Alkali production associated with malolactic fermentation by oral streptococci and protection against acid, oxidative, or starvation damage.

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.

Malolactic fermentation by Streptococcus mutans.

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.

Enhanced acid resistance of oral streptococci at lethal pH values associated with acid-tolerant catabolism and with ATP synthase activity.

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.

Co-operative inhibition by fluoride and zinc of glucosyl transferase production and polysaccharide synthesis by mutans streptococci in suspension cultures and biofilms.

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.

Antimicrobial actions of benzimidazoles against the oral anaerobes Fusobacterium nucleatum and Prevotella intermedia.

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.

Multi-target antimicrobial actions of zinc against oral anaerobes.

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.

[A comparison of the activities of membrane-bound, proton translocating ATPases between Streptococcus mutans fluoride-resistant and their parent strains].

PURPOSE: To figure out the reason for increased acid tolerance of Ingbritt-FR, the fluoride-resistant strain of Streptococcus mutans Ingbritt, by determining and comparing the H(+)-ATPase activities of both fluoride-resistant and their parental strains. METHODS: The permeabilized cells of S. mutans Ingbritt and Ingbritt-FR were prepared by treating them with 10% toluene and then two cycles of freezing and thawing. The permeabilized cells were used for ATPase assay by adding them to the reaction mixture which contained 50mM Tris-maleate buffer (pH 6.0), 10 mM MgSO4 and 5 mM ATP. ATPase activity was assessed by measuring inorganic phosphate released from ATP hydrolysis.Two-way ANOVA was used for statistical analysis. RESULTS: The activities of H(+)-ATPase of Ingbritt-FR were 308.48, 136.67, and 82.80 micromol Pi/g cell dry weight/min, at 10, 20, and 60 minutes respectively, significantly higher than those of their parent strain: 104.77, 64.69, and 30.7 (P<0.01). The enzyme activities were decreasing with time. CONCLUSIONS: The higher ATPase activity of fluoride-resistant mutant of S. mutans Ingbritt may account for the increased acid tolerance of this organism, and the increment of ATPase activity and acid tolerance of fluoride-resistant strain is likely to increase the cariogenic potential of S. mutans after fluoride-resistant mutation.

[Comparison of effect on S.mutans producing acid ability between organic weak acid and fluoride].

OBJECTIVE: To figure out the effects of organic weak acids, benzoate, citric acid and malic acid on the main cariogenic bacteria S.mutans Ingbritt. To evaluate the possibility of above weak acids as anti-caries reagents. METHODS: S.mutans Ingbritt was inoculated into tryptic soy broth containing different concentrations of fluoride, benzoate, citric acid and malic acid, grew in anaerobic conditions at 37 degrees centigrade for 48h. The final pH values of suspension were measured. RESULTS: Fluoride had best inhibitory effect on S.mutans Ingbritt in low concentrations, whereas other 3 weak acids, benzoate, citric acid and malic acid could inhibit S.mutans Ingbritt in a higher concentrations. The order of effectiveness was fluoride>malic acid>benzoate>citric acid. CONCLUSION: Fluoride has the best inhibitory effect on S.mutans producing acid ability. Though weak acids benzoate, malic acid and citric acid in high concentration had different inhibitory effects on S.mutans Ingbritt, however high concentration of organic acids can make enamel demineralization.