Amylase-binding as a discriminition among oral streptococci

The ability of 51 strains, belonging to Streptococcus sanguis, 'S. mitior', S. oralis and related groups, to bind salivary amylase was studied. Most strains were grouped according to their DNA-relatedness and then compared using 14 phenotypic tests. S. mitis, 'S. mitior' and three relatively new groups of strains ('CR', 'MGH' and 'Tufted mitior') bound salivary amylase, while strains of S. sanguis and S. oralis did not. The ability of strains to bind amylase or not was remarkably consistent within groups and the test proved to be reproducible, rapid and easy to perform. Combination of the amylase-binding test with 6 other conventional physiological tests allowed the construction of a dichotomous identification key which correctly identified 95% of strains for which genetic data was available. These findings suggest that the ability of organisms to bind salivary amylase could become a key test in identification schemes for certain oral streptococci.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.     

Clinical isolates of Enterococcus faecalis aggregate human platelets

Many endocarditis pathogens activate human platelets and this has been proposed to contribute to virulence. Here we report for the first time that many clinical isolates of Enterococcus faecalis, a common pathogen in infective endocarditis, aggregate human platelets. 84 isolates from human blood and urine were screened for their ability to aggregate platelets from four different donors. Platelet aggregation occurred for between 11 and 65% of isolates depending on the donor. In one donor, a significantly larger proportion of isolates from blood than from urine caused platelet aggregation. Median time to aggregation was 11 min and had a tendency to be shorter for blood isolates as compared to urine isolates. Immunoglobulin G (IgG) was shown to be essential in mediating activation and aggregation. Platelet aggregation could be abolished by an IgG-specific proteinase (IdeS), by an antibody blocking FcRγIIa on platelets, or by preabsorption of plasma with an E. faecalis isolate. Fibrinogen binding to bacteria or platelets does not contribute to platelet activation or aggregation under our experimental conditions. These results indicate that platelet activation and aggregation by E. faecalis is dependent on both host and bacterial factors and that it may be involved in the pathogenesis of invasive disease with this organism.     

Relationship between the ability of oral streptococci to interact with platelet glycoprotein Ibalpha and with the salivary low-molecular-weight mucin, MG2

The oral streptococci Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis are common aetiological agents of infective endocarditis, and their ability to adhere to and induce the aggregation of platelets is thought to be a virulence trait. The platelet glycoprotein GPIbalpha has been implicated as the adhesion receptor for S. sanguinis and S. gordonii, but it is not known if this is the case for S. oralis and other species. The aim of this study was to determine the GPIbalpha-interactive capability of a range of oral streptococci and to determine the relationship between this capability and their ability to interact with the salivary constituents that they would encounter in their normal habitat. All platelet-adhesive S. sanguinis strains and most S. gordonii strains adhered in a GPIbalpha-dependent manner, but strains of S. oralis, Streptococcus cristatus, Streptococcus parasanguinis and Streptococcus mitis had no direct affinity for platelets. Those strains that were able to bind GPIbalpha also bound to the low-molecular-weight submandibular salivary mucin, MG2, and this interaction was sialic acid-dependent. The data suggest that S. sanguinis and S. gordonii may be efficient colonizers of platelet vegetations because of their adaptation to recognize sialylated salivary mucins. In contrast, S. oralis does not interact with platelets and so is likely to colonize vegetations through an as yet unidentified mechanism.     

Surface structures, co-aggregation and adherence phenomena of Streptococcus oralis and related species

Seven strains of Streptococcus oralis were found to possess surface structures. Four strains possessed long fimbriae which ranged in length from 266-366 nm, while the remaining three strains possessed shorter peritrichously distributed fibrils which ranged in length from 80-197 nm. The fibrillar strains were morphologically similar to strains of Streptococcus sanguis I and II and Streptococcus mitis. No strain of S. oralis produced tufts of fibrils like certain strains of S. sanguis I. Strains of Streptococcus milleri produced peritrichously distributed fimbriae which were morphologically dissimilar to the fimbriae produced by S. oralis strains. S. oralis strains were moderately to highly hydrophobic, but hydrophobicity could not be related to adhesion parameters or type or length or density of surface structures. Furthermore, there appeared to be no correlation between type of surface structures and adhesion parameters. Two of the three fibrillar strains of S. oralis and the peritrichously fibrillar strains of S. sanguis, together with one strain of S. milleri, were able to co-aggregate with actinomycetes.     

Cell surface components of Streptococcus sanguis: relationship to aggregation, adherence, and hydrophobicity.

Cell surfaces of aggregation, adherence, and hydrophilic variants of Streptococcus sanguis were compared with cell surfaces of the parent strain with regard to their protein and antigenic constituents. Cell surface molecules were released by digestion with mutanolysin. Extraction with sodium dodecyl sulfate (SDS) urea, lithium diiodosalicylate, and boiling water did not solubilize any material which stained with AgNO3 in an SDS-polyacrylamide gel electrophoresis gel. The parent organism S. sanguis 12, which aggregates in saliva, adheres to saliva-coated hydroxyapatite and is hydrophobic, was found to possess a prominently staining 160,000 molecular weight (MW) protein. This protein was almost completely absent from strain 12na, a hydrophobic nonaggregating variant, and was completely absent from the hydrophilic nonaggregating strain 12L. Trypsinization of strain 12 resulted in the coincident loss of the 160,000-MW protein and the ability to aggregate in saliva. Trypsin treatment reduced but did not eliminate the hydrophobic character of the cells. Boiling destroyed their ability to aggregate, but did not alter their hydrophobicity. Cell wall digests of strain 12 contained a number of proteins which were absent from strains 12na and 12L. Mutanolysin digests of cell walls of the hydrophilic strains contained almost no material that was visible in a silver-stained SDS-polyacrylamide gel electrophoresis gel. Culture supernatants contained a number of proteins which were immunologically cross-reactive with cell surface proteins. The hydrophilic organisms released a number of 60,000- to 90,000-MW proteins not seen in culture supernatants from the parent strain.     

Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

Cross-reactive immunodeterminants on Streptococcus sanguis and collagen. Predicting a structural motif of platelet-interactive domains.

Cross-reactive immunodeterminants on a fibril-associated surface antigen of Streptococcus sanguis and types I and III collagen participate in the induction of aggregation of human platelets. To further understand the basis for this apparent molecular mimicry, antitype-specific collagen antibodies, anti-KPGEPGPK (an analogue of platelet-interactive domains on collagen) and a panel of KPGEPGPK-like synthetic peptides were used as probes. When collagen or S. sanguis cells were pretreated with the anti-collagen antisera, the induction of aggregation of platelet-rich plasma was greatly delayed or abrogated. These anti-collagen antibodies also neutralized KPGEPGPK and purified S. sanguis platelet-interactive antigens as inhibitors of S. sanguis or collagen-induced aggregation of platelets in plasma. In immunoblot analyses, these anti-collagen antibodies reacted with S. sanguis platelet-interactive antigens. Additionally, antisera against the platelet-interactive antigen of S. sanguis selectively reacted with undigested type I collagen and with fragments CB3 and CB6 of cyanogen bromide-treated type I collagen. Finally, when platelets were pretreated with synthetic peptides containing specific amino acid substitutions within the KPGEPGPK sequence, the time to onset of platelet-rich plasma aggregation by both agonists was altered. The hierarchical pattern of responses of platelets to these peptides and predictions of the structural changes produced by simulated insertions of each peptide into the CB4 sequence of type III collagen suggested conformational requirements for interactions with platelets. Thus, these data show that cross-reactive immunodeterminants of S. sanguis and collagen induce platelet aggregation. The platelet-interactive domains are predicted to be characterized by a structural motif with the consensus sequence X-P-G-E-P/Q-G-P-X.     

血链球菌与疾病研究进展

血链球菌是早期定植在口腔内的细菌之一,也是口腔内的常驻菌.血链球菌作为牙周有益菌对大多数牙周可疑致病菌具有拮抗作用.其主要机制为产生过氧化氢和血链素.由于各种原因进入血液循环后可引起感染性心内膜炎,主要机制为血链球菌的表面抗原使血小板产生黏附、聚集,其在血小板表面的结合位点在血小板膜糖蛋白Ib附近.单核细胞在血链球菌的刺激下表达大量的组织因子,激活外源性凝血途径形成血栓.     

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.     

血链球菌产生过氧化氢机理研究

作者在国内首次建立两种确定细菌产生过氧化氦的方法,可以定性和定量分析。本文探讨了血链球菌产生过氧化氢的条件,研究其在牙周微生态环境中的作用。     

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.     

Streptococcus crista sp. nov., a viridans streptococcus with tufted fibrils, isolated from the human oral cavity and throat.

We studied strains of an unusual streptococcus that superficially resembles Streptococcus sanguis but has fibrils that are arranged in lateral tufts. These strains were originally isolated from human throats and oral cavities and have been referred to previously as "Streptococcus sanguis I," the "CR group," and the "tufted-fibril group." Until now, insufficient phenotypic data have been available to allow reliable differentiation of these strains from other viridans streptococcal species, particularly the species in the S. sanguis group. Recently, workers have proposed a scheme of phenetic tests that is based on 4-methylumbelliferyl-linked substrates and conventional biochemical tests and allows the tufted-fibril group to be differentiated; these organisms differ from other viridans species in being able to hydrolyze arginine but not esculin and in producing alpha-L-fucosidase but not beta-glucosidase or alkaline phosphatase. These data, together with the results of our DNA-DNA hybridization experiments and the unusual ultrastructure of the tufted-fibril strains as determined by electron microscopy, demonstrate that these organisms represent a new species, for which the name Streptococcus crista is proposed. The DNA base composition is 42.6 to 43.2 mol% G + C. The type strain is strain CR311 (= NCTC 12479).     

Molecular basis for the association of glucosyltransferases with the cell surface of oral streptococci

The expression of Streptococcus mutans mutant glucosyltransferase-I enzymes in S. sanguis and S. milleri suggests that cell-associated glucosyltransferase activity is dependent upon both glucan synthesis and glucan binding by the carboxyl-terminal repeating units of the enzyme. Mutant enzymes lacking these repeating units were only present in the extracellular fluids of these transformed streptococcal strains.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis , dextran-positive Streptococcus mitior , dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

Comparative adhesion of seven species of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis to fibrin-platelet clots in vitro

The adhesion to fibrin-platelet clots in vitro of 21 strains of streptococci isolated from the blood of patients with sub-acute bacterial endocarditis (SABE) was measured. The species, in order of greatest adhesion, were Streptococcus faecalis, Streptococcus mutans, Streptococcus milleri, Streptococcus sanguis, dextran-positive Streptococcus mitior, dextran-negative Streptococcus mitior and Streptococcus salivarius. Individual strains within species, however, cannot be assumed to be representative of their species and may exhibit unusually high or low adhesion. Adhesion depended upon both bacterial concentration and period of contact. There was no simple relationship between ability to adhere and liability to cause endocarditis. Formation of dextran did not increase adhesion. The streptococci were more adhesive than strains of Escherichia coli and Neisseria sicca and less adhesive than strains of Staphylococcus aureus and Streptococcus pyogenes.     

Lack of correlation between fibrils, hydrophobicity and adhesion for strains of Streptococcus sanguis biotypes I and II

Fifteen strains of Streptococcus sanguis biotype I and eight strains of Streptococcus sanguis biotype II with peritrichous fibrils, tufts of fibrils or a mixture of fibrils and fimbriae on the cell surface, were tested for their ability to adhere to saliva coated spheroidal hydroxyapatite (S-SHA) in a radiolabelled assay. S. sanguis I strains adhered better than S. sanguis II strains and peritrichously fibrillar strains generally adhered better than tufted strains. There was no correlation between the density of fibrillation and adhesion. The only highly adherent strain of S. sanguis II carried fimbriae in addition to fibrils. No correlation was observed between cell surface hydrophobicity as measured by phase partitioning with hexadecane and adhesion to S-SHA.     

New serotype of mutans streptococci isolated from pig oral cavity

Gram-positive streptococcal mutans-like strains, but with clearly different colony formation than S. orisuis on Mitis Salivarius agar, were isolated from the pig oral cavity and identified by 16S rRNA sequencing, G+C content, DNA-DNA homology and extensive biochemical and serological testing. The phenotypic data showed that the strains were similar to S. orisuis except for susceptibility to bacitracin. DNA-DNA homology between the isolates and S. orisuis was 72∼81%. However, serological data showed that they have a different sero-specific antigen from S. orisuis and other mutans streptococci. A new serotype, designated p, strains are classified in a serovar of S. orisuis, one of mutans streptococci.