ABSTRACT
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.
Subject(s)
Gene Deletion , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial , Mutation , Streptococcus mutans/genetics , Animals , Bacterial Proteins/genetics , Biofilms/growth & development , DNA Barcoding, Taxonomic , Genetic Fitness , Genomics , Hydrogen-Ion Concentration , Mouth/microbiology , Oxidative Stress/genetics , Rats , Streptococcus mutans/growth & development , Streptococcus mutans/pathogenicityABSTRACT
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.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enzyme Inhibitors/pharmacology , Glycolysis/drug effects , Omeprazole/analogs & derivatives , Omeprazole/pharmacology , Streptococcus/drug effects , 2-Pyridinylmethylsulfinylbenzimidazoles , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/metabolism , Biofilms/drug effects , Cell Membrane Permeability , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Hydrogen-Ion Concentration , Lansoprazole , Mouth/microbiology , Omeprazole/chemistry , Omeprazole/metabolism , Phosphoenolpyruvate Sugar Phosphotransferase System/antagonists & inhibitors , Proton Pump Inhibitors , Proton-Translocating ATPases/antagonists & inhibitors , Streptococcus/enzymologyABSTRACT
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.
Subject(s)
Anti-Infective Agents/pharmacology , Biofilms/drug effects , Streptococcus/drug effects , Zinc/pharmacology , Acids/antagonists & inhibitors , Adenosine Triphosphatases/antagonists & inhibitors , Alkalies/antagonists & inhibitors , Anti-Infective Agents/administration & dosage , Arginine/metabolism , Cell Membrane Permeability/drug effects , Chelating Agents/pharmacology , Citric Acid/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/pharmacology , Glycolysis/drug effects , Humans , Hydrolases/antagonists & inhibitors , Mouth/microbiology , Phosphoenolpyruvate Sugar Phosphotransferase System/drug effects , Streptococcus/classification , Streptococcus/physiology , Streptococcus mutans/drug effects , Streptococcus mutans/physiology , Urease/antagonists & inhibitors , Zinc/administration & dosageABSTRACT
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.
