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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., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

NADH oxidase (Nox) is a flavin-containing enzyme used by Streptococcus mutans to reduce dissolved oxygen encountered during growth in the oral cavity. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans. A nox-defective mutant strain of S. mutans and its parental strain, the genomic type strain UA159, were exposed to various oxygen concentrations at pH values of 5 and 7 to better understand the adaptive mechanisms used by the organism to withstand environmental pressures. With the loss of nox, the activities of oxygen stress response enzymes such as superoxide dismutase and glutathione oxidoreductase were elevated compared to those in controls, resulting in a greater adaptation to oxygen stress. In contrast, the loss of nox led to a decreased ability to grow in a low-pH environment despite an increased resistance to severe acid challenge. Analysis of the membrane fatty acid composition revealed that for both the nox mutant and UA159 parent strain, growth in an oxygen-rich environment resulted in high proportions of unsaturated membrane fatty acids, independent of external pH. The data indicate that S. mutans membrane fatty acid composition is responsive to oxidative stress, as well as changes in environmental pH, as previously reported (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). The heightened ability of the nox strain to survive acidic and oxidative environmental stress suggests a multifaceted response system that is partially dependent on oxygen metabolites.

Objectives: This study aimed to evaluate antimicrobial effects of an o-cymen-5-ol/zinc system., Methods: o-Cymen-5-ol and zinc gluconate minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined against Streptococcus mutans, Actinomyces viscosus, Porphyromonas gingivalis, Fusobacterium nucleatum and Candida albicans. Synergy was investigated by checkerboard MIC/MBC; inhibition of P. gingivalis protease activity and S. mutans glycolysis were investigated. Slurried toothpastes containing the system were assessed in kill time assays against S. mutans and E. coli., Results: o-Cymen-5-ol MIC was between 1.7 mM to 3.4 mM; MBC was 3.4 mM to 6.7 mM. Zinc gluconate MIC was 2.8 mM to 11 mM; MBC was between 11 mM and >44 mM. The two agents in solution showed synergy (FICI≤0.50) against P. gingivalis and F. nucleatum, with MIC of 0.42 mM/0.69 mM for o-cymen-5-ol/zinc gluconate, respectively. Zinc inhibited glycolysis and protease to a greater degree than o-cymen-5-ol; glycolysis inhibition by the two agents was additive. o-Cymen-5-ol/zinc chloride in toothpaste showed greater effects than placebo (120s log10 kill=7.35±0.40 and 4.02±0.40, respectively)., Conclusions: The zinc/o-cymen-5-ol system has direct antimicrobial effects and inhibits oral disease-related processes. Synergistic effects were seen against anaerobes. A system combining o-cymen-5-ol and zinc shows properties desirable for incorporation in toothpastes., (© 2011 FDI World Dental Federation.)

The increasing prevalence of dental caries is making it more of a major world health problem. Caries is the direct result of acid production by cariogenic oral bacteria, especially Streptococcus mutans. New and better antimicrobial agents active against cariogenic bacteria are badly needed, especially natural agents derived directly from plants. We have evaluated the inhibitory actions of α-mangostin, a xanthone purified from ethanolic extracts of the tropical plant Garcinia mangostana L., by repeated silica gel chromatography. α-Mangostin was found to be a potent inhibitor of acid production by S. mutans UA159, active against membrane enzymes, including the F(H+)-ATPase and the phosphoenolpyruvate - sugar phosphotransferase system. α-Mangostin also inhibited the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase. Glycolysis by intact cells in suspensions or biofilms was inhibited by α-mangostin at concentrations of 12 and 120 µmol·L⁻¹, respectively, in a pH-dependent manner, with greater potency at lower pH values. Other targets for inhibition by α-mangostin included (i) malolactic fermentation, involved in alkali production from malate, and (ii) NADH oxidase, the major respiratory enzyme for S. mutans. The overall conclusion is that α-mangostin is a multitarget inhibitor of mutans streptococci and may be useful as an anticaries agent.

The saliva proteome includes host defense factors and specific bacterial-binding proteins that modulate microbial growth and colonization of the tooth surface in the oral cavity. A multidimensional mass spectrometry approach identified the major host-derived salivary proteins that interacted with Streptococcus mutans (strain UA159), the primary microorganism associated with the pathogenesis of dental caries. Two abundant host proteins were found to tightly bind to S. mutans cells, common salivary protein-1 (CSP-1) and deleted in malignant brain tumor 1 (DMBT1, also known as salivary agglutinin or gp340). In contrast to gp340, limited functional information is available on CSP-1. The sequence of CSP-1 shares 38.1% similarity with rat CSP-1. Recombinant CSP-1 (rCSP-1) protein did not cause aggregation of S. mutans cells and was devoid of any significant biocidal activity (2.5 to 10 μg/mL). However, S. mutans cells exposed to rCSP-1 (10 μg/mL) in saliva displayed enhanced adherence to experimental salivary pellicle and to glucans in the pellicle formed on hydroxyapatite surfaces. Thus, our data demonstrate that the host salivary protein CSP-1 binds to S. mutans cells and may influence the initial colonization of this pathogenic bacterium onto the tooth surface.

Alkali production by oral streptococci is considered important for dental plaque ecology and caries moderation. Recently, malolactic fermentation (MLF) was identified as a major system for alkali production by oral streptococci, including Streptococcus mutans. Our major objectives in the work described in this paper were to further define the physiology and genetics of MLF of oral streptococci and its roles in protection against metabolic stress damage. L-Malic acid was rapidly fermented to L-lactic acid and CO(2) by induced cells of wild-type S. mutans, but not by deletion mutants for mleS (malolactic enzyme) or mleP (malate permease). Mutants for mleR (the contiguous regulator gene) had intermediate capacities for MLF. Loss of capacity to catalyze MLF resulted in loss of capacity for protection against lethal acidification. MLF was also found to be protective against oxidative and starvation damage. The capacity of S. mutans to produce alkali from malate was greater than its capacity to produce acid from glycolysis at low pH values of 4 or 5. MLF acted additively with the arginine deiminase system for alkali production by Streptococcus sanguinis, but not with urease of Streptococcus salivarius. Malolactic fermentation is clearly a major process for alkali generation by oral streptococci and for protection against environmental stresses.

The oral cavity harbors several hundred different bacterial species that colonize both hard (teeth) and soft tissues, forming complex populations known as microbial biofilms. It is widely accepted that the phenotypic characteristics of bacteria grown in biofilms are substantially different from those grown in suspensions. Because biofilms are the natural habitat for the great majority of oral bacteria, including those contributing to oral diseases, a better understanding of the physiology of adherent populations is clearly needed to control oral microbes in health and disease. In this chapter, we use oral streptococci as examples for studying the physiology of oral biofilms.

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.

Hydrogen peroxide (H2O2) is considered a major endogenous source of oxidative stress to oral bacteria and also is widely used in oral care products. Our study objectives were to identify specific targets for H2O2-induced damage to cells of Streptococcus mutans in suspensions and monospecies biofilms and to differentiate bacteriostatic and bactericidal actions of the peroxide. Streptococcus mutans was grown in suspension cultures and fed-batch biofilms for assessing relative sensitivities of viability, glycolysis, and protein synthesis to H2O2 damage. Biofilm cells were found to have essentially the same peroxide sensitivity as cells in suspensions. H2O2 at low concentrations of about 16.3 mmol/L was highly inhibitory for glycolysis and mainly bacteriostatic. The most sensitive target detected for glycolytic inhibition was glyceraldehyde-3-phosphate dehydrogenase with IC50 (50% inhibitory concentration) values of ca. 2.2 mmol/L for suspension cells and 2.3 mmol/L for biofilms with 15 min treatments. The phosphoenolpyruvate:glucose phosphotransferase pathway was less sensitive with an IC50 of ca. 10 mmol/L. Aldolase was not inhibited at bacteriostatic concentrations of the peroxide. For suspensions and biofilms, acidification somewhat diminished peroxide sensitivity, while increased temperature enhanced sensitivity. At concentrations above about 30 mmol/L, H2O2 became mainly bactericidal but not mutagenic for S. mutans. A major target for bactericidal damage was protein synthesis, thus rendering cells incapable of repairing or replacing oxidatively damaged proteins.

Streptococcus mutans and certain other oral lactic-acid bacteria were found to have the ability to carry out malolactic fermentation involving decarboxylation of L-malate to yield L-lactic acid and concomitant reduction in acidity. The activity was inducible by L-malate in S. mutans UA159 growing in suspensions or biofilms. The optimal pH for the fermentation was c. 4.0 for both suspensions and biofilms, although the pH optimum for malolactic enzyme in permeabilized cells of S. mutans UA159 was close to 5.5. Although malate did not serve as a catabolite for growth of S. mutans, it did serve to protect the organism against acid killing and to maintain ATP pool levels during starvation. Alkalinization associated with malolactic fermentation resulted in pH rise or increased need to add standardized HCl solution to maintain a set pH value in pH-stat experiments. The net conclusion is that malate has the potential to be effective for alkalinization of dental plaque, although the fermentation is sensitive to fluoride and triclosan, which are commonly added to oral care products.

Background/objective: Benzimidazoles are widely used as proton-pump inhibitors to control stomach hyperacidity and have been found also to have antimicrobial actions against Helicobacter pylori and oral streptococci. Our primary aim was to determine if they are active also against oral anaerobes associated with gingivitis. Our major focus was on catabolism because it leads to production of inflammatory metabolites such as butyrate and ammonia. The benzimidazoles are effective in the protonated form at acid pH values and cause irreversible inhibition of enzymes associated with formation of drug-target disulfide bonds., Methods: Fusobacterium nucleatum ATCC 25586 and Prevotella intermedia ATCC 25611 were grown anaerobically in suspension cultures, harvested, washed and exposed to the benzimidazole lansoprazole at pH values of 4 or 5 before being washed and used for standard assays to detect inhibition of catabolic functions, uptake of the agent and lethality., Results: Lansoprazole was found to be a bacteriostatic, multi-target antimicrobial against F. nucleatum under anaerobic conditions inhibitory for amino acid fermentation and also for glycolysis of glucose or fructose. ID(50) values for fermentation of amino acids and dipeptides by F. nucleatum ranged from 0.05 mM for lysine to 0.25 mM for serine. Fructose catabolism was highly sensitive with an ID(50) value of 0.03 mM apparently related to high sensitivity of the phosphoenolpyruvate:fructose phosphotransferase system, while the ID(50) for glucose catabolism by intact cells was some 0.07 mM. Fermentation of aspartate or aspartylaspartate by P. intermedia was found to be lansoprazole-sensitive with ID(50) values of about 0.18 and 0.20 mM, respectively., Conclusion: Catabolism of amino acids, dipeptides and sugars by oral anaerobes associated with gingivitis are sensitive to the inhibitory actions of lansoprazole. Thus, catabolic pathways are potential targets for use of benzimidazoles against bacteria involved in gingivitis.

Triclosan was found to be a potent inhibitor of the F(H+)-ATPase of the oral pathogen Streptococcus mutans and to increase proton permeabilities of intact cells. Moreover, it acted additively with weak-acid transmembrane proton carriers, such as fluoride or sorbate, to sensitize glycolysis to acid inhibition. Even at neutral pH, triclosan could inhibit glycolysis more directly as an irreversible inhibitor of the glycolytic enzymes pyruvate kinase, lactic dehydro genase, aldolase, and the phosphoenolpyruvate:sugar phosphotransferase system (PTS). Cell glycolysis in suspensions or biofilms was inhibited in a pH-dependent manner by triclosan at a concentration of about 0.1 mmol/L at pH 7, approximately the lethal concentration for S. mutans cells in suspensions. Cells in intact biofilms were almost as sensitive to triclosan inhibition of glycolysis as were cells in suspensions but were more resistant to killing. Targets for irreversible inhibition of glycolysis included the PTS and cytoplasmic enzymes, specifically pyruvate kinase, lactic dehydrogenase, and to a lesser extent, aldolase. General conclusions are that triclosan is a multi-target inhibitor for mutans streptococci, which lack a triclosan-sensitive FabI enoyl-ACP reductase, and that inhibition of glycolysis in dental plaque biofilms, in which triclosan is retained after initial or repeated exposure, would reduce cariogenicity.

Caries-causing oral bacteria such as Streptococcus mutans are protected by the actions of F-ATPases against acid damage in dental plaque acidified by glycolytic acid production or ingestion of acids foods and beverages. Catabolites such as glucose and sucrose were found to enhance the protection of S. mutans and also other oral lactic-acid bacteria against acid killing at lethal pH values as low as 2.5. Protection involved glycolysis with the production of lactate and ATP, which is a substrate for F-ATPases. ATP could also be produced by starved cells apparently through synthase activity of the F-ATPase associated with acid decline. Fluoride and the organic weak-acid indomethacin acted to diminish this protection, as did F-ATPase inhibitors such as dicyclohexylcarbodi-imide. Protection against acid killing involving catabolism and synthase activity is likely to be important for plaque cariogenicity.

Fluoride and zinc, alone or in combination at concentrations of 0.2 mM, inhibited production-secretion of glucosyltranferases by Streptococcus mutans UA159 growing in suspension cultures. Inhibition did not involve growth inhibition or starvation. Fluoride and zinc also inhibited glucan production, especially insoluble glucan, in fed-batch biofilms. Inhibition of biofilms appeared to be associated with starvation as indicated by markedly decreased ATP pools and iodophilic polysaccharide levels in biofilm cells. As insoluble glucans are important for virulence of mutans streptococci, the inhibitory actions of fluoride and zinc could significantly affect cariogenicity.

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.

Background/aims: Fluoride is known to be a potent inhibitor of bacterial ureases and can also act in the form of hydrofluoric acid as a transmembrane proton conductor to acidify the cytoplasm of intact cells with possible indirect, acid inhibition of urease. Our research objectives were to assess the inhibitory potencies of fluoride for three urease-positive bacteria commonly found in the mouth and to determine the relative importance of direct and indirect inhibition of ureases for overall inhibition of intact cells or biofilms., Methods: The experimental design involved intact bacteria in suspensions, mono-organism biofilms, cell extracts, and dental plaque. Standard enzymatic assays for ammonia production from urea were used., Results: We found that ureolysis by cells in suspensions or mono-organism biofilms of Staphylococcus epidermidis, Streptococcus salivarius or Actinomyces naeslundii was inhibited by fluoride at plaque levels of 0.1-0.5 mm in a pH-dependent manner. The results of experiments with the organic weak acids indomethacin and capric acid, which do not directly inhibit urease enzyme, indicated that weak-acid effects leading to cytoplasmic acidification are also involved in fluoride inhibition. However, direct fluoride inhibition of urease appeared to be the major mechanism for reduction in ureolytic activity in acid environments. Results of experiments with freshly harvested supragingival dental plaque indicated responses to fluoride similar to those of S. salivarius with pH-dependent fluoride inhibition and both direct and indirect inhibition of urease., Conclusion: Fluoride can act to diminish alkali production from urea by oral bacteria through direct and indirect mechanisms.

Objective: Zinc is used in oral care products as an antiplaque/antigingivitis agent. Our objective was to assess the antimicrobial actions of zinc against oral anaerobes associated with gingivitis, specifically Fusobacterium nucleatum and Prevotella intermedia, with focus on catabolism and oxidative metabolism., Methods: The oral anaerobes were grown in complex medium in an anaerobic chamber, harvested by centrifugation and used directly for experiments with suspensions. Biofilm growth involved super-infection by F. nucleatum of an initial biofilm formed by Streptococcus sanguis., Results: Zn(2+) inhibited catabolism of glutamate, glutamyl-glutamate, glucose and fructose by F. nucleatum cells in suspensions with ID(50) values, respectively, of 0.05, 0.005, 0.01 and 0.01 mM. The ID(50) value for inhibition of glutamate catabolism by biofilms was 0.10 mM. Inhibition of glutamate catabolism could be related to inhibition of substrate uptake and of 2-oxoglutarate reductase. Zn(2+) also inhibited catabolism of aspartate or aspartyl-aspartate by P. intermedia with ID(50) values of 0.07 and about 0.03 mM, respectively. Respiration of intact cells of F. nucleatum and NADH oxidase in cell extracts were sensitive to zinc with ID(50) values, respectively, of about 1.0 and 1.4 mM. Zinc also inhibited production of hydrogen peroxide by F. nucleatum (ID(50) = ca. 0.04 mM.) but at high concentrations acted to potentiate and enhance peroxide killing of the anaerobe., Conclusion: Zn(2+) is a potent inhibitor of catabolism by F. nucleatum and P. intermedia, including catabolism of peptides, which can be degraded to yield inflammatory metabolic end products. Zn(2+) also inhibits O(2) metabolism of F. nucleatum by about 50% and hydrogen peroxide production nearly completely but also enhances killing by peroxide added to cells.

Background/aim: Benzimidazoles, such as lansoprazole and omeprazole, are used extensively as proton-pump inhibitors to control stomach acid secretion and also have antimicrobial actions against Helicobacter pylori. Our objective was to determine whether they are potentially useful antimicrobials against oral bacteria., Methods: Streptococcus mutans was our main test organism. It was grown in suspension cultures and biofilms. Standard physiologic assays were used to assess inhibitory actions of benzimidazoles., Results: Benzimidazoles inhibited acid production by S. mutans in suspensions or biofilms. In pH-drop experiments, lansoprazole at a level of only 0.025 mm irreversibly inhibited acid production from glycolysis. Cell uptake of lansoprazole was found to be very pH sensitive and occurred mainly at pH values below about 5, indicating that the protonated form was taken up. Lansoprazole inhibition of glycolysis could be blocked by 2-mercaptoethanol, which suggests that disulfide bonds form between benzimidazoles and protein targets. Identified targets for benzimidazole inhibition included the phosphoenolpyruvate : sugar phosphotransferase system, the glycolytic enzymes aldolase, glyceraldehyde-3-phosphate dehydrogenase, and lactic dehydrogenase, and enzymes such as urease and arginine deiminase. Lansoprazole increased proton permeabilities of S. mutans cells but did not inhibit F-ATPases. Although cells in biofilms were somewhat less sensitive to the agents than those in suspensions, biofilm glycolysis was still markedly inhibited by 0.1 mm lansoprazole. Benzimidazoles are bactericidal, and the oral anaerobes Fusobacterium nucleatum and Prevotella intermedia were more sensitive to killing than was S. mutans., Conclusion: Benzimidazoles appear to be useful inhibitors of oral bacteria in acid environments such as progressing caries lesions.

Glaucoma represents a major cause of vision loss throughout the world. Primary open-angle glaucoma, the most common form of glaucoma, is a chronic, progressive disease often, though not always, accompanied by elevated intraocular pressure (IOP). In this disorder, retinal ganglion cell loss and excavation of the optic nerve head produce characteristic peripheral visual field deficits. Patients with normal-tension glaucoma present with typical visual field and optic nerve head changes, without a documented history of elevated IOP. A variety of secondary causes, such as pigment dispersion syndrome and ocular trauma, can result in glaucoma as well. Treatment of all forms of glaucoma consists of reducing IOP. With proper treatment, progression of this disease can often be delayed or prevented. Treatment options for glaucoma include medications, laser therapy and incisional surgery. Laser techniques for the reduction of IOP include argon laser trabeculoplasty and selective laser trabeculoplasty. Both techniques work by increasing outflow of aqueous humour through the trabecular meshwork. Surgical options for glaucoma treatment include trabeculectomy, glaucoma drainage tube implantation and ciliary body cyclodestruction. While each of these types of procedures is effective at lowering IOP, therapy usually begins with medications. Medications lower IOP either by reducing the production or by increasing the rate of outflow of aqueous humour within the eye. Currently, there are five major classes of drugs used for the treatment of glaucoma: (i) cholinergics (acetylcholine receptor agonists); (ii) adrenoceptor agonists; (iii) carbonic anhydrase inhibitors (CAIs); (iv) beta-adrenoceptor antagonists; and (v) prostaglandin analogues (PGAs). Treatment typically begins with the selection of an agent for IOP reduction. Although beta-adrenoceptor antagonists are still commonly used by many clinicians, the PGAs are playing an increasingly important role in the first-line therapy of glaucoma. Adjunctive agents, such as alpha-adrenoceptor agonists and CAIs are often effective at providing additional reduction in IOP for patients not controlled on monotherapy. As with any chronic disease, effective treatment depends on minimising the adverse effects of therapy and maximising patient compliance. The introduction of a variety of well tolerated and potent medications over the past few years now allows the clinician to choose a treatment regimen on an individual patient basis and thereby treat this disorder more effectively.

Zinc is a known inhibitor of acid production by mutans streptococci. Our primary objective was to extend current knowledge of the physiologic bases for this inhibition and also for zinc inhibition of alkali production by Streptococcus rattus FA-1 and Streptococcus salivarius ATCC 13419. Zinc at concentrations as low as 0.01-0.1 mm not only inhibited acid production by cells of Streptococcus mutans GS-5 in suspensions or in biofilms but also sensitized glycolysis by intact cells to acidification. Zinc reversibly inhibited the F-ATPase of permeabilized cells of S. mutans with a 50% inhibitory concentration of about 1 mm for cells in suspensions. Zinc reversibly inhibited the phosphoenolpyruvate: sugar phosphotransferase system with 50% inhibition at about 0.3 mm ZnSO4, or about half that concentration when the zinc-citrate chelate was used. The reversibility of these inhibitory actions of zinc correlates with findings that it is mainly bacteriostatic rather than bactericidal. Zinc inhibited alkali production from arginine or urea and was a potent enzyme inhibitor for arginine deiminase of S. rattus FA-1 and for urease of S. salivarius. In addition, zinc citrate at high levels of 10-20 mm was weakly bactericidal.

Bacterial cells have adapted in a variety of ways to resist oxidative stresses and damage in their everyday lives in a predominantly aerobic world. The nearly universal occurrence of resistance mechanisms against oxidative stresses, particularly those due to reactive oxygen species (ROS), suggests that most, if not all, bacteria have to deal with oxidative assaults. A primary source of oxidative stress is aerobic metabolism, which leads to production of ROS such as hydrogen peroxide, superoxide radical, perhydroxyl radical, hydroxyl radical and a variety of other toxic metabolites, including organic peroxides and other organics or inorganics able to transfer electrons to sites of oxidative damage. Anaerobes as well as aerobic and facultative organisms are subject to oxidative stresses, often as a result of their own metabolism of O2 or that of associated facultative organisms. If anaerobes would just ignore oxygen instead of metabolizing it, they would not have to deal with toxic metabolites of their own making. Another major source of oxidative stress comes from the use of oxidative agents in the disinfection-sterilization industry. Notable examples are hypochlorite for water purification and hydrogen peroxide used for industrial sterilization. Antimicrobials such as isoniazide and mitomycin C also act oxidatively to cause damage. In this article, aseptic packaging and processing involving use of hydrogen peroxide for sterilization of packaging materials is reviewed as an example of oxidative stress imposed on bacterial spores and vegetative cells from outside the organisms or the microbial community. The other example considered is related to oral microbiology and infectious disease in which oxidative stress may arise from the metabolism of the oral microbiota or may come from outside through use of oral care products.

Objective: To test in vitro the anti-plaque/ antimicrobial efficacy of a new toothpaste formulation containing a 2% zinc citrate/ 0.3% Triclosan anti-microbial system compared with a 0.75% zinc citrate/ 0.3% Triclosan system and where appropriate, against controls of a standard fluoride paste and a 0.3% Triclosan/ 2% copolymer product., Methods: The anti-metabolic activity was assessed using a range of assays measuring the ability of the active systems to inhibit bacterial glycolysis. The antibacterial/ anti-plaque activity was assessed in an in vitro multispecies biofilm assay., Results: Both zinc formulations were shown to have significantly superior activity at inhibiting glycolysis compared with the 0.3% Triclosan/ 2% copolymer formulation and the standard fluoride paste, particularly in reducing the pH drop after sugar challenge, the new formulation having the greatest activity. Likewise, in the antibacterial assay, both zinc formulations were found to have significantly superior activity over a standard fluoride paste and the 2% zinc citrate/ 0.3% Triclosan formulation was shown to be significantly better than 0.75% zinc citrate/ 0.3% Triclosan formulation., Conclusion: These data provide support for the enhanced performance of the 2% zinc citrate/ 0.3% Triclosan formulation.

Advantage is taken in many sterilization processes, especially for food packaging materials, of the synergy between H2O2 and UV irradiation for spore killing. The nature of the synergy is currently not well defined in terms of targets and mechanisms. We found that under some experimental conditions, the synergistic killing of spores of Bacillus megaterium ATCC 19213 appeared to be mainly UV-enhanced peroxide killing, while under other conditions, it appeared to be mainly peroxide-enhanced UV killing. Lethal combinations of H2O2 and UV irradiation for spores resulted in only modest increases in auxotrophic mutations among survivors, indicative of little DNA damage, in contrast to higher mutation levels after dry-heat damage at 115 degrees C. However, the combination of UV light and peroxide did lead to major inactivation of glucose 6-phosphate dehydrogenase, an enzyme that was used to monitor the damage to bacterial protein. Synergistic UV-H2O2 killing was reduced by agents such as pyruvate, thiosulfate, and iron or copper cations, which appeared to act at least in part by reacting chemically with H2O2, and was only slightly affected by the use of UV light at a wavelength of 222 nn rather than 254 nm. Hydrogen peroxide treatment can precede UV irradiation for synergistic killing by some hours with an interim of drying for spores of Bacillus subtilis A, a spore type used commonly for the validation of aseptic processes. Synergistic killing of dried spores or those in suspensions was accelerated at higher temperatures (50 degrees C) rather than at lower temperatures (25 degrees C).

Fluoride is widely used as an anticaries agent in drinking water and a variety of other vehicles. This use has resulted in major health benefits. However, there are still open questions regarding the mechanisms of anticaries action and the importance of antimicrobial effects in caries reduction. Fluoride acts in multiple ways to affect the metabolism of cariogenic and other bacteria in the mouth. F(-)/HF can bind directly to many enzymes, for example, heme-containing enzymes or other metalloenzymes, to modulate metabolism. Fluoride is able also to form complexes with metals such as aluminum or beryllium, and the complexes, notably AlF(4)(-) and BeF(3)(-).H(2)O, can mimic phosphate with either positive or negative effects on a variety of enzymes and regulatory phosphatases. The fluoride action that appears to be most important for glycolytic inhibition at low pH in dental plaque bacteria derives from its weak-acid properties (pK(a)=3.15) and the capacity of HF to act as a transmembrane proton conductor. Since many of the actions of fluoride are related to its weak-acid character, it is reasonable to compare fluoride action to those of organic weak acids, including metabolic acids, food preservatives, non-steroidal anti-inflammatory agents and fatty acids, all of which act to de-energize the cell membrane by discharging DeltapH. Moreover, with the realization that the biofilm state is the common lifestyle for most microorganisms in nature, there is need to consider interactions of fluoride and organic weak acids with biofilm communities. Hopefully, this review will stimulate interest in the antimicrobial effects of fluoride or other weak acids and lead to more effective use of the agents for disease control and other applications.

Propolis, a resinous hive product secreted by Apis mellifera bees, has been shown to reduce the incidence of dental caries in rats. Several compounds, mainly polyphenolics, have been identified in propolis. Apigenin and tt-farnesol demonstrated biological activity against mutans streptococci. We determined here their effects, alone or in combination, on glucosyltransferase activity, biofilm viability, and development of caries in rats. Sprague-Dawley rats were infected with Streptococcus sobrinus 6715 and treated topically twice daily as follows: (1) tt-farnesol, (2) apigenin, (3) vehicle control, (4) fluoride, (5) apigenin +tt-farnesol, and (6) chlorhexidine. Apigenin (1.33 mM) inhibited the activity of glucosyltransferases in solution (90-95%) and on the surface of saliva-coated hydroxyapatite beads (35-58%); it was devoid of antibacterial activity. tt-Farnesol (1.33 mM) showed modest antibacterial activity against biofilms and its effects on glucosyltransferases were minimal. The incidence of smooth-surface caries was significantly reduced by apigenin +tt-farnesol (60%), fluoride (70%), and chlorhexidine (72%) treatments compared to control (P < 0.05).

The respiratory activities of oral streptococci grown in biofilms were found to be markedly repressed compared with those of cells from aerobic culture, or for Streptococcus mutans GS-5, even for those grown in static culture. Respiration rates generally reflected levels of NADH oxidase activities in cell extracts. Superoxide dismutase levels were somewhat reduced in biofilm cells. However, sensitivities to oxidative damage caused by H2O2, t-butylhydroperoxide, or 8-hydroxyquinoline were not greatly different for cells from suspension cultures and those from either intact or dispersed biofilms. The capacities of S. sanguis and S. gordonii to produce H2O2 also were markedly repressed by biofilm growth, and presumably this repression would affect the ecology of dental plaque by reducing oxidative stresses under crowded conditions.

Oxygen metabolism (respiration) of Streptococcus mutans GS-5 involving NADH oxidases, mainly of the H(2)O-producing type, was found to be acid sensitive, as was NADH oxidase activity of cell extracts. Respiration of intact cells in acidified media was also highly sensitive to fluoride, with a 50% inhibitory concentration of about 0.02 mM at pH 4. In contrast, NADH oxidases in cell extracts were fluoride insensitive. Fluoride inhibition of respiration of intact cells was related to weak-acid effects leading to enhanced proton permeability of cells, cytoplasmic acidification and resultant acid inhibition of NADH oxidases and glycolysis. Organic weak acids, such as indomethacin and benzoate, were also effective inhibitors. H(2)O(2) production by intact cells of Streptococcus sanguis NCTC 10904, a peroxide producer, was similarly inhibited by fluoride or organic weak acids in acidified media. Thus, weak acids act as respiratory inhibitors for oral streptococci indirectly by acidifying the cytoplasm rather than acting as direct inhibitors of NADH oxidases.

Fluoride and sulfide are known inhibitors of heme catalases in acid environments. Staphylococcus aureus H cells were found to be sensitized by fluoride or sulfide to H2O2 killing at acid pH values in the range of 3.5 to 4.0, and catalase activity was reduced concomitantly. In contrast, fluoride had little effect on H2O2 killing of Streptococcus mutans GS-5, which has fluoride-insensitive peroxidase activity, but still is more sensitive to H2O2 than is S. aureus in the absence of fluoride. Fluoride but not sulfide was inhibitory also for the Mn-containing, non-heme pseudocatalase of Lactobacillus plantarum ATCC 14431 over a wide pH range, and this inhibitory effect was reflected in enhanced H2O2 killing in the presence of fluoride. In addition, we found that catalase-positive S. aureus or Neisseria sicca could protect catalase-negative S. mutans against killing by H2O2 in mixed suspensions, but protection was compromised by fluoride or sulfide under acid conditions. Thus, catalase-positive organisms could protect a catalase-negative organism against peroxide damage, but inhibition of catalase reduced protection. These findings are pertinent to the widespread use of fluoride and peroxide in oral health care products.

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.

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.

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.

Fluoride and other weak acids, such as benzoate, indomethacin, salicylate and sorbate, were found to be sensitizers for acid killing of cells of Actinomyces naeslundii ATCC 19246 and Streptococcus sanguis NCTC 10904 in suspensions or in mono-organism biofilms on glass slides. These bacteria are among the more acid-sensitive organisms from dental plaque and were killed when acidified to pH values between 3.5 and 4.0. Biofilm cells were more resistant than cells in suspensions, especially in terms of the fraction of the initial population surviving acidification. The mechanism for sensitization to acid killing by fluoride and the other weak acids involved enhanced transmembrane transport of protons, reflected by increases in measured proton permeabilities of the cells. Thus, the weak acids thwarted the functions of F(H+)-ATPases in extruding protons and protecting cells against acid damage. Fluoride sensitization of biofilms or cells in suspensions to acid damage occurred rapidly. There was a delay in sensitization of biofilms by indomethacin and higher molecular weight acids which was interpreted in terms of diffusion limitation of sensitizer penetration. Overall, it seemed that weak-acid sensitization to acid killing is a general phenomenon that occurs not just for oral bacteria but also for organisms in food, soil, and other acidified environments.

Parabens were found to be potent inhibitors of alkali production from arginine by oral streptococci such as Streptococcus rattus, Streptococcus sanguis and Streptococcus gordonii. For example, 2 mumol butylparaben per ml completely and irreversibly inhibited arginolysis by intact cells of S. rattus FA-1 and was lethal for the organism. In contrast, butylparaben was not a very effective inhibitor of ureolysis by intact cells of Streptococcus salivarius 57.I, although it did kill the cells. Butylparaben irreversibly inhibited the cytoplasmic enzymes arginine deiminase, carbamate kinase and urease in permeabilized cells or isolated form. However, inhibition of arginolysis by intact cells appeared to be due primarily to irreversible inhibition of transport systems for arginine uptake, because butylparaben added to intact cells did not reduce levels of arginine deiminase when the cells were subsequently permeabilized after washing. The insensitivity of ureolysis by intact cells to butylparaben can be related to the known high permeability of cell membranes to urea and the cytoplasmic location of urease. The potency of butylparaben as an inhibitior of arginolysis or glycolysis and as a lethal agent was found to be greater at acid pH that at neutral or alkaline pH.

Treponema denticola strains ATCC 35405 and ASLM were found to have moderately active oxygen metabolism and consumed some 0.46 mumol O2/h/mg cell protein in anaerobic growth medium or about ten times this amount in aerobic medium. There appeared to be no differences between the two strains in their oxidative metabolism. The spirochetes showed significant endogenous O2 utilization, which was stimulated only slightly by added glucose or arginine, moderately by glycine, but markedly by casamino acids or brain-heart infusion broth. O2 metabolism by intact cells was insensitive to cyanide and so did not appear to involve cyanide-sensitive cytochrome oxidases. Moreover, difference spectra of cell extracts and membranes did not reveal heme profiles. However, the spirochetes did have very active reduced nicotinamide adenine dinucleotide (NADH) oxidase(s) and also contained the protective enzymes NADH peroxidase and superoxide dismutase. Both the oxidase(s) and the peroxidase had rather broad substrate specificities. Either NADH or reduced nicotinamide adenine dinucleotide phosphate could serve as reductant, and the enzymes were active with a variety of oxidants. Enzyme activity in fresh cell extracts was only somewhat stimulated by added flavins, but after frozen storage, the activity became much more activated by flavin adenine nucleotide, and to a lesser extent, by flavin mononucleotide. The enzymes were insensitive to fluoride, which inhibits heme-based but not flavin-based oxidases at low pH values. Clearly, these anaerobic spirochetes have significant oxygen metabolism, even at the low levels of O2 measured in periodontal pockets and contain enzymes that offer at least moderate protection against damage by reactive oxygen species.

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.

The arginine deiminase system in oral streptococci is highly regulated. It requires induction and is repressed by catabolites such as glucose or by aeration. A comparative study of regulation of the system in Streptococcus gordonii ATCC 10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 showed an increase in activity of the system in S. sanguis of some 1467-fold associated with induction-depression of cells previously uninduced-repressed. The activity of the system was assayed in terms of levels of arginine deiminase, the signature enzyme of the system, in permeabilized cells. Increases in enzyme levels associated with induction-depression were less for the other two organisms, mainly because of less severe repression, especially for S. rattus FA-1, which was the least sensitive to catabolite repression or aeration. Regulation of the arginine deiminase system involving induction and catabolite repression was demonstrated also with monoorganism biofilms composed of cells of S. sanguis adherent to glass slides. Fully uninduced-repressed cells from suspension cultures or biofilms were compromised in their abilities to catabolize arginine to protect themselves against acid damage. However, it was found that the system can be rapidly turned on or turned off, although induction-depression did appear to require cell growth. Still, the system could respond rapidly to the availability of arginine to reestablish high capacity for alkali production.

The organic hydroperoxides t-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores of bacillus megaterium ATCC 19213 concomitant with mortality. Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays. The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of the differences in the types of radicals generated. However, each agent inactivated each of the enzymes, albeit at different rates. Comparative assessments of enzyme inactivation by lethal levels of H2O2 or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant. The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat. Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.

Reduced, transition metal cations commonly enhance oxidative damage to cells caused by hydroperoxides formed as a result of oxygen metabolism or added externally. As expected, the cations Fe2+ and Cu+ enhanced killing of Streptococcus mutans GS-5 by hydroperoxides. However, unexpectedly, they also induced lethal damage under fully anaerobic conditions in a glove box with no exposure to O2 or hydroperoxides from initial treatment with the cations. Sensitivities to anaerobic killing by Fe2+ varied among the organisms tested. The oral streptococci Streptococcus gordonii ATCC 10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 were approximately as sensitive as S. mutans GS-5. Enterococcus hirae ATCC 9790, Actinomyces viscosus OMZ105E, and Actinomyces naeslundii WVU45 had intermediate sensitivity, while Lactobacillus casei ATCC 4646 and Escherichia coli B were insensitive. Killing of S. mutans GS-5 in response to millimolar levels of added Fe2+ occurred over a wide range of temperatures and pH. The organism was able to take up ferrous iron, but ferric reductase activity could not be detected. Chelators, uric acid, and thiocyanate were not effective inhibitors of the lethal damage. Sulfhydryl compounds, ferricyanide, and ferrocyanide were protective if added prior to Fe2+ exposure. Fe2+, but not Fe3+, acted to reduce the acid tolerance of glycolysis by intact cells of S. mutans. The reduction in acid tolerance appeared to be related directly to Fe2+ inhibition of F-ATPase, which could be assayed with permeabilized cells, isolated membranes, or F1 enzyme separated from membranes. Cu+ and Cu2+ also inhibited F-ATPase and sensitized glycolysis by intact cells to acid. All of these damaging actions occurred anaerobically and thus did not appear to involve reactive oxygen species.

When a hair cell is stimulated by positive deflection of its hair bundle, increased tension in gating springs opens transduction channels, permitting cations to enter stereocilia and depolarize the cell. Ca2+ is thought to be required in mechanoelectrical transduction, for exposure of hair bundles to Ca2+ chelators eliminates responsiveness by disrupting tip links, filamentous interstereociliary connections that probably are the gating springs. Ca2+ also participates in adaptation to stimuli by controlling the activity of a molecular motor that sets gating-spring tension. Using a flexible glass fiber to measure hair-bundle stiffness, we investigated the effect of Ca2+ concentration on stiffness before and after the disruption of gating springs. The stiffness of intact hair bundles depended nonmonotonically on the extracellular Ca2+ concentration; the maximal stiffness of approximately 1200 microN.m-1 occurred when bundles were bathed in solutions containing 250 microM Ca2+, approximately the concentration found in frog endolymph. For cells exposed to solutions with sufficient chelator capacity to reduce the Ca2+ concentration below approximately 100 nM, hair-bundle stiffness fell to approximately 200 microN.m-1 and no longer exhibited Ca2+-dependent changes. Because cells so treated lost mechanoelectrical transduction, we attribute the reduction in bundle stiffness to tip-link disruption. The results indicate that gating springs are not linearly elastic; instead, they stiffen with increased strain, which rises with adaptation-motor activity at the physiological extracellular Ca2+ concentration.

The mechanoelectrical-transduction channel of the hair cell is permeable to both monovalent and divalent cations. Because Ca2+ entering through the transduction channel serves as a feedback signal in the adaptation process that sets the channel's open probability, an understanding of adaptation requires estimation of the magnitude of Ca2+ influx. To determine the Ca2+ current through the transduction channel, we measured extracellular receptor currents with transepithelial voltage-clamp recordings while the apical surface of a saccular macula was bathed with solutions containing various concentrations of K+, Na+, or Ca2+. For modest concentrations of a single permeant cation, Ca2+ carried much more receptor current than did either K+ or Na+. For higher cation concentrations, however, the flux of Na+ or K+ through the transduction channel exceeded that of Ca2+. For mixtures of Ca2+ and monovalent cations, the receptor current displayed an anomalous mole-fraction effect, which indicates that ions interact while traversing the channel's pore. These results demonstrate not only that the hair cell's transduction channel is selective for Ca2+ over monovalent cations but also that Ca2+ carries substantial current even at low Ca2+ concentrations. At physiological cation concentrations, Ca2+ flux through transduction channels can change the local Ca2+ concentration in stereocilia in a range relevant for the control of adaptation.

The temperature ranges for growth of Streptococcus mutans GS-5 and S. sobrinus 6715 were found to be very narrow, from about 30 to 47 degrees C, with optimal growth around 37 degrees C. Thus, the organisms showed little potential to grow in the environment outside of the animal host. In contrast wider ranges were found for Enterococcus hirae, S. rattus and S. sanguis. Detailed study of S. mutans GS-5 showed that energetic coupling, reflected in yields of biomass per mol of glucose utilized, were not greatly affected by changes in temperature within the growth range. However, since glycolysis occurred over a wider temperature range (about 10 to 52 degrees C) than growth, yield values dropped to zero at temperatures above or below the growth range. The temperature range for glycolysis could be related to temperature sensitivity of the phosphoenolypyruvate: sugar phosphotransferase system for sugar uptake. F-ATPases were active over a similar range of temperatures, but with a broad optimal range from about 30 to 50 degrees C. Proton permeability of S. mutans increased steadily with temperature in a manner similar to that of other mesophilic bacteria, such as Escherichia coli. Growth of the bacteria in media supplemented with various fatty acids had major effects on proton permeabilities but the effects were not well reflected by changes in growth or glycolysis of the bacteria. The overall conclusions were that S. mutans is a typical mesophile in relation to membrane and catabolic functions but its narrow temperature range for growth is related to temperature sensitivities of anabolic systems.

Acid-adaptive responses could be induced readily in oral lactic-acid bacteria by growing them in batch cultures with excess sugar or more conveniently and rapidly by transferring cells to acidified growth media for the time required for biomass doubling. The response of Streptococcus mutans GS-5 was induced in a progressive rather than all-or-nothing way, and the extent of acid tolerance was inversely related to the pH of the inducing medium over a range from 8.5 to 5. The weak acids fluoride, acetate, or lactate did not measurably enhance acid adaptation, and so the response did not appear to depend primarily on changes in delta pH or the proton motive force across the cell membrane. Transcription and translation to form new proteins did appear to be necessary, as indicated by inhibition of adaptation by rifampin or chloramphenicol and by lack of adaptation by cells suspended in phosphate buffer at pH 5. Streptococcus salivarius and Lactobacillus casei were acid adapted by the rapid method, and the method appeared to be generally useful for oral lactic-acid bacteria. The rapid induction of the response in multiple oral lactic-acid bacteria suggests that it is of general importance for maintaining a diversity of organisms in the oral microbiota, which is regularly subjected to acid stresses.

Parabens were found to inhibit irreversibly glycolysis by the cariogenic dental plaque bacterium Streptococcus mutans GS-5 and to decrease the capacity of the bacterium to lower the pH in dense cell suspensions containing excess glucose. The hierarchy of effectiveness was butyl > propyl > ethyl > methyl paraben. Results of studies of the nature of glycolytic inhibition by butyl paraben indicated that it could act at millimolar concentrations as an irreversible inhibitor of the phosphotransferase system for sugar uptake and was lethal for the bacterium at these same levels. Butyl paraben acted also as a reversible inhibitor of the F-ATPase of the organism. Overall, it appeared that the lethal actions of parabens can be interpreted at least in part as due to irreversible damage to key enzymes, such as those of the phosphotransferase system.

Hair bundles, the mechanically sensitive organelles of hair cells in the auditory and vestibular systems, are elastic structures that are deflected by sound or acceleration. To examine rapid mechanical events associated with mechanoelectrical transduction, we stimulated individual hair bundles with flexible glass fibers and measured their responses with a temporal resolution of 400 microsec. When a hair bundle from the bullfrog's sacculus was abruptly deflected in the positive direction, the bundle's motion in the direction of stimulation was interrupted within the initial few milliseconds by an active movement, or twitch. This response was biphasic, with an initial component in the direction of the stimulus and a second component in the opposite direction. The amplitude and duration of the twitch depended on the bundle's initial position and the size and rise time of the stimulus; the twitch was largest over the range of bundle deflections in which transduction was most sensitive. Under displacement clamp conditions, in which a hair bundle's position was changed and then held constant with negative feedback, the twitch manifested itself as a biphasic force exerted by the bundle. Some hair bundles produced twitches in response to negatively directed stimuli, exhibited stimulus-evoked damped oscillations, or twitched spontaneously. The hair bundle's ability to perform work against an external load and to oscillate in response to stimulation indicates that the bundle could supply feedback for mechanical amplification in vertebrate auditory organs.

Although peracetic acid (PAA) is used widely for cold sterilization and disinfection, its mechanisms of sporicidal action are poorly understood. PAA at high concentrations (5-10%) can cause major loss of optical absorbance and microscopically-visible damage to bacterial spores. Spores killed by lower levels of PAA (0.02-0.05%) showed no visible damage and remained refractile. Treatment of spores of Bacillus megaterium ATCC 19213 with PAA at concentrations close to the lethal level sensitized the cells to subsequent heat killing. In addition, PAA was found to act in concert with hypochlorite and iodine to kill spores. Antioxidant sulfhydryl compounds or ascorbate protected spores against PAA killing. Trolox, a water-soluble form of alpha-tocopherol, was somewhat protective, while other antioxidants, including alpha-tocopherol, urate, bilirubin, ampicillin and ethanol were not protective. Chelators, including dipicolinate, were not protective, but transition metal ions, especially the reduced forms (Co2+, Cu+ and Fe2+) were highly protective. The net conclusions are that organic radicals formed from PAA are sporicidal and that they may act as reducing agents for spores that are normally in a highly oxidized state, in addition to their well known actions as oxidizing agents in causing damage to vegetative cells.

Streptococcus rattus FA-1 and Streptococcus sanguis NCTC 10904 underwent phenotypic acid adaptation in batch cultures toward the end of sugar-fueled growth after the culture pH had dropped to triggering values. The bacteria could be derepressed or induced for arginine deiminase independently of acid adaptation, and arginolysis afforded protection against acid killing over and above that of acid adaptation.

Fluoride is widely used as a highly effective anticaries agent. Although it is felt that its anticaries action is related mainly to effects on mineral phases of teeth and on the process of remineralization, fluoride also has important effects on the bacteria of dental plaque, which are responsible for the acidification of plaque that results in demineralization. The results of recent studies have shown that fluoride can affect bacterial metabolism through a set of actions with fundamentally different mechanisms. It can act directly as an enzyme inhibitor, for example for the glycolytic enzyme enolase, which is inhibited in a quasi-irreversible manner. Direct action seems also to occur in inhibition of heme-based peroxidases with binding of fluoride to heme. The flavin-based peroxidases of many oral bacteria are insensitive to fluoride. Another mode of action involves formation of metal-fluoride complexes, most commonly AlF4-. These complexes are responsible for fluoride inhibition of proton-translocating F-ATPases and are thought to act by mimicking phosphate to form complexes with ADP at reaction centers of the enzymes. However, the actions of fluoride that are most pertinent to reducing the cariogenicity of dental plaque are those related to its weak-acid character. Fluoride acts to enhance membrane permeabilities to protons and compromises the functioning of F-ATPases in exporting protons, thereby inducing cytoplasmic acidification and acid inhibition of glycolytic enzymes. Basically, fluoride acts to reduce the acid tolerance of the bacteria. It is most effective at acid pH values. In the acidic conditions of cariogenic plaque, fluoride at levels as low as 0.1 mM can cause complete arrest of glycolysis by intact cells of Streptococcus mutans. Overall, the anticaries actions of fluoride appear to be complex, involving effects both on bacteria and on mineral phases. The antibacterial actions of fluoride appear themselves to be complex but to be dominated by weak-acid effects.

Fluoride and a variety of other weak acids acted to reduce reversibily the acid tolerance of glycolysis by intact cells of Streptococcus mutans GS-5 as shown by higher final pH values in acid-drop experiments with glucose in excess. The order of effectiveness was fluoride > indomethacin > ibuprofen > ketoprofen > salicylate > sorbate > cinnamate > p-hydroxybenzoate > benzoate > ascorbate. Only fluoride also acted as an inhibitor of the glycolytic enzyme enolase. However, enolase in permeabilized cells was also inhibited by acidification with a sharp drop-off in activity between pH 6 and 5. It was proposed that the weak acids, including fluoride, acted to reduce glycolytic acid tolerance by enhancing cytoplasmic acidification and thereby inhibiting enzymes such as enolase. The potencies of the acids could not be predicted accurately from knowledge of pKa values, octanol-water partition coefficients, and molecular weights. It was concluded that their modes of action in acid sensitization involved perturbations of membrane function in addition to their acting as transmembrane carriers of protons. Methylparaben (methyl ester of p-hydroxybenzoate) was also a sensitizer but was less effective than the parent acid.

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.