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Defining antibody targets in Streptococcus oralis infection.

Immunoblotting of sera from 12 neutropenic patients with Streptococcus oralis septicemia and 18 patients with endocarditis due to viridans group streptococci revealed immunodominant S. oralis antigens at 85 and 180 kDa. The former cross-reacted with a mouse monoclonal antibody to hsp90. The latter was identified by sequencing positive clones obtained by screening a genomic expression library of S. oralis with pooled sera from patients who had been infected with S. oralis. Antibody eluted from one of these clones reacted with the 180-kDa antigen of S. oralis. Southern blotting confirmed the origin of the clone from S. oralis. The derived amino acid sequence showed 76.2% homology with the PAc protein precursor of Streptococcus mutans and 73.8% homology with the SpaA protein precursor of Streptococcus sobrinus. Epitope mapping of the derived amino acid sequence with sera from patients with viridans group streptococcal endocarditis delineated nine epitopes. Peptides 1 (TMYPNRQPGSGWDSS) and 2 (WYSLNGKIRAVDVPK), representing two of these epitopes, and peptide 3 (YEVEKPLEPAPVAPS), representing the repeat proline region, were synthesized. These three peptides were used to screen a phage antibody display library derived from a patient who had recovered from S. oralis infection. Two of the human recombinant antibodies produced (SORAL 3 and SORAL 4 against peptide 3) and a human recombinant antibody (B3.7) against the conserved epitope (LKVIRK) of hsp90 gave statistically significant protection, compared with control groups, in a mouse model of lethal S. oralis infection.

Amino Acid Sequence↗

The caries inhibitory effects of GOS-sugar in vitro and in rat experiments.

The caries inhibitory activity of GOS-sugar (panose- and maltose-rich sugar mixture) was examined and compared with that of sucrose, maltose, or glucose in in vitro and in vivo experiments. Streptococcus mutans MT8148R (serotype c) and Streptococcus sobrinus 6715 (g) did ferment GOS-sugar and produce acid in a similar way as with maltose and glucose. However, GOS-sugar could not be a substrate for the glucosyltransferases (GTases) of these mutans streptococci to synthesize the water-insoluble glucan. Also, it significantly inhibited not only the synthesis of water-insoluble glucan from sucrose by the crude GTases but also the sucrose-dependent adherence of these cells to a glass surface. In particular, adherence of growing cells of 6715 was markedly inhibited by the presence of GOS-sugar. GOS-sugar was found to induce significant but minimal dental caries in SPF rats infected with either MT8148R or 6715. Furthermore, the replacement of half of the dietary sucrose content with GOS-sugar resulted in a significant reduction of caries development in rats infected with strain 6715.

Animals↗

Intracellular and extracellular pHs of Streptococcus mutans after addition of acids: loading and efflux of a fluorescent pH indicator in streptococcal cells.

A pH-sensitive fluorescent dye, 2', 7'-bis-(2-carboxyethyl)-5 and 6-carboxyfluorescein (BCECF), was used to determine intracellular pH (pH(in)). The efflux of BCECF loaded into oral streptococcal cells was determined after incubation of the cells at 35 degrees C for 20 min in the presence and absence of glucose. In the absence of glucose, the fluorescence of intracellular BCECF in Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius and Streptococcus sobrinus decreased only very slightly, indicating that the dye could be useful for pH(in) determination. In the presence of glucose, however, the fluorescence decreased by 57%. Thus, the pH(in) of S. mutans cells was measured by the BCECF method in the absence of glucose at various acidic pH levels by adding lactic, acetic and hydrochloric acids to the cell suspensions. The pH(in) was almost equal to the extracellular pH (pH(out)) for pH(out) values of between 8 and 5, indicating that protons permeated easily across the S. mutans cell membrane. For pH(out) between 5 and 4, pH(in) was constant at around 5, suggesting that the cell membrane was impermeable to protons, or that a cytoplasmic buffering system functioned. pH(in) decreased at pH(out) values of < 4. The constant pH(in) at acidic pH(out) levels could protect intracellular components, such as proteins, against acidification by sugar fermentation.

Acetic Acid↗

Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II), in Escherichia coli.

Antigen P1, also called I/II, is one of the most abundant cell wall proteins of the mutans streptococci. It has been suggested that P1 may be involved in cell adherence to tooth surfaces and in sucrose-induced cell aggregation. As a first step toward fully understanding its biological functions, the P1 gene, which has been designated spaP1, from Streptococcus mutans NG5 (serotype c) has been cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. The recombinant strain expressing P1 carries a 5.2-kilobase DNA insert whose restriction map has been determined. This map is completely different from that of spaA of Streptococcus sobrinus (serotype g), even though P1 and SpaA are antigenically related. Southern hybridization revealed that DNA sequences closely homologous to spaP1 were present in serotypes c, e, and f, and similar sequences also existed in strains of serotypes a and d. The expression of the cloned spaP1 was found to be independent of the lac inducer and the orientation of the DNA insert, suggesting that it carries its own promoter. Western blotting (immunoblotting) revealed at least 20 bands reacting with a mixture of three anti-P1 monoclonal antibodies. The highest-molecular-weight reactive band was comparable in size to the parent P1 (185 kilodaltons [kDa]); however, the major reactive bands were smaller (approximately 160 kDa). Expression of cloned P1 in E. coli LC137 (htpR lonR9) resulted in the increased prominence of the 185-kDa protein reactive band. Ouchterlony immunodiffusion showed partial identity between the parent and cloned P1. In E. coli, P1 was detected primarily in the periplasm and extracellular fluid.

Antigens, Bacterial↗

Evaluation of antimicrobial properties of orthodontic composite resins combined with benzalkonium chloride.

An antimicrobial agent, benzalkonium chloride (BAC), was added to a chemically cured composite resin, and the antimicrobial benefits and physical properties of the modified composite were evaluated. BAC was added to Reliance Phase II composite to create modified composites with BAC concentrations ranging from 0.25 to 2.50 wt%. Specimen disks of the modified composite were incubated with Streptococcus mutans for 48 hours, and an agar disk diffusion assay was used to measure zones of bacterial inhibition. Larger disks were suspended in brain-heart infusion medium containing 2 x 10(4) colony-forming units/mL Streptococcus sobrinus (10 mL, 2 wt% sucrose, 24 hours) to measure bacterial adherence to the adhesive; adherent cells were removed from the surface with 1 N NaOH, and the optical density of the cells was measured at 550 nanometers. Traction hooks were bonded to bovine teeth with the modified composite, and tensile bond strength was evaluated with a universal testing machine. Diametral tensile stress was also measured. The modified composite samples showed that antimicrobial activity increased with higher BAC content; no antimicrobial activity was measured for the original compound in either the disk diffusion or the bacterial adherence test. There were no significant differences (P <.05) in either tensile bond strength or diametral tensile stress among the modified composite groups and the original product. The incorporation of BAC in composite material added antimicrobial properties to the original compound without altering its mechanical properties.

Analysis of Variance↗

The complex oral microflora of high-risk individuals and groups and its role in the caries process.

The involvement of the oral biofilm in the caries process requires re-evaluation. The essential role of mutans streptococci (Streptococcus mutans and Streptococcus sobrinus) in the caries process is not proven. Acid production by dental plaque is not dependent upon the presence of mutans streptococci; caries occurs in the absence of these species and their presence does not necessarily indicate caries activity. Other oral bacteria, non-mutans streptococci, Actinomyces spp. and Bifidobacterium spp., are acidogenic and aciduric. They outnumber mutans streptococci in dental plaque, and there are data which support a role for these bacteria in the initiation and progression of caries. Molecular studies demonstrate the great diversity and complexity of the flora associated with caries. Many taxa identified have not been cultured and the role of these taxa is not known. We have, in mutans streptococci, good markers of disease but not necessarily the aetiological agents of the disease. Considerably more research is required to investigate the transition of tooth surfaces from being intact and sound to the white spot lesion stage. A combination of conventional and molecular approaches are required to elucidate the involvement of an individual taxon and of microbial populations with particular traits in the caries process.

Acids↗

Chlorhexidine susceptibilities of mutans streptococcal serotypes and ribotypes.

The susceptibilities of 379 clinical mutans streptococcal isolates to chlorhexidine (CHX) were tested by agar dilution according to the standards of the National Committee for Clinical Laboratory Standards. Isolates were obtained from saliva samples of 34 young mothers who had high or moderate salivary levels of mutans streptococci at baseline. Samples were collected on three occasions, before childbirth, when each child was 6 months old, and 1 year later. Of these isolates, 50% were inhibited at 1 microgram of CHX per ml, 90% were inhibited at 2.0 micrograms/ml, and all were inhibited at 4.0 micrograms/ml. The MICs for Streptococcus mutans isolates (serotypes c, e, and f) were lower than those for Streptococcus sobrinus isolates (serotypes d and g). In some subjects, the MICs for isolates of the same serotype were different. This phenomenon was studied by ribotyping isolates (n = 45) from selected subjects (n = 7). It was found that if there were intraindividual differences in the MICs for isolates of the same serotype, then the ribotypes of these isolates were different. In order to decrease the mutans streptococcal infection risk for children, 24 mothers (test group) brushed their teeth periodically with a gel that contained 0.3% CHX digluconate and 0.2% NaF, pH 5.8, between the second and third sampling occasions. The gel was used twice a day for the first 10 days of each month. Development of resistant strains during CHX-NaF gel use was not detected. The serotype distribution of isolates from the test group after 1 year of periodic CHX-NaF gel use did not differ from that at baseline. Periodic CHX-NaF gel brushing did not lead to lower salivary mutans streptococcal counts.

Adolescent↗

Competitive displacement of mutans streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R.

The ability of Streptococcus salivarius TOVE-R to displace virulent representatives of the most prevalent human mutans streptococci from the teeth of rats, and thereby to inhibit caries, was studied. Streptococcus mutans 10449S- or Streptococcus sobrinus 6715-13WT-infected specific-pathogen-free rats consuming a high-sucrose diet were inoculated by TOVE-R. The infectants were differentially recovered from swabs of the teeth over the time course of infection and from sonically treated material of extracted teeth and excised tongues. Despite initial colonization of the teeth by the mutans streptococci, TOVE-R colonized the teeth, unlike other essentially nonvirulent plaque formers already described. It did not colonize the tongues of the rats. TOVE-R emerged and persisted as a prominent member of the plaque ecology. There was an associated decline in the mutans streptococci on the teeth, and this decline was associated with significant inhibition of the caries component attributable to 10449S infection (56%) and to 6715-13WT infection (52%). TOVE-R did not reliably inhibit the component of fissure caries attributable to the nonmutans indigenous flora of the rats. TOVE-R itself induced no detectable decay. The data suggest the potential therapeutic utility of TOVE-R to inhibit caries by displacement of mutans streptococci from the teeth. These results supplement the already reported ability of TOVE-R to preempt initial colonization of teeth by the mutans streptococci.

Animals↗

Plaque composition, fluoride tolerance and acid production of mutans streptococci before and after the suspension of the use of fluoride toothpastes.

Although fluoride toothpastes are widely used for caries prevention, little is known about the impact of fluoride dentifrices on plaque composition. Also the issue of adaptation of mutans streptococci to grow in vivo in a fluoride environment has received little attention. Such an adaptation may be of interest as it has been suggested that adapted mutans streptococci may show reduced glycolytic activity thereby being less cariogenic. In the present experiments the impact of the suspension of the use of fluoride toothpastes on plaque composition, fluoride tolerance and acid production of mutans streptococci was studied. Pooled plaque samples from the lingual surfaces of the lower incisors were collected from individuals (n = 13) just before and 7 weeks after they had replaced their fluoride toothpastes (0.1-0.15% F) with a non-fluoride one. The samples were analysed for fluoride and the numbers and proportions of streptococci, Streptococcus mutans, Streptococcus sobrinus, Actinomyces species, and lactobacilli, respectively. The fluoride tolerance of the mutans streptococci was estimated by culture of the plaque samples on TYCSB agar supplemented with of 0, 1, 2, 3, 4, and 5 mmol/l fluoride (NaF) at pH 7.2. From each plaque sample six S. mutans strains were isolated for the measurement of the rate of acid production (Vap) at pH 7 in the presence of 0, 5, and 10 mmol/l F. The overnight final pH was measured in cultures of the S. mutans strains with excess of glucose and 0, 5, and 10 mmol/l F. The results showed that the removal of the fluoride pressure from plaque did not affect the numbers or proportions of the various species and genera of bacteria. The fluoride tolerance of the mutans streptococci, and the Vap or the overnight final pH of the isolated strains had not changed. These results suggest that the use of fluoride toothpaste had not affected plaque composition, nor fluoride tolerance or acidogenicity of mutans streptococci. Probably the amount of fluoride delivered by fluoride dentifrices to dental plaque is too low to induce such adaptations.

Adaptation, Physiological↗

Bicarbonate-based powder and paste dentifrice effects on caries.

The effects of sodium bicarbonate-based powder and paste dentifrices on dental caries and Streptococcus sobrinus recoveries in rats were studied. Three S. sobrinus 6715-13WT infected groups of specific pathogen-free weanling rats, consuming a diet which contains 56% sucrose, were topically treated with either demineralized water, dentifrice paste, or dentifrice powder. Both the paste and powder contained 0.22% (w/w) NaF. All topical treatments were given for one min daily per rat, five days a week. Animals' teeth were swabbed for recovery of 6715-13WT and total recoverable flora. Recoveries of 6715-13WT were 75% lower from both powder-treated and paste-treated dentifrice than from water-treated rats' teeth (p less than 0.001). Total caries scores were 45% lower for the powder-treated and 43% lower for the paste-treated rats than the water-treated ones (p less than 0.01). The major portion of the caries inhibition was evident on smooth tooth surfaces, 68% for powder-treated and 65% for paste-treated by comparison with water-treated animals (p less than 0.01), although a lesser caries reduction was also evident in tooth fissures (sulci). Thus, both powder and paste formulations of a sodium bicarbonate-based dentifrice containing 0.22% NaF equally and potently inhibited caries in rats, challenged bacteriologically and dietarily to develop severe caries.

Animals↗

The influence of a novel propolis on mutans streptococci biofilms and caries development in rats.

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.

Adenosine Triphosphatases↗

Genetic basis for mutacin N and of its relationship to mutacin I.

BACKGROUND & OBJECTIVES: The mutans streptococci (MS) are a group of 7 species of dental cariesassociated bacteria of which Streptococcus mutans and Streptococcus sobrinus are the most important in humans. Many MS produce bacteriocin-like inhibitory substances (BLIS), some of which have been characterised as small peptides capable of inhibiting the growth of closely-related species. These peptides have most commonly been referred to as mutacins. S. mutans strains N and UA140 appear to have closely similar BLIS activities. Both produce mutacins that seem to target the same species of bacteria. On closer analysis however, these two strains have been shown to produce distinctly different mutacins, known as mutacin N and mutacin I respectively. In the present study the mutacin N structural gene (mutN) was cloned and compared with the mutacin I structural gene (mutA). METHODS: Cloning and sequencing of S. mutans N was done. The distribution of mutN using DNA from 216 streptococcal strains was determined by dot blotting. RESULTS: Mut N was cloned and sequenced from an 1800 bp Bam HI/Eco RI fragment. PCR with the mutN primers mutNF and mutNR on the four mutN-positive strains identified identical bands to S. mutans N. The location of mutN differs significantly from that of mutA in that it is directly upstream of comC, a gene encoding a putative competence stimulating factor. INTERPRETATION & CONCLUSION: The close upstream proximity of mutN to comC suggests a link between mutacin N production and competence development. Further studies need to be done to detect competence-related genes in S. mutans strain N.

Bacteriocins↗

Protection of gnotobiotic rats against dental caries by passive immunization with bovine milk antibodies to Streptococcus mutans.

A multivalent vaccine consisting of whole cell antigens of seven strains, representing four serotypes (b, c, d and g), of mutans streptococci was used to hyperimmunize a group of cows. Serum samples from these animals contained immunoglobulin G1 (IgG1) antibody activity to seven serotypes (a to g) of mutans streptococci. Whey obtained from the animal with the highest serum antibody activity, which also contained high levels of IgG1 antibody, was used in passive caries immunity studies. Gnotobiotic rats monoinfected with Streptococcus mutans MT8148 serotype c or Streptococcus sobrinus OMZ176 (d) or 6715 (g) and provided a caries-promoting diet containing immune whey had lower plaque scores, numbers of streptococci in plaque, and degree of caries activity than similarly infected animals given a diet containing control whey obtained from nonimmunized cows. To establish the nature of the protective component(s) present in the immune whey, an ultrafiltrate fraction of the whey was prepared. This preparation contained higher levels of IgG1 anti-S. mutans antibody activity than the immune whey. Rats monoinfected with S. mutans MT8148 and provided with a diet supplemented with 0.1% of this fraction exhibited a degree of caries protection similar to that seen in animals provided a diet containing 100% immune whey. In fact, a diet containing as little as 0.01% of the ultrafiltrate fraction gave some degree of protection against oral S. mutans infection. The active component in the immune whey was the IgG1 anti-S. mutans antibody, since rats monoinfected with S. mutans MT8148 and provided a diet supplemented with purified immune whey IgG1 had significantly reduced plaque scores, numbers of S. mutans in plaque, and caries activity compared with control animals. Prior adsorption of the IgG fraction with killed S. mutans MT8148 whole cells removed antibody activity and abrogated caries protection.

Animals↗

Effect of local deposition of antigen on salivary immune responses and reaccumulation of mutans streptococci.

In this study glucosyltransferase (GTF) from Streptococcus sobrinus was topically administered onto the lower lips of young adults. The effect of this route of antigenic exposure on labial and parotid gland and serum antibody levels to GTF and on the reaccumulation of indigenous mutans streptococci after a dental prophylaxis was then measured. Young adults between 18 and 42 years of age were screened for levels of antibody activity to GTF in parotid and labial gland salivas and levels of mutans streptococci in their whole saliva. Prior to antigen administration, two groups were formed which had similar distributions of mutans streptococci in their whole saliva. Antigen (GTF) or placebo, each combined with aluminum phosphate (AP), was then administered to the lower lip daily for 5 days. Immediately prior to topical application of GTF or placebo to the labial salivary glands, all subjects were given a dental prophylaxis. Statistically significant differences in anti-GTF IgA antibody activity in parotid or labial salivas were not observed between the GTF-administered and the placebo groups during the 6 weeks following topical application. However, the proportions of indigenous mutans streptococci/total streptococcal flora, or total cultivable flora, were always lower in the whole salivas of the GTF, compared with the placebo group. These differences were statistically significant on days 13, 20, 34, and 41 after initial topical application. Delays in reaccumulation were significantly associated (P less than 0.025) with elevations in parotid saliva IgA antibody levels of all subjects. Seven of 10 of the subjects demonstrating this association were in the group to which GTF was topically administered.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Topical↗

Haptenated streptococcal antigens elicit either T cell-dependent type 1 or T cell-independent type 2 immune responses.

Antigens of Streptococcus mutans 6715 (alternatively designated serotype g Streptococcus sobrinus), including whole cells (WC g), cell walls (CW g), peptidoglycan (PG g) and serotype carbohydrate (Ml g) were coupled with trinitrophenyl (TNP), and the nature of the immune response to each immunogen was determined in normal and X-linked immunodeficient (xid) murine spleen cell cultures. Responses to TNP-WC g, -CW g and -PG g and to the classical type 1 antigen TNP-Brucella abortus occurred in both xid and normal splenic cultures, while TNP-Ml g only triggered immune responses in normal spleen cell cultures, suggesting that the former three antigens are type 1 and the latter type 2. Further support for the type 2 nature of TNP-Ml g was the finding that Peyer's patch cell cultures from both xid (which contain mature B cells) and normal mice supported responses to TNP-Ml g and TNP-Ficoll, while xid splenic cultures failed to support responses to either type 2 antigen. The three type 1 TNP-S. mutans antigens induced responses in nude spleen cell and in purified splenic B cell cultures, but required T cells for in vitro responses to lower doses of immunogen. On the other hand, TNP-Ml g induced anti-TNP PFC responses at several antigen concentrations in purified B cell cultures, without requirement for added T cells. These studies show that the intact S. mutans cell, as well as CW g and PG g, acts as a T cell-dependent (TD) type 1 antigen, while the serotype carbohydrate (Ml g) induces a T cell-independent (TI) type 2 response. Thus, the intact bacterium is a TD type 1 antigen, whereas its purified components are either type 1 or type 2 antigens and differ significantly in terms of their T cell dependence.

Animals↗

Multiplex FISH analysis of a six-species bacterial biofilm.

Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.

Biofilms↗

In vitro evaluation of antibacterial effects of photo-cured glass ionomer liners and dentin bonding agents during setting.

Two dentin bonding agents (Optibond and Syntac), an enamel bonding agent (Heliobond), and two photo-cured glass ionomer cements (Vitremer and Variglass VLC) and their inhibitory effects on bacterial growth during setting were examined. Cultures of eight bacterial species were used to test these materials, except for Vitremer glass ionomer cement, which was tested against only five species. The photo-cured glass ionomer cements and the Syntac dentin bonding system with glutaraldehyde demonstrated a significant inhibitory effect on the growth of several bacteria. Meanwhile, Variglass VLC glass ionomer cement did not exhibit this effect on Lactobacillus casei and Streptococcus sobrinus. Optibond light-cured dentin bonding agent, with fluoride and fillers in its composition, and Heliobond (negative control group) did not demonstrate any inhibitory effect.

Acrylates↗

Avirulent Salmonella typhimurium delta cya delta crp oral vaccine strains expressing a streptococcal colonization and virulence antigen.

Salmonella typhimurium SR-11 strains lacking adenylate cyclase and the cyclic AMP receptor protein (CRP) due to deletion (delta) mutations in the cya and crp genes, respectively, are avirulent for mice and induce high level protective immunity against subsequent challenge with wild-type virulent S. typhimurium SR-11 cells. The avirulence of these delta cya delta crp mutants has been enhanced by elimination of the 100 kb virulence plasmid pStSR100 without impairing immunogenicity. The present report confirms the avirulence and immunogenicity of these mutant strains, demonstrates that immunization of both four- and eight-week-old mice has no adverse effect on weight gain, and that immunity lasts at least ninety days following initial immunization. Avirulent S. typhimurium strains have been endowed with the ability to produce several streptococcal colonization and virulence antigens for the purpose of constructing recombinant bivalent oral vaccine strains. Important antigenic determinants of the Streptococcus sobrinus surface protein antigen A (SpaA), presumed to be a critical colonization antigen of S. sobrinus, are expressed at high level by the delta cya delta crp S. typhimurium strains. The recombinant vaccine strains are stable in vitro and in animals (for a period of at least eight days) where they localize to the gut-associated lymphoid tissue (GALT).

Administration, Oral↗