Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po. gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu. nucleatum. However, strong co-adhesion between Fu. nucleatum and Po. gulae was able to overcome the inhibitory effects of Fr. canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr. canicola and Po. gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr. canicola and Po. gulae or Fu. nucleatum and Po. gulae were increased several-fold upon the addition of Pa. micra. This suggests that anaerobic cocci such as Pa. micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

Silver doped titanium dioxide nanoparticles as antimicrobial additives to dental polymers.

OBJECTIVE: The objectives of this in vitro study were to produce a filled resin containing Ag-TiO2 filler particles and to test its antibacterial properties. METHODS: Ag-TiO2 particles were manufactured using the ball milling method and incorporated into an epoxy resin using a high speed centrifugal mixer. Using UV/vis spectrophotometry investigations were performed to assess how the photocatalytic properties of the Ag-TiO2 particles are affected when encased in resin. Adopting the bacteria colony counting technique, the antibacterial properties of Ag-TiO2 particles and Ag-TiO2 containing resins were assessed using Streptococcus mutans under varying lighting conditions. RESULTS: Ag doping of TiO2 results in a band gap shift towards the visible spectrum enabling Ag-TiO2 to exhibit photocatalytic properties when exposed to visible light. Small quantities of Ag-TiO2 were able to produce a bactericidal effect when in contact with S. mutans under visible light conditions. When incorporated into the bulk of an epoxy resin, the photocatalytic properties of the Ag-TiO2 particles were significantly reduced. However, a potent bactericidal effect was still achieved against S. mutans. SIGNIFICANCE: Ag-TiO2 filled resin shows promising antimicrobial properties, which could potentially be used clinically.

Interactions between Streptococcus oralis, Actinomyces oris, and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle.

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual-species biofilms, or three-species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non-fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.

Transcriptome analysis of Streptococcus gordonii Challis DL1 indicates a role for the biofilm-associated fruRBA operon in response to Candida albicans.

Multiple levels of interkingdom signaling have been implicated in maintaining the ecological balance between Candida albicans and commensal streptococci to assure a state of oral health. To better understand the molecular mechanisms involved in the initial streptococcal response to the presence of C. albicans that can initiate oral surface colonization and biofilm formation, hypha-forming cells were incubated with Streptococcus gordonii cells for 30 min to assess the streptococcal transcriptome response. A genome-wide microarray analysis and quantitative polymerase chain reaction validation of S. gordonii transcripts identified a number of genes, the majority of which were involved in metabolic functions that were differentially expressed in the presence of hyphae. The fruR, fruB, and fruA genes encoding the transcriptional regulator, fructose-1-phosphate kinase, and fructose-specific permease, respectively, of the phosphoenolpyruvate-dependent fructose phosphotransferase system, were consistently upregulated. An S. gordonii mutant in which these genes were deleted by allelic replacement formed an architecturally distinct, less robust biofilm with C. albicans than did parental strain cells. Complementing the mutant with plasmid borne fruR, fruB, and fruA genes caused phenotype reversion, indicating that the genes in this operon played a role in dual-species biofilm formation. This genome-wide analysis of the S. gordonii transcriptional response to C. albicans has identified several genes that have potential roles in interkingdom signaling and responses.

Collagen-like peptide sequences inhibit bacterial invasion of root dentine.

AIM: To investigate the effects of peptides derived from the sequence of collagen to inhibit penetration of human or bovine dentine by species of streptococci and enterococci. METHODOLOGY: Blocks of human or bovine root dentine were infected for 14 days with bacterial cultures, in the presence or absence of various collagen-like peptide sequences. Invasion of dentinal tubules was determined from microscopic images of histochemically stained dentine thin sections. Extent of invasion was expressed as tubule invasion index (TI), or tubule invasion factor (TIF) which, in addition to the density of invasion, took into account the depth of invasion. Data were analysed by two-way anova. RESULTS: Streptococcus gordonii, Streptococcus mutans and Enterococcus faecalis were associated with heavy invasion (TI >2.5, TIF >4) of human or bovine root dentinal tubules, with E. faecalis being the most penetrative. Incorporation of peptides Gly-Pro-Ala or Gly-Pro-Hyp into the in vitro model system significantly reduced (P < 0.05) dentine invasion by the three species of highly invasive organisms. Inhibition of bacterial invasion by the peptides was dose dependent, and the peptides did not inhibit bacterial growth in culture. CONCLUSION: Specific collagen-like peptide sequences inhibited the invasion of dentine in vitro by a range of oral bacteria. The peptides likely act as competitive inhibitors blocking bacterial collagen receptors and could potentially allow for target-specific control of dentine infections.

Transcriptional landscape of trans-kingdom communication between Candida albicans and Streptococcus gordonii.

Recent studies have shown that the transcriptional landscape of the pleiomorphic fungus Candida albicans is highly dependent upon growth conditions. Here using a dual RNA-seq approach we identified 299 C. albicans and 72 Streptococcus gordonii genes that were either upregulated or downregulated specifically as a result of co-culturing these human oral cavity microorganisms. Seventy-five C. albicans genes involved in responses to chemical stimuli, regulation, homeostasis, protein modification and cell cycle were significantly (P ≤ 0.05) upregulated, whereas 36 genes mainly involved in transport and translation were downregulated. Upregulation of filamentation-associated TEC1 and FGR42 genes, and of ALS1 adhesin gene, concurred with previous evidence that the C. albicans yeast to hypha transition is promoted by S. gordonii. Increased expression of genes required for arginine biosynthesis in C. albicans was potentially indicative of a novel oxidative stress response. The transcriptional response of S. gordonii to C. albicans was less dramatic, with only eight S. gordonii genes significantly (P ≤ 0.05) upregulated at least two-fold (glpK, rplO, celB, rplN, rplB, rpsE, ciaR and gat). The expression patterns suggest that signals from S. gordonii cause a positive filamentation response in C. albicans, whereas S. gordonii appears to be transcriptionally less influenced by C. albicans.

Streptococcus gordonii DL1 adhesin SspB V-region mediates coaggregation via receptor polysaccharide of Actinomyces oris T14V.

Streptococcus gordonii SspA and SspB proteins, members of the antigen I/II (AgI/II) family of Streptococcus adhesins, mediate adherence to cysteine-rich scavenger glycoprotein gp340 and cells of other oral microbial species. In this article we investigated further the mechanism of coaggregation between S. gordonii DL1 and Actinomyces oris T14V. Previous mutational analysis of S. gordonii suggested that SspB was necessary for coaggregation with A. oris T14V. We have confirmed this by showing that Lactococcus lactis surrogate host cells expressing SspB coaggregated with A. oris T14V and PK606 cells, while L. lactis cells expressing SspA did not. Coaggregation occurred independently of expression of A. oris type 1 (FimP) or type 2 (FimA) fimbriae. Polysaccharide was prepared from cells of A. oris T14V and found to contain 1,4-, 4,6- and 3,4-linked glucose, 1,4-linked mannose, and 2,4-linked galactose residues. When immobilized onto plastic wells this polysaccharide supported binding of L. lactis expressing SspB, but not binding of L. lactis expressing other AgI/II family proteins. Purified recombinant NAVP region of SspB, comprising amino acid (aa) residues 41-847, bound A. oris polysaccharide but the C-domain (932-1470 aa residues) did not. A site-directed deletion of 29 aa residues (Δ691-718) close to the predicted binding cleft within the SspB V-region ablated binding of the NAVP region to polysaccharide. These results infer that the V-region head of SspB recognizes an actinomyces polysaccharide ligand, so further characterizing a lectin-like coaggregation mechanism occurring between two important primary colonizers.

Innocent until proven guilty: mechanisms and roles of Streptococcus-Candida interactions in oral health and disease.

Candida albicans and streptococci of the mitis group colonize the oral cavities of the majority of healthy humans. While C. albicans is considered an opportunistic pathogen, streptococci of this group are broadly considered avirulent or even beneficial organisms. However, recent evidence suggests that multi-species biofilms with these organisms may play detrimental roles in host homeostasis and may promote infection. In this review we summarize the literature on molecular interactions between members of this streptococcal group and C. albicans, with emphasis on their potential role in the pathogenesis of opportunistic oral mucosal infections.

The effects of different orthodontic appliances upon microbial communities.

OBJECTIVES: Orthodontic appliances can promote accumulation of dental plaque, with associated enamel decalcification or gingival inflammation. The aim of this study was to examine longer-term microbiological changes during orthodontic treatment with fixed appliances. MATERIALS AND METHODS: Twenty-four orthodontic patients aged 11-14 years undergoing fixed appliance therapy were recruited into the study. Each was randomized for cross-mouth assignment of molar bands and bonded molar tubes to contralateral quadrants of the mouth. All patients received self-ligating brackets, but again using randomization, one upper lateral incisor bracket (left or right) also received an elastomeric ligature. Plaque samples from the molars and upper lateral incisors were obtained at intervals during treatment and up to 1 year after appliance removal. Denaturing gradient gel electrophoresis and 16S rDNA microarray were used to compare plaque microbial fingerprints. RESULTS: Plaque populations changed within 3 months of commencing treatment at all sites. The greatest differences in plaque composition were seen with self-ligating brackets with an elastomeric ligature. Post-treatment plaque associated with both types of molar attachment contained increased levels of periodontal pathogens Porphyromonas gingivalis, Tannerella forsythia, and Eubacterium nodatum, while Campylobacter rectus, Parvimonas micra, and Actinomyces odontolyticus were also elevated with bonds. CONCLUSIONS: The results suggest that orthodontic treatment may cause sustained changes in plaque microbiotas and that molar bond-associated plaque may have raised disease potential.

Differential interactions of Streptococcus gordonii and Staphylococcus aureus with cultured osteoblasts.

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S. gordonii cells had typical features of phagocytosis, similar to S. aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S. gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S. gordonii were enhanced by expression of S. aureus fibronectin-binding protein A (FnBPA) on the S. gordonii cell surface. Lysosomal-associated membrane protein-1 phagosomal membrane marker accumulated with intracellular S. aureus and S. gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12 h, unless expressing FnBPA, whereas S. aureus showed extended survival times (>48 h). Both S. aureus and S. gordonii DL-1 elicited a rapid interleukin-8 response by osteoblasts, whereas S. gordonii FnBPA was slower. Only S. aureus elicited an interleukin-6 response. Hence, S. gordonii invades osteoblasts by a mechanism similar to that exhibited by S. aureus, and elicits a proinflammatory response that may promote bone resorption.

Microbial interactions in building of communities.

Establishment of a community is considered to be essential for microbial growth and survival in the human oral cavity. Biofilm communities have increased resilience to physical forces, antimicrobial agents and nutritional variations. Specific cell-to-cell adherence processes, mediated by adhesin-receptor pairings on respective microbial surfaces, are able to direct community development. These interactions co-localize species in mutually beneficial relationships, such as streptococci, veillonellae, Porphyromonas gingivalis and Candida albicans. In transition from the planktonic mode of growth to a biofilm community, microorganisms undergo major transcriptional and proteomic changes. These occur in response to sensing of diffusible signals, such as autoinducer molecules, and to contact with host tissues or other microbial cells. Underpinning many of these processes are intracellular phosphorylation events that regulate a large number of microbial interactions relevant to community formation and development.

Pneumococcal neuraminidase A: an essential upper airway colonization factor for Streptococcus pneumoniae.

Streptococcus pneumoniae colonizes the upper respiratory tract from where the organisms may disseminate systemically to cause life threatening infections. The mechanisms by which pneumococci colonize epithelia are not understood, but neuraminidase A (NanA) has a major role in promoting growth and survival in the upper respiratory tract. In this article we show that mutants of S. pneumoniae D39 deficient in NanA or neuraminidase B (NanB) are abrogated in adherence to three epithelial cell lines, and to primary nasopharyngeal cells. Adherence levels were partly restored by nanA complementation in trans. Enzymic activity of NanA was shown to be necessary for pneumococcal adherence to epithelial cells, and adherence of the nanA mutant was restored to wild-type level by pre-incubation of epithelial cells with Lactococcus lactis cells expressing NanA. Pneumococcal nanA or nanB mutants were deficient in biofilm formation, while expression of NanA on L. lactis or Streptococcus gordonii promoted biofilm formation by these heterologous host organisms. The results suggest that NanA is an enzymic factor mediating adherence to epithelial cells by decrypting receptors for adhesion, and functions at least in part as an adhesin in biofilm formation. Neuraminidase A thus appears to play multiple temporal roles in pneumococcal infection, from adherence to host tissues, colonization, and community development, to systemic spread and crossing of the blood-brain barrier.

Stick to your gums: mechanisms of oral microbial adherence.

Studies on the adherence properties of oral bacteria have been a major focus in microbiology research for several decades. The ability of bacteria to adhere to the variety of surfaces present in the oral cavity, and to become integrated within the resident microbial communities, confers growth and survival properties. Molecular analyses have revealed several families of Gram-positive bacterial surface proteins, including serine-rich repeat, antigen I/II, and pilus families, that mediate adherence to a variety of salivary and oral bacterial receptors. In Gram-negative bacteria, pili, auto-transporters, and extracellular matrix-binding proteins provide components for host tissue recognition and building of complex microbial communities. Future studies will reveal in greater detail the binding pockets for these adhesin families and their receptors. This information will be crucial for the development of new inhibitors or vaccines that target the functional regions of bacterial proteins that are involved in colonization and pathogenesis.

Characterization of a novel family of fibronectin-binding proteins with M23 peptidase domains from Treponema denticola.

Interactions with fibronectin are important in the virulence strategies of a range of disease-related bacteria. The periodontitis-associated oral spirochaete Treponema denticola expresses at least two fibronectin-binding proteins, designated Msp (major surface protein) and OppA (oligopeptide-binding protein homologue). To identify other T. denticola outer membrane fibronectin-binding proteins, the amino acid sequence of the Treponema pallidum fibronectin-binding protein Tp0155 was used to survey the T. denticola genome. Seven T. denticola genes encoding orthologous proteins were identified. All but two were expressed in Escherichia coli and purified recombinant proteins bound fibronectin. Using antibodies to the N-terminal region of Tp0155, it was demonstrated that T. denticola TDE2318, with highest homology to Tp0155, was cell surface localized. Like Tp0155, the seven T. denticola proteins contained an M23 peptidase domain and four (TDE2318, TDE2753, TDE1738, TDE1297) contained one or two LysM domains. M23 peptidases can degrade peptidoglycan whereas LysM domains recognize carbohydrate polymers. In addition, TDE1738 may act as a bacteriocin based on homology with other bacterial lysins and the presence of an adjacent gene encoding a putative immunity factor. Collectively, these results suggest that T. denticola expresses fibronectin-binding proteins associated with the cell surface that may also have cell wall modifying or lytic functions.

Invasive Streptococcus pneumoniae trigger platelet activation via Toll-like receptor 2.

BACKGROUND: Sepsis is the most common manifestation of invasive pneumococcal disease and is characterized by a severe systemic inflammatory state that leads to circulatory compromise or end organ malperfusion or dysfunction. Patients suffering from sepsis often display low platelet counts characterized by thrombocytopenia as a result of platelet activation. OBJECTIVE: To investigate the mechanism through which platelets become activated in sepsis upon binding to Streptococcus pneumoniae. PATIENTS AND METHODS: We determined S. pneumoniae inducible platelet reactivity using light transmission aggregometry. Dense granule secretion was measured by luminometry using a luciferin/luciferase assay. RESULTS: Streptococcus pneumoniae induced platelet aggregation in a strain-dependent manner. Induction of aggregation was not attributable to capsule serotype, as unencapsulated strains also induced platelet aggregation. Platelet aggregation was not associated with pneumolysin toxin, as a pneumolysin-deficient mutant of S. pneumoniae induced aggregation equally as well as the parent strain. Platelet aggregation also occurred in the absence of plasma proteins or antibody, and was GPIIbIIIa dependent but aspirin independent. Toll-like receptor 2 (TLR2) is present on platelets and acts as a receptor for gram-positive bacterial lipoteichoic acid and peptidoglycan. Inhibition of TLR2 but not TLR4 (also present on platelets) completely abolished platelet aggregation. S. pneumoniae-induced platelet aggregation resulted in activation of the PI3kinase/RAP1 pathway, leading to integrin GPIIbIIIa activation and dense granule release. CONCLUSIONS: Our results demonstrate a novel interaction between S. pneumoniae and TLR2, which results in platelet activation that is likely to contribute to the thrombotic complications of sepsis.

Pneumococcal protein PavA is important for nasopharyngeal carriage and development of sepsis.

Summary The pneumococcal cell surface protein PavA is a virulence factor associated with adherence and invasion in vitro. In this study we show in vivo that PavA is necessary for Streptococcus pneumoniae D39 colonization of the murine upper respiratory tract in a long-term carriage model, with PavA-deficient pneumococci being quickly cleared from nasopharyngeal tissue. In a pneumonia model, pavA mutants were not cleared from the lungs of infected mice and persisted to cause chronic infection, whereas wild-type pneumococci caused systemic infection. Hence, under the experimental conditions, PavA-deficient pneumococci appeared to be unable to seed from lung tissue into blood, although they survived in blood when administered intravenously. In a meningitis model of infection, levels of PavA-deficient pneumococci in blood and brain following intercisternal injection were significantly lower than wild type. Taken collectively these results suggest that PavA is involved in successful colonization of mucosal surfaces and in translocation of pneumococci across host barriers. Pneumococcal sepsis is a major cause of mortality worldwide so identification of factors such as PavA that are necessary for carriage and for translocation from tissue to blood is of clinical and therapeutic importance.

Fluid- or surface-phase human salivary scavenger protein gp340 exposes different bacterial recognition properties.

Salivary scavenger receptor cysteine-rich protein gp340 aggregates streptococci and other bacteria as part of the host innate defense system at mucosal surfaces. In this article, we have investigated the properties of fluid-phase gp340 and hydroxylapatite surface-adsorbed gp340 in aggregation and adherence, respectively, of viridans group streptococci (e.g., Streptococcus gordonii and Streptococcus mutans), non-viridans group streptococci (e.g., Streptococcus pyogenes and Streptococcus suis), and oral Actinomyces. Fluid-phase gp340 and surface-phase gp340 bioforms were differentially recognized by streptococci, which formed three phenotypic groupings according to their modes of interaction with gp340. Group I streptococci were aggregated by and adhered to gp340, and group II streptococci preferentially adhered to surface-bound gp340, while group III streptococci were preferentially aggregated by gp340. Each species of Streptococcus tested was found to contain strains representative of at least two of these gp340 interaction groupings. The gp340 interaction modes I to III and sugar specificities of gp340 binding strains coincided for several species. Many gp340 interactions were sialidase sensitive, and each of the interaction modes (I to III) for S. gordonii was correlated with a variant of sialic acid specificity. Adherence of S. gordonii DL1 (Challis) to surface-bound gp340 was dependent upon expression of the sialic acid binding adhesin Hsa. However, aggregation of cells by fluid-phase gp340 was independent of Hsa and involved SspA and SspB (antigen I/II family) polypeptides. Conversely, both gp340-mediated aggregation and adherence of S. mutans NG8 involved antigen I/II polypeptide. Deletion of the mga virulence regulator gene in S. pyogenes resulted in increased cell aggregation by gp340. These results suggest that salivary gp340 recognizes different bacterial receptors according to whether gp340 is present in the fluid phase or surface bound. This phase-associated differential recognition by gp340 of streptococcal species of different levels of virulence and diverse origins may mediate alternative host responses to commensal or pathogenic bacterial phenotypes.

From tooth to hoof: treponemes in tissue-destructive diseases.

With the advent of new molecular and immunological tools, there is better understanding of the roles that difficult to cultivate bacteria, and not-yet-cultivated bacteria such as spirochaetes, play in polymicrobial diseases. Only relatively recently have studies implicated Treponema spirochaetes in human periodontal disease, a destructive condition of the tissues supporting the teeth. A number of different Treponema species have been isolated and their surface protein components that mediate adhesion, cytotoxicity, and tissue damage have been characterized. More recently Treponema strains closely related to human oral isolates have been cultivated from active lesions of digital dermatitis, an ulcerative condition affecting the feet of cows and sheep. This condition, like periodontal disease, appears to have a polymicrobial aetiology in which enrichment for Treponema may play a crucial part. This article reviews the known mechanisms by which Treponema interact with eukaryotic host cells and tissue proteins, and how these interactions may contribute to pathogenic diversity.

Invasion of dentinal tubules by oral bacteria.

Bacterial invasion of dentinal tubules commonly occurs when dentin is exposed following a breach in the integrity of the overlying enamel or cementum. Bacterial products diffuse through the dentinal tubule toward the pulp and evoke inflammatory changes in the pulpo-dentin complex. These may eliminate the bacterial insult and block the route of infection. Unchecked, invasion results in pulpitis and pulp necrosis, infection of the root canal system, and periapical disease. While several hundred bacterial species are known to inhabit the oral cavity, a relatively small and select group of bacteria is involved in the invasion of dentinal tubules and subsequent infection of the root canal space. Gram-positive organisms dominate the tubule microflora in both carious and non-carious dentin. The relatively high numbers of obligate anaerobes present-such as Eubacterium spp., Propionibacterium spp., Bifidobacterium spp., Peptostreptococcus micros, and Veillonella spp.-suggest that the environment favors growth of these bacteria. Gram-negative obligate anaerobic rods, e.g., Porphyromonas spp., are less frequently recovered. Streptococci are among the most commonly identified bacteria that invade dentin. Recent evidence suggests that streptococci may recognize components present within dentinal tubules, such as collagen type I, which stimulate bacterial adhesion and intra-tubular growth. Specific interactions of other oral bacteria with invading streptococci may then facilitate the invasion of dentin by select bacterial groupings. An understanding the mechanisms involved in dentinal tubule invasion by bacteria should allow for the development of new control strategies, such as inhibitory compounds incorporated into oral health care products or dental materials, which would assist in the practice of endodontics.