Strains Of Streptococcus Salivarius Research Articles

The Ecology of Bacteriocin-producing Strains ofStreptococcus salivarius

Interest in bacteriocin-producing components of the human normal oral microbiota centres on their possible interference with colonisation by potentially pathogenic bacteria. Certain strains of Streptococcus salivarius produce bacteriocin-like agents displaying exceptional inhibitory activity toward Lancefield Group A streptococci. Four individuals were identified as naturally harbouring high proportions (> 90 per cent) of bacteriocin-producing strains of S. salivarius . Bacteriocinogenic isolates from three of these individuals and the previously described strain 20P3 displayed identical patterns of inhibition when tested by a deferred antagonism method against a series of ten indicator organisms. The fourth individual harboured S. salivarius strains producing a distinct, though possibly related, inhibitory pattern. All agents were of apparently low molecular weight (< 8000) and were stable to heating at 80°C for 30 min. The four individuals harbouring bacteriocinogenic S. salivarius strains possessed significantly greater proportions (p < 0.001) of bacteriocin-resistant Gram-positive alpha-haemolytic cocci than did 13 control individuals (not found to harbour bacteriocinogenic S. salivarius strains). These results accord with the idea that the production of active bacteriocins in situ should select a relatively bacteriocin-resistant accompanying microbiota. Keywords: Streptococcus salivarius ; Bacteriocin; Microbial interference; Indigenous microbiota; Colonisation resistance.

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.

The bacteria responsible for ureolysis in artificial dental plaque

The origin of ureolytic activity in artificial-mouth plaques was established by assessing the contribution to plaque ureolytic activity of the isolated bacteria. To overcome losses of ureolytic activity caused by the unstable presence of urease in oral bacteria, ureolytic bacteria were isolated from an exceptionally active plaque (1 μmol NH 3/min per mg protein) in which 63 per cent of the flora was ureolytic. After their ability to metabolize urea was stabilized, 13 ureolytic bacteria remained: seven strains of Streptococcus salivarius, one Streptococcus bovis, two Straphylococcus epidermidis and three Staphylococcus haemolyticus. Their urease activity, measured after growth into stationary phase, was reproducible and strain specific with a 20-fold range within each genus. The mean ureolytic activity of each species, when weighted by its calculated incidence in the original plaque, accounted for 40 per cent of the total plaque ureolytic activity. However, these values for urease levels were only a small fraction of the bacterial ureolytic potential. Urease per mg cell protein measured during the growth cycle of a selected Strep. salivarius, and Staph. epidermidis, varied 10-fold, and reached much higher activities (i.e. 6–8 μmol NH 3/ min per mg of cell protein) than under the growth conditions that were used to assess the contribution of these species to total plaque ureolysis. Thus urea metabolism in artificial plaque was due mainly to Strep. salivarius, with a small contribution from Staph. epidermidis. The presence of further unidentified species of ureolytic oral bacteria need not be invoked.

口腔Ｓｔｒｅｐｔｏｃｏｃｃｕｓ ｓａｌｉｖａｒｉｕｓの菌体外多糖の合成と付着に及ぼすグルカナーゼの影響

Synthesis of glucans and fructans from sucrose and adherence to glass by extracellular enzymes of two strains of oral Streptococcus salivarius were compared.The enzyme of strain Y5 produced a large amount of sticky deposits on a glass surface whereas the nzyme of strain S2 formed a much smaller amount of fragile deposits. Products of the S2 enzyme consisted of nearly equal amounts of water-insoluble and water-soluble glucans and fructans (IG, SG, IF, SF). Major products of the Y5 enzyme were IG with a trace of SG and IF. Methylation analysis together with glucanase susceptibility tests revealed that IGs and SGs of both strains consisted of an α-1, 3-linked linear chain as a backbone sequence possessing α-1, 6- and α-1, 4-linked side chains of which both may constitute a single molecule or either may be located on different molecules. The IG of strains Y5 contained higher proportions of α-1, 3, 6 branch and α-1, 6-linked residues than the S2-IG. Adherence of IG to glass was markedly inhibited by the presence of glucanases. Dextranase shortened the α-1, 6-linked, and mutanase the α-1, 3-linked linear chains in IG and thereby reduced the amounts of IG products and lowered their molecular size, which resulted in less stickiness and/or insolubility of the products.These results suggest that adhering deposits produced from sucrose by S. salivarius strains Y5 and S2 consist primarily of IG, and the α-1, 3- and α-1, 6-linked linear chains in IG, which are involved, respectively, in the water insolubility and adhesive property, act together to produce adherence of IG. Amounts of IG synthesized and relative proportion of the two linkages in IG determine the extent of adhesive mass formation. Thus, the glucanases would be effective to eliminate S. salivarius from various sites of the mouth, particularly the tongue dorsum and/or tooth surfaces.

Determination of the cell surface hydrophobicity of oral bacteria using a modified hydrocarbon adherence method

Hydrophobic interactions between bacterial cell surfaces and colonisable substrates have been implicated in the mechanisms of bacterial adherence. However, current methods of assessing bacterial hydrophobicity as a function of adherence to liquid hydrocarbons (especially hexadecane) do not always produce accurate or reproducible results. Therefore, the present technique was developed using xylene. The hydrophobic surface properties of fresh and type strains of Bacteriodes gingivalis, Bacteriodes intermedius, Capnocytophaga spp., Streptococcus salivarius and Streptococcus sanguis suspended either in saliva ions buffer (SIB) or in saliva diluted in SIB were measured. In SIB the test strains were predominantly hydrophobic. The addition of saliva caused a significant reduction (P < 0.05) in hydrophobicity compared to SIB alone, with 80% of the strains tested. Since oral bacteria will be suspended in saliva in vivo, it is concluded that bacteria in the oral cavity may be less hydrophobic than previous studies have suggested.

A comparison of the adhesion, coaggregation and cell-surface hydrophobicity properties of fibrillar and fimbriate strains of Streptococcus salivarius.

Fibrillar and fimbriate strains of Streptococcus salivarius were compared for their ability to adhere to buccal epithelial cells and saliva-coated hydroxyapatite beads, and for their ability to coaggregate with Veillonella strains. The fibrillar Lancefield group K strains adhered statistically significantly better to both buccal epithelial cells and saliva-coated hydroxyapatite beads than the fimbriate strains, which lacked the Lancefield group K antigen. After 1 h the fibrillar strains coaggregated statistically significantly better than the fimbriate strains with V. parvula strain V1, but after 24 h, coaggregation both of fibrillar and of fimbriate strains reached approximately 90%. Freshly isolated Veillonella strains all coaggregated with the S. salivarius strains, but the percentage coaggregation varied considerably after 1 h depending on the Veillonella strain. Coaggregation was independent of the presence of Ca2+. S. salivarius strain HB-V5, a mutant of strain HB that had lost the Veillonella-binding protein, coaggregated weakly with V. parvula strain V1, but coaggregated very well with other wild-type veillonellae, suggesting the presence of an alternative mechanism for Veillonella-binding for strain HB. Fibrillar strains were, therefore, more adhesive to oral surfaces and coaggregated with veillonellae after 1 h better than the fimbriate S. salivarius strains. Both fibrillar and fimbriate strains were highly hydrophobic in the hexadecane-buffer partition assay.

The hydrophobicity of Streptococcus salivarius strain HB and mutants deficient in adhesion to saliva-coated hydroxyapatite

The hydrophobicity and adhesion to saliva-coated hydroxyapatite of Streptococcus salivarius HB and the mutants HB7, HBV5 and HBV51 were measured. The mutants HB7 and HBV51 showed a significant reduction in adhesion to salivacoated hydroxyapatite and hydrophobicity compared with the mutant HBV5 and the parent strain. This supports the view that hydrophobic interactions are important for bacterial attachment in the oral cavity and is in contrast to previous studies on the hydrophobicity of these strains.

Bacteriocin-like inhibitory activity associated with beta-hemolytic strains of Streptococcus salivarius.

Seven beta-hemolytic Streptococcus salivarius isolates produced bacteriocin-like inhibitory activity in deferred antagonism tests using a set of nine indicator bacteria (I1-I9). Five of these S. salivarius strains (KWF, TOVE-R, K17, K21, and K26) were inhibitory to indicators I2, I5, I6, and I7. Mutated non-hemolytic derivatives showed concomitant loss of inhibitory activity against I2, I5, and I6, but retained activity against I7. Inhibitory activity against I2, I5, and I6 was restored in beta-hemolytic revertants of such mutants. Strain 3638 was inhibitory to all of the indicator organisms except I3, and this pattern of inhibitory activity was retained by non-hemolytic derivatives. It appeared that strain 3638 produced an additional broadly-active inhibitory agent, since a mutant (strain 3638A), which was apparently defective in the production of this inhibitor, retained both the beta-hemolytic and I2-, I5-, I6-, and I7-inhibitory activities. Non-hemolytic derivatives of strain 3638A were inhibitory only to I7. Strain 3638, therefore, appeared to produce at least three inhibitory agents: one active only on I7; another acting on I2, I5, and I6 (and associated with beta-hemolytic activity); and a third apparently active on all of the indicators other than I3. S. salivarius strain JH inhibited all nine indicator strains and possessed a beta-hemolytic character which differed from that of the other strains in being readily eliminated on treatment with the plasmid-curing agent novobiocin. Non-hemolytic derivatives of JH retained inhibitory activity against the complete set of indicators.(ABSTRACT TRUNCATED AT 250 WORDS)

Streptococcal and enterococcal inhibition of endocervical lactobacilli

26 endocervical lactobacilli were subdivided into 6 groups according to their patterns of sensitivity (S-types) to the bacteriocin-like inhibitory agents produced by a set of 6 streptococcal strains of Lancefield groups A, B, G and F. Further differentiation of the lactobacilli was achieved by assessing their sensitivity to bacteriocin-producing strains of Streptococcus salivarius, group C streptococcus. Streptococcus uberis, Streptococcus mutans and Enterococcus faecalis. Some of the anti-lactobacillus activity of E. faecalis ssp. liquefaciens strain T142 was shown to be due to its proteolytic digestion of hemoglobin in the blood-containing test medium with formation of inhibitory levels of hematin. It is suggested that the combined production of bacteriocins and hematin by proteolytic enterococci could afford them a competitive growth advantage over some indigenous lactobacilli in the vaginal milieu at the time of menses.

A simplified diagnostic system for cultural detection and enumeration of Streptococcus mutans.

A simple dip-slide test (Cariescreen SM) based on MSB selective agar was devised for detection and quantitation of Streptococcus mutans in oral samples. For this test, a bacitracin tablet is dissolved in a vial containing buffered saline diluent. Paraffin-stimulated saliva is collected in this diluent vial. A dip slide which incorporates a modified MSB agar (minus bacitracin) is immersed briefly in the diluted saliva. After addition of a CO2-generating tablet, the screw-cap dip slide is closed tightly in the vial and incubated for two days at 37 degrees C and one day at room temperature. S. mutans populations in saliva are estimated by comparison with a colony density chart. Growth of reference strains of S. mutans was equivalent on Cariescreen SM dip slides and on MSB agar plates. Reference strains of Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis, and Streptococcus milleri did not grow on Cariescreen SM dip slides. Aliquots of saliva from 50 schoolchildren and 51 adults were tested by the dip-slide method and by conventional plating methods in MSB agar. Very good correlation (0.93) between the two methods was obtained. This simplified S. mutans detection system is suitable for use by clinical personnel in dental clinics or other non-laboratory settings for identification of subjects potentially at risk for caries.

Isolation and characterization of a 60-kilodalton salivary glycoprotein with agglutinating activity against strains of Streptococcus mutans.

A bacterial agglutinin specific for strains of Streptococcus mutans was isolated from human saliva. Physiochemical analyses showed the agglutinin to be a glycoprotein with a molecular weight of 60,000. The agglutinin aggregated four of the eight strains of Streptococcus mutans tested but did not aggregate the strains of Streptococcus salivarius, Streptococcus sanguis, and Streptococcus mitis tested. Chemical modification of carbohydrate moieties of the agglutinin with sodium metaperiodate had no effect on aggregation, whereas modification of the polypeptide portion with trypsin abolished aggregating activity. A set of five murine hybridoma antibodies was employed to further analyze the agglutinin. Two carbohydrate-specific antibodies, directed against D-mannose and N-acetylgalactosamine moieties, respectively, failed to block agglutinin- or whole saliva-mediated aggregation of S. mutans cells. In contrast, two antibodies directed against pronase-sensitive antigenic sites blocked both agglutinin- and saliva-mediated aggregation of S. mutans cells. Western blot analysis with the agglutinin-specific hybridoma antibodies demonstrated the agglutinin in whole saliva and in artificial tooth pellicles formed on hydroxyapatite beads incubated with saliva. These results suggest that a 60-kilodalton glycoprotein of human saliva is a bacterial agglutinin with specificity for certain strains of S. mutans. They further suggest that aggregation is mediated by polypeptide rather than carbohydrate determinants of the glycoprotein.

Effect of monoclonal antibodies against lipoteichoic acid from the oral bacterium Streptococcus mutans on its adhesion and plaque-accumulation in vitro

Five monoclonal antibodies directed against Streptococcus mutans strain JBP lipoteichoic acid (LTA) were characterized. They were all similarly reactive with the immunizing LTA-containing extract, with intact Strep. mutans JBP cells and with LTA purified from Lactobacillus casei. Immobilized anti-LTA antibodies removes LTA from LTA-containing extracts. The binding of antibodies to LTA was inhibited by the aqueous extract but not by the organic extract of de-acylated LTA, indicating reactivity with the polyglycerol-phosphate portion of the molecule. Antibodies were reactive with all serotypes of Strep. mutans, as well as with strains of Streptococcus salivarius, Streptococcus sanguis and L. casei, but not with LTA-negative species Streptococcus mitis or Actinomyces viscosus. Anti-LTA antibodies at doses of 0.3 or 3.0 μg/ml, had no effect on the adherence of Strep. mutans JBP to experimental salivary pellicles formed on hydroxyapatite, but enhanced adherence 150–300 per cent at 30 μg/ml. There was no effect of anti-LTA antibodies in a chemostat model which measured sucrose-dependent plaque accumulation by Strep. mutans. The results argue against a major role for LTA in Strep. mutans adherence or plaque accumulation in vitro.

Fluoride uptake and fluoride resistance in oral streptococci.

Fluoride uptake was examined in the highly F-sensitive Streptococcus salivarius strain 25975, the F-resistant mutant Flr103, and the relatively insensitive S. sanguis H7PR3. F was taken up by all strains from media at neutral pH, containing 1 or 10 micrograms F/ml (0.053-0.526 mmol/L), and uptake was directly proportional to the delta pH that was maintained by the cells. The final intracellular concentrations of F in the F-resistant strains were higher than those in the F-sensitive strain. Similar differences in F uptake were observed in buffer at pH 5.5, although both delta pH and F uptake were higher in all strains at the lower pH. These experiments demonstrate that resistance to F does not necessarily involve the exclusion of F from the cells. The lower uptake of F by F-sensitive cells is attributed to the finding that F dissipates the transmembrane pH gradient, and this, in turn, limits the concentration of intracellular F.

Deoxyribonucleic Acid Hybridizations Among Strains of Streptococcus salivarius and Streptococcus bovis

The genetic relationships among 33 streptococci that were identified as Streptococcus salivarius or Streptococcus bovis and were isolated from humans were determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization on membrane filters. The phenotypic characteristics of these bacteria were determined by their action on 20 substrates in a commercially prepared system, as well as by colony morphology and conventional tests. The S. salivarius strains were biochemically heterogeneous but genetically homogeneous. Although there were some phenotypic similarities between S. bovis and S. salivarius, these two species were genetically distinct. Within S. bovis there was genetic heterogeneity. Typical S. bovis strains (S. bovis biotype I) were genetically homologous with some, but not all, of the S. bovis variant strains (S. bovis biotype II). Other S. bovis biotype II strains formed a separate genetic group, the members of which were biochemically somewhat different from other S. bovis biotype II strains.

Inhibition of ecological emergence of mutans streptococci naturally transmitted between rats and consequent caries inhibition by Streptococcus salivarius TOVE-R infection.

The ability of Streptococcus salivarius strain TOVE-R to inhibit the ecological emergence of virulent representatives of the most prevalent human mutans streptococci on the teeth of specific pathogen-free Osborne-Mendel rats was studied. Rats which were infected by TOVE-R, or either S. mutans 10449S or S. sobrinus 6715-13WT, or uninfected were transiently co-caged so as to allow natural fecal transfer of organisms due to coprophagy. The infectants were differentially recovered from swabs of the teeth over the time course of the experiments and from sonified teeth at termination. Data were expressed on both relative (percentage) and absolute (CFU) bases. Initial oral colonization of rats by TOVE-R inhibited the ecological emergence of fecally transmitted S. mutans 10449S and S. sobrinus 6715-13WT. There was a generally inverse relationship between the percentages and absolute numbers of TOVE-R and the mutans streptococci on the teeth, which strongly suggested their competition for tooth sites. Absolute numbers of total recoverable flora from the teeth upon sonification were correlated with caries scores, thus suggesting that total recoverable flora counts substantially reflect cavitation status. TOVE-R itself induced no apparent caries activity and its transmission to rats already infected by 10449S or its colonization of rats before 10449S infection inhibition caries induction by this S. mutans strain; similar anticaries effects were not statistically significant for TOVE-R against 6715-13WT in these experiments. These data on the inhibition of the ecological emergence of the mutans streptococci supplement the already reported ability of TOVE-R to preempt initial colonization of teeth and partially displace the colonization of teeth by the mutans streptococci.

Analytical pyrolysis of Streptococcus saliivarius as and aid to identification in bite-mark investigation

The use of pyrolysis mass spectrometry (Py-MS) and statistical analysis of mass spectra is introduced as a method for “finger-printing” strains of Streptococcus salivarius. The objective is to provide correlative evidence regarding the identity of suspects in cases of assault or rape involving bite-marks. The results of the analysis of isolates from two individuals are presented, illustrating the differentiation of S. salivarius at strain level according to the origin of the isolate.

Adhesive cell wall-associated glycoprotein of Streptococcus salivarius (K+) is a cell surface fibril

The structure of a cell wall-associated glycoprotein, supposed to be involved in host-related adherence of Streptococcus salivarius (K+) strains, was investigated. The Mr of the two major subclasses of the glycoprotein was estimated to be in the range of 230 000 to 295 000, both in the presence of SDS and under non-denaturating conditions. Circular dichroism spectra indicated an α-helix content of 45%. Electron microscopic observation of rotary shadowed individual molecules showed fibrillar structures with an average length of 870 ± 40 Å and a width of 75 ± 6 Å. The glycoprotein is presumably identical with a class of long fibrils found on the surface of these cells.

Adhesive cell wall-associated glycoprotein ofStreptococcus salivarius(K+) is a cell surface fibril

The structure of a cell wall-associated glycoprotein, supposed to be involved in host-related adherence of Streptococcus salivarius (K+) strains, was investigated. The Mr of the two major subclasses of the glycoprotein was estimated to be in the range of 230 000 to 295 000, both in the presence of SDS and under non-denaturating conditions. Circular dichroism spectra indicated an α-helix content of 45%. Electron microscopic observation of rotary shadowed individual molecules showed fibrillar structures with an average length of 870 ± 40 A and a width of 75 ± 6 A. The glycoprotein is presumably identical with a class of long fibrils found on the surface of these cells.

Streptococcus salivarius strains carry either fibrils or fimbriae on the cell surface.

Strains of Streptococcus salivarius were screened by negative staining for the presence of surface structures. Two structural subgroups were found, carrying either fibrils or fimbriae, projecting from the cell surface. Eight strains carried a very dense peritrichous array of fibrils of two distinct lengths. Long fibrils had an average length of 175 nm, and short fibrils had an average length of 95 nm. Two strains carried only long fibrils, one strain carried only short fibrils, and another strain carried a lateral tuft of very prominent fibrils of two lengths, with a fibrillar fuzz covering the remainder of the cell surface. In all the strains in which they were present, the long fibrils were unaffected by protease or trypsin treatment. In contrast, the short fibrils were completely digested by protease and partially removed by trypsin. Neither long nor short fibrils were affected structurally by mild pepsin digestion or by lipase. The Lancefield extraction procedure removed both long and short fibrils. These twelve fibrillar strains were therefore divisible into four structural subgroups. Extracts of all the fibrillar strains reacted with group K antiserum. The second main structural subgroup consisted of nine strains of S. salivarius, all of which carried morphologically identical, flexible fimbriae arranged peritrichously over the cell surface. The fimbriae were structurally distinct from fibrils and measured 0.5 to 1.0 micron long and 3 to 4 nm wide, with an irregular outline and no obvious substructure. There was no obvious reduction in the number of fimbriae after protease or trypsin treatment. Extracts of the fimbriated strains did not react with the group K antiserum. The two serological and structural subgroups could also be distinguished by colony morphology.

Growth and acid tolerance of human dental plaque bacteria

Pure cultures of representative strains of cariogenic and non-cariogenic plaque bacteria were assessed for their ability to initiate and maintain growth in broths, adjusted to initial pH levels of 7.0, 5.5 or 5.0, and to produce lactic acid from sucrose or glucose in resting-cell suspensions at pH 6.5, 5.0, 4.5 and 4.0. Streptococcus mutans, Lactobacillus casei and Streptococcus faecalis showed greater acid tolerance than strains of Streptococcus sanguis, Streptococcus salivarius, Streptococcus mitis and Actinomyces viscosus. For all species, growth initiation in broth was more acid sensitive than lactic-acid production in resting-cell suspensions. These data confirm and extend previous observations that the species of plaque bacteria most closely associated with the initiation or progression of dental caries are more aciduric than non-cariogenic species.