Extracellular Glycoside Hydrolase Activities in the Human Oral Cavity

Carbohydrate availability shifts when bacteria attach to a surface and form biofilm. When salivary planktonic bacteria form an oral biofilm, a variety of polysaccharides and glycoproteins are the primary carbon sources; however, simple sugar availabilities are limited due to low diffusion from saliva to biofilm. We hypothesized that bacterial glycoside hydrolase (GH) activities would be higher in a biofilm than in saliva in order to maintain metabolism in a low-sugar, high-glycoprotein environment. Salivary bacteria from 13 healthy individuals were used to grow in vitro biofilm using two separate media, one with sucrose and the other limiting carbon sources to a complex carbohydrate. All six GHs measured were higher in vitro when grown in the medium with complex carbohydrate as the sole carbon source. We then collected saliva and overnight dental plaque samples from the same individuals and measured ex vivo activities for the same six enzymes to determine how oral microbial utilization of glycoconjugates shifts between the planktonic phase in saliva and the biofilm phase in overnight dental plaque. Overall higher GH activities were observed in plaque samples, in agreement with in vitro observation. A similar pattern was observed in GH activity profiles between in vitro and ex vivo data. 16S rRNA gene analysis showed that plaque samples had a higher abundance of microorganisms with larger number of GH gene sequences. These results suggest differences in sugar catabolism between the oral bacteria located in the biofilm and those in saliva.     

Microbial Diversity in the Early In Vivo-Formed Dental Biofilm

Although the mature dental biofilm composition is well studied, there is very little information on the earliest phase of in vivo tooth colonization. Progress in dental biofilm collection methodologies and techniques of large-scale microbial identification have made new studies in this field of oral biology feasible. The aim of this study was to characterize the temporal changes and diversity of the cultivable and noncultivable microbes in the early dental biofilm. Samples of early dental biofilm were collected from 11 healthy subjects at 0, 2, 4, and 6 h after removal of plaque and pellicle from tooth surfaces. With the semiquantitative Human Oral Microbiome Identification Microarray (HOMIM) technique, which is based on 16S rRNA sequence hybridizations, plaque samples were analyzed with the currently available 407 HOMIM microbial probes. This led to the identification of at least 92 species, with streptococci being the most abundant bacteria across all time points in all subjects. High-frequency detection was also made with Haemophilus parainfluenzae, Gemella haemolysans, Slackia exigua, and Rothia species. Abundance changes over time were noted for Streptococcus anginosus and Streptococcus intermedius (P = 0.02), Streptococcus mitis bv. 2 (P = 0.0002), Streptococcus oralis (P = 0.0002), Streptococcus cluster I (P = 0.003), G. haemolysans (P = 0.0005), and Stenotrophomonas maltophilia (P = 0.02). Among the currently uncultivable microbiota, eight phylotypes were detected in the early stages of biofilm formation, one belonging to the candidate bacterial division TM7, which has attracted attention due to its potential association with periodontal disease.     

Molecular characterization of subject-specific oral microflora during initial colonization of enamel

The initial microbial colonization of tooth surfaces is a repeatable and selective process, with certain bacterial species predominating in the nascent biofilm. Characterization of the initial microflora is the first step in understanding interactions among community members that shape ensuing biofilm development. Using molecular methods and a retrievable enamel chip model, we characterized the microbial diversity of early dental biofilms in three subjects. A total of 531 16S rRNA gene sequences were analyzed, and 97 distinct phylotypes were identified. Microbial community composition was shown to be statistically different among subjects. In all subjects, however, 4-h and 8-h communities were dominated by Streptococcus spp. belonging to the Streptococcus oralis/Streptococcus mitis group. Other frequently observed genera (comprising at least 5% of clone sequences in at least one of the six clone libraries) were Actinomyces, Gemella, Granulicatella, Neisseria, Prevotella, Rothia, and Veillonella. Fluorescence in situ hybridization (FISH) confirmed that the proportion of Streptococcus sp. sequences in the clone libraries coincided with the proportion of streptococcus probe-positive organisms on the chip. FISH also revealed that, in the undisturbed plaque, not only Streptococcus spp. but also the rarer Prevotella spp. were usually seen in small multigeneric clusters of cells. This study shows that the initial dental plaque community of each subject is unique in terms of diversity and composition. Repetitive and distinctive community composition within subjects suggests that the spatiotemporal interactions and ecological shifts that accompany biofilm maturation also occur in a subject-dependent manner.     

Early Canine Plaque Biofilms: Characterization of Key Bacterial Interactions Involved in Initial Colonization of Enamel

Periodontal disease (PD) is a significant problem in dogs affecting between 44% and 63.6% of the population. The main etiological agent for PD is plaque, a microbial biofilm that colonizes teeth and causes inflammation of the gingiva. Understanding how this biofilm initiates on the tooth surface is of central importance in developing interventions against PD. Although the stages of plaque development on human teeth have been well characterized little is known about how canine plaque develops. Recent studies of the canine oral microbiome have revealed distinct differences between the canine and human oral environments and the bacterial communities they support, particularly with respect to healthy plaque. These differences mean knowledge about the nature of plaque formation in humans may not be directly translatable to dogs. The aim of this study was to identify the bacterial species important in the early stages of canine plaque formation in vivo and then use isolates of these species in a laboratory biofilm model to develop an understanding of the sequential processes which take place during the initial colonization of enamel. Supra-gingival plaque samples were collected from 12 dogs at 24 and 48 hour time points following a full mouth descale and polish. Pyrosequencing of the 16S rDNA identified 134 operational taxonomic units after statistical analysis. The species with the highest relative abundance were Bergeyella zoohelcum, Neisseria shayeganii and a Moraxella species. Streptococcal species, which tend to dominate early human plaque biofilms, had very low relative abundance. In vitro testing of biofilm formation identified five primary colonizer species, three of which belonged to the genus Neisseria. Using these pioneer bacteria as a starting point, viable two and three species communities were developed. Combining in vivo and in vitro data has led us to construct novel models of how the early canine plaque biofilm develops.     

Impact of Early Colonizers on In Vitro Subgingival Biofilm Formation

The aim of this study was to investigate the impact of early colonizing species on the structure and the composition of the bacterial community developing in a subgingival 10-species biofilm model system. The model included Streptococcus oralis, Streptococcus anginosus, Actinomycesoris, Fusobacterium nucleatum subsp. nucleatum, Veillonella dispar, Campylobacter rectus, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. Based on literature, we considered Streptococcus oralis, Streptococcus anginosus, and Actinomyces oris as early colonizers and examined their role in the biofilms by either a delayed addition to the consortium, or by not inoculating at all the biofilms with these species. We quantitatively evaluated the resulting biofilms by real-time quantitative PCR and further compared the structures using confocal laser scanning microscopy following fluorescence in situ hybridisation. The absence of the early colonizers did not hinder biofilm formation. The biofilms reached the same total counts and developed to normal thickness. However, quantitative shifts in the abundances of individual species were observed. In the absence of streptococci, the overall biofilm structure appeared looser and more dispersed. Moreover, besides a significant increase of P. intermedia and a decrease of P. gingivalis , P. intermedia appeared to form filamented long chains that resembled streptococci. A. oris, although growing to significantly higher abundance in absence of streptococci, did not have a visible impact on the biofilms. Hence, in the absence of the early colonizers, there is a pronounced effect on P. intermedia and P. gingivalis that may cause distinct shifts in the structure of the biofilm. Streptococci possibly facilitate the establishment of P. gingivalis into subgingival biofilms, while in their absence P. intermedia became more dominant and forms elongated chains.     

Exploring bacterial flora in oral squamous cell carcinoma: a microbiological study

The oral cavity contains a unique and diverse micro?ora. While most of these organisms exhibit commensalism, shifts in bacterial community dynamics cause pathological changes within the oral cavity and at distant sites. We assessed the microbial flora using cultured saliva and oral swabs from subjects with oral squamous cell carcinoma (OSCC) and healthy controls. Microbial samples were collected from the carcinoma site, contralateral healthy mucosa, and saliva of the study group and samples were collected from healthy mucosa and saliva of controls. Samples were stored on ice and transported to the laboratory for culture. The median number of colony forming units (CFU)/ml at carcinoma sites was significantly greater than at the contralateral healthy mucosa. Similarly,?in saliva of carcinoma subjects, the median number of CFU/ml was significantly greater than in saliva of control subjects.     

Differences in microbiological composition of saliva and dental plaque in subjects with different drinking habits

Several foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dental crown caries, as well as inhibition of glucosyltransferases, which are the S. mutans enzymes involved in the synthesis of an adherent, water-insoluble glucan from sucrose. Other studies have demonstrated an in vitro action on oral plaque biofilm formation and desorption. This study evaluated whether the activity displayed in vitro by food compounds could affect the microbiological composition of saliva and dental plaque of subjects with a diet rich in these foods, comparing the results with those obtained from subjects with a different diet. The foods considered were: coffee, barley coffee, tea and wine. A total of 93 subjects were recruited into the study. Six samples of both plaque and saliva were collected from each subject at roughly one-monthly intervals. Total bacteria, total streptococci, S. mutans and lactobacilli counts were determined by culture in both saliva and dental plaque. The highest bacterial titres were recorded for the control population, while each drinking habit subgroup showed counts roughly one log lower than the controls. These differences in bacterial counts proved statistically significant (P<0.05). As far as dental plaque was concerned, while total counts did not significantly vary per mg of plaque in the subjects belonging to the different drinking habit subgroups, a significant decrease (P<0.05) was observed in those subjects drinking coffee, tea, barley coffee and wine when mutans streptococci and lactobacilli were evaluated. In several cases a more than one log decrease was observed. Plaque indices were also determined, and a significant (P<0.05) reduction in values was recorded in the subjects belonging the specific drinking habit subgroups compared to the control group. This study indicates that there is a correlation between consumption of specific foods and oral health in terms of reduced plaque deposition and lower counts of odontopathogens.     

Characterization of Actinomyces species isolated from failed dental implant fixtures

In the oral cavity, Actinomyces form a fundamental component of the indigenous microflora, being among initial colonizers in polymicrobial biofilms. However, some differences may exist between different species in terms of their attachment not only to teeth but also to biomaterials. In this study we investigated the distribution of Actinomyces in 33 dental implant fixtures explanted from 17 patients. The identification was based on comprehensive biochemical testing and gas-liquid chromatography and when needed, 16S rRNA sequencing. Actinomyces was the most prevalent bacterial genus in these failed implants, colonizing 31/33 (94%) of the fixtures. Proportions of Actinomyces growth of the total bacterial growth in the Actinomyces-positive fixtures varied from 0.01% up to 75%. A. odontolyticus was the most common Actinomyces finding, present in 26/31 (84%) Actinomyces-positive fixtures. Actinomyces naeslundii and A. viscosus were both detected in 10/31 (32%) and A. israelii in 7/31 (23%) fixtures. Other Actinomyces species, including A. georgiae, A. gerencseriae and A. graevenitzii, were detected less frequently. Our results suggest that Actinomyces species are frequent colonizers on failed implant surfaces, where A. odontolyticus was the far most prominent Actinomyces species.     

Dental plaque as a biofilm

Dental plaque is the diverse microbial community found on the tooth surface embedded in a matrix of polymers of bacterial and salivary origin. Once a tooth surface is cleaned, a conditioning film of proteins and glycoproteins is adsorbed rapidly to the tooth surface. Plaque formation involves the interaction between early bacterial colonisers and this film (the acquired enamel pellicle). To facilitate colonisation of the tooth surface, some receptors on salivary molecules are only exposed to bacteria once the molecule is adsorbed to a surface. Subsequently, secondary colonisers adhere to the already attached early colonisers (co-aggregation) through specific molecular interactions. These can involve protein-protein or carbohydrate-protein (lectin) interactions, and this process contributes to determining the pattern of bacterial succession. As the biofilm develops, gradients in biologically significant factors develop, and these permit the co-existence of species that would be incompatible with each other in a homogeneous environment. Dental plaque develops naturally, but it is also associated with two of the most prevalent diseases affecting industrialised societies (caries and periodontal diseases). Future strategies to control dental plaque will be targeted to interfering with the formation, structure and pattern of development of this biofilm.     

PCR指纹图谱技术分析人体口腔内微生物菌群结构

目的 应用PCR-DGGE指纹图谱技术对人体口腔微生物菌群结构进行系统性研究.方法 对1例健康人唾液周期性采集的样品和8例健康人个体的唾液与牙菌斑采集的样品,进行微生物群落总DNA的抽提.以此为模板扩增16S rRNA V3可变区,产物经DGGE指纹图谱分析其组成结构,并运用UVIBAND/MAP等软件比较所得群落指纹图谱的相似性指数.结果 同一健康人个体不同采样时间的唾液菌群结构相似性系数＞74%,通过对不同健康个体口腔样本的研究,发现同一个体的唾液与牙菌斑菌群结构存在差异（84%～95%）.结论 同一健康个体其唾液微生物菌群在一定时间内基本稳定,仅有微小的变化;唾液与同个体牙菌斑的微生物组成虽然存在差异,但这种差异要明显小于个体间的差异.     

Osteopontin reduces biofilm formation in a multi-species model of dental biofilm

Background

Combating dental biofilm formation is the most effective means for the prevention of caries, one of the most widespread human diseases. Among the chemical supplements to mechanical tooth cleaning procedures, non-bactericidal adjuncts that target the mechanisms of bacterial biofilm formation have gained increasing interest in recent years. Milk proteins, such as lactoferrin, have been shown to interfere with bacterial colonization of saliva-coated surfaces. We here study the effect of bovine milk osteopontin (OPN), a highly phosphorylated whey glycoprotein, on a multispecies in vitro model of dental biofilm. While considerable research effort focuses on the interaction of OPN with mammalian cells, there are no data investigating the influence of OPN on bacterial biofilms.

Methodology/Principal Findings

Biofilms consisting of Streptococcus oralis, Actinomyces naeslundii, Streptococcus mitis, Streptococcus downei and Streptococcus sanguinis were grown in a flow cell system that permitted in situ microscopic analysis. Crystal violet staining showed significantly less biofilm formation in the presence of OPN, as compared to biofilms grown without OPN or biofilms grown in the presence of caseinoglycomacropeptide, another phosphorylated milk protein. Confocal microscopy revealed that OPN bound to the surface of bacterial cells and reduced mechanical stability of the biofilms without affecting cell viability. The bacterial composition of the biofilms, determined by fluorescence in situ hybridization, changed considerably in the presence of OPN. In particular, colonization of S. mitis, the best biofilm former in the model, was reduced dramatically.

Conclusions/Significance

OPN strongly reduces the amount of biofilm formed in a well-defined laboratory model of acidogenic dental biofilm. If a similar effect can be observed in vivo, OPN might serve as a valuable adjunct to mechanical tooth cleaning procedures.     

Ex vivo gingival-biofilm consortia

AIMS: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. METHODS AND RESULTS: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. CONCLUSIONS: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.     

Isolation and characterization of an early colonizing Rhizobium sp. R8 from a household toilet bowl

The bacterial community structure was compared between the third days’, one week’, and three weeks’ biofilm samples from the surface of a household toilet bowl. It was found that the PCR-DGGE band pattern of 16S rRNA gene was dramatically changed after the third day and was not further changed until three weeks. This result suggests that there are early and late colonizing bacterial groups. One of the early colonizers isolated from the third days’ sample was Rhizobium sp. R8, a closest relative to Rhizobium giardinii, which exhibited the highest biofilm formation activity in an artificial urine condition. R8 produced extracellular polysaccharides containing galactose, glucose, and mannose at the molar ratio of 8:1:1, which were probably responsible for the biofilm formation. Its excelled biofilm formation and urease activities together with the lack of nodulation and nitrogen fixing genes in R8 suggest that this strain has been specifically adapted to urine condition in a toilet bowl.     

Crystalline anatase-rich titanium can reduce adherence of oral streptococci

Dental implant abutments that emerge through the mucosa are rapidly covered with a salivary protein pellicle to which bacteria bind, initiating biofilm formation. In this study, adherence of early colonizing streptococci, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Streptococcus sanguinis to two saliva-coated anodically oxidized surfaces was compared with that on commercially pure titanium (CpTi). Near edge X-ray absorption (NEXAFS) showed crystalline anatase was more pronounced on the anodically oxidized surfaces than on the CpTi. As revealed by fluorescence microscopy, a four-species mixture, as well as individual bacterial species, exhibited lower adherence after 2?h to the saliva-coated, anatase-rich surfaces than to CpTi. Since wettability did not differ between the saliva-coated surfaces, differences in the concentration and/or configuration of salivary proteins on the anatase-rich surfaces may explain the reduced bacterial binding effect. Anatase-rich surfaces could thus contribute to reduced overall biofilm formation on dental implant abutments through diminished adherence of early colonizers.     

Possible variation of the human oral bacterial community after wearing removable partial dentures by DGGE

Although it is well-known that variations of the microbial community in a specific location of human body may be associated with some diseases, the developing change of the oral microbiota related to oral diseases before and after wearing the removable partial dentures (RPD) is not completely understood. In this study, three kinds of samples (saliva, supra- and subgingival plaque, and oral mucosal surfaces) were collected from the 10-patients group at three different times: before, 1-month and 6-months after the treatment. Ten healthy adults were also selected as the control group. Denaturing gradient gel electrophoresis was applied to identify the bacterial profiles and to analyze the dynamics of the oral microbial population in the pre- and post-therapy. The ANOVA of Repeated Measurement Data indicated that, in the saliva and mucosal surfaces, wearing RPDs caused significant change of numbers of amplicons. As many as 607 amplicons were chosen to cut out and re-amplify by PCR. After cloning and sequencing, a total of 16 bacterial genera were identified. The health-associated genera such as Streptococcus, Neisseria, Rothia, Corynebacterium, Leptotrichia, Gemella, Veillonella, Selenomona and Actinomyces tended to decrease, whereas the disease-associated species including Streptococcus mutans tended to increase. In general, wearing RPDs influenced the diversity of the bacterial species in the oral microbial ecosystem. It is noteworthy that the oral environment will be changed from the healthy status towards the disease status after the treatment.     

Growth and molecular characterization of dental plaque microcosms

AIMS: (i) To compare the effects of feeding protocols upon the composition and stability of dental plaque microcosms formed in constant-depth film fermenters (CDFF). (ii) To evaluate the utility of denaturing gradient gel electrophoresis (DGGE) and culture methodologies for the investigation of such models. METHODS AND RESULTS: Microcosms were established anaerobically in the CDFFs from freshly collected saliva. These were fed either with artificial saliva alone (famine) or combined with discontinuous feeding (feast-famine). Culture and 16s rDNA sequencing indicated that supplemental feeding gave ca. 2 log increases in Lactobacillus rhamnosus and Prevotella buccae. Feast-famine microcosms were then further characterized by DGGE using primers specific for the V2-V3 region of eubacterial rDNA. These gave single major bands with pure cultures (eight species) and resolved all strains apart from Lact. rhamnosus and Actinomyces naeslundii. Whilst culture with selective media indicated a degree of stability and reproducibility between replicate microcosms, DGGE showed a considerable degree of variability that related to several putatively uncultured bacteria. CONCLUSIONS: Feast-famine regimes altered community composition. DGGE analyses identified putatively unculturable species and demonstrated variability between replicate fermenters. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the utility of DGGE for the analysis of dental plaque, especially with respect to unculturable bacteria. Results question the assumptions of reproducibility of plaque microcosms established in non-replicated CDFFs made on the basis of selective media. Feeding regimes, particularly those involving complex nutrients, will dramatically affect population dynamics.     

Interactions between salivary Bifidobacterium adolescentis and other oral bacteria: in vitro coaggregation and coadhesion assays

Coaggregation assays were performed to investigate interactions between oral Bifidobacterium adolescentis and other oral bacterial species. Bifidobacterium adolescentis OLB6410 isolated from the saliva of healthy humans did not coaggregate with Actinomyces naeslundii JCM8350, Streptococcus mitis OLS3293, Streptococcus sanguinis JCM5708, Veillonella parvula ATCC17745 or Porphyromonas gingivalis OB7124, but it did coaggregate with Fusobacterium nucleatum JCM8532. Subsequent examination of biofilm formation on saliva-coated hydroxyapatite discs using FISH revealed that B. adolescentis OLB6410 could not directly adhere to the coated discs. It did, however, adhere to biofilms of A. naeslundii, V. parvula, and F. nucleatum, although it did not coaggregate with A. naeslundii nor with V. parvula. These results suggest that the adhesion of B. adolescentis to tooth surfaces is mediated by other oral bacteria. Heat- or proteinase K-treated F. nucleatum could not coaggregate with B. adolescentis. Similarly, the coaggregation and coadhesion of proteinase K-treated B. adolescentis were strongly inhibited. It is therefore probable that proteinaceous factors on the cellular surface of B. adolescentis and F. nucleatum are involved in their interaction. The data presented in this study add to our understanding of bifidobacterial colonization in the human oral cavity.     

Microbiota in the oral subgingival biofilm is associated with obesity in adolescence

To test the hypothesis whether microbiota in oral biofilm is linked with obesity in adolescents we designed this cross-sectional study. Obese adolescents (n = 29) with a mean age of 14.7 years and normal weight subjects (n = 58) matched by age and gender were examined with respect to visible plaque index (VPI%) and gingival inflammation (bleeding on probing (BOP%)). Stimulated saliva was collected. They answered a questionnaire concerning medical history, medication, oral hygiene habits, smoking habits, and sociodemographic background. Microbiological samples taken from the gingival crevice was analyzed by checkerboard DNA-DNA hybridization technique. The sum of bacterial cells in subgingival biofilm was significantly associated with obesity (P < 0.001). The link between sum of bacterial cells and obesity was not confounded by any of the studied variables (chronic disease, medication, VPI%, BOP%, flow rate of whole saliva, or meal frequency). Totally 23 bacterial species were present in approximately threefold higher amounts, on average, in obese subjects compared with normal weight controls. Of the Proteobacteria phylum, Campylobacter rectus and Neisseria mucosa were present in sixfold higher amounts among obese subjects. The association between obesity and sum of bacterial cells in oral subgingival biofilm indicates a possible link between oral microbiota and obesity in adolescents.     

Postnatal development of bacterial population in the gastrointestinal tract of calves

Bacterial 16S rDNA from fecal samples of two calves were amplified by PCR and analyzed by denaturing gradient gel electrophoresis; selected bands were sequenced. Escherichia coli and Bifidobacterium animalis were the initial colonizers, followed by species closely related to the genera Bacteroides, Clostridium and Faecalibacterium. Change of diet was connected with shifts of bacterial population and with the occurrence of many bacterial species that have not been cultured up to now. The diet change corresponded with an alteration in a volatile-fatty-acid concentration in fecal samples.     

Autoinducer‐2 is produced in saliva‐fed flow conditions relevant to natural oral biofilms

Aims: We evaluated the ability of a dual‐species community of oral bacteria to produce the universal signalling molecule, autoinducer‐2 (AI‐2), in saliva‐fed biofilms. Methods and Results: Streptococcus oralis 34, S. oralis 34 luxS mutant and Actinomyces naeslundii T14V were grown as single‐ and dual‐species biofilms within sorbarods fed with 25% human saliva. AI‐2 concentration in biofilm effluents was determined by the Vibrio harveyi BB170 bioluminescence assay. After homogenizing the sorbarods to release biofilm cells, cell numbers were determined by fluorometric analysis of fluorescent antibody‐labelled cells. After 48 h, dual‐species biofilm communities of interdigitated S. oralis 34 and A. naeslundii T14V contained 3·2 × 10⁹ cells: fivefold more than single‐species biofilms. However, these 48‐h dual‐species biofilms exhibited the lowest concentration ratio of AI‐2 to cell density. Conclusions: Oral bacteria produce AI‐2 in saliva‐fed biofilms. The decrease of more than 10‐fold in concentration ratio seen between 1 and 48 h in S. oralis 34–A. naeslundii T14V biofilms suggests that peak production of AI‐2 occurs early and is followed by a very low steady‐state level. Significance and Impact of the Study: High oral bacterial biofilm densities may be achieved by inter‐species AI‐2 signalling. We propose that low concentrations of AI‐2 contribute to the establishment of oral commensal biofilm communities.