Anti-idiotypic Vaccination against Group B Streptococci

We describe the antigenic properties of an anti-idiotypic single chain fragment variable (scFv) recombinant antibody mimicking the type III capsular polysaccharide of group B streptococci (GBS), an important cause of neonatal sepsis. This scFv could compete with the nominal antigen for binding to specific mouse or rabbit antibodies. Moreover, the scFv elicited, in mice, the production of antibodies which reacted against the type III polysaccharide and passively protected neonatal pups from GBS disease. Maternal immunization with the scFv also protected neonatal mice against GBS infection. Next, the scFv was expressed on the surface of the commensal bacterium Streptococcus gordonii. Intravaginal inoculation of mice with these recombinant bacteria induced significant elevations in serum titers of anti-GBS type III antibodies. Therefore, the expression scFv in commensal bacteria may be a convenient and effective way of delivering anti-idiotypic vaccines.     

Type-Specific Antigens of Group B Type Ic Streptococci

The type-specific antigens of group B type Ic (old designation type Ii) streptococci were extracted, purified, and characterized by serological and chemical methods. The Ia antigen, shared by types Ia and Ic, is a polysaccharide composed of 69% galactose and 25% glucosamine (i.e., 31% N-acetyl-glucosamine). However, these monosaccharides failed to inhibit significantly the quantitative precipitin reactions between purified antigen and type Ia antiserum. Indications are that the immunodominant group of this antigen consists of more than a simple monosaccharide. The Ic antigen, shared by types Ib and Ic, is a protein unrelated to the X and R protein antigens. Ic antigen consists of two serologically active determinants, one of which is susceptible to both trypsin and pepsin digestion and the other to pepsin but not to trypsin digestion. Acrylamide gel electrophoresis of the partially purified Ic antigen resulted in the occurrence of both determinants throughout the length of the gel, as shown by double gel diffusion slides.     

Properties of a Hemolysin Produced by Group B Streptococci

A hemolysin that appears to be responsible for the zones of beta-hemolysis surrounding colonies of group B streptococci (Streptococcus agalactiae) on blood agar plates has been isolated and partially purified. No soluble hemolysin was detectable in the supernatants of streptococcal cultures grown in several types of media. However, hemolytic activity was detected when streptococci were incubated with erythrocytes, and soluble hemolysin was isolated when bacterial suspensions were incubated in the presence of a variety of agents, including calf serum, albumin, Tween 80, and starch. Glucose and other fermentable carbohydrates stimulated hemolysin production, and metabolic inhibitors greatly reduced the titer of hemolysin that could be recovered, suggesting that cellular metabolism is necessary for hemolysin production or release. The soluble hemolysin was concentrated by ammonium sulfate precipitation and partially purified by gel filtration. Agents known to inhibit other streptococcal hemolysins, including phospholipids, trypan blue, proteases, and cholesterol, were tested for their effect on the group B hemolysin. Only the phospholipids inhibited hemolysin activity. The group B streptococcal hemolysin appears to be similar to, but distinct from, streptolysin S produced by Streptococcus pyogenes.     

Serotype Identification of Group B Streptococci by PCR and Sequencing

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution will be needed to guide the development and use of GBS conjugate vaccines. We designed sequencing primers based on the previously published sequences of the capsular polysaccharide (cps) gene clusters to further define partial cps gene clusters for eight of the nine GBS serotypes (serotypes Ia to VII). Subsequently, we designed and evaluated primers to identify serotypes Ia, Ib, III, IV, V, and VI directly by PCR and all eight serotypes (serotypes Ia to VII) by sequence heterogeneity. A total of 206 clinical GBS isolates were used to compare our molecular serotype (MS) identification method with conventional serotyping (CS). All clinical isolates were assigned an MS, whereas 188 of 206 (91.3%) were assigned a serotype by use of antisera. A small number of isolates (serosubtypes III-3 and III-4) showed different serotype specificities between PCR and sequencing, but the PCR results correlated with those obtained by CS. The overall agreement between the MS identification method and CS for isolates for which results of both tests were available was 100% (188 of 188 isolates). The MS identification method is a specific and practical alternative to conventional GBS serotyping and will facilitate epidemiological studies.     

Method for Rapid Detection of Group B Streptococci by Coagglutination

A quick and reliable technique for the identification of group B streptococci has been developed. The method requires no elaborate equipment or expensive reagents and can be used to detect the group B organisms in mixed broth cultures or to identify suspect colonies selected from agar plates. The method is a coagglutination technique in which 1 drop of specifically sensitized protein A-containing Staphylococcus aureus is mixed with 1 drop of supernatant of an actively growing culture. The soluble group-specific carbohydrate substance of the group B streptococci reacts with the staph particles to produce agglutination that is macroscopically readable. One colony of group B streptococci taken from an agar plate and inoculated into Todd-Hewitt broth will give a positive reaction within 6 h of incubation; with a larger inoculum, the positive reaction occurs within a shorter period. The method was applied for detection of group B streptococci in mixed broth cultures. In laboratory studies involving random mixtures of organisms, 59.3% of positive cultures were detected within the first 8 h of incubation, and 71.7% were found within 24 h. In clinical studies with mixed broth cultures grown directly from vaginal swabs, 78.6% of the positive cultures were detected within the first 8 h of incubation, and 92.9% were found within 24 h.     

Rapid Hippurate Hydrolysis Method for Presumptive Identification of Group B Streptococci

A rapid test to detect the hydrolysis of sodium hippurate by beta-hemolytic streptococci within 2 h was developed. All group B streptococci tested were positive using this method and all other groups were negative.     

Localization and Characterization of the Hippuricase Activity of Group B Streptococci

Studies are presented on the isolation, localization, and characterization of hippuricase activity of group B streptococci. Washed, intact cells, live or heat killed at 56 C, exhibited hydrolysis of hippuric acid, but cell-free filtrates of the organism did not. Excellent hippuricase activity was recoverable from supernatant fluids of mechanically disrupted cells, and evidence suggests that it exists largely intracellularly. Characteristics of the hippuricase preparation are consistent with the view that the biologically active principle is an enzyme. A quantitative microtiter technique has been developed which is useful in titrating enzymatic activity and antibody neutralization. Sera from rabbits immunized with filtered preparations neutralized hippuricase activity.     

Role of Toll-Like Receptor 13 in Innate Immune Recognition of Group B Streptococci

Murine Toll-like receptor 13 (TLR13), an endosomal receptor that is not present in humans, is activated by an unmethylated motif present in the large ribosomal subunit of bacterial RNA (23S rRNA). Little is known, however, of the impact of TLR13 on antibacterial host defenses. Here we examined the role of this receptor in the context of infection induced by the model pathogen group B streptococcus (GBS). To this end, we used bacterial strains masked from TLR13 recognition by virtue of constitutive expression of the ErmC methyltransferase, which results in dimethylation of the 23S rRNA motif at a critical adenine residue. We found that TLR13-mediated rRNA recognition was required for optimal induction of tumor necrosis factor alpha and nitrous oxide in dendritic cell and macrophage cultures stimulated with heat-killed bacteria or purified bacterial RNA. However, TLR13-dependent recognition was redundant when live bacteria were used as a stimulus. Moreover, masking bacterial rRNA from TLR13 recognition did not increase the ability of GBS to avoid host defenses and replicate in vivo. In contrast, increased susceptibility to infection was observed under conditions in which signaling by all endosomal TLRs was abolished, i.e., in mice with a loss-of-function mutation in the chaperone protein UNC93B1. Our data lend support to the conclusion that TLR13 participates in GBS recognition, although blockade of the function of this receptor can be compensated for by other endosomal TLRs. Lack of selective pressure by bacterial infections might explain the evolutionary loss of TLR13 in humans. However, further studies using different bacterial species are needed to prove this hypothesis.     

Changing Molecular Epidemiology of Group B Streptococcus in Korea

The prevalence of group B streptococcus (GBS) among pregnant women and disease burdens in neonates and adults are increasing in Korea. Colonizing isolates, collected by screening pregnant women (n=196), and clinical isolates collected from clinical patients throughout Korea (n=234), were serotyped and screened for antibiotic resistance. Serotype III (29.8%) and V (27.7%) predominated, followed by Ia (17.0%). Antibiotic resistance was higher among clinical than colonizing isolates for erythromycin (35.1% and 26.9%; P=0.10) and for clindamycin (49.4% and 42.1%; P=0.17). erm(B) occurred in 91.9% of erythromycin resistant isolates, and 84.0% of isolates resistant to clindamycin. Only five isolates (4.2%) resistant to erythromycin were susceptible to clindamycin; by contrast, and unique to Korea, 34% of isolates resistant to clindamycin were erythromycin susceptible. Among these 60 erythromycin-susceptible & clindamycin-resistant isolates, 88% was serotype III, and lnu(B) was found in 89% of strains. Four fifths of the serotype V isolates were resistant to both erythromycin and clindamycin. Further characterization of the genetic assembly of these resistance conferring genes, erm(B) and lnu(B), will be useful to establish the clonal lineages of multiple resistance genes carrying strains.     

Growth and Amino Acid Requirements of Various Strains of Group B Streptococci

A chemically defined medium (FMC; B. Terleckyj, N. P. Willett, and G. D. Shockman, Infect. Immun. 11:649-655, 1975) was used to compare the growth and amino acid requirements of 16 strains of group B streptococci, consisting of both laboratory-passaged organisms and fresh clinical isolates from adult and neonatal infections. The 5 standard Lancefield immunizing strains of group B streptococci, 090 (Ia), H36B (Ib), A909 (Ic), 18RS21 (II), and D136C (III), had doubling times in FMC (28 to 36 min) similar to those observed in Todd-Hewitt glucose broth (24 to 30 min). Similar doubling times were obtained with 11 clinical isolates growing in Todd-Hewitt glucose broth and FMC. The optimum buffering capacity of FMC was provided by 0.06 M sodium phosphate, and 1% glucose gave maximum cell yield. The group B streptococci, with minor exceptions, were very homogeneous in their amino acid requirements under both aerobic and anaerobic growth conditions. Phenylalanine, tyrosine, tryptophan, glutamate, arginine, valine, leucine, lysine, methionine, isoleucine, cystine, and histidine were required by all 16 strains under both aerobic and anaerobic growth conditions. In addition, threonine was required by all strains under aerobic growth conditions, whereas only 9 strains required threonine under anaerobic conditions. Serine was required by only 3 type III fresh clinical isolates aerobically, but not anaerobically. A requirement for glycine varied from strain to strain, apparently influenced by the oxidation-reduction potential of the growth medium.     

Molecular Analysis of Group B Protective Surface Protein, a New Cell Surface Protective Antigen of Group B Streptococci

Group B streptococci (GBS) express various surface antigens designated c, R, and X antigens. A new R-like surface protein from Streptococcus agalactiae strain Compton R has been identified by using a polyclonal antiserum raised against the R protein fraction of this strain to screen a lambda Zap library. DNA sequence analysis of positive clones allowed the prediction of the primary structure of a 105-kDa protein designated BPS protein (group B protective surface protein) that exhibited typical features of streptococcal surface proteins such as a signal sequence and a membrane anchor region but did not show significant similarity with other known sequences. Immunogold electron microscopy using a BPS-specific antiserum confirmed the surface location of BPS protein on S. agalactiae strain Compton R. Anti-BPS antibodies did not cross-react with R1 and R4 proteins expressed by two variant type III GBS strains but reacted with the parental streptococcal strain in Western blot and immunoprecipitation analyses. Separate R3 and BPS immunoprecipitation bands were observed when a cell extract of strain Compton R was tested with an antiserum against Compton R previously cross-absorbed to remove R4 antibodies. Immunization of mice with recombinant BPS protein by the subcutaneous route produced an efficient antigen-specific response, and immunized animals survived challenge with a lethal dose of a virulent strain. Therefore, BPS protein represents a new R-like protective antigen of GBS.     

Nonhemolytic Group B Streptococci of Human, Bovine, and Ichthyic Origin

The biochemical and serological characteristics of nonhemolytic group B streptococci of human, bovine, and ichthyic origin were described.     

Clinical Application of Immunofluorescence III. Identification of Lancefield Group B Streptococci

The identification of Lancefield group B streptococci with immunofluorescence was studied after significant differences between the precipitin and immunofluorescent results were noted. It was found that strains of group B streptococci, unlike groups A, C, and G streptococci, do not reliably react with a fluorescein isothiocyanate-labeled conjugate that contains antibodies mainly against C polysaccharide. It was assumed that the difficulty probably resulted from a blocking of the antigen-antibody reaction between the group antigen and its antibody by the S antigen located in the capsule. On the basis of this assumption, a conjugate was prepared that contained antibodies against the various S antigens of group B and it was found to be effective.