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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nanoparticles and the control of oral infections.

The potential of antimicrobial nanoparticles to control oral infections is reviewed. Such particles can be classified as having a size no greater than 100 nm and are produced using traditional or more novel techniques. Exploitation of the toxic properties of nanoparticles to bacteria, fungi and viruses, in particular metals and metal oxides, as well as their incorporation into polymeric materials have increased markedly over the past decade. The potential of nanoparticles to control the formation of biofilms within the oral cavity, as a function of their biocidal, anti-adhesive and delivery capabilities, is now receiving close attention. Latest insights into the application of nanoparticles within this field, including their use in photodynamic therapy, will be reviewed. Possible approaches to alter biocompatibility and desired function will also be covered.

Maternal oral origin of Fusobacterium nucleatum in adverse pregnancy outcomes as determined using the 16S-23S rRNA gene intergenic transcribed spacer region.

Fusobacterium nucleatum, a common Gram-negative anaerobe prevalent in the oral cavity, possesses the ability to colonize the amniotic cavity and the fetus. However, F. nucleatum may also be part of the vaginal microbiota from where it could reach the amniotic tissues. Due to the heterogeneity of F. nucleatum, consisting of five subspecies, analysis at the subspecies/strain level is desirable to determine its precise origin. The aims of this study were: (i) to evaluate the use of the 16S-23S rRNA gene intergenic transcribed spacer (ITS) region as a tool to differentiate subspecies of F. nucleatum, and (ii) to design a simplified technique based on the ITS to determine the origin of F. nucleatum strains associated with adverse pregnancy outcomes. Amplified fragments of the 16S-23S rRNA gene ITS region corresponding to the five subspecies of F. nucleatum were subjected to cloning and sequencing to characterize the different ribosomal operons of the subspecies. Distinctive length and sequence patterns with potential to be used for identification of the subspecies/strain were identified. These were used to evaluate the origin of F. nucleatum identified in neonatal gastric aspirates (swallowed amniotic fluid) by sequence comparisons with the respective oral and vaginal maternal samples. A simplified technique using a strain-specific primer in a more sensitive nested PCR was subsequently developed to analyse ten paired neonatal-maternal samples. Analysing the variable fragment of the ITS region allowed the identification of F. nucleatum subsp. polymorphum from an oral origin as potentially being involved in neonatal infections. Using a strain-specific primer, the F. nucleatum subsp. polymorphum strain was detected in both neonatal gastric aspirates and maternal oral samples in cases of preterm birth from mothers presenting with localized periodontal pockets. Interestingly, the same strain was not present in the vaginal sample of any case investigated. The 16S-23S rRNA gene ITS can be a useful tool to determine the origin of F. nucleatum. The results of this study strongly indicate that F. nucleatum subsp. polymorphum of oral origin could be involved with pregnancy complications.

Antimicrobial activity of nanoparticulate metal oxides against peri-implantitis pathogens.

Dental plaque accumulation may result in peri-implantitis, an inflammatory process causing loss of supporting bone that may lead to dental implant failure. The antimicrobial activities of six metal and metal oxide nanoparticles and two of their composites against bacterial pathogens associated with peri-implantitis were examined under anaerobic conditions. The activities of nanoparticles of silver (Ag), cuprous oxide (Cu(2)O), cupric oxide (CuO), zinc oxide (ZnO), titanium dioxide (TiO(2)), tungsten oxide (WO(3)), Ag+CuO composite and Ag+ZnO composite were assessed by minimum inhibitory (bacteriostatic) concentration (MIC) and minimum bactericidal concentration (MBC) determination against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. Time-kill assays were carried out to examine the dynamics of the antimicrobial activity with ZnO nanoparticles. MIC and MBC values were in the range of <100 µg/mL to 2500 µg/mL and <100 µg/mL to >2500 µg/mL, respectively. The activity of the nanoparticles tested in descending order was Ag>Ag+CuO>Cu(2)O>CuO>Ag+ZnO>ZnO>TiO(2)>WO(3). Time-kill assays with ZnO demonstrated a significant decrease in growth of all species tested within 4h, reaching 100% within 2h for P. gingivalis and within 3h for F. nucleatum and P. intermedia. Coating titanium surfaces of dental and orthopaedic implants with antimicrobial nanoparticles should lead to an increased rate of implant success.

Identification of bacteria and potential sources in neonates at risk of infection delivered by Caesarean and vaginal birth.

Neonatal gastric aspirates (NGA) are routinely screened in UK hospitals to investigate fetal/neonatal infections associated with cases of adverse pregnancy outcome (APO). The aim of this study was to describe and compare the microbiology of NGA from Caesarean and vaginal deliveries using molecular methods, and to evaluate other possible clinical and non-clinical variables that may have determined the presence of the bacteria in the samples. The value of using NGA and molecular methods to investigate potential pathogens associated with the risk of early infection was also evaluated. Bacteria were identified by a combined molecular approach on the basis of the 16S rRNA gene using both clone analysis and denaturing gradient gel electrophoresis. A total of 43 and 34 different species were identified in the vaginal (n = 121) and Caesarean (n = 119) deliveries, respectively; 26 of the species observed (51 %) were common to both modalities, although usually less prevalent in the Caesarean cases. Multivariate analysis confirmed an association between infection and prolonged rupture of membranes in vaginal deliveries (odds ratio = 5.7, 95 % confidence interval = 1.1-29.0). Various associations between infection and given variables were also shown, including labour, intrapartum antibiotic prophylaxis, and time and place of sample collection. The molecular methods allowed identification of a range of bacteria and potential sources not previously observed in NGA, including possible genito-urinary, gastrointestinal and oral pathogens. NGA represents a valuable sample for investigating potential pathogens associated with APO and the risk of early infection in neonates using molecular methods.

Characterisation of copper oxide nanoparticles for antimicrobial applications.

Copper oxide (CuO) nanoparticles were characterised and investigated with respect to potential antimicrobial applications. It was found that nanoscaled CuO, generated by thermal plasma technology, contains traces of pure Cu and Cu2O nanoparticles. Transmission electron microscopy (TEM) demonstrated particle sizes in the range 20-95 nm. TEM energy dispersive spectroscopy gave the ratio of copper to oxygen elements as 54.18% to 45.26%. The mean surface area was determined as 15.69 m(2)/g by Brunau-Emmet-Teller (BET) analysis. CuO nanoparticles in suspension showed activity against a range of bacterial pathogens, including meticillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, with minimum bactericidal concentrations (MBCs) ranging from 100 microg/mL to 5000 microg/mL. The ability of CuO nanoparticles to reduce bacterial populations to zero was enhanced in the presence of sub-MBC concentrations of silver nanoparticles. Studies of CuO nanoparticles incorporated into polymers suggest release of ions may be required for optimum killing.

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.

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.