Periodontal and Microbiological Profile of Intensive Care Unit Inpatients.

INTRODUCTION: The bidirectional relationship between the periodontal diseases and systemic diseases was attributed to the focal infection concept. The aims of this study were to assess the periodontal and microbiological profile of intensive care unit (ICU) inpatients submitted to orotracheal intubation, and classify them regarding gender, age group, ethnic, hospitalization reason and period, nosocomial infection occurrence, and death. MATERIALS AND METHODS: Inpatients were assessed, distributed into toothed and toothless groups. The periodontal clinical condition was assessed 24 hours after the ICU admission through plaque index, gum index, probing depth, and clinical level of insertion. All microbiological samples were collected on the 6th day of admission. These samples were collected from different intraoral sites, depending on the group: In the toothed group, samples were collected from gingival sulcus and in the toothless group, from buccal mucosa and tongue. Identification for Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), and Tannerella forsythia (Tf) was accomplished and analyzed, using absolute quantification and specific primer pairs through an amplification system with probes. RESULTS: Forty subjects composed the sample: Gender characterized by 60% of male, 27.5% of all patients were older than 60, and 22.5% were hospitalized due to cerebrovascular accident. Regarding hospitalization period, 55% of patients were hospitalized for 6 days and 70% of them died during the period of hospitalization. Of inpatients, 40% presented periodontal disease and 100% presented dental biofilm on assessed sites. When assessing the microbiota, statistical significance was observed between Aa, Pg, and Tf, for both toothed and toothless group (p < 0.0001). CONCLUSION: Large quantities of Aa were found in samples of toothless inpatients, a fact that suggests that the oral environment, even without teeth, presents favorable conditions for bacterial biofilm formation with a related pathogenic potential. CLINICAL SIGNIFICANCE: The dental biofilm may comprise pulmonary pathogen colonies, promoting a perfect environment for their growth and development, facilitating the colonization of the lower airways, as well as colonization by bacteria originally from the oral cavity.

Pyrosequencing of supra- and subgingival biofilms from inflamed peri-implant and periodontal sites.

BACKGROUND: To investigate the microbial composition of biofilms at inflamed peri-implant and periodontal tissues in the same subject, using 16S rRNA sequencing. METHODS: Supra- and submucosal, and supra- and subgingival plaque samples were collected from 7 subjects suffering from diseased peri-implant and periodontal tissues. Bacterial DNA was isolated and 16S rRNA genes were amplified, sequenced and aligned for the identification of bacterial genera. RESULTS: 43734 chimera-depleted, denoised sequences were identified, corresponding to 1 phylum, 8 classes, 10 orders, 44 families and 150 genera. The most abundant families or genera found in supramucosal or supragingival plaque were Streptoccocaceae, Rothia and Porphyromonas. In submucosal plaque, the most abundant family or genera found were Rothia, Streptococcaceae and Porphyromonas on implants. The most abundant subgingival bacteria on teeth were Prevotella, Streptococcaceae, and TG5. The number of sequences found for the genera Tannerella and Aggregatibacter on implants differed significantly between supra- and submucosal locations before multiple testing. The analyses demonstrated no significant differences between microbiomes on implants and teeth in supra- or submucosal and supra- or subgingival biofilms. CONCLUSION: Diseased peri-implant and periodontal tissues in the same subject share similiar bacterial genera and based on the analysis of taxa on a genus level biofilm compositions may not account for the potentially distinct pathologies at implants or teeth.

Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions.

The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes.

How is the development of dental biofilms influenced by the host?

BACKGROUND: The host provides environmental conditions that support diverse communities of microorganisms on all environmentally-exposed surfaces of the body. MATERIALS AND METHODS: To review the literature to determine which properties of the host substantially influence the development of dental biofilms. RESULTS: The mouth facilitates the growth of a characteristic resident microbiota. The composition of the oral microbiota is influenced by temperature, pH, and atmosphere, as well as by the host defences and host genetics. In addition, the host supplies endogenous nutrients and a variety of surfaces for biofilm formation. In health, the resident oral microbiota forms a symbiotic relationship with the host, regulated by active host-microbe cross talk. This resident microbiota is sensitive to perturbations in the host environment, especially to changes in nutrient supply and pH, so that previously minor components of the microbiota can become more competitive (and vice versa), resulting in reorganization of biofilm community structure. CONCLUSION: The host environment dictates the composition and gene expression of the resident microbiota. Changes in oral environmental conditions can disrupt the normal symbiotic relationship between the host and its resident microbes, and increase the risk of disease.

Genotypic and phenotypic analysis of S. mutans isolated from dental biofilms formed in vivo under high cariogenic conditions

The oral cavity harbors several Streptococcus mutans genotypes, which could present distinct virulence properties. However, little is known about the diversity and virulence traits of S. mutans genotypes isolated in vivo under controlled conditions of high cariogenic challenge. This study evaluated the genotypic diversity of S. mutans isolated from dental biofilms formed in vivo under sucrose exposure, as well as their acidogenicity and aciduricity. To form biofilms, subjects rinsed their mouths with distilled water or sucrose solution 8 times/day for 3 days. S. mutans collected from saliva and biofilms were genotyped by arbitrarily-primed PCR. Genotypes identified in the biofilms were evaluated regarding their ability to lower the suspension pH through glycolysis and their acid susceptibility and F-ATPase activity. Most subjects harbored only one genotype in saliva, which was detected in almost all biofilm samples at high proportions. Genotypes isolated only in the presence of sucrose had higher acidogenicity than those isolated only in the presence of water. Genotypes from biofilms formed with sucrose were more aciduric after 30 and 60 min of incubation at pH 2.8 and 5.0, respectively. The present results suggest that biofilms formed under high cariogenic conditions may harbor more aciduric and acidogenic S. mutans genotypes.

A cavidade oral apresenta vários genótipos de Streptococcus mutans, que podem possuir diferentes capacidades de virulência. Entretanto, pouco se sabe sobre a diversidade e virulência de genótipos de S. mutans isolados in vivo sob uma condição controlada de alto desafio cariogênico. Este estudo avaliou a diversidade genotípica de S. mutans identificados no biofilme dental formado in vivo na presença de sacarose, assim como a acidogenicidade e aciduricidade desses genótipos. Para possibilitar formação de biofilme, voluntários bochecharam com água destilada ou solução de sacarose 8x/dia durante 3 dias. S. mutans isolados da saliva e do biofilme dental foram genotipados por PCR com primers-arbitrários. Genótipos isolados do biofilme foram avaliados em relação à habilidade de reduzir o pH da suspensão devido à glicólise, em relação à susceptibilidade a ácidos e também atividade F-ATPase. A maioria dos voluntários apresentou apenas 1 genótipo na saliva, que foram detectados em quase todas as amostras de biofilme em altas proporções. Genótipos isolados somente na presença de sacarose apresentaram maior acidogenicidade do que aqueles genótipos isolados apenas na presença de água. Genótipos de biofilmes formados na presença de sacarose foram mais acidúricos após 30 e 60 min de incubação em pH 2,8 e 5,0, respectivamente. Os resultados do presente estudo sugerem que biofilmes formados sob condição de alto desafio cariogênico podem apresentar genótipos de S. mutans mais acidúricos e mais acidogênicos.

Nanotechnology: role in dental biofilms.

Biofilms are surface-adherent populations of microorganisms consisting of cells, water and extracellular matrix material Nanotechnology is promising field of science which can guide our understanding of the role of interspecies interaction in the development of biofilm. Streptococcus mutans with other species of bacteria has been known to form dental biofilm. The correlation between genetically modified bacteria Streptococcus mutans and nanoscale morphology has been assessed using AFMi.e atomic force microscopy. Nanotechnology application includes 16O/18O reverse proteolytic labeling,use of quantum dots for labeling of bacterial cells, selective removal of cariogenic bacteria while preserving the normal oral flora and silver antimicrobial nanotechnology against pathogens associated with biofilms. The future comprises a mouthwash full of smart nanomachines which can allow the harmless flora of mouth to flourish in a healthy ecosystem.

Longitudinal changes in microbiology and clinical periodontal variables after placement of fixed orthodontic appliances.

BACKGROUND: In the past few decades, more patients have been treated orthodontically, but no longitudinal study has compared orthodontic bands and brackets microbiologically and clinically. METHODS: This longitudinal trial (split-mouth design) included 24 patients. Microbiology (sub- and supragingival), probing depth (PD), bleeding on probing (BOP), and gingival crevicular fluid flow (GCF) were assessed at baseline (band placement) and at weeks 18 (bracket bonding) 20, 24, and 36. A statistical comparison was made over time and among the banded, bonded, and control sites. RESULTS: The aerobe/anaerobe ratio of sub- and supragingival colony forming units decreased significantly (relatively more anaerobes) over the study period for the banded and bonded sites (P <0.001). This decrease was accompanied by significant elevations in PD, BOP, and GCF. These changes occurred faster after bonding compared to banding. No significant changes were observed 18 weeks after banding with the exception of increased PD (P <0.001). At week 36, all microbial and clinical variables at the bonded site had changed significantly in the negative direction (P <0.001) compared to week 18. The control sites did not show any significant changes over time, indicating that the effects were localized. CONCLUSIONS: The placement of fixed orthodontic appliances had a significant impact on microbial and clinical variables. The changes occurred faster at the bonded sites compared to the banded sites, probably because wire insertion caused difficulties in approximal cleaning. Over the long term, banding did not lead to more adverse microbial and periodontal effects than bonding.

Clinical and microbiological benefits of systemic metronidazole and amoxicillin in the treatment of smokers with chronic periodontitis: a randomized placebo-controlled study.

AIM: The aim of this study was to evaluate the clinical and microbiological effects of scaling and root planing (SRP) alone or combined with metronidazole (MTZ) or with MTZ and amoxicillin (AMX) in the treatment of smokers with chronic periodontitis. METHODS: A double-blind, placebo-controlled, randomized clinical trial was conducted in 43 subjects who received SRP alone (n=15) or combined with MTZ (400 mg 3 x per day, n=14) or with MTZ+AMX (500 mg 3 x per day, n=14) for 14 days. Clinical and microbiological examinations were performed at baseline and 3 months post-therapy. Subgingival samples were analysed by checkerboard DNA-DNA hybridization. RESULTS: Subjects receiving MTZ+AMX showed the greatest improvements in mean probing depth and clinical attachment level. Both antibiotic therapies led to additional clinical benefits over SRP alone in initially shallow, intermediate, and deep sites. The SRP+MTZ+AMX therapy led to the most beneficial changes in the subgingival microbial profile. These subjects showed significant reductions in the mean counts and proportions of periodontal pathogens such as Tannerella forsythia, Porphyromonas gingivalis and Treponema denticola, and the greatest increase in proportions of host-compatible species. CONCLUSION: Significant advantages are observed when systemic antibiotics are combined with SRP in the treatment of smokers with chronic periodontitis. The greatest benefits in clinical and microbiological parameters are achieved with the use of SRP+MTZ+AMX.

Adhesion of oral streptococci to all-ceramics dental restorative materials in vitro.

In recent years, patients have benefited from the development of better and more esthetic materials, including all-ceramics dental restorative materials. Dental plaque formation on teeth and restorative materials plays an important role in the pathogenesis of oral diseases. This study investigates initial adhesion of stationary phase streptococcal species to different all-ceramics dental restorative materials. The saliva-coated materials were incubated with the bacteria for 1 h in an in vitro flow chamber which mimics environmental conditions in the oral cavity. Number and vitality of adhering bacteria were determined microscopically after staining. Surface roughness and the composition of the materials had no distinctive influence on bacterial adhesion. However, S. mutans and S. sobrinus adhered about tenfold less numerous to all materials than the other streptococcal species. Further, there was a correlation between bacterial vitality and materials' glass content. The results showed that early plaque formation was influenced predominantly by the presence of the salivary pellicle rather than by material dependent parameters whereas the composition of the all-ceramics appeared to have influenced the percentage of viable cells during the adhesion process. This presented in vitro technique may provide a useful model to study the influence of different parameters on adherence of oral streptococcal species.

[Biofilm formation on root canal--review]. / Biofilm w kanalach korzeniowych w swietle pismiennictwa.

It seems likely that one of the reasons for failures in the endodontic treatment is the presence of biofilm in root canals. Biofilm bacteria have a slower metabolism and higher resistance and virulence due to phenotypic changes. The occurrence of biofilms has been reported both inside the canal and on the external root surface. The results of many studies suggest that biofilm may be associated with refractory periapical periodontitis and is often caused by the coronal leakage.

Effect of xylitol on an in vitro model of oral biofilm.

PURPOSE: The aim of the present study was to examine whether xylitol, at different concentrations, inhibits the formation of an experimental model of oral biofilm. MATERIALS AND METHODS: Biofilms of six bacterial species (Streptococcus mutans, Streptococcus sobrinus, Lactobacillus rhamnosus, Actinomyces viscosus, Porphyromonas gingivalis and Fusobacterium nucleatum) were prepared on hydroxyapatite (HA) discs according to the Zürich Biofilm Model. Xylitol was tested at two concentrations, 1% and 3%. At the end of their designated incubation times, some HA discs were destined for confocal laser scanning microscopy (CLSM) and the others were harvested using a sterile surgical instrument. Aliquots of harvested biofilms were diluted and plated onto specific media. After a 48-h anaerobic incubation at 37 degrees C, the colony-forming units (CFUs) were counted. RESULTS: CLSM images showed that only a small amount of isolated bacteria was observed on the surface of HA discs. Culture of harvested biofilms showed an inhibition in the growth of different species included in the biofilms. CONCLUSIONS: Xylitol has a clear inhibitory effect on the formation of the experimental biofilms. This study shows that xylitol is not only efficient in inhibiting the acid production of cariogenic bacteria, but also in preventing the formation of a multispecies biofilm; it confirms the relevance of the use of this polyol for the prevention of oral diseases caused by dental plaque.

Rate of reformation of tongue coatings in young adults.

BACKGROUND: Reviewing the literature, no study on the rate of regrowth of tongue coatings after tongue cleaning was found. Therefore, the purpose of this study in young adults was to study the rate of reformation of tongue coatings after mechanical removal. MATERIAL AND METHODS: Thirty-five dental students participated in the present study. Following preparatory study instructions, baseline examinations were carried out followed by 3 days of observation. At baseline, tongue coating scores (prescraping) were obtained followed by tongue scrapings and determination of the wet weights of the coatings. A second tongue coating score was then obtained within 5 min of the first score (immediate post-scraping). The subjects returned for repeated tongue coating scores after 1 and 2 days and for final examination after 3 days, which included both tongue coating scores (prescraping and immediate post-scraping) and determination of the wet weights of the coatings. RESULTS: Prior to scraping the tongue at day 0 (baseline), mean tongue coating amounted to a surface extension of 33% of the entire dorsum of the tongue. Scraping the tongue reduced the score to 9%. On average, tongue coating scores had returned to baseline levels on day 2. The mean wet weights of tongue scrapings at days 0 and 3 were similar and amounted to 0.09 +/- 0.07 and 0.09 +/- 0.06 g, respectively. CONCLUSION: If tongue cleaning is to be recommended, the results of this study in dental students indicate that tongue cleaning should be performed on a daily basis.

SEM evaluation of in situ early bacterial colonization on a Y-TZP ceramic: a pilot study.

This study aimed to evaluate the effect of surface glazing and polishing of yttrium-stabilized tetragonal zirconia polycrystal ceramic on early dental biofilm formation, as well as the effect of brushing on the removal of adhered bacteria. Two subjects used oral appliances with polished and glazed samples fixed to the right and left sides. After 20 minutes, 1 hour, and 6 hours, the subjects manually brushed the samples on the right side. The samples were analyzed using scanning electron microscopy. Granular material was verified on the samples, especially on irregular surfaces. After 1 hour, there was no significant difference between glazed and polished surfaces in terms of bacterial presence. However, glazed surfaces tended to accumulate more biofilm, and brushing did not completely remove the biofilm. Polished surfaces seem to present a lower tendency for biofilm formation.

Localization of Porphyromonas gingivalis-carrying fimbriae in situ in human periodontal pockets.

Fimbriae, which are involved in adherence, constitute an important pathogenic factor of Porphyromonas gingivalis. In vivo, however, the distribution of P. gingivalis-carrying fimbriae is unknown. The localization of P. gingivalis-carrying fimbriae was examined in situ. From 19 patients with severe periodontitis and P. gingivalis, we obtained 20 teeth with periodontal tissue attached, with and without immunolocalized fimbriae. Eleven teeth were subjected to light microscopy, 9 to electron microscopy. In 6 of the 11 samples examined, we detected positive reactions with an anti-P. gingivalis-fimbriae serum, located in the cementum-attached plaque area in the deep pocket zones. In the so-called 'plaque-free zones', P. gingivalis-carrying fimbriae were immunocytochemically observed to reside in contact with the dental cuticle in 6 of the 9 samples examined. These findings suggest that P. gingivalis-carrying fimbriae are strongly related to adherence to the root surface at the bottoms of human periodontal pockets.

A preliminary investigation into the ultrastructure of dental calculus and associated bacteria.

INTRODUCTION: Though dental calculus is generally recognised as comprising mineralised bacteria, areas of non-mineralised bacteria may be present. AIM: To investigate the ultrastructure of non-decalcified young and mature supragingival calculus and subgingival calculus, and the possible presence of internal viable bacteria. MATERIALS AND METHODS: Supragingival calculus was harvested from five patients, 9-10 weeks after scaling and root debridement. Five samples of mature supragingival and subgingival calculus were taken from patients presenting with adult periodontitis. Specimens were fixed and embedded for transmission electron microscopy. RESULTS: The ultrastructure of young and mature supragingival calculus was similar with various large and small crystal types. Non-mineralised channels were observed extending into the calculus, often joining extensive lacunae, both containing intact non-mineralised coccoid and rod-shaped microorganisms. Subgingival calculus possessed more uniform mineralisation without non-mineralised channels and lacunae. CONCLUSION: Supragingival calculus contains non-mineralised areas which contain bacteria and other debris. The viability of the bacteria, and their identification could not be determined in this preliminary investigation. As viable bacteria within these lacunae may provide a source of re-infection, further work needs to be done to identify the bacteria in the lacunae, and to determine their viability.

Human minor and major gland saliva proteins and ability to mediate Actinomyces naeslundii adherence.

Bacteria-binding components and the ability to mediate bacterial adhesion to the tooth surface have been thoroughly studied in major salivary gland secretions. Our knowledge on the bacteria binding activity in minor gland saliva is, however, limited. In this study, proteins were examined in parallel in minor (palatal, buccal and labial) and major (parotid and submandibular/sublingual) salivary gland secretions in one subject using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. The adherence of early colonizing Actinomyces naeslundii to pellicles formed from the secretions on hydroxyapatite beads was also examined. Amylase, IgA, proline-rich proteins and the high-molecular-weight glycoproteins, agglutinins, were detected in all saliva tested. Carbohydrate-reactive antibodies recognized the low-molecular weight mucin, MUC 7 in submandibular/sublingual saliva only. A. naeslundii strain 12104 adhered to all pellicles and especially to the buccal gland saliva pellicles. Strain LY7 adhered in highest numbers to the submandibular/sublingual saliva pellicles. It also bound in considerable numbers to parotid and palatal saliva pellicles but not to the ones formed from buccal and labial gland saliva. Our findings indicate that several bacteria-binding components are secreted in both minor and major gland saliva. The adherence-promoting ability of the various gland secretions differs, however.

Non-contact removal of coadhering and non-coadhering bacterial pairs from pellicle surfaces by sonic brushing and de novo adhesion.

Coadhesion between oral microbial pairs is an established factor in the spatiotemporal development and prevalence of mixed-species communities in early dental plaque in vivo. This study compares removal and de novo adhesion of pairs of coadhering and non-coadhering oral actinomyces and streptococci by sonic brushing on salivary pellicles in a non-contact mode as a function of the distance between the brush and the pellicle surface in vitro. First, actinomycetes were adhered to a pellicle surface, after which streptococci suspended in saliva were allowed to adhere. Removal was examined by non-contact, sonic brushing with a wetted brush on a either a wetted or a substratum immersed to a depth of 7 mm. After brushing, de novo adhesion of streptococci to brushed pellicles was studied. For coadhering and non-coadhering pairs, 34% and 9%, respectively, of the adhering bacteria were involved in aggregates comprising more than 10 organisms. Non-contact, sonic brushing removed up to 99% of the adhering bacteria, regardless of the state of immersion of the substratum. Bacterial removal decreased with increasing distance of up to 6 mm between brush and pellicle surface. For the non-coadhering pair, subsequent exposure of pellicles to a streptococcal suspension yielded about 6% of bacteria involved in large aggregates. Alternatively, de novo adhesion of the coadhering streptococcal strain to pellicles brushed on the wetted substratum yielded 31% of bacteria involved in large aggregates, but after brushing the immersed substratum only 12% of the adhering bacteria were found in large aggregates. It is concluded that non-contact sonic brushing, under immersion, removes high percentage of adhering bacterial pairs up to a distance of 6 mm between the brush and the pellicle surface. However, non-contact, sonic brushing with only a thin wet film on the substratum may leave footprints to which streptococci preferentially adhere.

Bacteria-binding plasma proteins in pellicles formed on hydroxyapatite in vitro and on teeth in vivo.

Previous studies on dental pellicle formation and bacterial adherence have focussed on saliva and its components. The tooth surface is, however, also exposed to the plasma-derived crevicular fluid. In the present study, (i). plasma proteins in in vitro and in vivo pellicles were examined using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting and image analysis and (ii). the adherence of periodontopathogenic bacteria to experimental plasma and saliva pellicles was examined using radio-labelled bacteria and liquid scintillation counting. The plasma components fibrinogen, fibronectin, albumin and IgG were incorporated from plasma in the experimental pellicle and mediated the adherence of Porphyromonas gingivalis, Fusobacterium nucleatum and Actinomyces naeslundii. These proteins were also readily detected in in vivo pellicles and were found to a higher extent in pellicles formed at the gingival part of the tooth surface than at the incisal part.

Bacterial colonization during de novo plaque formation.

OBJECTIVE: To determine microbial changes that occur during plaque formation in a dentition free of gingival inflammation. MATERIAL AND METHODS: Ten subjects were recruited. The study included one preparatory period (2 weeks) and a plaque accumulation period (4 days). The volunteers exercised proper tooth cleaning methods, were scaled and received repeated professional mechanical tooth cleaning during the preparatory period. During the plaque accumulation period, the participants abstained from plaque control measures. Plaque was scored on the approximal surfaces of maxillary and mandibular premolars on Days 0, 1, 2 and 4 using a scale from 0 to 5 and according to the criteria of the Quigley and Hein Plaque Index (QHI). Supragingival plaque samples were obtained from the same intervals and surfaces and evaluated using a checkerboard DNA-DNA hybridization technique. RESULTS: The mean QHI increased from 0 to 1.6 (Day 4). The total number of organisms on Day 0 averaged 140 x 10(5) and increased to about 210 x 10(5) after 4 days without oral hygiene. The most dominant species on Day 0 were members of the genus Actinomyces. These organisms comprised almost 50% of the microbiota evaluated. None of the Actinomyces species increased significantly during the 4 days. Some Streptococcus species increased significantly over time as well as species of the genera Capnocytophaga, Campylobacter, Fusobacteria and Actinomyces actinomycetemcomitans. CONCLUSION: In the present investigation, the preparatory phase established a situation with minimal gingival inflammation and close to zero amounts of dental plaque. The Day 0 plaque samples exhibited high proportions of Actinomyces species. During the 4 days of no oral hygiene, there was a small increase in total numbers of organisms as well as a modest increase in the proportion of "disease-associated" taxa such as species of the "orange complex" species.