Hemagglutination Activity and Localization of Fc Receptor of Group A and G Streptococci

The IgG-Fc binding activity and binding sites on the cell surface of streptococci, strains AR1 (group A) and G148 (group G), and Staphylococcus aureus strain Cowan I were examined by hemagglutination (HA) and immunoelectron microscopic methods. No distinct difference was observed in the HA activity among these three strains. However, the strains differed in the distribution of Fc receptor. Cowan I cells (having protein A) were heavily covered with two layers of ferritin particles, whereas AR1 cells were heavily covered with a single, rough layer of ferritin particles. G148 cells (having protein G) were labeled with a relatively thin, rough ferritin layer. The trypsin susceptibility of the Fc receptors of the AR1 strain was much higher than that of the G148 strain. These results suggest that both streptococcal strains are distinctly different in the arrangement or in the conformation of the Fc receptor from the Cowan I strain. It is also suggested that the Fc receptor molecules of the streptococcal strains differ from each other not only in conformation but also in trypsin susceptibility.     

Selective Broth Medium for Isolation of Group B Streptococci

A selective medium containing Todd-Hewitt broth, sheep blood, nalidixic acid, and gentamicin sulfate was found to enhance significantly the isolation of group B streptococci from vaginal cultures. Preparation of the medium, which is stable for up to 4 weeks at 4 C, is simple and inexpensive. Use of such a medium should facilitate identification of vaginal colonization with group B streptococci.     

Production of Double Zones of Hemolysis by Certain Strains of Hemolytic Streptococci of Groups A, B, C, and G on Heart Infusion Agar

This report describes the appearance of a double zone of hemolysis around surface colonies of certain strains of streptococci of groups A, B, C, and G incubated aerobically on sheep blood-Heart Infusion Agar. The occurrence of the altered hemolytic pattern was related to peroxide production by the organism. Anaerobic conditions and the incorporation of catalase into the agar abolished the double-zone pattern and caused reversion of the organisms to a beta-hemolytic pattern. The double-zone pattern can be confused with alpha hemolysis on surface growth.     

Serological Relationships of Type I Antigens of Group B Streptococci

Some of the complex antigenic relationships of type I group B streptococci from various clinical sources were defined by means of immunodiffusion, absorption, and precipitin tests. Three predominant types are described: Ia, Ib, and Ii. Methods for preparing antisera for differentiating type I strains are presented.     

Expression of Group B Protective Surface Protein (BPS) by Invasive and Colonizing Isolates of Group B Streptococci

Group B protective surface protein (BPS) is expressed on the cell surface of some group B streptococcal (GBS) (Streptococcus agalactiae) strains and adds to the identification by capsular polysaccharide (CPS), and c or R proteins. We investigated the prevalence of BPS among GBS clinical isolates (303 invasive, 4122 colonizing) collected over 11 years in four American cities. Hot HCl cell extracts were tested by immunoprecipitation in agarose with rabbit antisera to BPS; the alpha (α) and beta (β) components of c protein; R1, R3, and R4 species of R protein; and CPS serotypes Ia–VIII. BPS was found in 155 isolates (seven invasive, 148 colonizing). Of these, 87 were Ia, 37 II, 20 V; none were III. BPS was expressed usually with another protein: a species of R by 87 or a component of c by 39. The predominant CPS/protein profiles with BPS were Ia/R1,BPS and II/c(α + β),BPS. Thus, along with CPS serotype and other surface proteins, BPS can be a valuable marker for precise strain characterization of unique GBS clinical isolates with complex surface protein profiles.     

Identification of Group A Streptococci by Direct Fluorometry

A simple direct fluorometric method for rapid identification of group A streptococci is described. The method permits the detection of the organism in mixed cultures without the aid of a microscope and is amenable to automated processing of specimens. Experience with the indirect fluorometric method revealed that nontrypsinized cells from a 10-fold dilution of overnight broth cultures could be stained with uniform brilliance with fluorescent antibody (1:15 dilution) and that fluorescent antibody dissociated from such cells at 55 C for 20 min gave serologically specific fluorometric values. With this information, it was possible to develop a simpler fluorometric test which gave results comparable to those obtained by conventional cultural-precipitin grouping techniques. In the direct test described, cultures from throat swabs were incubated overnight, and cells from a 10-fold dilution were stained with specific fluorescent antibody (1:50 dilution) and then rinsed. The stained specimens were transferred to a continuous-filter paper strip (Whatman 3 MM) and read serially in a Turner 110 fluorometer with Corning 5840 and Wratten 2A filters in place. The reagents used required careful standardization and testing to assure that fluorometric readings above a specified value would be indicative of the presence of group A streptococci.     

Comparative characteristics of protein G gene in Streptococci of groups C and G isolated from humans

The treatment of streptococci, groups C and G, with bromocyanogen made it possible to isolate surface G protein, capable of binding human serum albumin (HSA) and polyclonal IgG. In this work the presence of G protein in all staphylococcal strains, groups C and G, is shown. The differences between the strains by the level of expression, molecular weight and functional activity of G protein, extracted from streptococci of groups C and G, permitted the identification of 3 groups of strains, containing the molecules of G protein with different numbers of IgG- and HSA-binding domains: with 3 IgG- and HSA-binding domains, with 2 IgG- and HSA-binding domains and with only 2 IgG-binding domains. Each strain under study expressed only one of the molecule of G protein. The work shows the possibility of the identification of streptococci, groups C and G, by the molecular characteristics of G proteins themselves and their respective coding genes.     

Human Monocytes and Monocyte-Derived Macrophage Phagocytosis of Serotype III Group B Streptococci Strains

In this report group B streptococci (GBS) strains 90356 and 80340 isolated from liquor and vagina, respectively, were placed into contact with human peripheral blood monocytes (PBM) and macrophages derived from monocytes (MDM) by differentiation in vitro. The increased expression of CD16 and CD68 by macrophages cultured for 7 days compared with adherent monocytes supported the distinct maturation status of these cells. The number of viable intracellular bacteria of the 90356 strain was observed after 2 h of incubation with PBM (P < 0.001) and 0.5 h with MDM (P < 0.001). MDM cells seemed to present a more efficient mechanism of bacterial destruction of GBS type III, isolated from a case of meningitis. Viable cells of strain 80340, isolated from the vagina, were not detected in significant numbers in PBM and MDM phagocytic cells. These findings add to our current understanding of the roles played by multiple receptor-ligand systems in the uptake and pathogenesis of group B streptococci infection. Survival strategies of GBS, which interfere with macrophage bactericidal functions, might exist. Received: 25 September 2000 / Accepted: 20 October 2000     

Identification of Fluorescent-Antibody Labeled Group A Streptococci by Fluorometry

A quantitative system, amenable to automation, for determining the presence of group A streptococci in broth culture is described. After separation from the broth, the cells' protein coats are removed by digestion with 0.1% trypsin, and they are then stained with anti-A fluorescent antibody (FA). Excess FA is removed, and bound FA is put into solution by dissociation with demineralized distilled water. The amount of FA bound to the cells is quantitated by fluorometry of the solution. The level of nonspecific staining is measured by staining the cells with fluorescein-conjugated normal rabbit globulin absorbed with group A cells, dissociating, and quantifying, as above. The two quantities are subtracted to measure specific binding of FA to group A cells. A clinical trial showed 92% agreement with microscopists.     

Binding of collagen to group A, B, C, D and G streptococci

Abstract Binding of ¹²⁵I-labelled collagen type II to group A, B, C, D and G streptococci was studied. Strains of all five serogroups were found to bind. Binding to one high-binding strain (group G, strain 12127) was characterised. This was reversible, saturable with time and inhibited by unlabelled type II collagen, but not by other proteins such as fibronectin and ovalbumin. However, binding was inhibited by unlabelled type I, II and III collagens and gelatin, suggesting that a common structure of various collagens is involved in binding.     

Time, Cost, and Efficacy Study of Identifying Group A Streptococci with Commercially Available Reagents

During the 12-month period primary throat, wound, and skin cultures, tentatively identified as B streptococci, were submitted by 10 different clinical laboratories for evaluation. A total of 692 beta-hemolytic streptococci were isolated from cultures submitted and examined in parallel by the fluorescent-antibody, precipitin, and bacitracin techniques. An evaluation of the specificity and sensitivity in conjunction with basic and personnel costs was determined for each method. The standard Lancefield precipitin method was established as the standard by which the bacitracin and flourescent antibody techniques were compared. With some variation depending on the commerical source of the disc, approximately 7% of the strains examined produced false reactions with the bacitracin disc. False-negative reactions were rarely noted by the group A fluorescent antibody technique (0.5%), but an appreciable number of other Lancefield groups (B, C, and G) were nonreactive with homologous conjugates.     

Antigenic Variation in Group A Streptococci: Types 11 and 9

A strain of group A Streptococcus which was virulent but M-nontypable was isolated from patients in a hospital nursery during an epidemic. This strain, Boston 11, reacted in T-agglutination tests with antisera for types 9 and 11, an unusual combination. A comparison of this strain with Lancefield's M-11 strain (NCDC SS-721) and Alabama 11 (Provisional 61) revealed three serologically related but distinct strains. Antiserum produced with the Boston 11 strain exhibited similar reactivity with all three "11" strains as well as with M-9 (SS-501) as demonstrated in precipitin tests. Immunodiffusion studies indicated that the Boston 11 antigen was partially identical with the M-11 and M-9 strains and shared at least one antigen with the Alabama 11 strain. The Boston 11 antiserum could be made specific for precipitin tests, but bactericidal activity for the Alabama 11, M-11, and Boston 11 strains was essentially negative.     

Isolation of ginkgolides A, B, C, J and bilobalide from G. biloba extracts

Ginkgolides A, B, C and J, together with bilobalide, are unique terpenoid components of the Ginkgo biloba tree. Due to similar chemical properties, their separation is quite tedious. We have developed an efficient and rapid protocol for separation of individual ginkgolides and bilobalide from G. biloba extracts. The procedure takes advantage of enhanced susceptibility of ginkgolides B and C to benzylation and the ease of separation of these products from ginkgolides A and J which do not react. The protocol is applicable to the previously reported enriched extracts prepared from G. biloba leaves. A single chromatographic step prior to benzylation provides bilobalide and mixture of ginkgolides A, B, C, and J. After benzylation, the individual ginkgolides are separated by chromatography.     

Detection of Group A Streptococci in Throat Swabs by Direct Fluorometry

A direct fluorometric test for the rapid detection of group A streptococci from throat swabs was compared with the microscopic fluorescent-antibody test. Formalinized throat swab cultures (490) were examined by the two methods, and the results agreed on 84% of the specimens. In another comparison, 15-hr broth cultures of 103 freshly taken throat swabs were tested by both methods. Of the specimens tested, 101 (98%) were either positive or negative by both methods. In all cases, the latter results correlated with the demonstration of presence or absence of group A streptococci in the specimens by cultural isolation and precipitin grouping tests. It may be feasible to use the direct fluorometric test in a diagnostic laboratory as described or possibly to adapt it for automatic processing of throat swab cultures.     

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.