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.

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.

Life below the gum line: pathogenic mechanisms of Porphyromonas gingivalis.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent in the initiation and progression of severe forms of periodontal disease. An opportunistic pathogen, P. gingivalis can also exist in commensal harmony with the host, with disease episodes ensuing from a shift in the ecological balance within the complex periodontal microenvironment. Colonization of the subgingival region is facilitated by the ability to adhere to available substrates such as adsorbed salivary molecules, matrix proteins, epithelial cells, and bacteria that are already established as a biofilm on tooth and epithelial surfaces. Binding to all of these substrates may be mediated by various regions of P. gingivalis fimbrillin, the structural subunit of the major fimbriae. P. gingivalis is an asaccharolytic organism, with a requirement for hemin (as a source of iron) and peptides for growth. At least three hemagglutinins and five proteinases are produced to satisfy these requirements. The hemagglutinin and proteinase genes contain extensive regions of highly conserved sequences, with posttranslational processing of proteinase gene products contributing to the formation of multimeric surface protein-adhesin complexes. Many of the virulence properties of P. gingivalis appear to be consequent to its adaptations to obtain hemin and peptides. Thus, hemagglutinins participate in adherence interactions with host cells, while proteinases contribute to inactivation of the effector molecules of the immune response and to tissue destruction. In addition to direct assault on the periodontal tissues, P. gingivalis can modulate eucaryotic cell signal transduction pathways, directing its uptake by gingival epithelial cells. Within this privileged site, P. gingivalis can replicate and impinge upon components of the innate host defense. Although a variety of surface molecules stimulate production of cytokines and other participants in the immune response, P. gingivalis may also undertake a stealth role whereby pivotal immune mediators are selectively inactivated. In keeping with its strict metabolic requirements, regulation of gene expression in P. gingivalis can be controlled at the transcriptional level. Finally, although periodontal disease is localized to the tissues surrounding the tooth, evidence is accumulating that infection with P. gingivalis may predispose to more serious systemic conditions such as cardiovascular disease and to delivery of preterm infants.

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.

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.

Adherence and accumulation of oral streptococci.

Oral streptococci adhere to human salivary components and coadhere with specific partner oral bacteria. These interactions may favour the ordered development of plaque communities. The primary sequences of several streptococcal polypeptide adhesins are conserved, indicating that similar colonization mechanisms may have evolved. Critical amino acid changes within binding domains of adhesins might account for species- and site-specific adherence and accumulation.

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.

Cell surface protein receptors in oral streptococci.

Streptococci have a vast repertoire of adherence properties which include binding to human tissue components, epithelial cells and to other bacterial cells. These interactions are determined by the expression of cell-surface receptors some of which are species-specific. In the oral streptococci, two families of surface protein receptors with highly conserved amino acid sequences have been identified. The antigen I/II family of polypeptides are wall-associated high molecular mass proteins (158-166 kDa) with several binding functions that may be attributed to different domains of the receptor molecules. The LraI family of polypeptides are surface-associated lipoproteins (32-33 kDa) involved in adherence of streptococci to salivary glycoprotein pellicle and to oral Actinomyces. A region of amino acid sequence similarity is evident amongst members of the two protein families in Streptococcus gordonii. Ligand-binding specificities of these receptor polypeptides may account for species-specific adherence and site-directed colonization of streptococci within the human oral cavity.

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Ins and outs of antimicrobial resistance: era of the drug pumps.

Over the past five years, concerns have heightened over the escalating numbers of pathogenic micro-organisms isolated that are resistant to many antibiotics and drugs. This phenomenon poses major problems in the treatment of patients with hospital- or community-acquired infections caused by bacteria, fungi, or parasitic organisms. Microbial cells have acquired resistances to specific antibiotics and drugs by mechanisms that include antibiotic inactivation, target alteration, or drug exclusion. More recently, the importance of another mechanism, that of drug expulsion, has been recognized as contributing significantly to antibiotic and drug resistance in microbes. Drug expulsion, mediated by membrane-associated drug efflux pumps, can protect cells from a range of toxic compounds and therefore may confer single-step multidrug resistance. It is imperative that new drugs be designed or discovered that will poison the pumps or bypass the efflux mechanisms.

Interactions of Actinomyces naeslundii strains T14V and ATCC 12104 with saliva, collagen and fibrinogen.

Approximately similar numbers of actinomyces cells adhered to hydroxylapatite beads coated with saliva, collagen or fibrinogen. Adherence generally was unaffected by the presence of free saliva. Binding of cells to collagen- or fibrinogen-coated beads was reduced in the presence of either free collagen or fibrinogen. Glucan inhibited bacterial adherence only to collagen-coated hydroxylapatite beads. It is suggested that actinomyces bind to saliva-, collagen- or fibrinogen-coated surfaces by different mechanisms, but that these mechanisms involve some common bacterial cell-surface components.

Adherence of Streptococcus sanguis.

The presence of adhesins on the cell surface of S. sanguis enables the organism to grow and survive in the oral cavity. Many workers are now actively involved in attempting to characterise these adhesins and the molecular basis for adherence of various streptococci. The complexity of the adhesion processes is underlined by the many varied opinions as to the nature of the molecular components and biochemical interactions that are involved. Understanding the molecular mechanisms involved in bacterial adherence and in dental plaque formation will have significant impact on the prevention and treatment of oral diseases.

The microbiology of periodontal disease.

Periodontal disease result from mixed bacterial infections, in which both host resistance barriers and bacterial interactions are important. Approximately ten bacterial species are strongly implicated with various forms of periodontal disease, although species that cannot yet be cultivated are likely also to be relevant. New technologies have shown that pathogenic bacterial species are present in defined complexes within subgingival plaque, thus identifying specific targets for therapeutic intervention. In light of increasing antibiotic resistance amongst oral bacteria, new strategies for control of periodontal bacterial complexes must be developed that inhibit the bacterial factors necessary for colonization and destruction of host tissues.

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.

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.