Microbial differences between dental plaque and historic dental calculus are related to oral biofilm maturation stage

Dental calculus, calcified oral plaque biofilm, contains microbial and host biomolecules that can be used to study historic microbiome communities and host responses. Dental calculus does not typically accumulate as much today as historically, and clinical oral microbiome research studies focus primarily on living dental plaque biofilm. However, plaque and calculus reflect different conditions of the oral biofilm, and the differences in microbial characteristics between the sample types have not yet been systematically explored. Here, we compare the microbial profiles of modern dental plaque, modern dental calculus, and historic dental calculus to establish expected differences between these substrates.- Background - Results -- Authentication of a preserved oral biofilm in calculus samples -- Dental calculus and plaque biofilm communities are distinct -- Health-associated communities of dental plaque and calculus are distinct -- Signatures of health and of disease are shared in modern and historic calculus samples -- Microbial community differences between health and disease in calculus are poorly resolved -- Absence of caries-specific microbial profiles in dental calculus -- Microbial co-exclusion patterns in plaque and calculus reflect biofilm maturity -- Microbial complexes in plaque and calculus -- Functional prediction in calculus is poorly predictive of health status -- Proteomic profiles of historic healthy site calculus -- Correlations between taxonomic, proteomic, and metabolomic profiles - Discussion - Conclusions - Materials and methods --Historic and modern calculus sample collection DNA extraction -- DNA library construction and high-throughput sequencing -- DNA sequence processing -- Genetic assessment of historic calculus sample preservation -- Genetic microbial taxonomic profiling -- Principal component analysis -- Assessment of differentially abundant taxa -- Sparse partial least squares-discriminant analysis -- Assessment of microbial co-exclusion patterns -- Gene functional categorization with SEED -- Proteomics -- Metabolomics -- Regularized canonical correlation analysi

Macrophage Subset Sensitivity to Endotoxin Tolerisation by Porphyromonas gingivalis

Macrophages (MΦs) determine oral mucosal responses; mediating tolerance to commensal microbes and food whilst maintaining the capacity to activate immune defences to pathogens. MΦ responses are determined by both differentiation and activation stimuli, giving rise to two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2- MΦs. M2-like subsets predominate tolerance induction whereas M1 MΦs predominate in inflammatory pathologies, mediating destructive inflammatory mechanisms, such as those in chronic P.gingivalis (PG) periodontal infection. MΦ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by bacterial pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the susceptibility of MΦ subsets to suppression by P. gingivalis. CD14hi and CD14lo M1- and M2-like MΦs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and vitamin D3, respectively. MΦ subsets were pre-treated with heat-killed PG (HKPG) and PG-LPS prior to stimulation by bacterial PAMPs. Modulation of inflammation was measured by TNFα, IL-1β, IL-6, IL-10 ELISA and NFκB activation by reporter gene assay. HKPG and PG-LPS differentially suppress PAMP-induced TNFα, IL-6 and IL-10 but fail to suppress IL-1β expression in M1 and M2 MΦs. In addition, P.gingivalis suppressed NFκB activation in CD14lo and CD14hi M2 regulatory MΦs and CD14lo M1 MΦs whereas CD14hi M1 pro-inflammatory MΦs were refractory to suppression. In conclusion, P.gingivalis selectively tolerises regulatory M2 MΦs with little effect on pro-inflammatory CD14hi M1 MΦs; differential suppression facilitating immunopathology at the expense of immunity

Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules

Background: Inflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties.<p></p> Methods: An in vitro multi-species biofilm containing <i>S. mitis, F. nucleatum, P. Gingivalis</i> and <i>A. Actinomycetemcomitans</i> was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level.<p></p> Results: CHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA.<p></p> Conclusions: CHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.<p></p&gt

Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation

BackgroundPeriodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 ¿subgingival¿ bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition.ResultsFollowing 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant.ConclusionsThe gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola

A bacterial glycan core linked to surface (S)-layer proteins modulates host immunity through Th17 suppression

Tannerella forsythia is a pathogen implicated in periodontitis, an inflammatory disease of the tooth-supporting tissues often leading to tooth loss. This key periodontal pathogen is decorated with a unique glycan core O-glycosidically linked to the bacterium's proteinaceous surface (S)-layer lattice and other glycoproteins. Herein, we show that the terminal motif of this glycan core acts to modulate dendritic cell effector functions to suppress T-helper (Th)17 responses. In contrast to the wild-type bacterial strain, infection with a mutant strain lacking the complete S-layer glycan core induced robust Th17 and reduced periodontal bone loss in mice. Our findings demonstrate that surface glycosylation of this pathogen may act to ensure its persistence in the host likely through suppression of Th17 responses. In addition, our data suggest that the bacterium then induces the Toll-like receptor 2–Th2 inflammatory axis that has previously been shown to cause bone destruction. Our study provides a biological basis for pathogenesis and opens opportunities in exploiting bacterial glycans as therapeutic targets against periodontitis and a range of other infectious diseases

Bacterial 16S rRNA/rDNA Profiling in the Liquid Phase of Human Saliva

Human saliva can be separated by centrifugation into cell pellet and cell-free supernatant, which are called cellular phase and liquid phase in this study. While it is well documented that the cellular phase of saliva contains hundreds of oral bacteria species, little is known whether the liquid phase of saliva contains any information related to oral microbiota. In this study, we analyzed the bacterial nucleic acid contents of the liquid phase of saliva. Using primers universal to most eubacterial 16S rDNA, we detected large amounts of bacterial 16S rRNA and rDNA in the cell-free phase of saliva. Random sequencing analysis of forty PCR amplicons from the cell-free phase of saliva led to 15 operational taxonomic unit (OTU) groups. Furthermore, using denaturing gradient gel electrophoresis (DGGE), we compared 16S rRNA/rDNA profiles derived from liquid phases and cellular phases of saliva samples, and found positive correlations (Pearson Correlation=0.822, P<0.001) between these sample groups. These findings indicate that the liquid phase of saliva contains numerous bacterial 16S rRNA/rDNA molecules that have correlations with bacteria existing in the cellular phase

The vaginal microflora in relation to gingivitis

<p>Abstract</p> <p>Background</p> <p>Gingivitis has been linked to adverse pregnancy outcome (APO). Bacterial vaginosis (BV) has been associated with APO. We assessed if bacterial counts in BV is associated with gingivitis suggesting a systemic infectious susceptibilty.</p> <p>Methods</p> <p>Vaginal samples were collected from 180 women (mean age 29.4 years, SD ± 6.8, range: 18 to 46), and at least six months after delivery, and assessed by semi-quantitative DNA-DNA checkerboard hybridization assay (74 bacterial species). BV was defined by Gram stain (Nugent criteria). Gingivitis was defined as bleeding on probing at ≥ 20% of tooth sites.</p> <p>Results</p> <p>A Nugent score of 0–3 (normal vaginal microflora) was found in 83 women (46.1%), and a score of > 7 (BV) in 49 women (27.2%). Gingivitis was diagnosed in 114 women (63.3%). Women with a diagnosis of BV were more likely to have gingivitis (p = 0.01). Independent of gingival conditions, vaginal bacterial counts were higher (p < 0.001) for 38/74 species in BV+ in comparison to BV- women. Counts of four lactobacilli species were higher in BV- women (p < 0.001). Independent of BV diagnosis, women with gingivitis had higher counts of <it>Prevotella bivia </it>(p < 0.001), and <it>Prevotella disiens </it>(p < 0.001). <it>P. bivia, P. disiens, M. curtisii </it>and <it>M. mulieris </it>(all at the p < 0.01 level) were found at higher levels in the BV+/G+ group than in the BV+/G- group. The sum of bacterial load (74 species) was higher in the BV+/G+ group than in the BV+/G- group (p < 0.05). The highest odds ratio for the presence of bacteria in vaginal samples (> 1.0 × 10⁴cells) and a diagnosis of gingivitis was 3.9 for <it>P. bivia </it>(95% CI 1.5–5.7, p < 0.001) and 3.6 for <it>P. disiens </it>(95%CI: 1.8–7.5, p < 0.001), and a diagnosis of BV for <it>P. bivia </it>(odds ratio: 5.3, 95%CI: 2.6 to 10.4, p < 0.001) and <it>P. disiens </it>(odds ratio: 4.4, 95% CI: 2.2 to 8.8, p < 0.001).</p> <p>Conclusion</p> <p>Higher vaginal bacterial counts can be found in women with BV and gingivitis in comparison to women with BV but not gingivitis. <it>P. bivia </it>and <it>P. disiens </it>may be of specific significance in a relationship between vaginal and gingival infections.</p

Biogenesis and functions of bacterial S-layers.

The outer surface of many archaea and bacteria is coated with a proteinaceous surface layer (known as an S-layer), which is formed by the self-assembly of monomeric proteins into a regularly spaced, two-dimensional array. Bacteria possess dedicated pathways for the secretion and anchoring of the S-layer to the cell wall, and some Gram-positive species have large S-layer-associated gene families. S-layers have important roles in growth and survival, and their many functions include the maintenance of cell integrity, enzyme display and, in pathogens and commensals, interaction with the host and its immune system. In this Review, we discuss our current knowledge of S-layer and related proteins, including their structures, mechanisms of secretion and anchoring and their diverse functions