Plaque Organisms Research Articles

Stratification of Fusobacterium nucleatum by local health status in the oral cavity defines its subspecies disease association

The ubiquitous inflammophilic oral pathobiont Fusobacterium nucleatum (Fn) is widely recognized for its strong association with inflammatory dysbiotic diseases and cancer. Fn is subdivided into four subspecies, which are historically considered functionally interchangeable in the oral cavity. To test this assumption, we analyzed patient-matched dental plaque and odontogenic abscess clinical specimens and examined whether an inflammatory environment selects for/against particular Fn subspecies. Dental plaque harbored a greater diversity of fusobacteria, with Fn. polymorphum dominating, whereas odontogenic abscesses were exceptionally biased for the largely uncharacterized organism Fn. animalis. Comparative genomic analyses revealed significant genotypic distinctions among Fn subspecies that correlate with their preferred ecological niches and support a taxonomic reassignment of each as a distinct Fusobacterium species. Despite originating as a low-abundance organism in dental plaque, Fn. animalis typically outcompetes other oral fusobacteria within the inflammatory abscess environment, which may explain its prevalence in other oral and extraoral diseases.

A fogágybetegség és a krónikus obstruktív tüdőbetegség kapcsolatának jelentősége

A fogágybetegség és a krónikus obstruktív tüdőbetegség (COPD) is a leggyakrabban megjelenő krónikus betegségekcsoportjába tartozik. A fogelvesztéssel járó előrehaladott fogágybetegség a világ 6. leggyakrabban előforduló krónikusbetegsége, mely kb. 743 millió embert érint, míg a COPD világszerte évente átlagosan 3 millió emberéletet követel (ez5,3%-a az összes haláloknak világszerte) és 2016 óta a világ 3. vezető halálokává vált. Egy norvég tanulmány szerinta nagyon súlyos COPD-ben szenvedő, tüdőtranszplantáció előtt álló betegek esetén a parodontitisz prevalenciája megközelítia 44%-ot, szemben a nem COPD-s kontrollcsoport 7,3%-os gyakoriságával. A két betegség összefüggése jelenlegdirekt módon nem bizonyított, ám egyre több tanulmány támasztja alá a két betegség közti kapcsolat lehetőségét.Tanulmányunkban összegezni szeretnénk a jelenleg elérhető szakirodalmi adatokat és ez által betekintést nyújtania két betegség kölcsönhatásába és feltételezett patomechanizmusába. Szükség lenne olyan klinikai vizsgálatokra, melyekbennagy esetszámmal vizsgálnak parodontálisan érintett COPD-s betegeket, így megerősítve vagy cáfolva azta feltevést, miszerint a fogágybetegek parodontális terápiája hozzájárulhat-e a COPD-ben szenvedő betegek életminőségénekjavításához.

Antimicrobial and Antibiofilm Activity of Essential Oil of Cymbopogon citratus against Oral Microflora Associated with Dental Plaque

Aims: Dental biofilms are complex, multi-species microorganism communities that inhabit the oral cavity in the form of dental plaque which causes dental caries and periodontal diseases. The present study aims to explore the potential of Lemon Grass Essential Oil (LGEO) extracted from Cymbopogon citratus as antimicrobial and antibiofilm agent against the microorganisms responsible for dental plaque.
 Study Design: Observational and comparison study.
 Place and Duration of Study: Research centre, Department of Microbiology, Abasaheb Garware college, Pune, India, between Dec 2012 to Jan 2017.
 Methodology: Three bacterial species primarily responsible for the biofilm formation were isolated from dental plaque and identified using 16S ribosomal RNA sequences. Five most primary colonizer of dental plaque organisms were acquired from the Microbial Type Culture Collection cultures. Antimicrobial as well as antibiofilm activity of LGEO, was determined against these eight biofilm forming microorganism. The antibiofilm activity of LGEO was evaluated against oral flora individually, as well as in consortium.
 Results: LGEO displayed excellent antimicrobial activity against eight test organisms associated with dental plaque, representing four genera namely Streptococcus, Staphylococcus, Lactobacillus and Candida. MIC of LGEO for all test organisms was determined as 1.5% (v/v). The LGEO was found to exhibit as high as 76% biofilm inhibitory activity even in the consortium, where the biofilm formation sometimes has been noted to be comparatively more than that of the individual organism, making LGEO a very promising antibiofilm agent.
 Conclusion: LGEO present in rampantly grown plant, Cymbopogon citratus, has remarkable antimicrobial and antibiofilm activity against the dental plaque organism and thus can be the economical, convenient, natural and nontoxic herbal material to effectively control the oral microflora associated with dental plaque.

Identification of Redox Partners of the Thiol-Disulfide Oxidoreductase SdbA in Streptococcus gordonii.

We previously identified a novel thiol-disulfide oxidoreductase, SdbA, in Streptococcus gordonii that formed disulfide bonds in substrate proteins and played a role in multiple phenotypes. In this study, we used mutational, phenotypic, and biochemical approaches to identify and characterize the redox partners of SdbA. Unexpectedly, the results showed that SdbA has multiple redox partners, forming a complex oxidative protein-folding pathway. The primary redox partners of SdbA that maintain its active site in an oxidized state are a surface-exposed thioredoxin family lipoprotein called SdbB (Sgo_1171) and an integral membrane protein annotated as CcdA2. Inactivation of sdbB and ccdA2 simultaneously, but not individually, recapitulated the sdbA mutant phenotype. The sdbB-ccdA2 mutant had defects in a range of cellular processes, including autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release. AtlS, the natural substrate of SdbA produced by the sdbB-ccdA2 mutant lacked activity and an intramolecular disulfide bond. The redox state of SdbA in the sdbB-ccdA2 mutant was found to be in a reduced form and was restored when sdbB and ccdA2 were knocked back into the mutant. In addition, we showed that SdbB formed a disulfide-linked complex with SdbA in the cell. Recombinant SdbB and CcdA2 exhibited oxidase activity and reoxidized reduced SdbA in vitro Collectively, our results demonstrate that S. gordonii uses multiple redox partners for oxidative protein folding.IMPORTANCEStreptococcus gordonii is a commensal bacterium of the human dental plaque. Previously, we identified an enzyme, SdbA, that forms disulfide bonds in substrate proteins and plays a role in a number of cellular processes in S. gordonii Here, we identified the redox partners of SdbA. We showed that SdbA has multiple redox partners, SdbB and CcdA2, forming a complex oxidative protein-folding pathway. This pathway is essential for autolysis, bacteriocin production, genetic competence, and extracellular DNA (eDNA) release in S. gordonii These cellular processes are considered to be important for the success of S. gordonii as a dental plaque organism. This is the first example of an oxidative protein-folding pathway in Gram-positive bacteria that consists of an enzyme that uses multiple redox partners to function.

Effects of lactoferrin and lactoperoxidase-containing food on the oral microbiota of older individuals.

The oral microbiota influences health and disease states. Some gram-negative anaerobic bacteria play important roles in tissue destruction associated with periodontal disease. Lactoferrin (LF) and lactoperoxidase (LPO) are antimicrobial proteins found in saliva; however, their influence on the whole oral microbiota currently remains unknown. In this randomized, double-blinded, placebo-controlled study, the effects of long-term ingestion of LF and LPO-containing tablets on the microbiota of supragingival plaque and tongue coating were assessed. Forty-six older individuals ingested placebo or test tablets after every meal for 8 weeks. The relative abundance of bacterial species was assessed by 16S rRNA gene high-throughput sequencing. Most of the bacterial species in supragingival plaque and tongue coating that exhibited significant decreases in the test group were gram-negative bacteria, including periodontal pathogens. Decreases in the total relative abundance of gram-negative organisms in supragingival plaque and tongue coating correlated with improvements in assessed variables related to oral health, such as oral malodor and plaque accumulation. Furthermore, there was significantly less microbiota diversity in supragingival plaque at 8 weeks in the test group than in the placebo group and low microbiota diversity correlated with improvements in assessed variables related to oral health. These results suggest that LF and LPO-containing tablets promote a shift from a highly diverse and gram-negative-dominated to a gram-positive-dominated community in the microbiota of supragingival plaque and tongue coating. This microbial shift may contribute to improvements in oral health, including oral malodor and state of the gingiva.

An In vitro Study to Compare the Effect of Different Types of Tea with Chlorhexidine on Streptococcusmutans.

Tea is the second most commonly consumed beverage in the world after water. The leaf and bud of the plant Camellia sinensis produces tea. The different forms of tea are 'non-fermented' green tea, 'semi-fermented' oolong tea and 'fermented' black tea according to the manufacturing process. Streptococcus mutans is the main causative organism in dental caries and plaque formation. The present study was undertaken to determine the antibacterial effectiveness of aqueous and ethanol extracts of green tea, black tea and oolong tea against S. mutans in comparison with 0.2% chlorhexidine. An in vitro study was conducted to compare the effectiveness of aqueous and ethanol extracts of green tea, black tea and oolong tea with 0.2% chlorhexidine against S. mutans. Chlorhexidine 0.2% commercially available as mouthwash was used as such for comparison. The antimicrobial activity was determined using agar well diffusion method. About 50 µl of the aqueous and ethanol extracts of tea and 0.2% chlorhexidine were inoculated into the wells prepared on blood agar plates smeared with S. mutans. The agar plates were incubated for 24 hours after which the diameter of the zone of inhibition was measured. Analysis of Variance (ANOVA) followed by Tukey's post hoc test were used for statistical analysis. The mean zone of inhibition of the aqueous extracts of green tea, black tea, oolong tea and chlorhexidine was found to be 16.33 mm, 10.33 mm, 19.66 mm and 22 mm respectively. The mean zone of inhibition of the ethanol extracts of green tea, black tea, oolong tea and chlorhexidine was found to be 14 mm, 9 mm, 20.66 mm and 22 mm respectively. The study result state that the inhibitory effect of chlorhexidine is almost similar to that of oolong tea followed by green tea and black tea. From the present study, it can be concluded that the aqueous and ethanol extracts of oolong tea showed highest antimicrobial activity compared to green tea and black tea.

English

Helicobacter pylori is a spiral gram negative bacterium that normally grows in the stomach, and is the main causal agent of gastritis and peptic ulcers. In 1994, The International Agency for Research on Cancer recognized H. pylori as a carcinogenic factor for stomach cancer. The oral cavity has been proposed as a reservoir for H. pylori and several authors have demonstrated the presence in adult patients of the organism in saliva and dental plaque. However, isolation of H. pylori in children is still questionable. Several investigators have reported the presence of H. pylori in the dental plaque of children, sometimes associated with gastro-esophageal reflux. These isolations has been realized by polymerase chain reaction were gold standard is the culture. On the other hand, many authors have failed to isolate H. pylori by culture in samples obtained from the saliva or dental plaque. We performed an extensive review of the literature to facilitate future research in this controversial topic. Key words: Helicobacter pylori, children, dental plaque, polymerase chain reaction, urease breathe test.

Salivary pH Levels and Caries among Siblings and Parents within Families

High level of caries activity is related to organisms in the dental plaque with high acidogenesis capacity. To test salivary pH in children of the same family and compare it with their caries status. To compare pH levels between children and their parents. To examine the relationship between pH and caries status among children of the same family and their parents. We examined 123 children and adolescents aged 3-18, (73 boys and 50 girls) and 33 adults, (12 men and 21 women), parents of these children. Caries status was examined clinically, using DMF index. Salivary pH measurements were made by a digital pH meter. Among adults, increase in patient age led to increased DMF (p = 0.005). The higher the pH, the lower the DMF (p = 0.037). Among men, DMF was lower by 3 compared to women (p = 0.049). Children's pH correlated with the parents' (p = 0.004). Children's DMF correlated to their pH (p = 0.001). Children's pH was the best predictor of their DMF (R2 = 0.309, p = 0.001). Among children, the higher the pH, the lower the DMFT. Children's pH was the best predictor of their DMF.

A study to assess and compare the peripheral blood neutrophil chemotaxis in smokers and non smokers with healthy periodontium, gingivitis, and chronic periodontitis

Background:Chronic periodontitis is the inflammation within the supporting tissues of the teeth resulting in attachment loss and bone loss. There are certain environmental factors such as smoking that can modify the host response to plaque organisms; hence can account for the aggressive progression of the disease. Smokers show a decreased expression of clinical inflammation even in the presence of abundant plaque accumulation. Neutrophils are the predominant host defense cells which protect the periodontal tissues from plaque organisms, deficiencies of neutrophil function, such as chemotaxis and phagocytosis, often result in increased susceptibility to periodontitis. Smoking can induce alteration in the neutrophil function; therefore, it is of importance to know the changes caused by smoking on neutrophil chemotaxis. This study will provide an essential basis for evaluating the role of nicotine in pathogenesis of periodontal disease by assessing the neutrophil activity.Materials and Methods:A total of 60 smokers and 60 non smokers were examined for this study. Both the groups included 20 subjects with gingivitis, periodontitis, and healthy periodontium. The periodontal status of the study subjects were assessed by gingival index, Russels periodontal index, sulcus bleeding index, and clinical attachment level. The blood sample was taken from each individual for the chemotactic analysis using agarose method.Results:In this study, there was a significant decrease in the neutrophil chemotaxis in smokers with gingivitis, periodontitis, and healthy periodontium, compared to non smokers with similar findings.Conclusion:Delayed neutrophil chemotaxis was found in smokers compared to non smokers with same periodontal status.

Presence of Helicobacter pylori in subgingival plaque of periodontitis patients with and without dyspepsia, detected by polymerase chain reaction and culture

Background:Helicobacter pylori is an important gastrointestinal pathogen that is strongly associated with gastritis as well as peptic ulcer disease. Antimicrobial therapy frequently fails to cure H. pylori infection, which suggests there may be sanctuary sites where the organism resides. This study was aimed to assess the role of oral cavity as a reservoir of H. pylori by evaluating the occurrence of the organism in subgingival plaque of dyspeptic patients by polymerase chain reaction as well as culture.Materials and Methods:Thirty chronic periodontitis patients whose biopsy specimens were found to be H. pylori positive with rapid urease test and histopathologic examination were considered as cases and 20 chronic periodontitis patients who never had any symptoms of gastritis or peptic ulcer were taken as controls. Subgingival plaque samples were collected and sent to microbiological laboratory for detection of H. pylori by 16S rRNA based polymerase chain reaction as well as culture.Results:60% of the samples were found to be positive with polymerase chain reaction in the case group when compared to 15% in the controls. Also, 30% of the cases were found to be positive with culture compared to none in controls.Conclusion:A higher frequency of detection of H. pylori in those patients with positive antral biopsy report was seen. Also, polymerase chain reaction was found to be more sensitive than culture for detection. Thus, we conclude that detection of H. pylori in dental plaque of dyspeptic patients cannot be neglected and might represent a risk factor for recolonization of stomach after systemic eradication therapy.

Detection of Helicobacter Pylori using PCR in dental plaque of patients with and without gastritis

Helicobacter pylori (H.pylori) accounts for gastritis, peptic ulcer and is a probable cause of gastric cancer. Since its detection in the oral cavity, concerns have been raised about dental plaque as a reservoir for reinfection. The aim of this study was to detect the organism in the dental plaque and to determine the association, if any, between H. pylori gastritis and dental plaque contamination causing H.pylori. A polymerase chain reaction-based method was used for detection of H. pylori in clinical specimens. Supra and subgingival samples were collected from 67 patients with chronic periodontitis, 23 of whom were also suffering from gastritis. The data were analyzed with Chi square and Fisher exact test and the statistical significance was set to 0.05. H.pylori was scarce in patients with periodontitis(5.9%). There was a significant association between the presence of H.pylori in the dental plaque and gastritis (p=0.012). Although rarely seen, H.pylori infected dental plaque may be a source for reinfection. It is therefore suggested that professional plaque removal and oral hygiene procedures be performed, along with the antibiotic treatment of H.pylori.

Interaction of Porphyromonas gingivalis with Oral Streptococci Requires a Motif That Resembles the Eukaryotic Nuclear Receptor Box Protein-Protein Interaction Domain

Porphyromonas gingivalis initially colonizes the oral cavity by interacting with organisms in supragingival plaque, such as the oralis group of oral streptococci. This interaction involves the association of the streptococcal antigen I/II with the minor fimbrial antigen (Mfa1) of P. gingivalis. Our previous studies showed that a peptide (BAR) derived from antigen I/II inhibits P. gingivalis adherence and subsequent biofilm formation on streptococcal substrates. In addition, screening a combinatorial peptide library identified select amino acid substitutions in the NITVK active region of BAR that increased the adherence of P. gingivalis to streptococci. Here we report that incorporating these residues in a synthetic peptide results in more-potent inhibition of P. gingivalis adherence and biofilm formation (I(50) [50% inhibition] at 0.52 microM versus I(50) at 1.25 microM for BAR). In addition, a second structural motif in BAR, comprised of the amino acids KKVQDLLKK, was shown to contribute to P. gingivalis adherence to streptococci. Consistent with this, the KKVQDLLKK and NITVK motifs are conserved only in antigen I/II proteins expressed by the oralis group of streptococci, which interact with P. gingivalis. Interestingly, the primary and secondary structures and the functional characteristics of the amphipathic VQDLL core alpha-helix resemble the consensus nuclear receptor (NR) box protein-protein interacting domain sequence (LXXLL) of eukaryotes. BAR peptides containing amino acid substitutions with the potential to disrupt the secondary structure of VQDLL were less-effective inhibitors of P. gingivalis adherence and biofilm formation, suggesting that the alpha-helical character of VQDLL is important. Furthermore, replacing the lysines that flank VQDLL with acidic amino acids also reduced inhibitory activity, suggesting that the association of VQDLL with Mfa1 may be stabilized by a charge clamp. These results indicate that the Mfa1-interacting interface of streptococcal antigen I/II encompasses both the KKVQDLLKK and NITVK motif and suggest that the adherence of P. gingivalis to streptococci is driven by a protein-protein interaction domain that resembles the eukaryotic NR box. Thus, both motifs must be taken into account in designing potential peptidomimetics that target P. gingivalis adherence and biofilm formation.

The microbiological profiles of saliva, supragingival and subgingival plaque and dental caries in adults with and without type 2 diabetes mellitus

The relationships between suspected bacteria in saliva, yeasts in oral rinse, and supragingival and subgingival plaque versus root surface and coronal caries in adults with type 2 diabetes mellitus and a non-diabetic group were explored. One-hundred and five patients with type 2 diabetes and 103 non-diabetic subjects were recruited; their periodontal status, plaque index and magnitude of root surface and coronal caries were assessed. Saliva and an oral rinse were cultured for mutans streptococci, lactobacilli and yeasts. Toothbrush samples of supragingival plaque and curette samples of subgingival plaque were assessed for 17 bacterial species using the checkerboard DNA-DNA hybridization method. Type 2 diabetes patients had significantly more severe periodontitis, a higher plaque index and a higher prevalence and magnitude of root surface caries than non-diabetic subjects. Significantly more diabetic subjects had higher levels of Treponema denticola, Prevotella nigrescens, Streptococcus sanguinis, Streptococcus oralis and Streptococcus intermedius in their supragingival plaque than non-diabetic subjects. No significant difference was found for the organisms in saliva, oral rinse and subgingival plaque between the two groups. After adjustment for diabetic status, root surface caries was associated with an increased count of mutans streptococci, lactobacilli and yeasts in saliva and of Streptococcus mutans in supragingival plaque samples. Coronal caries was only associated with lactobacilli and yeasts in saliva. The number of cariogenic organisms in saliva and oral rinse estimated by culture demonstrated a stronger association with both root surface and coronal caries compared to those 17 species assessed with the checkerboard method in supragingival and subgingival plaque.

Hyperactivity and reactivity of peripheral blood neutrophils in chronic periodontitis

Some evidence exists that peripheral neutrophils from patients with chronic periodontitis generate higher levels of reactive oxygen species (ROS) after Fcgamma-receptor stimulation than those from healthy controls. We hypothesized that peripheral neutrophils in periodontitis also show both hyper-reactivity to plaque organisms and hyperactivity in terms of baseline, unstimulated generation and release of ROS. Peripheral neutrophils from chronic periodontitis patients and age/sex/smoking-matched healthy controls (18 pairs) were assayed for total ROS generation and extracellular ROS release, with and without stimulation (Fcgamma-receptor and Fusobacterium nucleatum), using luminol and isoluminol chemiluminescence. Assays were performed with and without priming with Escherichia coli lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Phox gene expression (p22, p47, p67, gp91) was investigated using reverse transcription-polymerase chain reaction (RT-PCR). Neutrophils from patients produced higher mean levels of ROS in all assays. Total generation and extracellular release of ROS by patients' cells were significantly greater than those from controls after FcgammaR-stimulation, with (P = 0.023) and without (P < or = 0.023) priming with GM-CSF. Differences in unstimulated total ROS generation were not significant. By contrast, patients' cells demonstrated greater baseline, extracellular ROS release than those from controls (P = 0.004). This difference was maintained after priming with LPS (P = 0.028) but not GM-CSF (P = 0.217). Phox gene expression was similar in patient and control cells at baseline and stimulation with F. nucleatum (3 h) consistently reduced gp91(PHOX) transcripts. Our data demonstrate that peripheral neutrophils from periodontitis patients exhibit hyper-reactivity following stimulation (Fcgamma-receptor and F. nucleatum) and hyperactivity in terms of excess ROS release in the absence of exogenous stimulation. This hyperactive/-reactive neutrophil phenotype is not associated with elevated phox gene expression.

Prevalence of periodontal pathogens in dental plaque of children.

Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, and Tannerella forsythensis have been implicated as the main etiological agents of periodontal disease. The purpose of this work was to estimate the prevalence of these organisms in plaque from children without gingivitis (group 1; n = 65) and from those with gingivitis (group 2; n = 53). Extracted DNA from plaque was subjected to two rounds of PCR targeting the 16S rRNA gene using both universal primers and species-specific primers. The results were as follows: group 1, P. gingivalis, 49%; A. actinomycetemcomitans, 55%; and T. forsythensis, 65%; group 2, P. gingivalis, 47%; A. actinomycetemcomitans, 59%; and T. forsythensis, 45%. T. forsythensis was detected more frequently in children with no gingivitis than in those with gingivitis (P = 0.03). There was no significant difference between the two groups with respect to the presence of P. gingivalis or A. actinomycetemcomitans in either group (P > 0.05). Logistic regression analysis revealed that the odds of a patient having gingivitis were 2.3 times greater in the absence of T. forsythensis. In conclusion, the results of this study have shown that the three pathogens can be detected in the dental plaque of healthy children and of those with gingivitis and that T. forsythensis is associated with dental plaque at sites with no gingivitis.

TaqMan real-time polymerase chain reaction assay for the correlation of Treponema denticola numbers with the severity of periodontal disease.

Treponema denticola has been implicated in periodontitis, and the presence of this organism in periodontal pockets has been investigated. However, qualitative analysis is insufficient for the clinical evaluation of periodontal treatments, and quantification of T. denticola populations is essential for monitoring therapeutic efficacy. Therefore, we developed a quantitative method for T. denticola that uses the TaqMan real-time polymerase chain reaction assay. Using this system, we evaluated the relative and absolute numbers of this organism in saliva and subgingival plaque. Furthermore, we analyzed the relationship between the numbers of T. denticola and pocket depth, and found a significant positive correlation (P < 0.0001) between these parameters. This report demonstrates the broad potential of real-time polymerase chain reaction applications in periodontology.

The rag locus of Porphyromonas gingivalis: a novel pathogenicity island.

Previous studies in our laboratories of the serum IgG antibody response of periodontal patients have demonstrated the presence of an immunodominant surface antigen (Mr 55 kDa) in the outer membrane of Porphyromonas gingivalis W50. Genetic analysis of this antigen revealed that the corresponding gene forms part of a small operon which may have arisen via horizontal gene transfer into the genome of this strain. On the basis of sequence homology, the 55 kDa antigen (RagB) and the product of a cotranscribed gene (RagA) may act in concert at the surface of the bacterium to facilitate active transport, mediated through the periplasmic spanning protein, TonB, or form part of a signal transduction system in this organism. The rag locus is present in only a proportion of P. gingivalis laboratory strains and clinical isolates. Analysis of the distribution of ragB in subgingival samples by PCR demonstrated that rag+ P. gingivalis are more frequently detected in deep periodontal pockets than shallow sites in periodontal patients. These findings indicate that the rag genes may influence the virulence potential of P. gingivalis strains which harbour this locus and may thus be considered a novel pathogenicity island. Furthermore, horizontal gene transfer between organisms in subgingival plaque may represent a significant force in the evolution of these bacteria with ramifications for both diagnosis and targeted treatment of periodontal disease.

Inactivation of the gbpA gene of Streptococcus mutans alters structural and functional aspects of plaque biofilm which are compensated by recombination of the gtfB and gtfC genes.

Inactivation of the gbpA gene of Streptococcus mutans increases virulence in a gnotobiotic rat model and also promotes in vivo accumulation of organisms in which gtfB and gtfC have recombined to reduce virulence (K. R. O. Hazlett, S. M. Michalek, and J. A. Banas, Infect. Immun. 66:2180-2185, 1998). These changes in virulence were hypothesized to result from changes in plaque structure. We have utilized an in vitro plaque model to test the hypothesis that the absence of GbpA alters S. mutans plaque structure and that the presence of gtfBC recombinant organisms within a gbpA background restores a wild-type (wt)-like plaque structure. When grown in the presence of sucrose within hydroxyapatite-coated wells, the wt S. mutans plaque consisted primarily of large aggregates which did not completely coat the hydroxyapatite surface, whereas the gbpA mutant plaque consisted of a uniform layer of smaller aggregates which almost entirely coated the hydroxyapatite. If 25% of the gbpA mutants used as inoculum were also gtfBC recombinants (gbpA/25%gtfBC), a wt-like plaque was formed. These changes in plaque structure correlated with differences in susceptibility to ampicillin; gbpA plaque organisms were more susceptible than organisms in either the wt or gbpA/25%gtfBC plaques. These data allow the conclusion that GbpA contributes to S. mutans plaque biofilm development. Since the changes in plaque structure detailed in this report correlate well with previously observed changes in virulence, it seems likely that S. mutans biofilm structure influences virulence. A potential model for this influence, which can account for the gtfBC recombination compensating gbpA inactivation, is that the ratio of glucan to glucan-binding protein is a critical factor in plaque development.

Genetic and physiologic characterization of urease of Actinomyces naeslundii.

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and the ecological balance of oral microbial populations. In this study we cloned and characterized the urease gene cluster of Actinomyces naeslundii, which is one of the pioneer organisms in the oral cavity and a significant constituent of supragingival and subgingival dental plaque in children and adults. An internal fragment of the ureC gene of A. naeslundii WVU45 was initially amplified by PCR with degenerate primers derived from conserved amino acid sequences of the large catalytic subunit of urease in bacteria and plants. The PCR product was then used as a probe to identify recombinant bacteriophages carrying the A. naeslundii urease gene cluster and roughly 30 kbp of flanking DNA. Nucleotide sequence analysis demonstrated that the gene cluster was comprised of seven contiguously arranged open reading frames with significant homologies at the protein and nucleotide sequence levels to the ureABCEFGD genes from other organisms. By using primer extension, a putative transcription initiation site was mapped at 66 bases 5' to the start codon of ureA. A urease-deficient strain was constructed by insertion of a kanamycin resistance determinant within the ureC gene via allelic replacement. In contrast to the wild-type organism, the isogenic mutant was unable to grow in a semidefined medium supplemented with urea as the nitrogen source and was not protected by the addition of urea against killing in moderately acidic environments. These data indicated that urea can be effectively utilized as a nitrogen source by A. naeslundii via a urease-dependent pathway and that ureolysis can protect A. naeslundii against environmental acidification at physiologically relevant pH values. Therefore, urease could confer to A. naeslundii critical selective advantages over nonureolytic organisms in dental plaque, constituting an important determinant of plaque ecology.

Implications of resource-ratio theory for oral microbial ecology.

The ability to compete for the limited nutrients available to the microorganisms of dental plaque is a strong ecological determinant of the structure of the subgingival ecosystem. This paper introduces a new concept from the field of ecology, resource-ratio theory, and applies it to the dynamics of microbial dental plaque with emphasis on the putative periodontal pathogens. Resource-ratio theory is a mechanistic theory of resource competition that utilizes pairs of growth-limiting nutrients in a stoichiometric fashion to predict zones of competitive dominance, exclusion, and coexistence for organisms competing for these resources. Once these resource pairs are identified for plaque organisms, resource-ratio theory may provide predictions of changes in the microbial community structure of plaque based on directional changes in their resource supply ratios.