Fluoride, triclosan and organic weak acids as modulators of the arginine deiminase system in biofilms and suspension cells of oral streptococci.

INTRODUCTION: The arginine deiminase system (ADS) of oral bacteria is a major generator of alkali (ammonia) in dental plaque and is considered to have anticaries effects. However, many of the antimicrobial agents used in oral care products may reduce alkali production by the ADS. The objective of our work was to assess the sensitivity of the ADS of oral streptococci to commonly used antimicrobials, fluoride, triclosan and organic weak acids. METHODS: Streptococcus sanguinis NCTC 10904 and Streptococcus ratti FA-1 were grown in suspension cultures and mono-organism biofilms. ADS activity at pH values of 4, 5 and 6 was assessed, and the actions of the agents was determined in terms of reduced production of alkali from arginine, inhibition of ADS enzymes and changes in uptake of arginine. RESULTS: ADS activity was not greatly affected by pH changes between 4 and 6 and was greater per unit of biomass for cell suspensions than for biofilms. NaF was a poor inhibitor, while triclosan was highly effective with a 50% inhibitory dose for the two organisms between 0.03 and 0.05 and between 0.10 and 0.15 mm-h for suspension cells and biofilms, respectively. The weak acid indomethacin was nearly as potent at pH 4.0 as triclosan, while capric and lauric acids were less potent, especially for biofilms. The methyl ester of lauric acid was slightly stimulatory. The major targets for the inhibitors appeared to be transport systems for arginine uptake, although carbamate kinase was a secondary target. CONCLUSION: Triclosan, indomethacin, caprate and laurate can reduce ADS activity in dental plaque.

The influence of a novel propolis on mutans streptococci biofilms and caries development in rats.

A flavonoids-free Brazilian propolis (type 6) showed biological effects against mutans streptococci and inhibited the activity of glucosyltransferases. This study evaluated the influence of the ethanolic extract of a novel type of propolis (EEP) and its purified hexane fraction (EEH) on mutans streptococci biofilms and the development of dental caries in rats. The chemical composition of the propolis extracts were examined by gas chromatography/mass spectrometry. The effects of EEP and EEH on Streptococcus mutans UA159 and Streptococcus sobrinus 6715 biofilms were analysed by time-kill and glycolytic pH drop assays. Their influence on proton-translocating F-ATPase activity was also tested. In the animal study, the rats were infected with S. sobrinus 6715 and fed with cariogenic diet 2000. The rats were treated topically twice a day with each of the extracts (or control) for 5 weeks. After the experimental period, the microbial composition of their dental plaque and their caries scores were determined. The results showed that fatty acids (oleic, palmitic, linoleic and stearic) were the main compounds identified in EEP and EEH. These extracts did not show major effects on the viability of mutans streptococci biofilms. However, EEP and EEH significantly reduced acid production by the biofilms and also inhibited the activity of F-ATPase (60-65%). Furthermore, both extracts significantly reduced the incidence of smooth surface caries in vivo without displaying a reduction of the percentage of S. sobriuns in the animals' plaque (P < 0.05). However, only EEH was able to reduce the incidence and severity of sulcal surface caries (P < 0.05). The data suggest that the cariostatic properties of propolis type 6 are related to its effect on acid production and acid tolerance of cariogenic streptococci; the biological activities may be attributed to its high content of fatty acids.

Mineralization and responses of bacterial spores to heat and oxidative agents.

Mineralization of bacterial spores with Ca2+ and a variety of other mineral cations enhances resistance to heat damage. Part of the enhancement is associated with increased dehydration of the mineralized protoplast or spore core, while part is independent of dehydration and effective for resistance even to dry heat. Spore mineralization was found also to enhance resistance to oxidative damage caused by agents such as tertiary butyl hydroperoxide or H2O2. In contrast, mineral cations in the environment increased oxidative damage, presumably by catalyzing radical formation. Metal ion chelators such as o-phenanthroline protected spores against such damage.

Elastic, flexible peptidoglycan and bacterial cell wall properties.

The peptidoglycan sacculus serves as a mechanical framework for the cell walls of most eubacteria and largely determines cell shape. The notion that the structure is a rigid shell is contradicted by findings that peptidoglycan can expand and contract. Thus, the sacculus functions as an elastic, flexible, polyionic, amphoteric, restraining network.

Growth of Streptococcus faecalis under high hydrostatic pressure and high partial pressures of inert gases.

Growth of Streptococcus faecalis in a complex medium was inhibited by xenon, nitrous oxide, argon, and nitrogen at gas pressures of 41 atm or less. The order of inhibitory potency was: xenon and nitrous oxide > argon > nitrogen. Helium appeared to be impotent. Oxygen also inhibited streptococcal growth and it acted synergistically with narcotic gases. Growth was slowed somewhat by 41 atm hydrostatic pressure in the absence of narcotic gases, but the gas effects were greater than those due to pressure. In relation to the sensitivity of this bacterium to pressure, we found that the volume of cultures increased during growth in a volumeter or dilatometer, and that this dilatation was due mainly to glycolysis. A volume increase of 20.3 +/- 3.6 ml/mole of lactic acid produced was measured, and this value was close to one of 24 ml/mole lactic acid given for muscle glycolysis, and interestingly, close to the theoretic volume increase of activation calculated from the depression of growth rate by pressure.

Functional profiling in Streptococcus mutans: construction and examination of a genomic collection of gene deletion mutants.

A collection of tagged deletion mutant strains was created in Streptococcus mutans UA159 to facilitate investigation of the aciduric capability of this oral pathogen. Gene-specific barcoded deletions were attempted in 1432 open reading frames (representing 73% of the genome), and resulted in the isolation of 1112 strains (56% coverage) carrying deletions in distinct non-essential genes. As S. mutans virulence is predicated upon the ability of the organism to survive an acidic pH environment, form biofilms on tooth surfaces, and out-compete other oral microflora, we assayed individual mutant strains for the relative fitness of the deletion strain, compared with the parent strain, under acidic and oxidative stress conditions, as well as for their ability to form biofilms in glucose- or sucrose-containing medium. Our studies revealed a total of 51 deletion strains with defects in both aciduricity and biofilm formation. We have also identified 49 strains whose gene deletion confers sensitivity to oxidative damage and deficiencies in biofilm formation. We demonstrate the ability to examine competitive fitness of mutant organisms using the barcode tags incorporated into each deletion strain to examine the representation of a particular strain in a population. Co-cultures of deletion strains were grown either in vitro in a chemostat to steady-state values of pH 7 and pH 5 or in vivo in an animal model for oral infection. Taken together, these data represent a mechanism for assessing the virulence capacity of this pathogenic microorganism and a resource for identifying future targets for drug intervention to promote healthy oral microflora.

Biofilm acid/base physiology and gene expression in oral bacteria.

Environmental pH is one the major factors affecting the composition, biological activities, and pathogenic potential of the biofilms colonizing supragingival surfaces. In periodontal diseases, small changes in pH from the metabolism of amino acids and urea may influence the activity of proteolytic enzymes of host and bacterial origin. Still, there is a significant void in the understanding of pH-dependent gene expression in bacteria, in general, and this is of course a more acute problem when one considers there is virtually no information about gene expression in response to pH in biofilms. The development of new methods and applications of some of the techniques detailed above should help to ameliorate this situation and to generate much-needed data about the role of pH in biofilm composition, stability, and activity.

Physiologic homeostasis and stress responses in oral biofilms.

Studies performed since the early, 1970s have yielded tremendous amounts of information about the physiology, genetics, and interactions of oral bacteria. This pioneering work has provided a solid foundation to begin to apply the knowledge and technologies developed using suspended populations for studying oral bacteria under conditions that more closely mimic conditions in the oral cavity, in biofilms. Our current understanding of phenotypic capabilities of individual and complex mixtures of adherent oral bacteria is in its infancy. There is ample evidence that oral streptococci have different patterns of gene expression than planktonic cells, but we have little understanding of the basis for these observations. Even in biofilmforming bacteria with very well-developed genetic systems it is only very recently that genetic loci involved in biofilm formation and responses to surface growth have been identified. A comprehensive study of the physiology and gene expression characteristics of adherent oral bacteria not only will enhance our abilities to control oral diseases, but it will provide critical information that can be applied to a variety of other pathogenic microorganisms.

Alkali production by oral bacteria and protection against dental caries.

pH is a key environmental factor affecting the physiology, ecology and pathogenicity of the oral biofilms colonizing the hard tissues of the human mouth. Much attention has been focused on the production of organic acids through the metabolism of carbohydrates by pathogenic oral bacteria. Now, evidence is emerging that alkali generation, particularly through ammonia production from arginine and urea, plays major roles in pH homeostasis in oral biofilms and may moderate initiation and progression of dental caries. This short review highlights recent progress on understanding molecular genetic and physiologic aspects of ammonia generation by prominent oral bacteria.

Quasi-irreversible inhibition of enolase of Streptococcus mutans by fluoride.

Fluoride at concentrations greater than 0.01 mM was found to be a quasi-irreversible inhibitor of enolase of permeabilized cells of Streptococcus mutans GS-5 and also of isolated yeast enolase. The inhibition appeared to be of the type that has been described for P-ATPases, but was not dependent on added Al3+ or Be2+ ions. Fluoride inhibition of enolase was not reversed by repeatedly washing the permeabilized cells in chilled fluoride-free medium but could be reversed by the product, phosphoenolpyruvate, or by very high levels of the substrate, 2-phosphoglycerate. Irreversible inhibition of glycolysis was not evident after fluoride treatment of intact cells, washing to remove unbound or loosely bound fluoride and addition of glucose, presumably because intracellular levels of phosphoenolpyruvate were sufficiently high to preclude irreversible fluoride inhibition of enolase.

Acid adaptation in Streptococcus mutans UA159 alleviates sensitization to environmental stress due to RecA deficiency.

A RecA-deficient stain of Streptococcus mutans, isolated previously, was found to be more susceptible than the prototroph organism to acid killing and also showed reduced colony-forming ability on sucrose-containing medium. The deficient strain was able to grow in chemostat culture at a low pH value of 5 and did not show reduced capacity to produce acid in standard pH-drop experiments with excess glucose. Moreover, it was able to undergo an adaptive response when grown at a low pH to become more resistant to acid killing and also to killing by ultraviolet radiation or hydrogen peroxide. In fact, after adaptation, it was nearly as resistant as the prototroph strain. These findings were interpreted, in part, in terms of an acid-inducible DNA repair system which functions independently of RecA.

Rapid artificial restoration of electrical continuity across a crush lesion of a giant axon.

Action potentials never conducted through a crush lesion to the medial giant axon in the earthworm (Lumbricus terrestris) if the axon was exposed to normal or hypotonic salines that did not contain polyethylene glycol. However, action potentials, as well as electrotonic potentials, often conducted through a crush lesion exposed for 1 min to polyethylene glycol in hypotonic saline.

Diminished acid tolerance of plaque bacteria caused by fluoride.

Fluoride acts to reduce acid tolerances of plaque bacteria by upsetting normal proton currents across cell membranes. Streptococcus mutans was found to be unusually sensitive to fluoride, in part because its F1F0, proton-translocating ATPase is directly inhibited by fluoride at plaque levels. Thus, not only does fluoride serve in the HF form to bring extruded protons back into the cell, but it also reduces the capacity of the cell to extrude protons. Reductions in acid tolerance caused by fluoride would be expected to result in concomitant reductions in cariogenic potential.

Membrane-associated and solubilized ATPases of Streptococcus mutans and Streptococcus sanguis.

The proton-translocating, membrane ATPases of oral streptococci have been implicated in cytoplasmic pH regulation, acidurance, and cariogenicity. Membranes were isolated from Streptococcus mutans GS-5 and Streptococcus sanguis NCTC 10904 following salt-induced lysis of cells treated with lysozyme and mutanolysin. The ATPase activities of these membranes were 1.8 and 1.1 units per mg membrane protein, respectively. F1 ATPases were washed free from the membranes and purified by fast protein liquid chromatography (FPLC). Hydrolytic activities of the F1 ATPases were maximal at pH values between 6.0 and 6.6, whereas the membrane-bound enzymes had pH maxima of 7.5 (S. sanguis) and 6.0 (S. mutans). The F1 ATPases of the streptococci were similar to the well-characterized enzyme of Escherichia coli; they consisted of five different polypeptides and had apparent, aggregate molecular weights of from 335 to 350 Kd. The membrane-bound ATPases were characterized biochemically and found to be similar to those of proton-translocating ATPases of E. coli and Streptococcus faecalis. Km values for the membranes with respect to ATP were found to be 0.9 and 1.0 mmol/L for S. mutans and S. sanguis, respectively. Both enzymes had specificities for purine triphosphates and were active with a variety of divalent cations, although optimal activity occurred with ATP and Mg. The membrane-associated enzymes were sensitive to the inhibitors dicyclohexylcarbodiimide (DCCD) and azide, but insensitive to ouabain and vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid sensitivity of glycolysis in normal and proton-permeable cells of Streptococcus mutans GS-5.

Gramicidin, an ionophoric antibiotic which enhances proton permeability of cell membranes, was found to increase the acid sensitivity of glycolysis by intact cells of Streptococcus mutans GS-5, to inhibit uptake of 2-deoxyglucose at pH values of 5.5 or less, and to cause a decrease in the intracellular levels of early intermediates of glycolysis. The inhibitory effects of the antibiotic on glycolysis at low pH values were related to inhibition of sugar uptake via the phosphotransferase system.

Uptake of fluoride by cells of Streptococcus mutans in dense suspensions.

Fluoride uptake by nongrowing cells of Streptococcus mutans GS-5 was assessed by means of the space technique. Uptake was highly concentrative at low fluoride concentrations or low pH. In all, it appeared that fluoride uptake is predictably related to its weak-acid properties and that fluoride can be used, as certain other weak acids are, to estimate intracellular pH.

Reduction of acidurance of streptococcal growth and glycolysis by fluoride and gramicidin.

The acidurance of glycolysis by intact cells of Streptococcus mutans GS-5, Streptococcus salivarius ATCC 25925, and Streptococcus sanguis NCTC 10904 was found to be highly dependent on membrane functions affected by gramicidin, which increases the proton permeability of cell membranes. Plots of % glucose utilized during two hours against suspension pH values for cells suspended in 100 mM phosphate buffer plus 1 mM MgCl2 plus 13.9 mM glucose indicated, for 50% glucose utilization, pH values of 5.0 for S. mutans, 5.7 for S. salivarius, and 6.2 for S. sanguis. Gramicidin treatment shifted these values to 6.0, 6.3, and 6.9, respectively. Growth of S. mutans and S. salivarius in complex media proved to be more acid-sensitive than was glycolysis, and in batch cultures, there was a well-defined, post-growth phase of glycolysis. Minimum pH values for growth and for glycolysis in medium with excess glucose were approximately 4.8 and 4.4, respectively, for S. mutans, and 4.9 and 4.3 for S. salivarius. S. sanguis was less aciduric and showed little differential acid sensitivity, with minimum pH values of about 5.2 for both growth and glycolysis. Fluoride acted to eliminate the differences in acidurance of growth and glycolysis for S. mutans or S. salivarius and to render both processes more acid-sensitive. Thus, glycolysis was more fluoride-sensitive than was growth. Growth was found to be acid-limited in media with initial glucose levels greater than 0.2, 0.3, and 0.5% (weight/volume) for S. sanguis, S. mutans, and S. salivarius, respectively, and to be glucose-limited at lower levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition and killing of oral bacteria by silver ions generated with low intensity direct current.

Silver cations generated by passing low intensity direct current through pure silver electrodes were found to be sufficiently antibacterial to cause sterilization of samples of infected dentin. The optimal procedure involved a 5 microA current applied for 20 minutes with the anode then left in contact with the sample. Minimal inhibitory concentrations of electrically generated silver ions for representative oral bacteria were essentially equal to those for silver ions added as nitrate or fluoride salts, and medium constituents, including sodium thioglycolate, antagonized antibacterial action. A major advantage to the use of the electrode method is that it allows for continuous, focal application of antibacterial silver cations.

pH-dependent fluoride inhibition of peroxidase activity.

Fluoride was found to be a potent inhibitor of bovine lactoperoxidase and of salivary peroxidase at acid pH values. Inhibition was reversible at neutral pH, and appeared to involve HF binding by the enzyme. Fluoride inhibition of lactoperoxidase occurred with all reductants tested, including thiocyanate, iodide, and guaiacol. Fluoride concentrations for 50% inhibition of enzymatic activity with iodide as reductant were: less than 0.05 mM at a pH value of 4.0, 0.3 mM at 5.0, 4.0 mM at pH 6.0, and more than 10.0 mM at pH 7.0. Salivary peroxidases were found to have lower pH optima but to be approximately as sensitive to acid-dependent fluoride inhibition as was purified bovine lactoperoxidase. The findings suggest that the fluoride in dental plaque may be inhibitory to the antimicrobial peroxidase system.

Method to sensitize bacterial spores to subsequent killing by dry heat or ultraviolet irradiation.

Hydrogen peroxide and ultraviolet irradiation are known to interact synergistically for killing of bacterial spores. Synergy could be demonstrated with spores of Bacillus megaterium ATCC19213 adsorbed to filter paper strips or glass coverslips treated first with the peroxide and then dried for as long as 48 h prior to UV irradiation. This delayed action was considered to be due to absorption of the peroxide by the spores in an active but not readily vaporized form, which could become sporicidal also if the spores were heated to 50 degrees C. B. megaterium spores mixed with 0.1% (32.6 mM) H(2)O(2) solution appeared to absorb as much as 15 micromol/mg dry weight or about 0.5 mg/mg, but only a third to half of the peroxide could be recovered by water washing. A part of the unrecovered peroxide was degraded in reactions resulting in measurable production of oxygen. Degradation was not reduced by heating the spores to 65 degrees C or by azide and so appeared to be non-enzymatic. Spores of the anaerobe Clostridium sporogenes were also sensitized to ultraviolet killing by H(2)O(2) treatment followed by drying. They appear to absorb less peroxide, only about 2 micromol/mg, but had lower capacities to degrade H(2)O(2) so that nearly all of the peroxide could be recovered by washing with water. The findings presented should be helpful in the design of new methods for synergistic killing of spores by H(2)O(2) and UV irradiation or dry heat, especially involving, for example, packaging materials.