Factors affecting peptide catabolism by oral streptococci

The binding of a number of unsubstituted peptides to Streptococcus sanguis and Streptococcus mutans and their subsequent degradation by such cells were examined. Peptides were added to cell suspensions prepared from glucose-limited growth in a chemostat and, at appropriate time intervals, cell-free filtrates were analyzed for peptides and their constituent amino acid residues by high-pressure liquid chromatography techniques. The results indicated that peptide hydrophobicity plays a limited role in peptide binding, but that charge and chain-length are probably important. In S. sanguis, carboxypeptidase activity rapidly released C-terminal arginine (Arg); this amino acid was less rapidly released from the N-terminus but a number of other residues were also released by aminopeptidase activity. When Arg is buried in the peptide, the rate of its release depends upon the number and type of residues between it and the N-terminus. In contrast, S. mutans possessed only weak peptidase activities. The nature of its peptidase activities indicates that S. sanguis can obtain the metabolically important Arg from a variety of peptides.     

Studies on fusobacteria associated with periodontal diseases

The physiological and metabolic characteristics of representative isolates of the various subspecies of Fusobacterium nucleatum were investigated by growing them in continuous culture in chemically-defined media. Behaving almost identically, these organisms were found to obtain energy from the fermentation of simple carbohydrates such as glucose or fructose or from the fermentation of certain amino acids, free or in the form of small peptides. The latter can be attacked by aminopeptidase activity which was shown to be essential for the growth of the organism in an environment lacking fermentable carbohydrate and free amino acids but replete with small peptides. This metabolic versatility may explain the presence of F nucleatum in both supra- and sub-gingival dental plaque and why it is often found together with organisms such as Porphyromonas gingivalis which display powerful endopeptidase activities. Using the technique of allozyme electrophoresis, the current subspeciation of F. nucleatum was shown to be of doubtful validity and evidence, based upon physiological and metabolic properties, for differences in pathogenicity between isolates was not detected. While this organism is a member of various bacterial consortia associated with periodontal diseases, its contribution to the disease process remains unclear.     

Influence of hydrophobicity on oligopeptide utilization by oral streptococci

The growth responses of Streptococcus mutans VA-29R, Streptococcus sanguis ATCC 10556, and Streptococcus mitior NIH to hydrophilic and hydrophobic peptides obtained following isopropanol fractionation of Trypticase were compared. Although the two fractions contained peptides of similar molecular size, differences were observed with respect to amino acid compositions. S. mutans VA-29R showed a pronounced difference in growth response to hydrophilic vs. hydrophobic peptides. While growth of this micro-organism on hydrophilic peptides was indistinguishable from that on unfractionated Trypticase, only very slow growth occurred on the hydrophobic peptides. S. sanguis ATCC 10556 and S. mitior NIH also displayed some selectivity, as evidenced by their faster relative growth rates on hydrophilic, as compared with hydrophobic, peptides. The results of this study support the conclusion that the properties of the substrate, as defined by its amino acid composition, may be more important than molecular size as a factor influencing recognition and subsequent utilization of oligopeptides as sources of amino acids for growth by these three oral streptococci.     

Influence of saliva from 'heavy' and 'light' plaque formers on the colloidal stability of bacterial suspensions

The influence of parotid saliva on the colloid stability of suspensions of Streptococcus sanguis and S. salivarius was studied in groups of previously identified 'heavy' and 'light' plaque formers. For S. sanguis it was observed that addition of parotid saliva from light plaque formers had more pronounced negative influence on the colloid stability than addition of such saliva from heavy plaque formers. No differences were observed for S. salivarius. The results indicate that saliva and bacteria might be regarded as a biological colloidal system and that the individual rate of plaque formation can perhaps be partially related to the colloid-chemical properties of bacteria and saliva.     

A conceptual model for the co-existence of Streptococcus spp. and Actinomyces spp. in dental plaque

One of the most important questions in ecology is how to explain the co-existence of the variety of physiologically related organisms in the same habitat. A model is presented for the co-existence of Streptococcus species and Actinomyces species in dental plaque. The hypothesis is that these organisms co-exist because they simultaneously utilize several carbon and energy substrates. The hypothesis follows from the observation that the growth yield of oral streptococci and actinomyces in saliva is limited by carbohydrate. Preliminary experiments were undertaken to test the hypothesis using mixed chemostat cultures and gnotobiotic rats. Competition between S. mutans K1R and A. viscosus Ut2 in mixed chemostat cultures on glucose and asparagine was hampered by the early appearance of high-glucose-affinity variants of A. viscosus. From the physiological characteristics of S. sanguis and S. milleri, it might be predicted that simultaneous utilization of carbohydrate and arginine would enable these organisms to co-exist with S. mutans in an ecosystem. To test this mechanism under natural conditions, germ-free rats were inoculated with a combination of S. mutans K1R and S. sanguis P4A7 or the combination S. mutans K1R and S. milleri B448. The rats were fed on three different diets: (1) 58% cornstarch; (2) 48% cornstarch and 10% sucrose; and (3) 53% cornstarch and 5% arginine. The results of this experiment demonstrated that dietary arginine caused a significant decrease of the ratios K1R/P4A7 and K1R/B448 in dental plaque.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane locus and pH sensitivity of paraben inhibition of alkali production by oral streptococci

Parabens were found to be potent inhibitors of alkali production from arginine by oral streptococci such as Streptococcus rattus, Streptococcus sanguis and Streptococcus gordonii. For example, 2 mumol butylparaben per ml completely and irreversibly inhibited arginolysis by intact cells of S. rattus FA-1 and was lethal for the organism. In contrast, butylparaben was not a very effective inhibitor of ureolysis by intact cells of Streptococcus salivarius 57.I, although it did kill the cells. Butylparaben irreversibly inhibited the cytoplasmic enzymes arginine deiminase, carbamate kinase and urease in permeabilized cells or isolated form. However, inhibition of arginolysis by intact cells appeared to be due primarily to irreversible inhibition of transport systems for arginine uptake, because butylparaben added to intact cells did not reduce levels of arginine deiminase when the cells were subsequently permeabilized after washing. The insensitivity of ureolysis by intact cells to butylparaben can be related to the known high permeability of cell membranes to urea and the cytoplasmic location of urease. The potency of butylparaben as an inhibitior of arginolysis or glycolysis and as a lethal agent was found to be greater at acid pH that at neutral or alkaline pH.     

Constituents of salivary supernatant responsible for stimulation of oxygen uptake by the bacteria in human salivary sediment

The 10,000 g supernatant of wax-stimulated whole saliva was fractionated by gel filtration and its components were tested along with amino acids, small peptides and urea for their ability to stimulate this oxygen uptake, and for their effects on pH. Fractions containing the larger components, the proteins and large peptides, stimulated much less oxygen uptake than unfractionated supernatant, and caused a small decrease in pH. Analysis with anthrone indicated that both these effects were due mainly to the carbohydrate associated with these constituents. In contrast, fractions containing the remaining lower molecular-weight components stimulated substantial oxygen uptake and a rise in pH; both effects were like those seen with whole saliva supernatant. The oxygen effects were attributed mainly to certain amino acids and small peptides in the small molecular-weight fractions. Ornithine, arginine, proline and glutamic acid consistently stimulated oxygen uptake by the oral microflora in a test of 23 amino acids with the sediments of 13 subjects. Ornithine and arginine at the same time stimulated a significant rise in pH, whereas the other two amino acids showed no such effect. Variable and sometimes significant oxygen uptake was seen with alanine, aspartic acid, asparagine, glutamine and cysteine in 4-7 of the subjects; infrequent or no effects were seen with the remainder of the amino acids tested. There was some evidence to suggest that amino acid stimulation of oxygen uptake may be inducible. Urea had no effect on uptake but did contribute significantly to the pH rise. Small peptides containing those amino acids that could stimulate oxygen uptake also stimulated such uptake; peptides without such acids did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the adherence of Porphyromonas gingivalis to oral streptococci

Adherence of Porphyromonas gingivalis to strains of Streptococcus sanguis and Streptococcus mitis deposited on nitrocellulose paper was investigated. A variety of laboratory strains and clinical isolates of P. gingivalis bound to both S. sanguis and S. mitis. Binding of P. gingivalis to all but one of the streptococci was not inhibited by salivary molecules. Pretreatment of P. gingivalis with periodate and pretreatment of S. sanguis and S. mitis with pronase decreased binding, suggesting that adherence may be mediated by a protein on the streptococci interacting with a carbohydrate on P. gingivalis. Binding was not inhibited by a selection of simple sugars. The ability to adhere to early plaque bacteria such as S. sanguis and S. mitis may be important in the colonization of the mouth by P. gingivalis.     

Susceptibility of some plaque microorganisms to chemotherapeutic agents.

Investigators have used chemotherapeutic agents topically for plaque control without knowing the drug concentration necessary to inhibit the growth of odontopathic microorganisms. S mutans, S sanguis, A viscosus and A naeslundii are important components of the plaque flora. The minimal inhibitory concentrations (MIC) and minimal bactericidal concentrations (MBC) of niddamycin, vancomycin, bacitracin, and kanamycin were determined for each organism in liquid culture. These antibiotics were selected because of their low absorption from the gastrointestinal tract. Niddamycin, vancomycin, and bacitracin had the lowest MIC, from 0.2 to 10 units/ml. Kanamycin was inhibitory only at much higher concentrations (130 to 500 units/ml). The corresponding MBC was generally higher than the MIC. A viscosus was the most resistant organism tested. These data are important in designing controlled release devices for delivering a suitable antibiotic on a continuous basis intraorally.     

Some aspects of protease production by a strain of Streptococcus sanguis

Our previous studies indicated that Arginine (Arg) plays a key nutritional role in Streptococcus sanguis P4A7 and that this organism can grow on whole casein as the sole nitrogen source. Its protease activities were therefore studied after glucose-limited continuous culture in a chemically-defined medium with either free amino acids or casein as the nitrogen source. Both culture supernatant and cell-associated endopeptidase (EP) and exopeptidase (amino-AP and carboxy-CP) activities were determined. Growth rate (mu) had little effect on EP, 75% of which was consistently in culture supernatants; AP and CP both decreased as mu was increased and both were predominantly cell-associated. At high growth pH, EP was substantially increased while AP and CP activities were optimal at pH 7. The most striking nutritional effect occurred under nitrogen limitation (glucose excess) when EP and AP were greatly increased and CP greatly decreased. It was concluded that S. sanguis is well equipped to scavenge its environment for Arg under a wide range of growth conditions.     

Acid-induced acid tolerance and acidogenicity of non-mutans streptococci

Acid tolerance and acidogenicity of non-mutans streptococci and their capacity of acid adaptation were studied. The cells of non-mutans streptococci (Streptococcus sanguis [Streptococcus sanguinis], Streptococcus gordonii, Streptococcus oralis and Streptococcus mitis) grown at pH 7.0 showed 0.0088% to 71% viability after acidification at pH 4.0 for 60 min, whereas the cells of mutans streptococci (Streptococcus mutans) were not killed by the acidification. Washed cells of non-mutans streptococci lowered pH to 4.04-4.33 in the presence of glucose, while mutans streptococci cells lowered pH to 3.70. When the growth pH was shifted to 5.5 for 30-90 min, the viability of non-mutans streptococci after the acidification at pH 4.0 for 60 min increased (0.25% to 91%) and the minimum pH values of the cells in the presence of glucose decreased (3.90 4.19). Along with the increase in acid tolerance and acidogenicity, non-mutans streptococci increased activities of H(+)-ATPase and arginine deiminase and amounts of stress proteins cross-reacting with 60 kDa and 70 kDa heat shock proteins (Hsp60 and Hsp70). These results indicate that non-mutans streptococci were capable of increasing their acid tolerance and acidogenicity in response to environmental acidification. Furthermore, it is suggested that the acid adaptation observed in non-mutans streptococci cells could involve the induction of H(+)-ATPase, arginine deiminase and stress protein syntheses. The strains of non-mutans streptococci, which are pioneer bacteria for dental plaque formation and predominant in plaque microbial flora, may play a significant role in shifting the dental plaque environment toward acidic and consequently promoting the colonization of more acid-tolerant and acidogenic bacteria such as mutans streptococci and lactobacilli.     

Effect of SnF2, administered as mouthrinses or topically applied, on Streptococcus mutans, Streptococcus sanguis and lactobacilli in dental plaque and saliva

Abstract – Mouthrinsing with SnF₂ reduced the Streptococcus mutans population in plaque and saliva and the proportion of Streptococcus sanguis in plaque. The effect was of short duration: 2 weeks after treatment the values of S. mutans in plaque and saliva were even higher than the pretreatment values. Topical SnF₂ applications reduced the S. mutans population in plaque and saliva but did not reduce the proportion of S. sanguis in plaque. The eflect was more prolonged: 4 weeks after treatment the S. mutans population in interproximal plaque remained significantly reduced and the salivary levels of the organism had not fully returned to pretreatment levels. Both SnF₂ treatments significantly increased the salivary levels of lactobacilli. The values of laclobacilli in saliva remained signilicantly increased 4 weeks after the SnF₂ mouthrinsing but had almost returned to pretreatment levels within 2 weeks after the topical SnF₂ applications. The findings suggest that the cariogenic potential of dental plaque is differently affected depending on whether a drug is administered as a mouthrinse or is applied topically.     

Ecology of viridans streptococci in the oral cavity and pharynx

Recently published taxonomic studies of viridans streptococci have resulted in several changes in the nomenclature and definition of oral streptococcal species. With this background, the ecology of streptococci in the oropharyngeal cavities was reinvestigated. The results based on the examination of 1426 streptococcal isolates confirmed and extended earlier findings. Apart from mature supragingival plaque, which contained a mixture of all orally encountered streptococci, each site showed a characteristic streptococcal flora. Initial dental plaque formation is primarily associated with Streptococcus sanguis, Streptococcus mitis biovar 1 and Streptococcus oralis. Our investigation showed that S. sanguis and S. mitis biovar 1 were the most prominent streptococci, also on buccal mucosa. In contrast, S. oralis was almost exclusively found in initial dental plaque. Streptococcus gordonii, formerly part of S. sanguis, was found in small numbers on the oropharyngeal mucosa and in mature supragingival plaque. The dorsum of the tongue was dominated by S. mitis biovar 2 and Streptococcus salivarius, the latter of which was predominant also on the pharyngeal mucosa. Streptococcus anginosus was by far the most predominant streptococcus in subgingival plaque. Immunoglobulin A1 (IgA1) protease-producing streptococci were primarily isolated from initial dental plaque and from the buccal mucosa. This lends further support to the concept of IgA1 proteases being important for the ability of streptococci to evade the local immune defence during their initial colonization of certain oral surfaces.     

Biofilm-specific surface properties and protein expression in oral Streptococcus sanguis

OBJECTIVE: Oral streptococci are primary colonisers of the tooth surface and are abundant in dental plaque biofilms. Bacteria growing in these relatively dense, surface-associated communities are phenotypically quite distinct from their planktonic counterparts. The purpose of the present study was to develop a method to investigate biofilm-specific surface protein expression by Streptococcus sanguis to help provide a better understanding of the critical events in plaque development. DESIGN: Biofilm cells were grown on the surface of glass beads in a biofilm device fed with mucin-containing artificial saliva. Planktonic cells were grown in continuous culture at approximately the same growth rate. Surface hydrophobicity of biofilm and planktonic cells was determined by hexadecane partitioning, and expression of streptococcal fibronectin adhesin CshA was determined in ELISA using specific antiserum. Antisera raised to glutaraldehyde-fixed whole biofilm or planktonic grown cells were used to screen an expression library of S. sanguis genomic DNA, and isolated clones were sequenced. RESULTS: Phenotypic analysis of biofilm and planktonic cells confirmed that mode of growth affected surface properties of S. sanguis. Thus, hydrophobicity and CshA expression was significantly elevated in biofilm cells. Library screening with biofilm antiserum yielded 32 recombinant clones representing 21 different S. sanguis proteins involved in adhesion and colonisation, carbohydrate utilisation or bacterial metabolism. In differential analysis of four selected Escherichia coli clones, biofilm antiserum reacted five times stronger than planktonic antiserum with cell-free extracts of clones encoding homologues of CshA and Cna collagen adhesin of Staphylococcus aureus, suggesting that these surface proteins are up-regulated in biofilm cells. In contrast, both antisera reacted equally strongly with cell-free extracts of the remaining two clones (encoding dihydrofolate synthase and an unknown protein). CONCLUSIONS: The method described represents a useful means for determining bacterial protein expression in biofilms based on a combination of molecular and immunological techniques. Surface expression of putative fibronectin and collagen adhesins was up-regulated in biofilm cells.     

Serum immunoglobulin G antibodies to Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis during experimental gingivitis in young adults

Twenty-eight young, healthy adults completed an experimental gingivitis study in which blood and clinical recordings were obtained at baseline; after a 4-week period of thorough oral hygiene; after a subsequent 3-week period of plaque accumulation; and after another 2 weeks of thorough oral hygiene. Serum immunoglobulin G antibodies against whole cells of Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum and Streptococcus sanguis were determined using enzyme-linked immunosorbent assay. Mean serum immunoglobulin G antibody levels to P. intermedia, F. nucleatum and S. sanguis remained essentially constant during the experiment, whereas the immunoglobulin G antibodies to P. gingivalis declined during the initial period of oral hygiene and the subsequent period of plaque accumulation to an average of 84.5% of the baseline value. This reduction could be attributed to the people who developed marked gingival inflammation during the period of plaque accumulation, indicating that the systemic host response may be associated with local tissue responses to variations in oral hygiene. These people were, however, also characterized by higher initial serum immunoglobulin G responses to P. gingivalis than people who developed less pronounced gingival inflammation during the experiment. The variability and individuality noted in the host response to potential pathogens have important implications for attempts to use such measures for establishing a diagnosis or prognosis for the individual patient.     

Effect of chlorhexidine on the relative proportions of Streptococcus mutans and Streptococcus sanguis in hamster plaque

abstract — The effect of chlorhexidine on the proportions of Streptococcus mutans and Streptococcus sanguis in plaque was studied in hamsters fed a diet containing 28% sucrose. In animals given chlorhexidine in their drinking water for 10 d a decrease in the population of S. mutans and an increase of S. sanguis occurred in the plaque. Following the removal of chlorhexidine the population of S. mutans increased again in the presence of sucrose and the number of S. sanguis returned to initial values. When animals were given a sucrose-free diet the low proportion of S. mutans observed following the short-term chlorhexidine period persisted. These data indicate that there is an inverse relationship between the number of S. sanguis and S. mutans in plaque and that the sensitivity in vivo of S. mutans to chlorhexidine can be used to suppress the population of S. mutans with a concomitant rise in the proportion of S. sanguis .     

Effect of chlorhexidine on the relative proportions of Streptococcus mutans and Streptococcus sanguis in hamster plaque.

The effect of chlorhexidine on the proportions of Streptococcus mutans and Streptococcus sanguis in plaque was studied in hamsters fed a diet containing 28% sucrose. In animals given chlorhexidine in their drinking water for 10 d a decrease in the population of S. mutans and an increase of S. sanguis occurred in the plaque. Following the removal of chlorhexidine the population of S. mutans increased again in the presence of sucrose and the number of S. sanguis returned to initial values. When animals were given a sucrose-free diet the low proportion of S. mutans observed following the short-term chlorhexidine period persisted. These data indicate that there is an inverse relationship between the number of S. sanguis and S. mutans in plaque and that the sensitivity in vivo of S. mutans to chlorheximide can be used to suppress the population of S. mutans with a concomitant rise in the proportion of S. sanguis.     

Effect of penicillin on Streptococcus mutans, Streptococcus sanguis and lactobacilli in hamsters and in man

The effect of penicillin on the number of oral Streptococcus mutans, Streptococcus sanguis and lactobacilli in hamsters and in man was investigated. This is of interest as S. mutans and lactobacilli are involved in the carious process while S. sanguis is not. Hamsters infected with both S. mutans and S. sanguis or only S. sanguis received penicillin in their drinking water for 14 d. The treatment reduced the proportion of S. mutans and S. sanguis in dental plaque to undetectable levels. After the penicillin treatment the population of S. mutans and S. sanguis gradually increased. In man, the effect of oral penicillin therapy was examined in 21 adults with more than 2 X 10(5) S. mutans per ml saliva. The penicillin treatment had almost no effect on the numbers of S. sanguis and lactobacilli, but a pronounced decrease in the number of S. mutans was observed. The duration of this effect, however, was short. Consequently, such treatment alone is of limited value for the control of the oral infection of these microorganisms.     

Adherence of Streptococcus sanguis

The presence of adhesins on the cell surface of S. sanguis enables the organism to grow and survive in the oral cavity. Many workers are now actively involved in attempting to characterise these adhesins and the molecular basis for adherence of various streptococci. The complexity of the adhesion processes is underlined by the many varied opinions as to the nature of the molecular components and biochemical interactions that are involved. Understanding the molecular mechanisms involved in bacterial adherence and in dental plaque formation will have significant impact on the prevention and treatment of oral diseases.     

Effect of penicillin on Streptococcus mutans, Streptococcus sanguis and lactobacilli in hamsters and in man

Abstract – The effect of penicillin on the numbers of oral Streptococcus mutans, Streptococcus sanguis and lactobadlii in hamsters and in man was investigated. This is of interest as S. mutans and lactobacilli are involved in the carious process while S. sanguis is not. Hamsters infected with both S. mutans and S. sanguis or only S. sanguis received penicillin in their drinking water for 14 d. The treatment reduced the proportion of S. mutants and S. sanguis in dental plaque to undetectable levels. After the penicillin treatment the population of S. mutans and S. sanguis gradually increased. In man, the effect of oral penicillin therapy was examined in 21 adults with more than 2 × 10⁵ S. mutans per ml saliva. The penicillin treatment had almost no effect on the numbers of S. sanguis and lactobacilli, but a pronounced decrease in the number of S. mutans was observed. The duration of this effect, however, was short. Consequently, such treatment alone is of limited value for the control of the oral infection of these microorganisms.