Associations between Host Genetic Variants and Subgingival Microbiota in Patients with the Metabolic Syndrome.

Host genetic variants may affect oral biofilms, playing a role in the periodontitis-systemic disease axis. This is the first study to assess the associations between host genetic variants and subgingival microbiota in patients with metabolic syndrome (MetS); 103 patients with MetS underwent medical and periodontal examinations and had blood and subgingival plaque samples taken. DNA was extracted and processed, assessing a panel of selected single nucleotide polymorphisms (SNPs) first (hypothesis testing) and then expanding to a discovery phase. The subgingival plaque microbiome from these patients was profiled. Analysis of associations between host genetic and microbial factors was performed and stratified for periodontal diagnosis. Specific SNPs within RUNX2, CAMTA1 and VDR genes were associated with diversity metrics with no genome-wide associations detected for periodontitis severity or Mets components at p < 10-7. Severe periodontitis was associated with pathogenic genera and species. Some SNPs correlated with specific bacterial genera as well as with microbial taxa, notably VDR (rs12717991) with Streptococcus mutans and RUNX2 (rs3749863) with Porphyromonas gingivalis. In conclusion, variation in host genotypes may play a role in the dysregulated immune responses characterizing periodontitis and thus the oral microbiome, suggesting that systemic health-associated host traits further interact with oral health and the microbiome.

Interdental and subgingival microbiota may affect the tongue microbial ecology and oral malodour in health, gingivitis and periodontitis.

BACKGROUND AND OBJECTIVE: Oral malodour is often observed in gingivitis and chronic periodontitis patients, and the tongue microbiota is thought to play a major role in malodorous gas production, including volatile sulphur compounds (VSCs) such as hydrogen sulphide (H2 S) and methanethiol (CH3 SH). This study aimed to examine the link between the presence of VSCs in mouth air (as a marker of oral malodour) and the oral bacterial ecology in the tongue and periodontal niches of healthy, gingivitis and periodontitis patients. METHODS: Participants were clinically assessed using plaque index, bleeding on probing (BOP) and periodontal probing depths, and VSC concentrations in their oral cavity measured using a portable gas chromatograph. Tongue scrapings, subgingival and interdental plaque were collected from healthy individuals (n = 22), and those with gingivitis (n = 14) or chronic periodontitis (n = 15). The bacterial 16S rRNA gene region V3-V4 in these samples was sequenced, and the sequences were analysed using the minimum entropy decomposition pipeline. RESULTS: Elevated VSC concentrations and CH3 SH:H2 S were observed in periodontitis compared with health. Significant ecological differences were observed in the tongue microbiota of healthy subjects with high plaque scores compared to low plaque scores, suggesting a possible connection between the microbiota of the tongue and the periodontium and that key dysbiotic changes may be initiated in the clinically healthy individuals who have higher dental plaque accumulation. Greater subgingival bacterial diversity was positively associated with H2 S in mouth air. Periodontopathic bacteria known to be prolific VSC producers increased in abundance on the tongue associated with increased bleeding on probing (BOP) and total percentage of periodontal pockets >6 mm, supporting the suggestion that the tongue may become a reservoir for periodontopathogens. CONCLUSION: This study highlights the importance of the periodontal microbiota in malodour and has detected dysbiotic changes in the tongue microbiota in periodontitis.

In vitro effectiveness of pomegranate extract present in pet oral hygiene products against canine oral bacterial species.

Background and Aim: Pomegranate is known to possess antibacterial properties, partly because of its punicalagin content. However, its effect on canine oral bacterial species has not yet been elucidated. In this study, we evaluated the effect of pomegranate extract present in pet dental products on the growth and survival of five canine oral bacterial species in biofilms. Materials and Methods: Five bacterial species, Neisseria shayeganii, Neisseria canis, Porphyromonas gulae, Porphyromonas macacae, and Porphyromonas crevioricanis, were individually cultured for biofilm formation and exposed to pomegranate extract (or control) for 15 min. Cell survival was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and was compared between different conditions using a student's t-test. In addition, the individual strains were grown in planktonic suspensions and exposed to serial dilutions of the extract to determine the minimum inhibitory concentration. Results: At a concentration of 0.035% w/v, the extract significantly reduced the survival of P. gulae (-39%, p < 0.001) and N. canis (-28%, p = 0.08) in biofilms. At similar concentrations, the extract also completely or partially inhibited the growth of N. canis and Porphyromonas spp. in planktonic suspensions, respectively. Conclusion: The pomegranate extract found in some pet dental products can limit bacterial growth and survival in the biofilms formed by N. canis and P. gulae in vitro. As P. gulae is involved in periodontal disease progression, limiting its proliferation using products containing pomegranate extract could contribute to disease prevention. Further studies on dogs receiving such products are necessary to confirm these effects.

Novel anti-microbial therapies for dental plaque-related diseases.

Control of dental plaque-related diseases has traditionally relied on non-specific removal of plaque by mechanical means. As our knowledge of oral disease mechanisms increases, future treatment is likely to be more targeted, for example at small groups of organisms, single species or at key virulence factors they produce. The aim of this review is to consider the current status as regards novel treatment approaches. Maintenance of oral hygiene often includes use of chemical agents; however, increasing problems of resistance to synthetic antimicrobials have encouraged the search for alternative natural products. Plants are the source of more than 25% of prescription and over-the-counter preparations, and the potential of natural agents for oral prophylaxis will therefore be considered. Targeted approaches may be directed at the black-pigmented anaerobes associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Studies to date have demonstrated selective killing of Porphyromonas gingivalis and Prevotella intermedia in biofilms. Functional inhibition approaches, including the use of protease inhibitors, are also being explored to control periodontitis. Replacement therapy by which a resident pathogen is replaced with a non-pathogenic bacteriocin-producing variant is currently under development with respect to Streptococcus mutans and dental caries.

Tolerance of MRSA ST239-TW to chlorhexidine-based decolonization: Evidence for keratinocyte invasion as a mechanism of biocide evasion.

OBJECTIVES: Information on genetic determinants of chlorhexidine tolerance (qacA carriage and MIC) in vitro is available, although evidence of the clinical impact and mechanisms remain poorly understood. We investigated why, following chlorhexidine intervention, prevalent epidemic MRSA ST22 and ST36 clones declined at an ICU, whilst an ST239-TW clone did not. The chlorhexidine tolerant ST239-TW phenotypes were assessed for their protein binding, cell adhesion and intracellular uptake potential. METHODS: Six ST22, ST36 and ST239-TW bloodstream infection isolates with comparable chlorhexidine MICs were selected from a 2-year outbreak in an ICU at Guy's and St. Thomas' Hospital. Isolates were tested for fibrinogen and fibronectin binding, and adhesion/internalization into human keratinocytes with and without biocide. RESULTS: Binding to fibrinogen and fibronectin, adhesion and intracellular uptake within keratinocytes (P < 0.001) and intracellular survival in keratinocytes under chlorhexidine pressure (ST22 3.18%, ST36 4.57% vs ST239-TW 12.79%; P < 0.0001) was consistently higher for ST239-TW. CONCLUSIONS: We present evidence that MRSA clones with similarly low in vitro tolerance to chlorhexidine exhibit different in vivo susceptibilities. The phenomenon of S. aureus adhesion and intracellular uptake into keratinocytes could therefore be regarded as an additional mechanism of chlorhexidine tolerance, enabling MRSA to evade infection control measures.

Host defence peptides-a bridge between the innate and adaptive immune responses.

At the interface of innate and adaptive immunity, host defence peptides have been shown to enhance the overall immune response, where peptide expression and activity map onto aspects of the response to infection. This includes the ability to chemoattract phagocytic and antigen-presenting cells, and regulate the host cytokine response. Effects of peptides on B- and T-lymphocyte function, including B-cell activation and antibody production, cytotoxic T-cell and natural-killer-cell killing, and T-helper cell function, are starting to demonstrate that some of these peptides are capable of directing a prolonged cellular and humoral response to a pathogen.

Potential impact of nanotechnology on the control of infectious diseases.

Nanotechnology encompasses those technologies used to fabricate materials, including sphere, cubic and needle-like nanoscaled particles (approximately 5-100nm), and near-nanoscaled devices (up to micrometres). In comparison, mycoplasma are approximately 200nm in length, and a nanometre is 10(-9) of a metre. The field of nanotechnology is experiencing rapid growth, with many and diverse potential applications being explored in the biomedical field, including the control of infectious diseases. Nanotechnology not only has the potential to offer improvements to current approaches for immunisation, drug design and delivery, diagnostics and cross-infection control, but is also unexpectedly delivering many new tools and capabilities.

Topographic distribution of bacteria associated with oral malodour on the tongue.

OBJECTIVE: To investigate the topographic distribution of bacterial types and loads associated with mid-morning oral malodour on the tongue surface. DESIGN: Fifty subjects with good oral health and at least 20 natural uncrowned teeth were included. Samples were taken with sterile brushes from the dorsal anterior (DA), dorsal middle (DM), dorsal posterior (DP), dorsal posterior to the circumvallate papillae (DPCP), lateral posterior (LP) and ventral posterior (VP) tongue surfaces. Samples were cultured on appropriate media for anaerobic bacteria, aerobic bacteria, Gram-negative anaerobic bacteria, volatile sulphur compound (VSC)-producing bacteria and Streptococcus saliuarius. Malodour was assessed by trained judges on an intensity basis. RESULTS: The counts of all bacterial groups were consistently highest at the DPCP surface. Mean VSC-producing bacterial counts (colony forming units/brush x10(5)) were 1.45, 5.67, 32.52, 88.94, 6.46 and 0.33 at DA, DM, DP, DPCP, LP and VP surfaces, respectively. Anaerobic, Gram-negative and VSC counts at DPCP surfaces increased with malodour intensity, whereas aerobic and S. saliuarius counts decreased; however these differences were not statistically significant. CONCLUSION: It is concluded that the DPCP area consistently carries the highest load of bacteria capable of contributing to oral malodour. The study demonstrates that tongue surfaces not accessible to routine oral hygiene procedures can significantly contribute to oral malodour.

Activity of a nitric oxide-generating wound treatment system against wound pathogen biofilms.

Wound bioburden plays an important role in impaired healing and development of infection-related complications. The objective of this study was to determine the efficacy of an innovative two-layer nitric oxide-generating system (NOx) to prevent and treat biofilms formed by bacterial and fungal pathogens commonly associated with wound infection, and activity against Pseudomonas aeruginosa virulence factors. Single- and mixed-species biofilms were grown for 24 h on nitrocellulose filters placed on agar. Filters were covered with either NOx or placebo, before and after biofilm formation. Populations of bacteria and yeasts were determined using viable counts. Pyocyanin and elastase production from P. aeruginosa were determined in supernatants derived from suspended biofilms. Efficacy of NOx was demonstrated against Staphylococcus aureus, P. aeruginosa, Acinetobacter baumannii, Escherichia coli and Candida spp. Population reductions between 2- and 10-log fold were observed. Pyocyanin and elastase activities from P. aeruginosa were reduced 1.9- and 3.2-fold, respectively. This study demonstrated activity of NOx against formation and treatment of single- and mixed-species biofilms, including multidrug-resistant strains. NOx represents a new generation of antimicrobial agent with potent, broad-spectrum activity, and with no evidence of resistance development.

Development of a 3D Collagen Model for the In Vitro Evaluation of Magnetic-assisted Osteogenesis.

Magnetic stimulation has been applied to bone regeneration, however, the cellular and molecular mechanisms of repair still require a better understanding. A three-dimensional (3D) collagen model was developed using plastic compression, which produces dense, cellular, mechanically strong native collagen structures. Osteoblast cells (MG-63) and magnetic iron oxide nanoparticles (IONPs) were incorporated into collagen gels to produce a range of cell-laden models. A magnetic bio-reactor to support cell growth under static magnetic fields (SMFs) was designed and fabricated by 3D printing. The influences of SMFs on cell proliferation, differentiation, extracellular matrix production, mineralisation and gene expression were evaluated. Polymerase chain reaction (PCR) further determined the effects of SMFs on the expression of runt-related transcription factor 2 (Runx2), osteonectin (ON), and bone morphogenic proteins 2 and 4 (BMP-2 and BMP-4). Results demonstrate that SMFs, IONPs and the collagen matrix can stimulate the proliferation, alkaline phosphatase production and mineralisation of MG-63 cells, by influencing matrix/cell interactions and encouraging the expression of Runx2, ON, BMP-2 and BMP-4. Therefore, the collagen model developed here not only offers a novel 3D bone model to better understand the effect of magnetic stimulation on osteogenesis, but also paves the way for further applications in tissue engineering and regenerative medicine.

Interaction of adrenomedullin and calcitonin gene-related peptide with the periodontal pathogen Porphyromonas gingivalis.

The nature of the interaction between Porphyromonas gingivalis and the multifunctional peptides adrenomedullin and calcitonin gene-related peptide (CGRP) was investigated. Growth of P. gingivalis was not inhibited in the presence of either of these peptides [minimal inhibitory concentration (MIC)>250 microg mL(-1)]. The ability of the arginine- and lysine-specific proteases from P. gingivalis to breakdown these peptides was investigated. Adrenomedullin and CGRP were incubated with culture supernatants from wild-type and protease gene knockout strains. No significant effect on antimicrobial activity against the indicator organism Escherichia coli BUE55 was found (MIC=6.25 microg mL(-1) in all cases). The role of anionic components on the surface of P. gingivalis, which may alter binding of these cationic peptides, was also investigated in relation to adrenomedullin. Growth of gene knockout strains lacking surface polysaccharide and capsule components was not inhibited (MIC>250 microg mL(-1)). It is suggested that a lack of sensitivity to adrenomedullin and CGRP may enable P. gingivalis to persist in the oral cavity and cause disease.

Antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

The aim of this study was to investigate cytokine release from oral keratinocytes and fibroblasts in response to AM and shortened derivatives previously characterised in terms of their antimicrobial activities. Cells were incubated with AM or its fragments (residues 1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52, and 34-52), and culture supernatants collected after 1, 2, 4, 8, and 24 hours. A time-dependant increase in production of interleukin1-alpha and interleukin 1-beta from keratinocytes in response to all peptides was demonstrated. However, exposure to fragments compared to whole AM resulted in reduced production of these cytokines (60% mean reduction at 24 hours, P<.001). No consistent differences were shown between the cytokine response elicited by antimicrobial and nonantimicrobial fragments. The production of interleukin-6 and interleukin-8 did not change significantly with time or peptide used. Fibroblast cells were relatively unresponsive to all treatments. This study demonstrates that antimicrobial activity does not predict cytokine response to adrenomedullin or its shortened derivatives.

Use of Probiotics and Oral Health.

PURPOSE OF REVIEW: The purpose of this study is to critically assess recent studies concerning the use of probiotics to control periodontal diseases, dental caries and halitosis (oral malodour). RECENT FINDINGS: Clinical studies have shown that probiotics when allied to conventional periodontal treatment can ameliorate microbial dysbiosis and produce significant improvement in clinical indicators of disease. However, this effect is often not maintained by the host after the end of probiotic use. Current probiotics also show limited effects in treating caries and halitosis. Novel approaches based up on replacement therapy and using highly abundant health-associated oral species, including nitrate-reducing bacteria, have been proposed to improve persistence of probiotic strains and maintain oral health benefits. SUMMARY: Probiotics have potential in the management of multifactorial diseases such as the periodontal diseases and caries, by more effectively addressing the host-microbial interface to restore homeostasis that may not be achieved with conventional treatments.

Mechanisms of adrenomedullin antimicrobial action.

The mechanism of antimicrobial action of the multifunctional peptide adrenomedullin (AM) against Escherichia coli and Staphylococcus aureus was investigated. AM (52 residues) and AM fragments (1-12, 1-21, 13-52, 16-21, 16-52, 22-52, 26-52 and 34-52 residues) were tested for activity. Carboxy-terminal fragments were shown to be up to 250-fold more active than the parent molecule. Minimum inhibitory concentration values of the most active fragments (13-52 and 16-52) and the parent molecule were 4.9 x 10(-2) and 12.5 microg/ml, respectively, with E. coli. Ultrastructural analyses of AM treated cells demonstrated marked cell wall disruption with E. coli within 0.5 h. Abnormal septum formation with no apparent peripheral cell wall disruption was observed with S. aureus after 2 h. Outer membrane permeabilisation assays with E. coli confirmed that the C-terminal fragments were significantly (P < 0.05) more active. It is suggested that postsecretory processing may generate multiple AM congeners that have enhanced antimicrobial activities against a range of potential targets.

Antibacterial effect of copper-bearing titanium alloy (Ti-Cu) against Streptococcus mutans and Porphyromonas gingivalis.

Formation of bacterial biofilms on dental implant material surfaces (titanium) may lead to the development of peri-implant diseases influencing the long term success of dental implants. In this study, a novel Cu-bearing titanium alloy (Ti-Cu) was designed and fabricated in order to efficiently kill bacteria and discourage formation of biofilms, and then inhibit bacterial infection and prevent implant failure, in comparison with pure Ti. Results from biofilm based gene expression studies, biofilm growth observation, bacterial viability measurements and morphological examination of bacteria, revealed antimicrobial/antibiofilm activities of Ti-Cu alloy against the oral specific bacterial species, Streptococcus mutans and Porphyromonas gingivalis. Proliferation and adhesion assays with mesenchymal stem cells, and measurement of the mean daily amount of Cu ion release demonstrated Ti-Cu alloy to be biocompatible. In conclusion, Ti-Cu alloy is a promising dental implant material with antimicrobial/antibiofilm activities and acceptable biocompatibility.

A novel coping metal material CoCrCu alloy fabricated by selective laser melting with antimicrobial and antibiofilm properties.

OBJECTIVE: The aim of this study was to fabricate a novel coping metal CoCrCu alloy using a selective laser melting (SLM) technique with antimicrobial and antibiofilm activities and to investigate its microstructure, mechanical properties, corrosion resistance and biocompatibility. METHODS: Novel CoCrCu alloy was fabricated using SLM from a mixture of commercial CoCr based alloy and elemental Cu powders. SLM CoCr without Cu served as control. Antibacterial activity was analyzed using standard antimicrobial tests, and antibiofilm properties were investigated using confocal laser scanning microscope. Cu distribution and microstructure were determined using scanning electron microscope, optical microscopy and X-ray diffraction. Corrosion resistance was evaluated by potential dynamic polarization and biocompatibility measured using an MTT assay. RESULTS: SLM CoCrCu alloys were found to be bactericidal and able to inhibit biofilm formation. Other factors such as microstructure, mechanical properties, corrosion resistance and biocompatibility were similar to those of SLM CoCr alloys. SIGNIFICANCE: The addition of appropriate amounts of Cu not only maintains normal beneficial properties of CoCr based alloys, but also provides SLM CoCrCu alloys with excellent antibacterial and antibiofilm capabilities. This material has the potential to be used as a coping metal for dental applications.

In Vitro Effect of Porphyromonas gingivalis Methionine Gamma Lyase on Biofilm Composition and Oral Inflammatory Response.

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p<0.001), whilst A. naeslundii (p<0.01) and Streptococcus spp. (p<0.05) decreased in mutant biofilms. Keratinocytes produced less IL-8, IL-6 and IL-1α when stimulated with the mutant biofilms compared to wild type. Lack of mgl in P. gingivalis has been shown to affect microbial ecology in vitro, giving rise to a markedly different biofilm composition, with a more pro-inflammatory cytokine response from the keratinocytes observed. A possible role for methanethiol in biofilm formation and cytokine response with subsequent effects on oral malodor and periodontitis is suggested.

Non-conventional therapeutics for oral infections.

As our knowledge of host-microbial interactions within the oral cavity increases, future treatments are likely to be more targeted. For example, efforts to target a single species or key virulence factors that they produce, while maintaining the natural balance of the resident oral microbiota that acts to modulate the host immune response would be an advantage. Targeted approaches may be directed at the black-pigmented anaerobes, Porphyromonas gingivalis and Prevotella intermedia, associated with periodontitis. Such pigments provide an opportunity for targeted phototherapy with high-intensity monochromatic light. Functional inhibition approaches, including the use of enzyme inhibitors, are also being explored to control periodontitis. More general disruption of dental plaque through the use of enzymes and detergents, alone and in combination, shows much promise. The use of probiotics and prebiotics to improve gastrointestinal health has now led to an interest in using these approaches to control oral disease. More recently the potential of antimicrobial peptides and nanotechnology, through the application of nanoparticles with biocidal, anti-adhesive and delivery capabilities, has been explored. The aim of this review is to consider the current status as regards non-conventional treatment approaches for oral infections with particular emphasis on the plaque-related diseases.

Nanoparticulate zinc oxide as a coating material for orthopedic and dental implants.

Orthopedic and dental implants are prone to infection. In this study, we describe a novel system using zinc oxide nanoparticles (nZnO) as a coating material to inhibit bacterial adhesion and promote osteoblast growth. Electrohydrodynamic atomisation (EHDA) was employed to deposit mixtures of nZnO and nanohydroxyapatite (nHA) onto the surface of glass substrates. Nano-coated substrates were exposed to Staphylococcus aureus suspended in buffered saline or bovine serum to determine antimicrobial activity. Our results indicate that 100% nZnO and 75% nZnO/25% nHA composite-coated substrates have significant antimicrobial activity. Furthermore, osteoblast function was explored by exposing cells to nZnO. UMR-106 cells exposed to nZnO supernatants showed minimal toxicity. Similarly, MG-63 cells cultured on nZnO substrates did not show release of TNF-α and IL-6 cytokines. These results were reinforced by both proliferation and differentiation studies which revealed that a substrate coated with exclusively nZnO is more efficient than composite surface coatings. Finally, electron and light microscopy, together with immunofluorescence staining, revealed that all cell types tested, including human mesenchymal cell (hMSC), were able to maintain normal cell morphology when adhered onto the surface of the nano-coated substrates. Collectively, these findings indicate that nZnO can, on its own, provide an optimal coating for future bone implants that are both antimicrobial and biocompatible.