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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.

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.

The basis for genotypic and phenotypic variation within Streptococcus mutans is poorly understood but the availability of the genome sequence of strain UA159 provides an opportunity for comparative studies. Genomic DNA prepared from nine strains of S. mutans was used to probe a microarray consisting of oligonucleotides representing 1948 open reading frames of S. mutans UA159. A total of 385 (20%) of the UA159 open reading frames were found to be absent from one or more of the test strains. Absent open reading frames frequently occurred in blocks of adjacent open reading frames and represented regions previously experimentally detected by polymerase chain reaction, predicted genomic islands and insertion sequence elements as well as novel open reading frames. Approximately half appear to involve foreign DNA acquired by horizontal transmission. The results indicate the existence of distinct core and dispensable genomes and may help explain the phenotypic and genotypic variation within S. mutans.

The genome of Streptococcus mutans UA159 contains two phospho-beta-glucosidase genes, bglA and celA, which occur in operon-like arrangements along with genes for components of phosphotransferase transport systems and a third phospho-beta-glucosidase encoded by the arb gene, which does not have its own associated transport system but relies on uptake by the bgl or cel systems. Targeted inactivation of each of the phospho-beta-glucosidase genes revealed that bglA is involved in aesculin hydrolysis, celA is essential for utilisation of cellobiose, amygdalin, gentobiose and salicin, and arb is required for utilisation of arbutin. Inactivation of genes for the phosphotransferase systems revealed an overlap of specificity for transport of beta-glucosides and also indicated that further, unidentified transport systems exist. The cel and arb genes are subject to catabolite repression by glucose, but the regM gene is not essential for catabolite repression. Screening a collection of isolates of S. mutans revealed strains with deletions affecting the msm, bgl and/or cel operons. The phenotypes of these strains could largely be explained on the basis of the results obtained from the knockout mutants of S. mutans UA159 but also indicated the existence of other pathways apparently absent from UA159. The extensive genetic and phenotypic variation found in beta-glucoside metabolism indicates that there may be extensive heterogeneity in the species.

Many isolates of Streptococcus mutans lack the ability to ferment melibiose and other sugars. We previously reported that this was commonly due to a chromosomal deletion and, in the present study, sequence information from the S. mutans genome project was used to design PCR primers to explore the nature and extent of the deletion. In all melibiose-negative strains examined, there was an incomplete insertion element, ISSmu3, in place of the 18-kb stretch of chromosome encoding the msm and gal operons. Strains that were also unable to utilise β-glucosides were found to have a separate 4 kb deletion in the bgl regulon that is proposed to be due to homologous recombination between two short stretches of identical sequence. The evidence is consistent with all the melibiose-negative strains examined being derived from a common ancestor.

Porphyromonas gingivalis produces virulence factors which can modify the molecular and cellular components of the host immune response. In the present work we investigated the role of specific virulence factors from P. gingivalis in the induction of apoptosis in Jurkat T cells. P. gingivalis culture supernatants mimicked the effect of butyric acid on T-cell apoptosis and this effect was associated with an increase in histone H4 acetylation. A role for proteases was excluded in experiments which demonstrated that neither protease inhibitors nor use of P. gingivalis mutants defective in protease synthesis had any effect on the stimulation of T-cell apoptosis in this system.

The ability of a range of potential inhibitors to affect the catalytic activity or binding of dextran by a glucosyltransferase (GTF-I) that synthesises insoluble α1,3-linked glucan was tested. Acarbose, deoxynojirimycin, N-dodecyldeoxynojirimycin and Tris, which are thought to interfere with the active site of the enzyme of GTF and related glycosidases, inhibited glucan synthesis but not glucan binding. Tris was found to act as a competitive inhibitor of GTF-I. The effectiveness of the active site inhibitors was not altered by immobilisation of GTF-I on saliva-coated hydroxyapatite. In contrast, three amine hydrofluorides were markedly less effective against immobilised GTF than soluble GTF. The pH of the reaction mixture was found to have a strong influence on inhibition by acarbose, Tris and amine hydrofluorides, a finding that is of direct relevance to use of inhibitors in vivo.

The activity of glucosyltransferases (GTF), a group of enzymes that synthesize water-soluble and -insoluble glucans from sucrose, significantly contributes to the cariogenicity of mutans streptococci. Streptococcus downei produces four glucosyltransferases, GTFI, which produces insoluble glucan, and GTFS, GTFT, and GTFU, which synthesize soluble glucans. We have previously reported that inactivation of gtfS results in altered adherence and have now examined its interaction with other enzymes by constructing mutants which were gtfS, gtfS/gtfT, gtfS/gtfI and gtfI. The mutants were tested for their ability to accumulate on wires and on plastic microtiter trays in the presence of sucrose. The gtfS mutant displayed a reduced ability to adhere compared to the wild type but there was no further reduction of adherence in a gtfS/gtfT mutant. In contrast, the gtfS/gtfI double mutant showed a drastic reduction in adherence and when gtfI alone was inactivated, bacteria were unable to adhere to a hard surface. The results confirmed that insoluble glucan is required for strong adherence to a smooth surface but that the amount and structure of this glucan is dependent upon the availability of soluble glucans to act as primer molecules.

Attempts to produce resin composite with antibacterial properties by incorporation of an antibacterial agent such as chlorhexidine have been reported, but problems can arise due to release of the inhibitory agent from the composite. Such problems may include toxic effects, influence on mechanical properties, and loss of effectiveness. A new monomer, methacryloyloxydodecylpyridinium bromide (MDPB), was synthesized by combining an antibacterial agent and methacryloyl group. The monomer was incorporated into resin composite to develop a non-releasing antibacterial composite. The ability of composite incorporating MDPB to inhibit growth and plaque accumulation by Streptococcus mutans in vitro was assayed, elution of antibacterial components from the material was investigated, and the influence of incorporation of MDPB on the mechanical properties of composite was studied. Uncured MDPB revealed antibacterial activity against S. mutans and six other species of oral streptococci, with the minimum inhibitory concentration for S. mutans being comparable with that of triclosan. After composite incorporating MDPB was cured, no elution of the antibacterial components was observed from the material, even after 90 days' immersion in water or other solvents. Growth of S. mutans on agar under specimens of MDPBcontaining composite was inhibited compared with controls. In a bacterial accumulation study, S. mutans accumulated to a lesser degree on the surface of composite incorporating MDPB (p < 0.05) than on control. Incorporation of MDPB had no significant influence on the mechanical properties of the composite.

The introduction of techniques for the manipulation of DNA in vitro has had an enormous impact on progress in every area of biological research. In the case of oral microbiology, the first reports on the application of molecular genetics to streptococci started to appear in the early 1980s, and it is now more than 10 years since the first paper describing cloning and expression of a gene from Streptococcus mutans in another bacterium was published. The purpose of this review is not to provide a resumé of all the work that has been done on the molecular biology of oral bacteria; indeed, we are approaching the stage when a comprehensive survey of work on all oral bacteria is no longer feasible--for instance, over 40 genes have now been cloned and sequenced from S. mutans alone--but rather to illustrate examples of how the new techniques have been applied to give novel approaches and insights into old problems, or expand into new directions. The great majority of published work so far relates to streptococci, and many aspects have been covered in previous review articles [Curtiss, 1985; Russell, 1991], the most recent thorough review being that by Kuramitsu [1993]. Discussion of molecular biology is now so all-pervasive that most readers will be familiar with many of the basic terms, but it may be worth clarifying some of the main points during the course of this article. For total novices, the short book by Brown [1990] forms a useful introduction. A number of recent articles have reviewed recent research on plaque formation [Kolenbrander and London, 1993] and the mutans streptococci in particular [Loesche, 1986], while Freedman et al. [1981] and Tanzer [1992] have summarised the information derived from a genetic approach to oral microbiology before the advent on in vitro manipulation of DNA.

Genome sequencing and other molecular techniques for the identification and characterisation of bacteria have provided us with a vast amount of new information, both on the detailed properties of certain species and on the total plaque microflora. Thus, from genomics, we have gained many valuable insights into the biology of Streptococcus mutans and the diversity within the species, while molecular techniques of identification have prompted a heightened awareness of the importance of species other than S. mutans to the caries process. S. mutans remains deserving of detailed study, both as an exemplar of an acidogenic, aciduric plaque bacterium and because it shows a strong association with caries. Nevertheless, its value as a target for inhibitors and its useful- ness as an indicator organism for monitoring the value of caries prediction or prevention need to be evaluated in the light of new discoveries.

Ethyleneglycol dimethacrylate monomers have been previously reported to stimulate the growth of certain caries-associated bacteria on the basis of turbidity measurements. To elucidate the detail of this effect, we examined the influence of resin monomers on the growth of Streptococcus sobrinus and Streptococcus sanguis by determination of bacterial numbers (colony-forming units), morphological observation, and chemical analysis. Although the absorbance values in the stationary phase of bacterial suspension were increased in the presence of ethyleneglycol monomers, no significant differences were observed for bacterial numbers throughout the incubation period. Scanning electron microscopy observation revealed the formation of sparse vesicular material surrounding bacterial cells when incubated with ethyleneglycol monomers, and these products were proved to be resin polymers. The results demonstrate that the apparent biomass increase during incubation with ethyleneglycol monomers is due not to promotion of bacterial multiplication, but to the polymerization of resin monomers to form vesicular structures attached to cells.

The polymerizable monomer methacryloyloxydodecylpyridinium bromide (MDPB) shows antibacterial activity when immobilized in a resin-based material. In this study, the antibacterial effect of a dentin primer incorporating MDPB was investigated. The influence of incorporation of MDPB on bond strength to dentin and on the curing performance of the adhesive system was also evaluated. Experimental primers were prepared by addition of MDPB into a proprietary primer at 1, 2, or 5%. Antibacterial effects of experimental primers were compared with those of control primer and two other proprietary primers by an agar disc-diffusion method and bactericidal activity test. Experimental primers produced greater inhibition zones against Streptococcus mutans, Actinomyces viscosus, and Lactobacillus casei than any of three proprietary primers, and inhibition increased as the concentration of MDPB was increased. Bactericidal activity of MDPB-containing primers against Streptococcus mutans was greater than those of the other three primers, with incorporation of MDPB at 5% showing complete killing of bacteria after 30 s contact. No decrease in tensile bond strength was observed for materials containing MDPB. On the contrary, the primer incorporating 1 and 2% MDPB showed higher bond strength than all the others, including the control (p < 0.05). When the degree of conversion of the complex of primer and adhesive resin was determined with Fourier Transform Infrared Spectroscopy, there were no significant differences between any of the experimental primers and the control (p > 0.05). These results indicate that incorporation of the antibacterial monomer MDPB enhanced the antibacterial effect of a proprietary dentin primer before curing, and had no adverse influence on bond strength to dentin and curing of the adhesive system.

Objectives: Previously, we have reported that dental composite incorporating the new monomer methacryloy-loxydodecylpyridinium bromide (MDPB) showed no release of antibacterial components after being cured but still exhibited antibacterial activity against Streptococcus mutans on its surface. In this study, in order to elucidate the mechanism of the antibacterial effect of immobilized MDPB, the bactericidal activity of MDPB polymer in water-soluble and -insoluble form was investigated, and the effect of MDPB polymer on bacterial attachment was estimated. Methods: Solutions of homo-polymer of MDPB and co-polymer of MDPB with acrylamide were prepared and the viability of seven major oral streptococci was determined after incubation with each polymer solution. For the estimation of bactericidal activity of insolubilized MDPB polymer, bacteria were kept in contact with cured unfilled Bis-GMA-based resin discs with or without MDPB, and the recovery of viable cells was measured. Attachment of streptococci to cured resin discs with or without MDPB was also compared using radiolabelled bacteria. Results: Water-soluble homo-polymer of MDPB and co-polymer with acrylamide showed bactericidal activity against oral streptococci. However, cured resin incorporating MDPB, which is in water-insoluble form, had little bactericidal activity. Attachment of streptococci, including species which are early colonizers in dental plaque formation, to the cured resin containing MDPB was significantly less than to the control without MDPB. Conclusions: These results indicate that the bactericidal activity of MDPB polymer is reduced after immobilization, but MDPB on the surface of a resin-based material still shows a bacteriostatic effect and antiadhesion property against oral streptococci.

Streptococcus mutans is frequently identified on the basis of phenotypic characteristics such as the ability to ferment carbohydrates. The usefulness of some of these identification tests may be limited in the case of isolates which are atypical with regard to their fermentation properties. We previously identified isolates of S. mutans which were unable to ferment melibiose, a characteristic which is included in some typing schemes. In all of these isolates there was a large chromosomal deletion which included the multiple sugar metabolism (msm) operon which encodes several genes involved in the uptake and metabolism of a number of sugars including melibiose. In the present study, sugar fermentation tests, ribotyping, colony hybridisation with DNA probes and polymerase chain reaction (PCR) were used to investigate the relatedness of these atypical isolates. The PCR and colony hybridisation procedures were based on amplification and detection of two genes: the wapA gene which encodes a surface protein found in all S. mutans strains and the gtfA gene which lies within the msm operon. The colony hybridisation and PCR results confirmed loss of the gtfA gene in the melibiose-negative isolates. Three new melibiose-negative isolates were also identified, but in only 2 of these was the gtfA gene absent, the third did not appear to have lost this region of the chromosome. Biotyping, as well as ribotyping based on an EcoRI digest of chromosomal DNA, revealed that the melibiose-negative isolates fell into a number of distinct groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The range of substrates transported by the sugar-binding protein-dependent msm (multiple sugar metabolism) system of S. mutans was investigated. By determining the ability of unlabeled sugar to compete with radiolabeled melibiose transport, we have demonstrated that the transported sugars included a number of carbohydrates structurally related to raffinose. A model accommodating these results has been devised which accounts for the sugars transported by the msm transport system. Competition with radiolabeled melibiose transport indicated sucrose to be an msm substrate. This was confirmed by examination of uptake of radiolabeled sucrose in scrAB mutants lacking the sucrose-specific phosphotransferase system.

Reports of the isolation of streptococci from dental abscesses have shown an association of the "S. milleri" group with such lesions. There has been considerable confusion regarding the taxonomy of these organisms, but the milleri group has recently been reclassified into three distinct species: Streptococcus anginosus, Streptococcus constellatus, and Streptococcus intermedius. In this study, 45 samples from dental periapical abscesses were examined. Milleri group streptococci were isolated from 16 patients (37%), 15 being identified as S. anginosus and one as S. intermedius. In one patient, S. anginosus was isolated in pure culture, and it would appear that this is the predominant species of milleri group streptococci associated with periapical abscesses.

Isolates from a collection of phenotypically melibiose-negative (Mel-) Streptococcus mutans from widely-scattered geographical locations were examined and found to lack the activities of the enzymes α-galactosidase and a-glucosidase, in addition to being unable to transport melibiose. Cloned fragments of S. mutans DNA from the region of the chromosome carrying the genes for a-galactosidase ( aga), sucrose phosphorylase ( gtfA), and dextranglucosidase ( dexB), as well as the genes encoding components of the binding-protein-dependent uptake system for raffinose and melibiose, were used in hybridization studies for investigation of the genetic basis of the Mel- phenotype. A region of at least 12 kilobases, containing all the above genes, was found to be deleted from the chromosome of the Mel- strains. It appears that this region of the chromosome is not essential for survival of S. mutans in the oral cavity. The reason for the frequent occurrence of deletions, as opposed to other forms of mutational events, is unknown.

Eleven strains of Streptococcus sanguis biotype I carrying peritrichous fibrils and 4 strains with lateral tufts of fibrils were screened for crossreactivity with Antigen B (AgB), a protein with a molecular weight of 190,000, isolated from Streptococcus mutans Ingbritt. An antigen of similar molecular weight, which cross-reacted serologically with AgB was detected by immunodiffusion, Western blotting and dot immuno-binding in strains of S. sanguis biotype I with peritrichous fibrils but not in tufted strains. One strain, S. sanguis LGR2 carried dense fibrils (5+ density) and the gold label appeared to be associated with the fibrils. However, over all the strains there was no correlation between the density of fibrils and the amount of AgB labelling. Immunonegative staining confirmed the absence of AgB cross reactivity in the tufted strains of S. sanguis I, although the gold labelled the tuft fibrils non-specifically. Antigen B was located on the cell surface of S. mutans Ingbritt and the label extended 45.9±9.9 nm from the cell wall although the strain was non-fibrillar.