Streptococcus Type Research Articles

Oral colonization by more than one clonal type of mutans streptococcus in children with nursing-bottle dental caries

By ribotyping the genetic diversity of mutans streptococci in six 1.5–3-yr-old children with nursing-bottle caries and in six caries-free, age-matched children and in their mothers was examined. The proportion of mutans streptococci in the dental plaque of the children and their levels in the saliva of the mothers were also examined. For ribotyping, chromosomal DNA of isolates obtained from the plaque of the children (3–12 isolates per child) and from the saliva of the mothers (4–13 isolates per mother) was digested with restriction endonuclease HindIII. The DNA fragments were hybridized to the plasmid pKK3535 which contains the rRNA operon of the Escherichia coli chromosome. The results showed that children with nursing-bottle caries exposed to frequent consumption of sucrose had a high proportion of mutans streptococci in plaque and four of them were colonized with more than one ribotype, whereas caries-free children had a low proportion of mutans streptococci in plaque and only one of them harboured more than one ribotype. Mothers of children with nursing bottle caries had similar levels and numbers of ribotypes of mutans streptococci in saliva as the mothers of the caries-free children. In both child groups, mothers were probably the main source of infection with mutans streptococci. Thus, children with nursing-bottle caries were not only heavily infected with mutans streptococci but also often colonized with more than one clonal type. In the child's acquisition of such clones, frequent sugar consumption may have an important role.

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Monoclonal antibodies (mAbs) have been produced by immunizing BALB C mice with whole M + bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M + and M − bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M + bacteria after treatment with N-acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M − bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.

Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M+ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M+ and M- bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M+ bacteria after treatment with N-acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1-5 and 198-202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M- bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.

Effect of surfactant on nitroblue tetrazolium reduction of polymorphonuclear leucocytes stimulated with type Ia group B streptococci.

Activation of polymorphonuclear leucocytes (PMN) was investigated after incubation of adult human PMN and group B streptococci (GBS) type Ia with a type-specific polyclonal antiserum and a modified porcine surfactant (Curosurf). The level of oxidative metabolism of PMN was studied using a micromethod modification of the nitroblue tetrazolium (NBT) reduction test. GBS alone did not stimulate significant oxygen metabolite release from PMN, and incubation of PMN with surfactant alone resulted in decreased NBT reduction. After opsonization of GBS with a specific antibody, PMN were activated and the increased oxygen metabolite release was not suppressed when surfactant was added to the system. We conclude that the encapsulated GBS strain investigated needs opsonization with specific antibody to increase oxidative metabolism of PMN, and that incubation of PMN and opsonized GBS with surfactant does not interfere with NBT reduction.

Purification of preparative quantities of group B Streptococcus type III oligosaccharides.

Many bacterial capsular polysaccharides are regularly repeating units of oligosaccharides. Bacterial oligosaccharides have been used in neoglycoconjugate vaccines and as reagents in the study of specific antibody binding. Unfortunately, separation methods have not been adequate for the purification of preparative quantities of bacterial oligosaccharides. Here we describe a size-exclusion procedure that resulted in the resolution of group B Streptococcus type III oligosaccharides composed of 4-25 sugars.

The effects of growth in human serum on an acapsular group BStreptococcusmutant

A Group B Streptococcus Type III (GBS) mutant which, when grown in Todd Hewitt broth (THB), does not produce any detectable capsule, produced a clearly visible polysaccharide capsule when grown in human serum. We isolated cytoplasmic membranes from GBS and separated the component membrane proteins by polyacrylamide gel electrophoresis. A significant change in membrane composition was found during growth in human serum. Several unique proteins were produced on serum growth and there was both up- and down-regulation of other proteins. We measured the intracellular levels of sialic acid for a variety of GBS serotype III isolates. Interestingly, while there was little difference between the intracellular sialic levels of most isolates, the sialic acid level of COH31-15 grown in THB was over 100% higher than that of any other isolate. When grown in serum this pool was reduced to a level similar to that in other strains. The concentration of bacterial cell sialic acid was directly correlated with the sialic acid content of the serum. Exogenous sialic acid content, in concert with other serum factors, plays a role in determining the capsular size in GBS.

Maternal immunization of mice with group B streptococcal type III polysaccharide-beta C protein conjugate elicits protective antibody to multiple serotypes.

Group B streptococcal infection is a major cause of neonatal mortality. Antibody to the capsular polysaccharide protects against invasive neonatal disease, but immunization with capsular polysaccharides fails to elicit protective antibody in many recipients. Conjugation of the polysaccharide to tetanus toxoid has been shown to increase immune response to the polysaccharide. In animal models, C proteins of group B streptococci are also protective determinants. We examined the ability of the beta C protein to serve in the dual role of carrier for the polysaccharide and protective immunogen. Type III polysaccharide was covalently coupled to beta C protein by reductive amination. Immunization of rabbits with the polysaccharide-protein conjugate elicited high titers of antibody to both components, and the serum induced opsonophagocytic killing of type III, Ia/C, and Ib/C strains of group B streptococci. Female mice were immunized with the conjugate vaccine and then bred; 93% of neonatal pups born to these dams vaccinated with conjugate survived type III group B streptococcal challenge and 76% survived type Ia/C challenge, compared with 3% and 8% survival, respectively, in controls (P < 0.001). The beta C protein acted as an effective carrier for the type III polysaccharide while simultaneously induced protective immunity against beta C protein--containing strains of group B streptococci.

Solid-phase antigen density and avidity of antibodies detected in anti-group B streptococcal type III IgG enzyme immunoassays

Two enzyme immunoassays which measure anti-group B streptococcal type III capsular carbohydrate IgG antibodies were compared. One utilised poly- L-lysine conjugated coating antigen while the other used tyraminated coating antigen. Both carbohydrate antigens appeared to be antigenically identical but the poly- L-lysine based assay gave significantly lower values for some sera. Sera were identified which had low and high avidity anti-group B streptococcal type III IgG antibodies by the thiocyanate elution method. These antibodies gave results on a dilution range of coating concentrations consistent with their relative avidity. Comparison of dilution ranges of the two conjugates used for coating suggests that the poly- L-lysine conjugate coats with a ten-fold lower efficiency than the tyramine conjugate and therefore detects only higher avidity antibodies. Four fractions containing different relative avidities of affinity-purified IgG were produced from a single serum. These fractions behaved in the same manner as sera containing antibodies of different avidities. The results of this study suggest that the method of polysaccharide conjugation in enzyme immunoassays may affect the antigen concentration on the solid phase and thence the detection of antibodies of various avidities.

Growth of group B streptococci in human serum leads to increased cell surface sialic acid and decreased activation of the alternative complement pathway.

Group B streptococcus type III is a major cause of neonatal death. The terminal sialic acid moiety of the group B streptococcus type specific capsule has been shown to be an important virulence factor. We demonstrate here that bacteria grown in human serum have increased cell surface sialic acid content compared with cells grown in common laboratory media. This sialic acid was removed by incubation with neuraminidase, showing that it was on the bacterial surface. Serum-dependent sialylation was dependent on metabolic activity, as the addition of chloramphenicol reduced the amount of added sialic acid by more than 90%. Probing the cell surface with an antibody specific for group B streptococcus type III capsular sialic acid showed an increase in antibody binding after growth in human serum. This effect could be lowered by incubating serum-grown cells in neuraminidase prior to antibody exposure. A group B streptococcus mutant that when grown in laboratory media lacks cell surface sialic acid showed significant cell surface sialic acid when grown in human serum. This increase was associated with a significantly decreased ability to bind C3 and hence activate the alternative complement pathway.

MACROPHAGE CANDIDACIDAL ACTIVITY IN HUMAN NEONATES: DECREASED SENSNIVITY TO MODULATION BY IFN-γ

In an attempt to understand the mechanisms of host defense in the term neonate against candida, we studied interaction between Calbicans and monocyte-derived macrophages (MDM) cultured for 5 days from the cord blood. In the presence of 25% normal adult serum, the extent of phagocutosis of candida was equivalent with cord or adult MDM. In contrast to candida, MDM did not phagocytose non-opsonized group B Streptococcus type III, Mannosylated BSA (Man BSA, molar ratio of mannose to BSA = 23:1) inhibited non-opsonic ingestion of candida by cord and adult MDM 74 and 87 inhibition by 64 and 320 pg mannose/ml, respectively, after 60 min incubation means, n=6), suggesting a role for the mannose receptor. Cord MDM killed both opsonized and unopsonized Calbicans as effectively as did adult MDM (n=7) However, exposure of adult and cord MDM to IFN-γ (10-500 U/ml) gave quantitativel different results in candida killing and O release maximal increase in these functions with adult MDM were achueved with 100 U/ml IFN-γ, whereas no enhancement with cord MDM could be detected after treatment with 100 Uml, and at 500 Uml there was still a significantly lower killing and O. release by and MDM compared to adult cells. These data suggest that: 1. clearance of candida both the blood stream may occur in the absence of full opsonization a condition that might exist in the newborn; 2 resistance of cord MDM to the effect of IFN-γ may represent a developmental immaturity of human Phagocytes.

Typing of pneumococci by using 12 pooled antisera.

A new simplified chessboard system for typing of Streptococcus pneumoniae is described. It is intended for typing or grouping of 90 to 95% of the pneumococcal strains most commonly isolated from blood or cerebrospinal fluid and is based on 12 pooled diagnostic antisera, each reacting with 7 to 11 single types, together covering the 23 different vaccine-related types as well as 25 other cross-reacting types. Worldwide surveillance of the type distribution is important in order to ensure an optimal formulation of pneumococcal polysaccharide vaccines and, in the future, of polysaccharide-protein conjugate vaccines. The simplified typing system described in this paper makes it easier to carry out surveillance in other than specialized reference laboratories. Finally, it takes advantage of the fact that some types cause disease more often in children--as opposed to adults--than do others.

Presumptive fecal streptococci in environmental samples characterized by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

The use of fecal streptococci as fecal indicators requires better knowledge of the ecology of these bacteria. We isolated 371 presumptive fecal streptococci from environmental samples--domestic wastewater, forest industry wastewater, contaminated surface and seawater, well water, cow dung, bird droppings, and pristine waters--and clustered them according to their protein profiles in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Some clusters could be tentatively identified with the help of reference strains. Samples from each environment had a typical composition of streptococcus types. Enterococcus faecalis was present, but not as a dominating enterococcal species, in samples in which fecal contamination was probable. Enterococcus faecium, Enterococcus durans, Enterococcus hirae, and Enterococcus mundtii had protein profiles that were difficult to distinguish from each other. These bacteria were found in a variety of samples. Enterococcus casseliflavus and Enterococcus gallinarum had identical protein profiles. On the basis of the maximum temperatures for growth and pigment production, isolates of this protein profile group common in forest industry wastewaters were identified as E. casseliflavus. Lactococcus lactis subsp. lactis was also found in this environment. Nearly all strains from pristine waters belonged to protein profile groups which could not be identified with the aid of known Aerococcus, Enterococcus, Lactococcus, or Streptococcus strains. The maximum temperatures for growth and the results of fatty acid analysis were in general agreement within each protein profile group.

Co-existent cross-infection with Streptococcus pneumoniae and group B streptococci on an adult oncology unit

An outbreak of respiratory infection in an adult oncology unit is described where there was evidence of co-existent cross-infection with Streptococcus pneumoniae serotype 14 and Lancefield group B streptococcus type Ia. The presumed route of transmission was droplet spread from patient to patient. No further cases arose after the ward had been closed to new admissions and all symptomatic patients were treated with erythromycin. We suggest that patients with pneumococcal pneumonia on units housing elderly debilitated patients should be isolated. Infection control teams should be aware of the ability of Lancefield group B streptococci to spread by cross-infection among adult patients.

Human B lymphocyte in vitro response to the group B streptococcal type III capsular carbohydrate.

Anti-group B streptococcal type III polysaccharide-specific human B cells from the peripheral circulation can be activated and detected in an in vitro culture system. It is possible to detect both IgM- and IgG- producing cells from both seropositive and seronegative donors. The specificity of the response was demonstrated by inhibition with excess liquid phase antigen and the use of related but antigenically distinct control antigens. The response was absent without the addition of T cells, optimal at 10% and 25% T cells respectively for IgM- and IgG-secreting cells, and undetectable using 50% T cells. The optimal antigen concentration for in vitro B-cell activation is 2.5 x 10(-4) micrograms/ml. Cells from 5 of 6 seropositive donors and 3 of 7 seronegative donors produced specific IgM antibody after culture with antigen. We conclude that the control of the human antibody response to the group B streptococcal type III polysaccharide is influenced by T cells. The response seen in the culture system may be of value in assessing future vaccine candidates designed to prevent neonatal infections.

Recurrent juvenile dermatomyositis and cutaneous necrotizing arteritis with molecular mimicry between streptococcal type 5 M protein and human skeletal myosin

An adult patient had a syndrome associating the features of juvenile dermatomyositis and cutaneous polyarteritis nodosa that followed a cyclic course from childhood; recurrences were always associated with a rise of serum antistreptococcal antibodies. Regions of homology between streptococcal type 5 M protein and skeletal myosin were found. These findings suggest that streptococcal infection, possibly through a molecular mimicry mechanism, played a role in the pathogenesis of the disease in our patient.

Capsular types of Streptococcus pneumoniae isolated from blood and CSF during 1982-1987.

Knowledge about the type distribution of Streptococcus pneumoniae is fundamental to ensure an effective formulation of pneumococcal vaccine, especially with the possibility of producing a polysaccharide-protein-conjugated vaccine for the prevention of invasive disease in children. During the 6-year period 1982-1987, we received and typed 10,298 isolates from patients with invasive pneumococcal disease: 7,812 (76%) from blood and 2,486 (24%) from CSF. Of all isolates, 81% were recovered from individuals in Europe and 23% were from children. In order of frequency, S. pneumoniae types 6A + 6B, 14, 18C, 19F, 1, 7F, 23F, 19A, 4, and 5 were most commonly isolated from children, and types 3, 1, 14, 7F, 4, 6A + 6B, 8, 23F, 9V, and 19F, from adults. The pneumococcal types in the currently available 23-valent vaccine represented 87% of all isolates in this study, but the proportion of vaccine types varied somewhat with age and source. In all pneumococcal groups included in the vaccine, the vaccine types represented > 80% of the isolates, except in groups 6, 15, and 18.

Production of monovalent antisera by induction of immunological tolerance for capsular typing of Streptococcus pneumoniae

Hyperimmune and high-titered polyclonal pneumococcal antisera, specific for cross-reactive types within groups, were produced in adult rabbits. Purified capsular polysaccharide was injected intravenously into adult rabbits. One week later, these rabbits were given multiple intravenous injections of formalin-inactivated pneumococci of the cross-reactive type by an established method. Each of the resultant antisera were specific for the cross-reactive type indicating that the previous injection of the polysaccharide had induced epitope-specific tolerance. This method was successful for production of antisera against pneumococcal types 6A, 6B, 9N, 9V, 19F and 19A. Polyclonal rabbit pneumococcal antisera have some advantages over murine monoclonal antibodies for serologic studies and this method should be applicable for producing type-specific antibodies to cross-reactive polysaccharides of clinical interest. Further, this method is simpler and generally produces higher titered monovalent (factor) reagents than absorbed antisera.

Production of monovalent antisera by induction of immunological tolerance for capsular typing ofStreptococcus pneumoniae

Production of monovalent antisera by induction of immunological tolerance for capsular typing of<i>Streptococcus pneumoniae</i>

Development of IgM antibody to group B Streptococcus type III in human infants.

An ELISA was developed to measure IgM antibody to albumin-coupled native capsular polysaccharide of type III group B streptococcus (GBS). The assay was standardized by two double-label methods that agreed within 33%. In quantitative assays, the range of IgM antibody to type III GBS in the sera of 94 adult pregnant women was 1.2-50.6 micrograms/ml (median, 5.4), while each of 38 cord serum samples contained less than 0.03 micrograms/ml IgM antibody. Neonatal rats were passively immunized with a serially diluted human serum containing 15 micrograms/ml IgM and undetectable IgG antibody to type III GBS. The rats were protected against lethal infection with an IgM antibody concentration of 0.5 micrograms/ml. In single serum samples from 31 healthy infants less than 2 years old and serial specimens from 5 infants with type III GBS infections, specific IgM antibody was detectable by 3 months of age. Levels greater than or equal to 0.5 micrograms/ml were present in all samples from infants greater than 7 months of age. The acquisition of specific IgM antibody is inversely correlated with the age-limited incidence of type III GBS infections in young children.

Food-Borne Outbreak of Group A ß-Hemolytic Streptococcal Pharyngitis

An outbreak of pharyngitis due to group A beta-hemolytic streptococci type T 12 occurred at a military base. An epidemiologic investigation indicated that the outbreak was food borne. Consumption of boiled egg salad at lunch was significantly associated with the illness. Immediate institution of antibiotic therapy and isolation of the patients prevented secondary respiratory spread of the infection. No cases of poststreptococcal suppurative and nonsuppurative complications were found during a 6-week period after the outbreak. Medical personnel should be aware of the possibility of food-borne streptococcal pharyngitis. Regular health surveillance of food handlers and food preparation processes are important for prevention of such outbreaks.