Microbial diagnostics in periodontal diseases.

Microbial analytical methods have been instrumental in elucidating the complex microbial etiology of periodontal diseases, by shaping our understanding of subgingival community dynamics. Certain pathobionts can orchestrate the establishment of dysbiotic communities that can subvert the host immune system, triggering inflammation and tissue destruction. Yet, diagnosis and management of periodontal conditions still rely on clinical and radiographic examinations, overlooking the well-established microbial etiology. This review summarizes the chronological emergence of periodontal etiological models and the co-evolution with technological advances in microbial detection. We additionally review the microbial analytical approaches currently accessible to clinicians, highlighting their value in broadening the periodontal assessment. The epidemiological importance of obtaining culture-based antimicrobial susceptibility profiles of periodontal taxa for antibiotic resistance surveillance is also underscored, together with clinically relevant analytical approaches to guide antibiotherapy choices, when necessary. Furthermore, the importance of 16S-based community and shotgun metagenomic profiling is discussed in outlining dysbiotic microbial signatures. Because dysbiosis precedes periodontal damage, biomarker identification offers early diagnostic possibilities to forestall disease relapses during maintenance. Altogether, this review highlights the underutilized potential of clinical microbiology in periodontology, spotlighting the clinical areas most conductive to its diagnostic implementation for enhancing prevention, treatment predictability, and addressing global antibiotic resistance.

Bacteriome and mycobiome dysbiosis in oral mucosal dysplasia and oral cancer.

It has long been considered that the oral microbiome is tightly connected to oral health and that dysbiotic changes can be detrimental to the occurrence and progression of dysplastic oral mucosal lesions or oral cancer. Improved understanding of the concepts of microbial dysbiosis together with advances in high-throughput molecular sequencing of these pathologies have charted in greater microbiological detail the nature of their clinical state. This review discusses the bacteriome and mycobiome associated with oral mucosal lesions, oral candidiasis, and oral squamous cell carcinoma, aiming to delineate the information available to date in pursuit of advancing diagnostic and prognostic utilities for oral medicine.

Systemic antibiotics in the surgical treatment of peri-implantitis: A randomized placebo-controlled trial.

AIM: To study the clinical, radiographic and microbiological outcomes after surgical treatment of peri-implantitis, with or without adjunctive systemic antibiotics. MATERIALS AND METHODS: Eighty-four patients (113 implants) with peri-implantitis were randomized into three groups (A, amoxicillin and metronidazole; B, phenoxymethylpenicillin and metronidazole; or C, placebo). Treatment included resective surgery and implant surface decontamination with adjunctive antibiotics or placebo. Primary outcomes were probing pocket depth (PPD) reduction and marginal bone level (MBL) stability. Secondary outcomes were treatment success (defined as PPD ≤ 5 mm, bleeding on probing [BOP] ≤ 1site, absence of suppuration on probing [SOP] and absence of progressive bone loss of >0.5 mm), changes in BOP/SOP, mucosal recession (REC), clinical attachment level (CAL), bacterial levels and adverse events. Outcomes were evaluated for up to 12 months. The impact of potential prognostic indicators on treatment success was evaluated using multilevel logistic regression analysis. RESULTS: A total of 76 patients (104 implants) completed the study. All groups showed clinical and radiological improvements over time. Statistically significant differences were observed between groups for MBL stability (A = 97%, B = 89%, C = 76%), treatment success (A = 68%, B = 66%, C = 28%) and bacterial levels of Aggregatibacter actinomycetemcomitans and Tannerella forsythia, favouring antibiotics compared to placebo. Multiple regression identified antibiotic use as potential prognostic indicator for treatment success. Gastrointestinal disorders were the most reported adverse events in the antibiotic groups. CONCLUSIONS: Adjunctive systemic antibiotics resulted in additional improvements in MBL stability. However, the potential clinical benefits of antibiotics need to be carefully balanced against the risk of adverse events and possible antibiotic resistance.

Periodontal microbiology and microbial etiology of periodontal diseases: Historical concepts and contemporary perspectives.

This narrative review summarizes the collective knowledge on periodontal microbiology, through a historical timeline that highlights the European contribution in the global field. The etiological concepts on periodontal disease culminate to the ecological plaque hypothesis and its dysbiosis-centered interpretation. Reference is made to anerobic microbiology and to the discovery of select periodontal pathogens and their virulence factors, as well as to biofilms. The evolution of contemporary molecular methods and high-throughput platforms is highlighted in appreciating the breadth and depth of the periodontal microbiome. Finally clinical microbiology is brought into perspective with the contribution of different microbial species in periodontal diagnosis, the combination of microbial and host biomarkers for this purpose, and the use of antimicrobials in the treatment of the disease.

Precision periodontal care: from omics discoveries to chairside diagnostics.

The interface of molecular science and technology is guiding the transformation of personalized to precision healthcare. The application of proteomics, genomics, transcriptomics, and metabolomics is shaping the suitability of biomarkers for disease. Prior validation of such biomarkers in large and diverse patient cohorts helps verify their clinical usability. Incorporation of molecular discoveries into routine clinical practice relies on the development of customized assays and devices that enable the rapid delivery of analytical data to the clinician, while the patient is still in session. The present perspective review addresses this topic under the prism of precision periodontal care. Selected promising research attempts to innovate technological platforms for oral diagnostics are brought forward. Focus is placed on (a) the suitability of saliva as a conveniently sampled biological specimen for assessing periodontal health, (b) proteomics as a high-throughput approach for periodontal disease biomarker identification, and (c) chairside molecular diagnostic assays as a technological funnel for transitioning from the laboratory benchtop to the clinical point-of-care.

Cytokine profiles and the dynamic of gingivitis development in humans.

AIM: To investigate the relationship between cytokine profiles and "fast" and "slow" patterns of gingival inflammation development. MATERIALS AND METHODS: Forty-two adults participated in an experimental gingivitis study, comprising a 2-week hygiene phase (clinical examination and professional cleaning); a 3-week induction phase (absence of oral hygiene); and a 2-week resolution phase (re-establishment of oral hygiene). Plaque and gingival inflammation scores were assessed. Interferon-gamma (IFN-γ), interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and tumour necrosis factor-alpha (TNF-α) from gingival crevicular fluid were collected and measured by multiplex ELISA. Group-based-trajectory-modelling (GBTM) was used to model cytokine profiles over the induction phase. The effect of gingival inflammation on cytokine levels over time was estimated with mixed-effects modelling. RESULTS: GBTM analysis revealed two cytokine profiles, "non-organized response" (IL-4, IL-6, IL-8, IL-12, and IL-13) and "organized response" (IL-2, IL-10, and TNF-α). Among the "slow" responders, neither cytokine profile was associated with gingivitis. In contrast, a "fast" response was associated with a higher "non-organized response" factor (coef. 0.14) and a lower "organized response" factor (coef. -0.03). CONCLUSION: A "fast" gingivitis development was associated with a higher "non-organized response" and a lower "organized response", which may elucidate the role of individual variability in gingivitis susceptibility.

A critical analysis of research methods to study clinical molecular biomarkers in Endodontic research.

The authors of this narrative review aimed to address various experimental methods and make recommendations for how research should move forward in the context of studying biomarkers in clinical Endodontic research. The approach adopted is exemplified using two prominent clinical problems, namely (a) the 'reversible' versus 'irreversible' pulpitis conundrum and (b) persistent idiopathic dentoalveolar pain (PIDAP). Pulpitis under deep caries or dentinal cracks is understood from a histological perspective, but clinical assessment tools to indicate irreversibly inflamed aspects of the dental pulp are elusive. PIDAP, on the other hand, is a diagnosis of exclusion; its pathophysiology is complex and not understood sufficiently to avoid unnecessary dental treatments. This review addresses how diagnostic biomarkers could further our understanding of those and other clinical problems, and how issues can be tackled from a methodological point of view. Hence, different methodological approaches to identify suitable diagnostic biomarker(s) or use known biomarkers are presented. The importance of asking a relevant research question, collecting the most suitable fluid and using the ideal collection vehicle for the research question under investigation is discussed based on the defined clinical problems.

Metaproteome and metabolome of oral microbial communities.

The emergence of high-throughput technologies for the comprehensive measurement of biomolecules, also referred to as "omics" technologies, has helped us gather "big data" and characterize microbial communities. In this article, we focus on metaproteomic and metabolomic approaches that support hypothesis-driven investigations on various oral biologic samples. Proteomics reveals the working units of the oral milieu and metabolomics unveils the reactions taking place; and so these complementary techniques can unravel the functionality and underlying regulatory processes within various oral microbial communities. Current knowledge of the proteomic interplay and metabolic interactions of microorganisms within oral biofilm and salivary microbiome communities is presented and discussed, from both clinical and basic research perspectives. Communities indicative of, or from, health, caries, periodontal diseases, and endodontic lesions are represented. Challenges, future prospects, and examples of best practice are given.

Metagenomic sequencing provides new insights into the subgingival bacteriome and aetiopathology of periodontitis.

"Open-ended" molecular techniques such as 16S rRNA sequencing have revealed that the oral bacteriome of subgingival plaque is more diverse than originally thought. 16S rRNA analysis has demonstrated that constituents of the overall bacterial community are qualitatively similar in health and disease, differing mainly in their relative proportions with respect to each other. Species in low abundance can also act as critical species, leading to the concept of global community dysbiosis which relates to shifts in community structure, rather than shifts in membership. Correlation analysis suggests that coordinated interactions in the community are essential for incipient dysbiosis and disease pathogenesis. The subgingival bacteriome also provides biomarkers that are useful for disease detection and management. Combined with clinical and biological parameters, these may assist clinicians in developing and implementing effective treatment strategies to restore microbial homeostasis and monitor disease. Identification of higher risk groups or poor responders to treatment using unique subgingival bacteriome signatures may also lead to early intervention.

The adjunctive use of host modulators in non-surgical periodontal therapy. A systematic review of randomized, placebo-controlled clinical studies.

BACKGROUND: Considering the role of the inflammatory host response in the pathogenesis of periodontitis, different host modulators have been proposed to enhance the outcomes of non-surgical periodontal therapy (NSPT), but their efficacy has not been fully clarified. OBJECTIVES: This systematic review investigated the efficacy of host modulators combined with NSPT in reducing probing pocket depth (PPD) in periodontitis patients. MATERIALS AND METHODS: Placebo-controlled RCTs with ≥6 months follow-up were searched. Meta-analysis was conducted when ≥5 studies using the same host modulator were identified. RESULTS: Fifty eight studies met the inclusion criteria. After 6 months, local administration of 1.2% statin gels as adjuncts to NSPT significantly improved PPD reduction (1.83 mm) in infrabony defects and systemic administration of sub-antimicrobial dose doxycycline (SDD) in addition to NSPT improved PPD reduction of deep pockets. Administration of probiotics conferred limited clinical benefits. Local bisphosphonate and metformin gels showed potential for clinical use in infrabony defects, which needs to be confirmed. CONCLUSIONS: Local delivery of statins in infrabony defects and systemic SDD for deep pockets may confer additional clinical benefits to NSPT. Their long-term effectiveness and safety need to be confirmed in independent multi-centred studies. Further studies are needed to confirm the benefit of other host modulators.

Salivary proteotypes of gingivitis tolerance and resilience.

AIM: This study aimed to characterize the salivary proteome during the induction and resolution of gingival inflammation in the course of human experimental gingivitis (EG), and to cluster the proteomic profiles based on the clinically defined "slow" and "fast" response patterns. MATERIALS AND METHODS: A total of 50 unstimulated whole saliva were obtained from the EG model which was induced over 21 days (days 0, 7, 14 and 21), followed by a two-week resolution phase (day 35). Label-free quantitative proteomics using liquid chromatography-tandem mass spectrometry was applied. Regulated proteins were subject to Gene Ontology enrichment analysis. RESULTS: A total of 804 human proteins were quantified by ≥ 2 peptides. Principal component analysis depicted significant differences between "fast" and "slow" responders. Despite gingival and plaque scores being similar at baseline among the two groups, "fast" responders presented with 48 proteins that were at > 4-fold higher levels than "slow" responders. These up-regulated proteins showed enrichment in "antigen presentation" and "proteolysis." CONCLUSIONS: Together, these findings highlight the utility of integrative systems-level quantitative proteomic approaches to unravel the molecular basis of "salivary proteotypes" associated with gingivitis dubbed as "fast" and "slow" responders. Hence, these differential responses may help prognosticate individual susceptibility to gingival inflammation.

Targeted Proteomics Guided by Label-free Quantitative Proteome Analysis in Saliva Reveal Transition Signatures from Health to Periodontal Disease.

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. Here we carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n = 67, including individuals with health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n = 82). The LFQ platform led to the discovery of 119 proteins with at least 2-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 functionally related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases with maximum area under the receiver operating curve >0.97 (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1). This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum leap brought into the field of periodontal diagnostics by this study is the application of the biomarker discovery-through-verification pipeline, which can be used for validation in further cohorts.

Advances in Oral Mucosal Immunity and the Microbiome.

The 1st International Conference on Oral Mucosal Immunity and the Microbiome (OMIM) took place at the Avra Imperial Hotel, Chania, Crete, Greece, between 26th and 30th September 2018, under the auspices of the Aegean Conferences. This was the first Aegean Conference of its kind in thematic oral research, and a unique blend of immunological and microbiological perspectives, which attracted leading scientists from around the world to discuss the latest advances in the field. The Conference was divided into eight sessions that spanned across 4 days and included the following topics: (a) mucosal barrier immunity; (b) host response and inflammation; (c) microbiome in homeostasis and dysbiosis; (d) fungal and viral pathogenesis; (e) oral microbiome and proteome; (f) microbial virulence and biofilms; (g) microbiome, cancer, and systemic disease; and (h) microbiota and inflammation. There was substantial thematic overlap among all sessions, which promoted constant involvement of the participating scientists. An important hallmark was the active debate between oral microbiologists and oral immunologists, who explored new ideas and potential research collaborations, a crucial aspect for bridging our understanding of oral diseases in the context of the whole body. Key findings are highlighted and thematically presented in the following sections.

Gingival crevicular fluid and its immune mediators in the proteomic era.

The discovery of biomarkers for periodontal disease requires an in-depth understanding of the molecular basis of the initiation and progression of the disease. The gingival crevicular fluid is a biological medium suitable for identifying and measuring such biomarkers because it can be easily and noninvasively sampled from the immediate vicinity of the affected tissues. An ever-expanding pool of gingival crevicular fluid proteins associated with periodontal health or disease has been catalogued over the years, particularly with the recent implementation of proteomic technologies. 'Proteomics' refers to the large-scale study of entire arrays of proteins expressed by a genome and present in a cell, tissue, biological fluid or organism. Hence, such technologies provide a broad qualitative and quantitative insight of the proteins present in gingival crevicular fluid. Pertinent studies have amassed on the information gathered to date on protein signatures in periodontal health and disease, and have confirmed the nature of the immunological host response. This review discusses the application of proteomic technologies in characterizing the molecular networks present in gingival crevicular fluid, their potential for discovery of biomarkers that are meaningful for clinical practice, and the associated technical challenges.

Phenalen-1-One-Mediated Antimicrobial Photodynamic Therapy and Chlorhexidine Applied to a Novel Caries Biofilm Model.

Antimicrobial photodynamic therapy (aPDT) may be useful as a supportive antimicrobial measure for caries-active subjects. In this study, the antimicrobial efficacy of aPDT with a phenalen-1-one photosensitizer was evaluated in a novel in vitro biofilm model comprising Actinomyces naeslundii, Actinomyces odontolyticus, and Streptococcus mutans and was compared to chlorhexidine. The proposed biofilm model allows high-throughput screening for antimicrobial efficacy while exhibiting a differentiated response to different antimicrobial approaches. While chlorhexidine 0.2% showed a reduction of ≈4 log10 for all species, aPDT led to a more pronounced reduction of S. mutans (2.8 log10) than of Actinomyces spp. (1.2 or 1.3 log10). A similar effect was also observed in monospecies biofilms. Therefore, aPDT may be more effective against S. mutans than against Actinomyces spp. when in biofilms, and this antimicrobial approach merits further investigations.

The epigenetic architecture at gene promoters determines cell type-specific LPS tolerance.

Synovial fibroblasts (SF) drive inflammation and joint destruction in chronic arthritis. Here we show that SF possess a distinct type of LPS tolerance compared to macrophages and other types of fibroblasts. In SF and dermal fibroblasts, genes that were non-tolerizable after repeated LPS stimulation included pro-inflammatory cytokines, chemokines and matrix metalloproteinases, whereas anti-viral genes were tolerizable. In macrophages, all measured genes were tolerizable, whereas in gingival and foreskin fibroblasts these genes were non-tolerizable. Repeated stimulation of SF with LPS resulted in loss of activating histone marks only in promoters of tolerizable genes. The epigenetic landscape at promoters of tolerizable genes was similar in unstimulated SF and monocytes, whereas the basal configuration of histone marks profoundly differed in genes that were non-tolerizable in SF only. Our data suggest that the epigenetic configuration at gene promoters regulates cell-specific LPS-induced responses and primes SF to sustain their inflammatory response in chronic arthritis.

Exploring the microbiome of healthy and diseased peri-implant sites using Illumina sequencing.

AIM: To compare the microbiome of healthy (H) and diseased (P) peri-implant sites and determine the core peri-implant microbiome. MATERIALS AND METHODS: Submucosal biofilms from 32 H and 35 P sites were analysed using 16S rRNA sequencing (MiSeq, Illumina), QIIME and HOMINGS. Differences between groups were determined using principal coordinate analysis (PCoA), t tests and Wilcoxon rank sum test and FDR-adjusted. The peri-implant core microbiome was determined. RESULTS: PCoA showed partitioning between H and P at all taxonomic levels. Bacteroidetes, Spirochetes and Synergistetes were higher in P, while Actinobacteria prevailed in H (p < .05). Porphyromonas and Treponema were more abundant in P while Rothia and Neisseria were higher in H (p < .05). The core peri-implant microbiome contained Fusobacterium, Parvimonas and Campylobacter sp. T. denticola, and P. gingivalis levels were higher in P, as well as F. alocis, F. fastidiosum and T. maltophilum (p < .05). CONCLUSION: The peri-implantitis microbiome is commensal-depleted and pathogen-enriched, harbouring traditional and new pathogens. The core peri-implant microbiome harbours taxa from genera often associated with periodontal inflammation.

Impact of implant-abutment connection on osteoimmunological and microbiological parameters in short implants: a randomized controlled clinical trial.

OBJECTIVES: The study aimed to determine the levels of soluble receptor activator of nuclear factor-кB ligand (sRANKL) and osteoprotegerin (OPG) as well as their relative calculated ratio in peri-implant crevicular fluid (PICF) obtained around two different types of implant-abutment connection on short implants following a 12-month monitoring period. Moreover, the levels of a number of oral bacterial species were investigated in the corresponding submucosal biofilm samples. MATERIALS AND METHODS: Thirty short implants were randomly placed in posterior maxillary edentulous sites using a split-mouth design in 15 periodontally healthy subjects. Tapered interference fit (TIF) and taper-integrated screwed-in (TIS) types of implant-abutment connections were selected for investigation. PICF and submucosal biofilm samples were collected 1 month after surgery and repeated 12 months after prosthetic loading. Clinical parameters, including probing depth, dichotomous presence of bleeding on probing, and plaque index, were recorded and digital periapical radiographs were taken at each time point. sRANKL and OPG levels in PICF were analyzed using an enzyme-linked immunosorbent assay. Total bacterial levels, as well as levels of Fusobacterium nucleatum, Porphyromonas gingivalis, Treponema denticola, Tannerella forsythia, Prevotella intermedia, and Streptococcus oralis, were analyzed in the corresponding submucosal biofilm samples using quantitative real-time polymerase chain reaction. RESULTS: The total amount of sRANKL in TIF implants was 2.64-fold lower than that in TIS implants at baseline (P < 0.001), whereas similar levels were found after 12 months (P > 0.05). Accordingly, OPG and RANKL/OPG ratio were similar between the groups at each time point (P > 0.05). Microbiological results were similar in both groups at each time point (P > 0.05). CONCLUSION: The results of this longitudinal study suggested that sRANKL and OPG in PICF, as well as microbiological parameters in submucosal biofilms, were similar between TIF and TIS implants, after a 12-month monitoring period, despite early differences in the former. Therefore, the type of implant-abutment connection does not appear to influence longitudinally the levels of osteoimmunological and microbiological markers in the peri-implant tissues of short implants.

Impact of aging on TREM-1 responses in the periodontium: a cross-sectional study in an elderly population.

BACKGROUND: Aging is associated with altered immune response, which increases susceptibility to infections. sTREM-1 is involved in the amplification of the inflammatory response to bacterial infection. The present cross-sectional study aims to investigate local sTREM-1 levels in gingival crevicular fluid (GCF) as well as key periodontal pathogen levels in the subgingival plaque in an elderly cohort with periodontal health, gingivitis, and chronic periodontitis (CP). METHODS: Subjects were 51 systemically healthy, elderly individuals (mean age, 68 ± 4.5 years) who had undergone full-mouth periodontal examinations. Subgingival plaque and GCF samples were collected from the healthy sites of participants without periodontal disease (n = 17), the sites with gingival inflammation from patients with gingivitis (n = 19), and the periodontitis sites of patients with CP (n = 15). GCF volumes were measured by an electronic impedance device, and total protein levels were assessed by a flouremetric assay. sTREM-1 levels in GCF were measured by enzyme-linked immunosorbent assay. The subgingival plaque total bacteria, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Fusobacterium nucleatum, and Prevotella intermedia levels were determined by quantitative real-time polymerase chain reaction. Statistical analysis was performed using nonparametric methods. RESULTS: GCF volume, total protein concentrations, and sTREM-1 levels in GCF were similar among the groups (p > 0.05). Significantly higher T. forsythia levels were observed in subgingival plaque samples harvested from patients with gingivitis and CP, than in those from healthy participants (p < 0.05). However, the subgingival levels of the other four periodontal pathogens and total bacteria were not statistically different among the groups (p > 0.05). CONCLUSIONS: Our findings suggest that there are no differences in GCF volume, total protein, and sTREM-1 levels between healthy and periodontally diseased elderly adults. We found only limited differences in the studied subgingival microbial profile. This finding indicates an already deregulated, local inflammatory response in this elderly cohort, on which bacterial biofilm challenge may have a limited further impact.

Incorporation of staphylococci into titanium-grown biofilms: an in vitro "submucosal" biofilm model for peri-implantitis.

OBJECTIVES: Staphylococcus spp. are postulated to play a role in peri-implantitis. This study aimed to develop a "submucosal" in vitro biofilm model, by integrating two staphylococci into its composition. MATERIALS AND METHODS: The standard "subgingival" biofilm contained Actinomyces oris, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Streptococcus anginosus, Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola, and was further supplemented with Staphyoccous aureus and/or Staphylococcus epidermidis. Biofilms were grown anaerobically on hydroxyapatite or titanium discs and harvested after 64 h for real-time polymerase chain reaction, to determine their composition. Confocal laser scanning microscopy and fluorescence in situ hybridization were used for identifying the two staphylococci within the biofilm. RESULTS: Both staphylococci established within the biofilms when added separately. However, when added together, only S. aureus grew in high numbers, whereas S. epidermidis was reduced almost to the detection limit. Compared to the standard subgingival biofilm, addition of the two staphylococci had no impact on the qualitative or quantitative composition of the biofilm. When grown individually in the biofilm, S. epidermidis and S. aureus formed small distinctive clusters and it was confirmed that S. epidermidis was not able to grow in presence of S. aureus. CONCLUSIONS: Staphyoccous aureus and S. epidermidis can be individually integrated into an oral biofilm grown on titanium, hence establishing a "submucosal" biofilm model for peri-implantitis. This model also revealed that S. aureus outcompetes S. epidermidis when grown together in the biofilm, which may explain the more frequent association of the former with peri-implantitis.