Effects of the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on bacterial viability and metabolism.

The antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) is a strong bactericide when unpolymerized and has the potential to be utilized in various resinous biomaterials. To analyze the antibacterial characteristics of this monomer in detail, the ability of high concentrations of unpolymerized MDPB to kill Streptococcus mutans in planktonic or biofilm forms within a short time-period of contact, and the inhibitory effects of low concentrations of MDPB on the metabolic function of S. mutans, were examined. High concentrations of MDPB showed effective killing of planktonic and biofilm S. mutans cells within 60 s, and complete killing was obtained by contact with 1,000 µg ml(-1) of MDPB for 60 s. At a concentration of 4-8 µg ml(-1) , MDPB demonstrated growth inhibition, inducing elongation of the lag phase and of the doubling time, when the bacterial number was low. Inhibition of the production of acid from S. mutans by 8 µg ml(-1) of MDPB may have been caused by the inhibition of lactate dehydrogenase activity. At high concentrations, MDPB is lethal to both planktonic and biofilm forms of S. mutans in a short time-period, and at low concentrations, MDPB inhibits metabolic enzymatic activity.

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

Changing concepts in caries microbiology.

Concepts and beliefs about the cause of dental caries have evolved over many centuries, with the involvement of microorganisms being recognized since the late 1800s. A main thrust of enquiry since then has been to tackle the question of the relative importance of different bacteria in the disease and this article will consider how technical advances in our ability to identify, cultivate and count different species has influenced our understanding. Over the last decade, molecular biological approaches have had a major impact on views of the relative contribution of particular species of plaque bacteria to the caries process. At a more detailed level, molecular genetic studies of species such as Streptococcus mutans have given new insights into the way in which particular genes and the functions that they encode may affect virulence.

Distribution and activity of IS elements in Streptococcus mutans.

Insertion sequence (IS) elements are widely distributed selfmobilizing genetic elements that can affect genetic changes by integrating into the chromosome, mediating genetic rearrangements and facilitating horizontal gene transfer. Three members of the IS3 family have been identified in Streptococcus mutans: IS199 was discovered by its transposition while ISSmu1 and ISSmu2 were predicted from the genome sequence of S. mutans UA159. Sixty-eight strains of S. mutans were screened by PCR for carriage of the IS elements IS199, ISSmu1 and ISSmu2. Twenty-seven (30%) of the strains were positive for IS199, 33 (49%) were positive for ISSmu1 and 51 (75%) carried ISSmu2. All three IS were found in 11 strains, two were found in various combinations in 31, one was found in 16, while only 10 strains had none of the three IS for which we screened. ISSmu1 was demonstrated to be capable of transposition at a low frequency but no transposition of ISSmu2 was observed.

Determination of bactericidal activity of antibacterial monomer MDPB by a viability staining method.

In this study, the bactericidal activity of antibacterial monomer MDPB (12-methacryloyloxydodecylpyridinium bromide) against Streptococcus mutans was tested by a rapid method for monitoring viability. To S. mutans culture containing fluorescence staining solution that distinguishes live from dead cells, MDPB was added at a concentration of 250, 100, 50, or 10 microg/ml. Bacterial cells were observed by fluorescence microscopy and the percentage of dead cells was calculated. After 10, 20, or 30 minutes' contact with MDPB, the live/dead ratio was measured by fluorometry and viable counts (CFU) determined by the conventional plating method. Viability staining revealed that MDPB exhibited significant bactericidal effects at 50 microg/ml or greater (ANOVA, Fisher's PLSD test), and complete killing of the cells at 250 microg/ml of MDPB was demonstrated in conjunction with a plating method. The staining method thus provided a sensitive means to determine loss of viability, and indicated the strong killing effects of MDPB on S. mutans.

Dispensable genes and foreign DNA in Streptococcus mutans.

A range of properties, including the ability to utilize various sugars, bind macromolecules and produce mutacins, are known to vary in their occurrence in different strains of Streptococcus mutans. In addition, insertion-sequence elements show a limited distribution and sequencing of the genome of S. mutans UA159 has revealed the presence of putative genomic islands of atypical base composition indicative of foreign DNA. PCR primers flanking regions suspected of having inserted DNA were designed on the basis of the genome sequence of S. mutans UA159 and used to explore variation in a collection of 39 strains isolated in various parts of the world over the last 40 years. Extensive differences between strains were detected, and similar insertion/deletion events appear to be present in the genomes of strains with very different origins. In two instances, insertion of foreign DNA appears to have displaced original S. mutans genes. Together with previous results on the occurrence of deletions in genes associated with sugar metabolism, the results indicate that S. mutans has a core genome and a dispensable genome, and that dispensable genes have become widely distributed through horizontal transfer.

Conserved repeat motifs and glucan binding by glucansucrases of oral streptococci and Leuconostoc mesenteroides.

Glucansucrases of oral streptococci and Leuconostoc mesenteroides have a common pattern of structural organization and characteristically contain a domain with a series of tandem amino acid repeats in which certain residues are highly conserved, particularly aromatic amino acids and glycine. In some glucosyltransferases (GTFs) the repeat region has been identified as a glucan binding domain (GBD). Such GBDs are also found in several glucan binding proteins (GBP) of oral streptococci that do not have glucansucrase activity. Alignment of the amino acid sequences of 20 glucansucrases and GBP showed the widespread conservation of the 33-residue A repeat first identified in GtfI of Streptococcus downei. Site-directed mutagenesis of individual highly conserved residues in recombinant GBD of GtfI demonstrated the importance of the first tryptophan and the tyrosine-phenylalanine pair in the binding of dextran, as well as the essential contribution of a basic residue (arginine or lysine). A microplate binding assay was developed to measure the binding affinity of recombinant GBDs. GBD of GtfI was shown to be capable of binding glucans with predominantly alpha-1,3 or alpha-1,6 links, as well as alternating alpha-1,3 and alpha-1,6 links (alternan). Western blot experiments using biotinylated dextran or alternan as probes demonstrated a difference between the binding of streptococcal GTF and GBP and that of Leuconostoc glucansucrases. Experimental data and bioinformatics analysis showed that the A repeat motif is distinct from the 20-residue CW motif, which also has conserved aromatic amino acids and glycine and which occurs in the choline-binding proteins of Streptococcus pneumoniae and other organisms.

A novel glucan-binding protein with lipase activity from the oral pathogen Streptococcus mutans.

Streptococcus mutans produces extracellular glucosyltransferases (GTFs) that synthesize glucans from sucrose. These glucans are important in determining the permeability properties and adhesiveness of dental plaque. GTFs and the GbpA glucan-binding protein are characterized by a binding domain containing a series of 33-amino-acid repeats, called 'A' repeats. The S. mutans genome sequence was searched for ORFs containing 'A' repeats, and one novel gene, gbpD, which appears to be unique to the mutans group of streptococci, was identified. The GbpD sequence revealed the presence of three 'A' repeats, in the middle of the protein, and a novel glucan-binding assay showed that GbpD binds to dextran with a K(D) of 2-3 nM. Construction of truncated derivatives of GbpD confirmed that the 'A' repeat region was essential for binding. Furthermore, a gbpD knockout mutant was modified in the extent of aggregation induced by polymers derived from sucrose. The N-terminus of GbpD has a signal sequence, followed by a region with no homologues in the public databases, while the C-terminus has homology to the alpha/beta hydrolase family (including lipases and carboxylesterases). GbpD contains the two regions typical of these enzymes: a GxSxG active site 'lipase box' and an 'oxyanion hole'. GbpD released free fatty acids (FFAs) from a range of triglycerides in the presence of calcium, indicating a lipase activity. The glucan binding/lipase bifunctionality suggested the natural substrate for the enzyme may be a surface macromolecule consisting of carbohydrate linked to lipid. The gbpD mutant was less hydrophobic than wild-type and pure recombinant GbpD reduced the hydrophobicity of S. mutans and another plaque bacterium, Streptococcus sanguinis. GbpD bound to and released FFA from lipoteichoic acid (LTA) of S. sanguinis, but had no effect on LTA from S. mutans. These results raise the intriguing possibility that GbpD may be involved in direct interspecies competition within the plaque biofilm.

Antibacterial activity of bactericide-immobilized filler for resin-based restoratives.

This study examined the antibacterial activity of prepolymerized resin filler, in which the bactericide quaternary ammonium was immobilized. The experimental filler was prepared by grinding prepolymerized resin blocks of methacrylate monomers, silica particles, and the antibacterial monomer methacryloyloxydodecylpyridinium bromide (MDPB). The number of Streptococcus mutans after incubation for 18h in contact with the experimental filler with or without protein adsorption by saliva treatment was determined, and adherence of bacteria to the filler surface was evaluated by scanning electron microscopy. Elution of unpolymerized MDPB from the filler and its influence on bacterial growth were also investigated. The growth of S. mutans was completely inhibited by contact with the experimental filler without saliva treatment. Although the effects were attenuated, the saliva-treated filler still exhibited growth inhibition at >99.9%. Less bacteria attached to the experimental filler than the control filler without MDPB, indicating that the reduction in bacterial number after contact with the experimental filler was not due to bacterial adherence to the particles. Unpolymerized MDPB at 1 microg/ml was eluted from the filler particles but was confirmed to have little effect on bacterial growth. The results indicate that the bactericide-immobilized filler containing MDPB shows significant bacteriostatic effects without releasing antibacterial components, and is useful for incorporation into various resin-based restoratives.

Comparison of antibacterial activity of simplified adhesive systems.

PURPOSE: To determine and compare the intrinsic antibacterial activity of commercially available simplified adhesive systems. MATERIALS AND METHODS: The antibacterial activity of five self-etching/priming one-step adhesives and three priming/bonding adhesives against Streptococcus mutans, Lactobacillus casei, and Actinomyces viscosus was assessed by the agar disc-diffusion test and determination of the minimum inhibitory/bactericidal concentration (MIC/MBC). Twenty microl of each adhesive was impregnated into a paper disc and placed on an agar plate inoculated with a bacterial suspension, with or without light-irradiation. The size of inhibition zones produced around the specimens was measured after 48 hours of incubation. The MIC values were measured by serial microdilution assays, visually examining the growth of bacteria after incubation with each adhesive for 24-48 hours. The subcultures were made on BHI agar plates from the wells showing no visible growth of bacteria, and the MBC values were determined based on production of colonies. Two primers in two-step self-etching systems and an experimental primer containing chlorhexidine were tested for reference. The results of disc-diffusion tests were analyzed by ANOVA and Fisher's PLSD test. RESULTS: The size of inhibition zones produced by adhesives varied among the brands. None of the available commercial adhesives showed significant inhibition against all three of the bacterial species tested. Light-irradiation reduced the size of inhibition zones for a few materials, but a similar trend to the effectiveness of non-irradiated specimens was observed. The antibacterial activity of commercial products determined in terms of MIC/MBC values were different from the results by disc-diffusion tests. The self-etching adhesives with low pH were not necessarily more effective than priming/bonding solutions, and no significant relationships between the acidity and antibacterial effects were found. Compared with commercial products, an experimental primer containing chlorhexidine produced significantly larger inhibition zones against all species (P < 0.05) and exhibited greater bacteriostatic/bactericidal activity, demonstrating lower MIC/MBC values.