were not effective in eliminating E. faecalis in the dentinal tubules. Laser activation of sodium hypochlorite resulted in clean dentine walls and undetectable levels of bacteria within dentinal tubules. Qualitatively, sonic or laser activation of 4% NaOCl resulted in greater bacterial reduction compared with syringe irrigation, with laser activation producing the greatest overall reduction.

Inability to form a biofilm of Streptococcus mutans on silver fluoride- and potassium iodide-treated demineralized dentin.

OBJECTIVE: The presence of a biofilm is necessary for both initiation and progression of dental caries. Silver-based preparations incorporated into, or applied onto, various materials designed for medical use have been shown to be effective in inhibiting biofilm formation. The purpose of this in vitro study was to measure whether a topical application of diamine silver fluoride (AgF) followed by potassium iodide (KI) on partially demineralized dentin affected the formation of a Streptococcus mutans biofilm. METHOD AND MATERIALS: Forty partially demineralized dentin disks were divided into 4 groups as follows: 10 disks as a control, 10 disks treated with AgF followed by KI, 10 disks treated with KI, and 10 disks treated with AgF. The outer surfaces of the disks were examined with a scanning electron microscope. Cross sections of the disks were subjected to electron probe microanalysis (EPMA) to determine the levels of calcium, phosphorous, silver, and fluoride in the dentin. RESULTS: An S mutans biofilm covered the entire exposed surfaces of all control and KI-treated disks. No discernible bacterial biofilm was detected on disks treated with AgF or AgF/KI. Detectable amounts of silver and fluoride were found up to 450 microm in the AgF and AgF/KI sections. CONCLUSIONS: Demineralized dentin disks treated with AgF and AgF/KI prevented the formation of an S mutans biofilm and were significantly more resistant to further demineralization than the control and KI-treated disks over the experimental period. The presence of silver and fluoride in the outer layers of the disks treated with AgF and AgF/KI was the likely cause of the prevention of biofilm formation. Additional studies are required before any clinical recommendations can be made.

The proteomic profile of Fusobacterium nucleatum is regulated by growth pH.

Fusobacterium nucleatum is a saccharolytic Gram-negative anaerobic organism believed to play an important role in the microbial succession associated with the development of periodontal disease. Its genome contains niche-specific genes shared with the other inhabitants of dental plaque, which may help to explain its ability to survive and grow in the changing environmental conditions experienced in the gingival sulcus during the transition from health to disease. The pH of the gingival sulcus increases during the development of periodontitis and this is thought to occur by the metabolism of nutrients supplied by gingival crevicular fluid. In comparison with other plaque inhabitants, F. nucleatum has the greatest ability to neutralize acidic environments. The differential expression of soluble cytoplasmic proteins induced by acidic (pH 6.4) or basic (pH 7.4 and 7.8) conditions, during long-term anaerobic growth in a chemostat, was identified by two-dimensional gel electrophoresis and image analysis software. Twenty-two proteins, found to have altered expression in response to external pH, were identified by tryptic digestion and mass spectrometry. Eight differentially expressed proteins associated with increased energy (ATP) production via the 2-oxoglutarate and Embden-Meyerhof pathways appeared to be directed towards either cellular biosynthesis or the maintenance of internal homeostasis. Overall, these results represent the first proteomic investigation of F. nucleatum and the identification of gene products which may be important in the organism's persistence during the transition from health to disease in vivo.