Streptococci and Actinomyces induce antibodies which cross react with epithelial antigens in periodontitis.

Perturbation of epithelial structure is a prominent but poorly understood feature of the immunopathological response to bacterial antigens which characterizes the destructive lesion of periodontitis. Western analysis of sera from 22 patients with periodontitis detected multiple antigens in extracts of epithelial cells whereas sera from 12 periodontally healthy subjects displayed only trace reaction with epithelial antigens. To investigate a possible relationship between the bacterial flora adjacent to diseased sites and the presence of antibodies reactive with epithelium, subgingival plaque samples were taken from deep periodontal pockets and cultured anaerobically. Gram positive bacteria containing antigens cross-reactive with epithelial cells were reproducibly isolated by probing membrane colony-lifts with affinity-isolated (epithelium-specific) antibodies and identified by 16S rDNA sequence homology as streptococci (S. mitis, S. constellatus and two S. intermedius strains) and Actinomyces (A. georgiae, and A. sp. oral clone). Conversely, when serum from patients with periodontitis was absorbed with the captured bacterial species the number of epithelial antigens recognized was specifically reduced. It was concluded that development of cross-reactive antibodies related to these organisms may contribute to perturbation of the epithelial attachment to the tooth and the progression of periodontitis. These autoreactive antibodies could also be a contributing factor in other diseases affecting epithelia.

pH-regulated secretion of a glyceraldehyde-3-phosphate dehydrogenase from Streptococcus gordonii FSS2: purification, characterization, and cloning of the gene encoding this enzyme.

Streptococcus gordonii and other viridans streptococci (VS) are primary etiologic agents of infective endocarditis, despite being part of the normal oral microflora. Recently, a surface-bound glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been found on the cells of all tested streptococcal species, where it has been implicated as a virulence factor. In contrast, we observed that a soluble extracellular GAPDH was the major secreted protein from S. gordonii FSS2, an endocarditis strain. The biochemical properties and gene sequence of S. gordonii GAPDH are almost identical to those of other streptococcal GAPDHs. Growth at defined pHs showed that secretion of GAPDH is regulated by environmental pH. GAPDH was primarily surface-associated at growth pH 6.5 and shifted to > 90% secreted at growth pH 7.5. Others have identified S. gordonii promoters that are up-regulated by a pH shift similar to that experienced by organisms entering the blood stream (neutral) from the oral cavity (slightly acid). Analysis of our results suggests that secretion of GAPDH may be a similar adaptation by S. gordonii.

In vitro adhesion and platelet aggregation properties of bacteremia-associated lactobacilli.

Eight bacteremia-associated Lactobacillus strains were evaluated in vitro for the ability to adhere to human intestinal mucosa and to aggregate platelets. Adherence varied significantly among the strains, and platelet aggregation was induced by three strains. In conclusion, strong binding ability does not appear to be a prerequisite for the involvement of lactobacilli in bacteremia or to their ability to aggregate platelets.

Candida-associated denture stomatitis. Aetiology and management: a review. Part 3. Treatment of oral candidosis.

Treatment of oral candidosis with topical antifungal agents such as nystatin and amphotericin B is effective initially. However, medication can produce side effects in some patients and when therapy is stopped the condition can recur. Alternative treatment involving the use of antiseptics and disinfecting agents has been shown to play an important role in the control of dental plaque. The use of sodium hypochlorite as an overnight denture soak has been shown to eliminate denture plaque and recent investigations have demonstrated that microwave irradiation of dentures at a specified setting and exposure time is bactericidal and candidacidal.

Effectiveness of two methods of denture sterilization.

Species of Candida and in particular Candida albicans may be involved in the aetiology of denture stomatitis. Studies have shown that Candida and other oral micro-organisms including Streptococcus gordonii are associated with denture plaque; hence denture hygiene is an important factor in the prevention and treatment of the disease. The aim of this investigation was to test in vitro the efficacy of two methods of denture sterilization: (1) microwave irradiation and (2) sodium hypochlorite soak. Twenty upper acrylic dentures were prepared for microbiological assay; 10 were inoculated with C. albicans H1 and 10 with S. gordonii LGR2. Within each group, five dentures were tested in a domestic microwave oven for optimal exposure time and temperature to ensure sterilization; the five control dentures were not microwaved. Microbiological analyses showed that the inoculated dentures became sterile after six min of irradiation at medium setting (2450 MHz, 350 W). Damage to the microorganisms after microwave irradiation was clearly visible by scanning electron microscopy (SEM). Following the same protocol as above, experimental dentures were soaked for 8 h in either 0.02%, or 0.0125% sodium hypochlorite solution and control dentures soaked in distilled water. Microbiological analyses showed that the experimental dentures inoculated with C. albicans H1 became sterile. By contrast, those inoculated with S. gordonii LGR2 did not become sterile, and the SEM procedures confirmed these findings. The results of this study indicate that microwaving may be a more effective method of denture sterilization than denture soaking in sodium hypochlorite. However, compared with microwaving, hypochlorite reduces the levels of residual non-viable micro-organisms attached to the denture surface.

Candida-associated denture stomatitis. Aetiology and management: a review. Part 2. Oral diseases caused by Candida species.

Certain systemic conditions and/or defects in the immune system may predispose the host to oral candidal infection and the commonest form of oral candidosis is candida-associated denture stomatitis. Until recently there has been controversy concerning the aetiology of the disease. Although some earlier investigators linked denture stomatitis with trauma or bacterial infection, others had isolated Candida albicans from the mouths of patients with the condition. Current studies indicate that denture stomatitis lesions are associated with the detection of candida species while other factors such as denture hygiene, trauma, systemic diseases and deficiencies of the immune system may be involved.

Candida-associated denture stomatitis. Aetiology and management: a review. Part 1. Factors influencing distribution of Candida species in the oral cavity.

Candida species are yeasts and within the oral cavity, Candida albicans is the most frequently isolated. There is clear evidence that C. albicans adheres to oral surfaces including acrylic dentures and mucosa. The mechanisms of attachment differ, with candidal adhesion to inert surfaces under the control of hydrophobic and electrostatic forces and adhesion to mucosa dependent on a number of complex ligand-recognition systems. Other factors within the oral environment such as saliva, pH, bacteria and hyphal formation have been shown to influence adhesion of candida species to surfaces in the mouth.

Interactions between Candida species and platelets.

Candida spp. are able to cause disseminated disease in immunocompromised patients. This study examined the interactions of Candida spp. with platelets, complement and polymorphonuclear leucocytes (PMNLs). With the exception of C. albicans, all other Candida spp., including a C. albicans strain previously classified as C. stellatoidia, aggregated human platelets at a ratio of yeast cells: platelets of 1:80. Usually, those species and strains that aggregated platelets were either killed or prevented from growing in platelet-rich plasma indicating that the aggregation released microbicidal or microbistatic substances that were active against Candida spp. All Candida spp. were resistant to attack by complement in 50% serum. However, all species activated complement as determined by the presence of C3 fragments on their surface, in particular a 195-kDa fragment corresponding to C3c, two fragments at 67 and 40 kDa corresponding to iC3b, and a 33-kDa fragment corresponding to C3d. When strains were tested for their ability to stimulate the release of pro-inflammatory substances from platelets and PMNLs, it was found that most strains stimulated PMNLs to release interleukin(IL)-8 but not IL-1beta or leucotriene B4. The ability of C. albicans to evade complement-mediated killing and not to aggregate platelets may contribute to the survival of this species in the blood during vascular infections.

The effect of sodium hypochlorite on potential pathogenic traits of Candida albicans and other Candida species.

Strains of Candida albicans, Candida krusei, Candida kefyr, Candida tropicalis, Candida parapsilosis and Candida guilliermondii were grown in the presence or absence of concentrations of sodium hypochlorite below the minimal inhibitory concentration and tested for a range of characteristics that may be associated with pathogenicity. Sodium hypochlorite is used routinely in hospitals in Australia for disinfection procedures, and these experiments were designed to assess the efficacy of hypochlorite as a sterilizing agent for acrylic dentures. Candida showed varying abilities to adhere to surfaces that may be present in the oral cavity. Sodium hypochlorite reduced the adhesion of all C. albicans strains and most other Candida species to both polystyrene and buccal epithelial cells. A biofilm of Streptococcus gordonii reduced the adhesion of most C. albicans strains and most other Candida species to polystyrene. However, Candida species were able to coaggregate with S. gordonii in suspension, with one strain of C. albicans, GDH 2346, showing greater coaggregating ability than the other strains or species. Sodium hypochlorite increased coaggregation of all C. albicans strains and most other Candida species. Examination of cell wall proteins from strains of C. albicans and a strain of C. parapsilosis showed that growth in hypochlorite increased the amount of protein in some existing bands and, in one strain of C. albicans, increased the number of detectable protein bands ranging from 56 to 26 kDa. Only 4 strains of C. albicans were able to produce hyphae, and 3 of these same strains and C. parapsilosis were able to produce proteinase. Hypochlorite increased the rate of blastospore to hyphal transition but had no effect on proteinase production or activity. It is concluded that hypochlorite at a concentration below the minimal inhibitory concentration may function as an anti-adhesin for Candida species but may not affect their more pathogenic characteristics.

Modulation of glycosidase and protease activities by chemostat growth conditions in an endocarditis strain of Streptococcus sanguis.

The effects of growth conditions on the properties of the endocarditis-producing oral bacterium Streptococcus sanguis FSS2 were studied. This strain produces a variety of proteases and glycosidases, including a thrombin-like activity that is a potential virulence factor for endocarditis. Cultures were grown with limiting glucose or galactose in chemostats over a range of dilution rates and pH levels, and the following activities were measured at pH 7.5: thrombin-like, Hageman factor-like, N-acetyl-beta-D-galactosaminidase, beta-D-glucosidase, and beta-D-galactosidase. At growth pH 6.5, specific activities generally decreased as the dilution rate increased from 0.05 to 0.40 h(-1). At a dilution rate of 0.1 h(-1), specific activities generally were highest at growth pH 6.5 and lower and approximately equal at growth pH 5.5 and 7.5. The major exception was the thrombin-like activity, for which the specific activity at growth pH 7.5 was approximately 5-fold higher than at growth pH 5.5. Hageman factor-like activity was apparently glucose catabolite repressible, as its activity was 3-fold higher in galactose cultures. The measured activities changed as functions of growth conditions and thus were modulated by environment. Environmental regulation of thrombin-like activity by pH is consistent with an activity that is less important on tooth surfaces than in tissues.

Phospholipids of Lactobacillus spp.

Lactobacillus phospholipid molecular species were analyzed by mass spectrometry. Prominent anions were consistent with presence of the phosphatidylglycerols PG(37:2), PG(36:2), PG(35:1), PG(34:1), and PG(33:1). Diglycosyldiacylglycerol molecular species were also observed, although nitrogen-containing phospholipids were absent. An anion of m/z 759 was derived from an apparently novel type of lipid.

Fibronectin binding by Streptococcus milleri group strains and partial characterisation of the fibronectin receptor of Streptococcus anginosus F4.

The Streptococcus milleri group were shown to bind fibronectin (Fn) to their cell-surface and this binding increased the adhesion of cells to hydroxyapatite. The binding of Fn to Streptococcus anginosus F4 was studied in more detail. Fn binding to bacterial cells increased the association of the bacteria with the polymorphonuclear leukocytes obtained from the peritoneal cavity of rats but did not increase killing of the bacteria. The cell-surface receptor was a protein of M(r) 14,000 which was released from cells after mutanolysin digestion. The binding was specific, with cells having a maximum number of binding sites per cell of 770. Electron microscopy, using gold-labelled Fn, localised the receptor to areas between daughter cells.

Enzyme production by lactobacilli and the potential link with infective endocarditis.

Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei, both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris. Commonly expressed glycosidase activities were alpha-D-galactosidase and beta-N-acetyl-D-glucosaminidase followed by beta-D-glucosidase and alpha-L-fucosidase. The combined production of beta-N-acetyl-D-glucosaminidase and alpha-D-galactosidase was a feature of the endocarditis isolates. In contrast, beta-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris. The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathogenic potential of lactobacilli.

Lactobacilli are often considered to be commensal or beneficial participants in human microbial ecology and considerable research is being carried out into the effects of the use of lactobacilli as additives in both human and animal diets. However, lactobacilli also cause some human diseases (e.g. dental caries, rheumatic vascular disease, septicaemia and infective endocarditis (IE)), and have recently been identified as potential emerging pathogens in elderly and immunocompromised patients, particularly those receiving broad spectrum antibiotic therapy. The identification of potential pathogenic traits amongst lactobacilli will therefore facilitate the use of the organisms for probiotic purposes. The ability to aggregate human platelets is considered to be a possible pathogenic trait in the progression of IE. A comparison of bacterial cell surface properties amongst L. rhamnosus strains showed that platelets were aggregated by 5/5 IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains the respective numbers were 2/5 and 2/9. However two strains, morphological mutants of a non-aggregating strain, which had been re-isolated after passaging through rats were found to aggregate platelets. No loss of aggregating function occurred on extensive subculturing of IE strains. Aggregation also occurred with 11/14 strains for five other species, namely, Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salvivarius, with each species being represented indicating that the property is not uncommon in the genus. A comparison of IE and oral isolates of L. rhamnosus and L. paracasei subsp. paracasei and seven other Lactobacillus species, has shown that the binding of both fibronectin and fibrinogen by lactobacilli is greatly increased, up to 50 fold, when the pH is reduced from 7.0 to 5.0. Re-exposing the lactobacilli to a neutral pH environment releases most of the bound proteins, but the amount still remaining bound to the cell is several times more than is bound at neutral pH. Lactobacilli will also bind to the proteins that make up the extracellular matrix of endothelial cells. Lactobacilli bound significantly better to collagen types I and V than to types III and IV (p < 0.01). Further, strains isolated from IE cases, particularly L. rhamnosus strains, bound significantly better to types I and V than did 'normal' strains (p < 0.02). Type V collagen has been demonstrated at the sites of endothelial damage. Thus the binding of lactobacilli, particularly L. rhamnosus to these collagen types may be of importance in the early stages of colonization of the damaged heart valve.(ABSTRACT TRUNCATED AT 400 WORDS)

Lancefield group C Streptococcus milleri group strains aggregate human platelets.

Lancefield group C Streptococcus milleri group (SMG) strains are the only SMG types that are able to aggregate human platelets. Complete aggregation occurred within 10 min of mixing bacterial cells and platelets together in the ratio 8:1. Substances which (i) chelated cations; (ii) inhibited the cycloxygenase pathway in platelets; (iii) reduced the availability of ADP and disrupted platelet membrane stability; (iv) reduced bacterial aggregation of platelets. The platelet-interacting substance on the surface of the SMG appeared to be proteinacious as digestion of bacterial cells with protease inhibited aggregation whereas treatment with lipase, periodate or antisera to Lancefield group C polysaccharide had no effect.

The aggregation of human platelets by Lactobacillus species.

The ability to aggregate human platelets was examined for five Lactobacillus rhamnosus strains and five Lactobacillus paracasei subsp. paracasei strains isolated from patients with infective endocarditis (IE), 25 laboratory isolates from the same two species, and 14 strains from five other oral species, namely Lactobacillus acidophilus, Lactobacillus fermentum, Lactobacillus oris, Lactobacillus plantarum and Lactobacillus salivarius. Amongst the L. rhamnosus strains, platelets were aggregated by all five IE strains and 8/16 laboratory strains. For the L. paracasei subsp. paracasei strains, the respective numbers were 2/5 and 2/9. Aggregation also occurred with 11/14 strains of the other five species; each species was represented. The optimal ratio of bacteria to platelets for aggregation was approximately 1:1, and there was considerable variation in the lag phase that preceded aggregation, depending on the source of the platelets. Overall, the lag phase varied between 0.25 +/- 0.1 and 20.4 +/- 3.2 min and the percentage aggregation ranged between 70 +/- 2.6 and 104 +/- 13.5%. Confirmation that aggregation was being observed came from studies with five strains on the inhibitory effects of EDTA, dipyridamole, apyrase, imipramine, acetylsalicylic acid and quinacrine. Inhibition of aggregation by L. rhamnosus strains by the peptide arginine-glycine-aspartic acid-serine (RGDS) further indicated a role for fibronectin and/or fibrinogen. Pronase treatment of cells for 1 h and extraction of bacterial surface components with 0.1 M-Tris/HCl (pH 8.5) at 37 degrees C for 1 h stopped aggregation in 8/9 IE strains. Extracted surface proteins (200 micrograms) completely inhibited platelet aggregation by 8/9 of the homologous strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Coaggregation of oral lactobacilli with streptococci from the oral cavity.

The ability of oral lactobacilli to coaggregate with streptococci and actinomycetes was investigated. Of the 7 species of lactobacilli studied, only two were capable of coaggregation and the coaggregation was restricted to streptococci. Lactobacillus salivarius strains (2/4) coaggregated with Streptococcus salivarius, Streptococcus gordonii, Streptococcus crista and tufted Streptococcus sanguis II strains. Lactobacillus fermentum (2/3) coaggregated with S. gordonii and S. sanguis. The coaggregation between L. salivarius and S. salivarius, S. gordonii or tufted S. sanguis II strains was mediated by a protein on the surface of the lactobacilli and was not inhibited by lactose. The coaggregation between L. fermentum and the streptococci was mediated by protein on the surface of the streptococci and was inhibited by lactose.

Serum esterase activities and hyperlipidaemia in the streptozotocin-diabetic rat.

The levels of activity of four serum esterases were measured in control and streptozotocin-diabetic rats for a period of 6 months. Pseudocholinesterase activity was significantly elevated in the diabetic rats at all time points tested, reaching 250% of the control activity at 6 months. Levels of paraoxonase activity progressively decreased with time in the diabetic rats, being 36% lower than in controls at 6 months. No significant differences in either serum arylesterase or carboxylesterase activity between control and diabetic rats were observed.

Expression of the surface properties of the fibrillar Streptococcus salivarius HB and its adhesion deficient mutants grown in continuous culture under glucose limitation.

Streptococcus salivarius HB and four adhesion deficient mutants, HB-7, HB-V5, HB-V51 and HB-B, were grown in continuous culture in a defined medium under glucose limitation over a range of growth rates from 0.1 to 1.1 h-1. The ability to coaggregate with Veillonella parvula V1 cells and the ability to adhere to buccal epithelial cells did not alter with increasing growth rate. Cell surface hydrophobicity decreased markedly with increasing growth rate for the non-fibrillar non-adhesive mutant HB-B but not for the other four strains which all carry different combinations of fibril classes. The thickness of the ruthenium red staining layer (RRL) also varied with growth rate for strain HB-B, ranging from 19.5 +/- 3.8 nm at high growth rate to a minimum of 12.3 +/- 4.8 nm at low growth rate. Low cell surface hydrophobicity correlated with a thicker RRL for strain HB-B. Strains HB-V5 and HB-7 also showed a significant increase in RRL thickness at high growth rates although to a lesser degree than HB-B. SDS-PAGE revealed a large number of protein bands common to all strains at all growth rates, with the major common protein occurring at 15.6 kDa. Protein bands at 70, 56, 40.5 and 39 kDa appeared stronger at high growth rates than at low. A protein band at 82 kDa showed strongly only at low growth rates. Therefore, adhesion and coaggregation are not phenotypically variable with increasing growth rate but RRL thickness, hydrophobicity and cell surface proteins may be phenotypically variable depending on the strain.

Ruthenium red staining reveals surface fibrils and a layer external to the cell wall in Streptococcus salivarius HB and adhesion deficient mutants.

Ruthenium red staining revealed both the long and short classes of cell surface fibril in thin sections of Streptococcus salivarius HB, indicating that the fibrils contained polyanionic polymers, probably polysaccharides. Also visible was a 16.2 +/- 2.2 nm thick ruthenium red staining layer (RRL) outside the 16.7 +/- 2.2 nm thick cell wall. The fibrils could not be seen after conventional glutaraldehyde and osmium fixation. The RRL was protease resistant and was not involved in septum formation. Loss of the fibrils after protease treatment coincided with a decrease of 54% in cell surface hydrophobicity, indicating that cell surface hydrophobicity was due partly to fibrils and partly to the RRL. There was no correlation between the lengths of fibrils as measured on whole cells after negative staining and on thin sections of ruthenium red stained cells. The thickness of the RRL was the same in three adhesion deficient mutants--strains HB-7, HB-V5 and HB-V51--with various fibril lengths. However, a completely bald mutant, HB-B, had a significantly thicker RRL than S. salivarius HB, although it was unable to adhere to buccal epithelial cells, and it could not co-aggregate with Veillonella parvula V1. The RRL therefore did not contain adhesins.