Probiotic bacteria affect the composition of salivary pellicle and streptococcal adhesion in vitro.

INTRODUCTION: The use of probiotic bacteria is increasing worldwide and at least some of them can transiently colonize the oral cavity. Several studies have shown that probiotic bacteria, which are often thought of in relation only to intestinal health, can also affect the oral ecology, but the mechanisms for this are largely unknown. The aim of this study was to investigate in vitro if the probiotic bacteria used in commercial products affect the protein composition of the salivary pellicle and the adherence of other oral bacteria. METHODS: Salivary pellicle on hydroxyapatite and the adhesion of two oral streptococci, Streptococcus mutans and Streptococcus gordonii, were used as a model. RESULTS: Probiotic bacteria that bound to saliva-coated hydroxyapatite reduced the adhesion of S. mutans but the inhibitory effect on the adherence of S. gordonii was weaker. Salivary pellicle protein composition was modified by all the strains tested. The modifications in the pellicle affected the adherence of S. mutans but not of S. gordonii. Two of the proteins missing from the pellicles made of saliva-treated with the probiotic bacteria were identified as salivary agglutinin gp340 and salivary peroxidase. All bacterial strains bound salivary agglutinin gp340. The ability of the probiotic bacteria to degrade peroxidase was demonstrated with purified bovine lactoperoxidase and two of the probiotic strains. CONCLUSION: This in vitro study showed that probiotic strains used in commercial products may affect the oral ecology by specifically preventing the adherence of other bacteria and by modifying the protein composition of the salivary pellicle.

Acid production from sugars and sugar alcohols by probiotic lactobacilli and bifidobacteria in vitro.

Some probiotic bacterial strains have been suggested to improve oral health. However, lactobacilli and bifidobacteria are associated with the progression of dental caries. The pH fall caused by 14 probiotic and dairy bacterial strains from glucose, lactose, sucrose, sorbitol and xylitol was followed. All strains used glucose, nine lactose and seven sucrose. Six of the lactobacilli caused a small decrease in pH with sorbitol and two with xylitol. None of the bifidobacteria fermented sugar alcohols. As all the strains could be considered acidogenic, more long-term clinical trials are needed before recommendations for oral health purposes can be made.

Dental caries increments and related factors in children with type 1 diabetes mellitus.

The aim of this study was to analyse possible associations between caries increments and selected caries determinants in children with type 1 diabetes mellitus and their age- and sex-matched non-diabetic controls, over 2 years. A total of 63 (10-15 years old) diabetic and non-diabetic pairs were examined for dental caries, oral hygiene and salivary factors. Salivary flow rates, buffer effect, concentrations of mutans streptococci, lactobacilli, yeasts, total IgA and IgG, protein, albumin, amylase and glucose were analysed. Means of 2-year decayed/missing/filled surface (DMFS) increments were similar in diabetics and their controls. Over the study period, both unstimulated and stimulated salivary flow rates remained significantly lower in diabetic children compared to controls. No differences were observed in the counts of lactobacilli, mutans streptococci or yeast growth during follow-up, whereas salivary IgA, protein and glucose concentrations were higher in diabetics than in controls throughout the 2-year period. Multivariable linear regression analysis showed that children with higher 2-year DMFS increments were older at baseline and had higher salivary glucose concentrations than children with lower 2-year DMFS increments. Likewise, higher 2-year DMFS increments in diabetics versus controls were associated with greater increments in salivary glucose concentrations in diabetics. Higher increments in active caries lesions in diabetics versus controls were associated with greater increments of dental plaque and greater increments of salivary albumin. Our results suggest that, in addition to dental plaque as a common caries risk factor, diabetes-induced changes in salivary glucose and albumin concentrations are indicative of caries development among diabetics.

Kinetics of hypothiocyanite production during peroxidase-catalyzed oxidation of thiocyanate.

We report here a kinetic study of the generation of hypothiocyanite (OSCN-), a product of lactoperoxidase-catalyzed oxidation of thiocyanate ion. Previous studies have measured OSCN- by reactions involving the oxidation of sulfhydryl compounds. Our results show that a more suitable kinetic analysis of OSCN- can be based on absorbance changes measured at 235 nm. About 90% of the oxidation products of SCN- observed at 235 nm were reactive with sulfhydryls and could be reduced with 2-mercaptoethanol. Both thiocyanate and peroxide were rate-limiting and the formation of OSCN- was proportional to the initial concentration of H2O2 until an equimolar concentration of H2O2 and SCN- was reached. This equimolar concentration gave the maximum generation of OSCN-. High concentrations (greater than 100 micrograms/ml) of lactoperoxidase decreased OSCN- generation, but only if the SCN- was added to the enzyme prior to addition of H2O2. With lactoperoxidase concentrations exceeding 1 microgram/ml, the reaction velocity was rapid, but the decay of OSCN- was slow. Free H2O2 in the reaction mixture always resulted in rapid decay of OSCN-. Addition of varying concentrations of peroxide to solutions containing 1 microgram/ml of enzyme and [SCN-] = 5 mM gave a family of hyperbolic A235 vs. time curves. Both the initial slopes and the plateaus of these curves increased linearly with increasing initial peroxide concentrations up to [H2O2] = 0.4 mM, remained relatively constant in the range [H2O2] = 0.4 to 0.8 mM, and decreased rapidly above [H2O2] = 0.8 mM. These results are consistent with the following kinetic model: Hydrogen peroxide reacts rapidly with lactoperoxidase to produce compound I. This compound I oxidizes SCN- to OSCN- and also oxidizes OSCN- to O2SCN-. The OSCN- also reacts with SCN-. The formation of OSCN- is associated with the appearance of an absorbance peak at 225-235 nm. The oxidation of OSCN- by excess peroxide or its decomposition at high concentrations is associated with a decrease in A235 and the appearance of a peak at 245-255 nm. The extinction coefficient for OSCN- was determined to be 1.29 x 10(3) M-1 . cm-1. The second-order rate constant for the oxidation of thiocyanate by compound I was estimated to be 2 x 10(5) M-1 . s-1.

Human milk peroxidase is derived from milk leukocytes.

Peroxidase enzymes present in human colostrum, saliva, polymorphonuclear leukocytes, and bovine milk were compared with respect to their molecular exclusion chromatographic behavior and immunological cross-reactivity. Human milk peroxidase gave an elution profile similar to myeloperoxidase derived from blood polymorphonuclear leukocytes. Human salivary peroxidase reacted with an antibody directed against bovine lactoperoxidase, but with the same antibody preparation no reaction was detected either with human milk peroxidase or leukocyte myeloperoxidase. We conclude that the peroxidase enzyme in human milk is different from the human salivary and the bovine enzymes and is probably derived from milk leukocytes.

Products of thiocyanate peroxidation: properties and reaction mechanisms.

The lactoperoxidase-catalyzed oxidation of thiocyanate (SCN-) was studied in the pH range 3-8. The ultraviolet spectra of the oxidation products, the hypothiocyanite ion, OSCN- (at pH 8) and hypothiocyanous acid, HOSCN (at pH 3), were recorded. The absorbance maxima for OSCN- and HOSCN were observed at 220 and 240 nm, respectively. The extinction coefficients for OSCN- and HOSCN were determined to be 3870 (at 220 nM) and 95 M-1 X cm-1 (at 240 nM), respectively. Pure solutions of OSCN- (at pH 8) and HOSCN (at pH 3) were stable, but the mixtures of these two species at intermediate pH values were unstable. The decomposition could be divided into two periods, an initial period of rapid increase in oxidizing equivalents and a second period of decomposition. Decomposition during the second period followed first-order kinetics, and the pH-dependence of the apparent first-order rate constant was consistent with a decomposition mechanism which involved HOSCN. The first-order rate constant for this step was estimated to be 6 X 10(-3) s-1 at 37 degrees C.

Is thiocyanate peroxidation at equilibrium in vivo?

The peroxidase-catalyzed oxidation of SCN- by H2O2 is an important in vivo reaction because it limits the accumulation of toxic H2O2 and provides significant concentrations of the antimicrobial agents, HOSCN and OSCN-. Data presented in this report suggest that the reaction: (Formula: see text) is in a state of dynamic equilibrium in vivo. Since OSCN- can form the weak acid HOSCN (pKa = 5.3), the equilibrium constant expression (Kox) for thiocyanate peroxidation is dependent on the concentration of hydrogen ions as well as the concentrations of H2O2, SCN-, HOSCN, OSCN- and water, and on the HOSCN ionization constant, Ka: (Formula: see text). The concentration of water is assumed to be constant and unaffected by the other components and is omitted from the Kox equation. The value of Kox was estimated from in vitro data to be 3.7 X 10(3) M-1 (S.D. = 0.8 X 10(3) M-1, n = 8). Using this value for Kox and observations of salivary concentrations of SCN- and HOSCN + OSCN- from several previous reports, the equilibrium concentrations of H2O2 in whole saliva were calculated to range from 8 to 13 microM. This range is consistent with reported estimates of 10 microM as the hydrogen peroxide tolerance limit for human cells.

Sensitive immunometric assays for secretory peroxidase and myeloperoxidase in human saliva.

We have developed specific immunoassays for secretory peroxidase (SP) and for myeloperoxidase (MP) (polymorphonuclear leukocyte-derived peroxidase) in human saliva. Antibodies against SP and MP were produced using bovine milk lactoperoxidase (LP) and human MP as the immunogens, respectively. The methods developed are non-isotopic immunometric assays using biotinylated antibodies and avidin-enzyme conjugate. The detection limit was 0.1 ng/ml and the performance time less than 3 h for both assays. The determination ranges were 0.5-100 ng LP (SP)/ml and 0.5-200 ng MP/ml with intra- and interassay CVs of 4.3% and 15.6% for SP and 3.7% and 10.8% for MP, respectively. The mean analytical recoveries were 108.9% (SP) and 91.5% (MP). These assays correlated well (r = 0.849-0.871) with the colorimetric assays based on the oxidation of thiocyanate or chloride by peroxidases. However, compared to the colorimetric methods the new immunometric assays are much more sensitive and specific for salivary SP and MP. The assays are also more rapid since extensive dialysis to remove endogenous thiocyanate is not required.

Inhibition of herpes simplex virus type 1, respiratory syncytial virus and echovirus type 11 by peroxidase-generated hypothiocyanite.

The human mouth is an important route of viral transmission and evidence exists that human saliva can neutralize some viruses, e.g. herpes simplex type 1 (HSV-1) and human immunodeficiency virus (HIV) in vitro. However, little is known of the actual antiviral agents in saliva. We have analyzed how hypothiocyanite (HOSCN/-OSCN) ions, present in human saliva and generated by salivary peroxidase systems, affect the viability of three different types of viruses; HSV-1 (capable of inducing oral lesions), respiratory syncytial virus (RSV, respiratory infections), and echovirus 11 (EV 11, enteric diseases). Viral suspensions were pretreated (30 min) with HOSCN/-OSCN concentrations up to 180 microM both at pH 6.0 and 7.1 and inoculated into human gingival fibroblasts. The cultures were incubated at 37 degrees C for 18-48 h, fixed and the infected cells were counted after immunoperoxidase staining. HSV-1 was most sensitive to HOSCN/-OSCN with an IC50 of 8.5 microM at pH 6.0 and an IC50 of 20 microM at pH 7.1, respectively. RSV was inhibited by HOSCN/-OSCN only at pH 6.0 with an IC50 of 8.0 microM. EV 11 was also resistant at neutral pH, but sensitive at pH 6.0 with an IC50 of 68 microM. In contrast to HSV-1 and RSV, the inhibition of EV 11 was not dependent on the concentration of HOSCN/-OSCN. The inhibition was in all cases stronger at pH 6.0 than at neutral pH. Our results suggest that hypothiocyanite, a normal component of human whole saliva, in physiological concentrations effectively inhibits HSV-1 and RSV at acidic pH, whereas EV 11 is more resistant in vitro.

Fungicidal effect of human lactoferrin against Candida albicans.

Human lactoferrin (LF) in its iron-free state (apo LF), killed Candida albicans in a time- and dose-dependent way. The lethal effect was stronger at pH 7.0 than at pH 5.5 and maximum inhibition at neutral pH was achieved in 25 min when the fungal cells were exposed to LF in 0.05 mM KCl at 37 degrees C. Fe(3+)-saturated LF had no fungicidal activity. Apo LF-mediated killing was also temperature-dependent with enhanced inhibition at higher temperatures (37 degrees, 42 degrees C). The presence of 1 mM D-glucose did not affect the candidacidal activity of apo LF but both phosphate and bicarbonate ions at physiological salivary concentrations completely blocked the anti-fungal effect. Therefore it seems unlikely that LF belongs to the major host defence factors against oral candidosis.

Colostral proteins from cows immunised with Streptococcus mutans/S. sobrinus support the phagocytosis and killing of mutans streptococci by human leucocytes.

Passive immunisation, based on bovine colostral preparations, is an area of active research. Specific bovine antibodies inhibit the virulence factors of target pathogens but the interactions between whey preparations and human immune defence cells are not well known. Bovine colostrum inhibits the phagocytic activity of bovine leucocytes and this may reflect the biological activity of immunoglobulins in it. Therefore, this study aimed to examine the effects of bovine whey protein preparations from the colostrum of Streptococcus mutans/S. sobrinus-immunised and sham-immunised cows on binding, ingestion and killing of these bacteria by human leucocytes. Binding and ingestion of FITC-labelled bacteria were estimated by flow cytometry and leukocyte activation was measured as chemiluminescence. Killing rate was estimated by plate counting and by measuring bioluminescence from S. mutans- containing the insect luciferase gene. Colostral whey protein preparation from hyperimmunised cows activated human leucocytes by opsonising specific bacteria. Neutrophils, eosinophils and monocytes weakly phagocytosed non-opsonised bacteria and bacteria opsonised with control product. On the contrary, binding and ingestion were efficient in the presence of the preparation from immunised cows. Thus, these results show that bovine colostral whey proteins are able to support the activation of human phagocytes against pathogenic microbes and that this property is related to specific antibodies in whey preparations. These whey proteins may also be clinically useful, especially in preventing the colonisation of newly erupted teeth by mutans streptococci.

The antibacterial action of the various components of the lactoperoxidase system on a cariogenic strain of Streptococcus mutans.

Physiological activity of lactoperoxidase and in vivo concentration of thiocyanate ions were shown to be inhibitory against a cariogenic strain of Streptococcus mutans. However, the amount of H2O2 in vivo may be too low for optimum inhibition by lactoperoxidase system. H2O2 alone also inhibited the growth of S mutants to some degree.

Antibacterial effect of salivary peroxidases on a cariogenic strain of Streptococcus mutans.

The antibacterial effect of purified human salivary lactoperoxidase on a cariogenic strain of Streptococcus mutans was demonstrated while another oral peroxidase, probably of leukocytic origin, did not affect the growth. Lactoperoxidase was rapidly adsorbed by bacterial cells indicating the necessity of the contact between the enzyme and the cells before inhibition.

Concentration of thiocyanate and ionizable iodine in saliva of smokers and nonsmokers.

The concentration of thiocyanate and ionizable iodine was determined with saliva samples from two test groups, smokers (N = 27) and nonsmokers (N = 92). The contents of both ions were also compared as to sex and the amount of cigarettes smoked. The concentrations of the ions studied were dependent on each other-the less iodine, the more thiocyanate in the samples. Females had significantly more iodine and less thiocyanate concentrations than males. Smoking had an increasing effect on the concentration of thiocyanate and a decreasing effect on the content of ionizable iodine in the saliva of the volunteers. The lower amount of iodine ions in the saliva of smokers may restrict the nonthyroidal metabolism of the thyroid hormone in humans.

Peroxidase antimicrobial system of human saliva: hypothiocyanite levels in resting and stimulated saliva.

The antimicrobial oxidizing agent hypothiocyanite ion (OSCN-) was measured in resting (drooling) and stimulated (expectorated) whole saliva. Stimulation of the saliva flow rate resulted in a rapid decrease in OSCN- concentration, whereas the thiocyanate ion (SCN-) concentration and peroxidase activity were increased. The decrease in OSCN- levels was greater than could be accounted for by dilution of the whole saliva volume. Assuming that the antimicrobial activity of the salivary peroxidase system is proportional to OSCN- concentration, this system may be more effective in resting saliva than in stimulated saliva.

Xylitol-induced increase of lactoperoxidase activity.

Whole saliva samples of volunteers who ate a strict diet for two years with regard to the type of sweeteners used (sucrose, fructose, and xylitol) showed considerable differences in the lactoperoxidase activity. The consumption of a xylitol diet increased the activity of this enzyme fourfold to tenfold when compared to the other two test groups. Lactoperoxidase belongs to the natural defense mechanisms of the oral cavity. However, the consumption of a xylitol diet also leads to a strong reduction in the incidence of dental caries. It is suggested that the xylitol-induced elevation of the salivary lactoperoxidase activity and the cariostatic properties of xylitol are partly interrelated phenomena.

Influence of maternal xylitol consumption on acquisition of mutans streptococci by infants.

Xylitol is effective as a non-cariogenic sugar substitute. Habitual xylitol consumption appears to select for mutans streptococci (MS) with impaired adhesion properties, i.e., they shed easily to saliva from plaque. One hundred sixty-nine mother-child pairs participated in a two-year study exploring whether the mothers' xylitol consumption could be used to prevent mother-child transmission of mutans streptococci. All mothers showed high salivary levels of mutans streptococci during pregnancy. The mothers in the xylitol group (n = 106) were requested to chew xylitol-sweetened gum (65% w/w) at least 2 or 3 times a day, starting three months after delivery. In the two control groups, the mothers received either chlorhexidine (n = 30) or fluoride (n = 33) varnish treatments at 6, 12, and 18 months after delivery. The children did not chew gum or receive varnish treatments. MS were assessed from the mothers' saliva at half-year intervals and from the children's plaque at the one- and two-year examinations. The MS were cultured on Mitis salivarius agars containing bacitracin. The salivary MS levels of the mothers remained high and not significantly different among the three study groups throughout the study. At two years of age, 9.7% of the children in the xylitol, 28.6% in the chlorhexidine, and 48.5% in the fluoride varnish group showed a detectable level of MS. In conclusion, therefore, habitual xylitol consumption by mothers was associated with a statistically significant reduction of the probability of mother-child transmission of MS assessed at two years of age. The effect was superior to that obtained with either chlorhexidine or fluoride varnish treatments performed as single applications at six-month intervals.

Longitudinal analysis of the association of human salivary antimicrobial agents with caries increment and cariogenic micro-organisms: a two-year cohort study.

Previous studies of the possible associations of salivary antimicrobial agents with dental caries have given controversial results, obviously mainly because almost all studies have been cross-sectional. Our aim was to find out, in a two-year longitudinal follow-up study, the associations among selected salivary non-immune and immune antimicrobial variables, cariogenic bacteria, and caries increment. The study population was comprised of 63 subjects, all of whom had their 13th birthday during the first study year. In addition to a comprehensive dental examination at baseline and after 2 yrs, paraffin-stimulated whole saliva samples were collected in a standardized way at six-month intervals. Saliva samples were analyzed for flow rate, buffer effect, lysozyme, lactoferrin, total peroxidase activity, hypothiocyanite, thiocyanate, agglutination rate, and total and specific anti-S. mutans IgA and IgG, as well as for numbers of total and mutans streptococci, lactobacilli, and total anaerobic bacteria. Cluster analysis and Spearman-Rank correlation coefficients were used to explore possible associations between and among the studied variables. During the two-year period, a statistically significant increase was observed in flow rate, thiocyanate, agglutination rate, anti-S. mutans IgA antibodies, lactobacilli, and total anaerobes, whereas lysozyme, lactoferrin, and total and anti-S. mutans IgG antibodies declined significantly. Based on various analyses, it can be concluded that, at baseline, total IgG and hypothiocyanite had an inverse relationship with subsequent two-year caries increment, anti-S. mutans IgG antibodies increased with caries development, and mutans streptococci and lactobacilli correlated positively with both baseline caries and caries increment. Total anaerobic microflora was consistently more abundant among caries-free individuals. In spite of the above associations, we conclude that none of the single antimicrobial agents as such has sufficiently strong power to have diagnostic significance in vivo with respect to future caries.

A mouthrinse which optimizes in vivo generation of hypothiocyanite.

We report here the properties of a mouthrinse which enhances one of the natural defense factors in human saliva, the salivary peroxidase system. Concentrations of the antimicrobial agent, the hypothiocyanite (OSCN-) ion, can be increased in vivo to bacteriostatic levels by use of a mouthrinse which is 4 mM (0.014%) in hydrogen peroxide and 1 mM (0.0097%) in potassium thiocyanate at pH 5.5. The volume of the rinse, the H2O2 concentrations, and the pH were shown to be determinants of the concentration of OSCN- generated by the rinse.

Salivary defense factors in herpes simplex virus infection.

Saliva may contribute to a lowering of the infectious herpes simplex virus (HSV) dose during transmission and consequently abrogate infection or lead to decreased reactivation. To test this hypothesis, we assayed saliva for innate defense factors, immunoglobulin content, and the capacity to interfere with HSV infection. Serum or salivary anti-HSV IgG levels did not correlate with control of recurrent labial herpes (RLH) and were significantly higher in subjects with RLH compared with asymptomatic seropositive subjects. Although no differences in levels or output rate of innate defense factors between the groups were observed, the salivary neutralizing activity correlated with lactoferrin and hypothiocyanite concentrations in the asymptomatic seropositive group. Our results suggest that saliva contains factors, in addition to anti-HSV immunoglobulins, that neutralize HSV and may indirectly contribute to the control of RLH.