Clinical Group B Streptococcus Isolates Research Articles

Immunization with a single-repeat alpha C protein may prevent escape of lower repeat mutants of group B Streptococcus.

Group B streptococci (GBS) are the leading cause of meningitis, pneumonia, and sepsis in neonates1. The alpha C protein, a protective surface protein is found in > 90% of non-serotype-III GBS. The prototype alpha C protein contains 9 identical tandem repeats5; clinical isolates of GBS showed variable number of repeats3. Previously, we showed in a mouse model that GBS with lower repeat number could escape the immune system when immunized with 9-repeat-elicited antiserum4. ELISA inhibition, using purified alpha C protein, indicated that 9- and 16-repeat-elicited antibodies recognize conformational epitopes present in higher repeat, but absent in lower repeat alpha C protein2. This may explain escape of the lower repeat mutants. Simultaneously, we observed equal binding affinities of 1- and 2-repeat-elicited antisera to all alpha C proteins, independent of their repeat number. This suggested that 1- and 2-repeat-elicited antibodies may prevent escape of lower repeat mutants. To study the importance of the repeat number for vaccine use, we immunized mice passively with rabbit antisera elicited to 1-, 2-, 9-, and 16-repeat alpha C protein and challenged their neonates with GBS strain A909 (expressing 9 repeats). We determined protection levels and analyzed the repeat number of the escape mutants, isolated from the spleens of these neonates, by western blotting.KeywordsRepeat NumberRabbit AntiserumStreptococcal InfectionBlood Agar PlateEscape MutantThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Cloning and expression of the gene for group B streptococcal hyaluronate lyase.

Group B streptococci (GBS) are a major cause of serious human perinatal infections. Most clinical isolates of GBS secrete hyaluronate lyase, and production of high levels of the enzyme has been associated with strain virulence. Degenerate oligonucleotide primers, designed on the basis of the amino acid sequences of tryptic peptides prepared from the purified enzyme, permitted the polymerase chain reaction amplification from GBS chromosomal DNA of a 363-base pair internal DNA fragment of the GBS hyaluronate lyase gene (hylB). This DNA fragment was used as a probe to screen a lambda phage library of GBS chromosomal DNA fragments. Sequence analysis of positive clones identified an open reading frame capable of coding for a 111-kDa protein. Since no single clone was found to contain the entire gene it was necessary to reconstruct the gene from two plasmids containing inserts with suitable overlapping sequences. When this reconstructed gene was transformed into Escherichia coli, high level expression of hyaluronate lyase activity was obtained.

Clonal Analysis of Streptococcus agalactiae Isolated from Infants with Neonatal Sepsis or Meningitis and their Mothers and from Healthy Pregnant Women

The purpose of this study was to evaluate if group B streptococci (GBS) isolated from infants with neonatal sepsis or meningitis and their mothers differ from GBS isolated from healthy pregnant women who gave birth to healthy infants. Danish clinical isolates of GBS (n = 118) were characterized by multilocus enzyme electrophoresis, a method recently used to identify virulent clones of GBS type III from North American infants. By analysis of allelic profiles of 11 metabolic enzyme loci, 43 different electrophoretic types were found. Clustering analysis revealed two primary phylogenetic divisions at a distance of > 0.8, both containing serotype III isolates. One division, comprising virtually all disease isolates, showed a population structure like that of traditional pathogens. The other division, containing the majority of carrier isolates, had a structure reminiscent of that of mucosal commensals, with each isolate being different.

Protection of neonatal mice from group B streptococcal infection by maternal immunization with beta C protein.

Group B streptococci (GBS) cause the majority of cases of neonatal sepsis and meningitis in the United States. Immunization of women of childbearing age is one strategy under consideration for the prevention of neonatal disease. The beta C protein, a 130-kDa antigen present in many clinical isolates of GBS, was purified from GBS by extraction into sodium dodecyl sulfate (SDS)-containing buffer, preparative SDS-polyacrylamide gel electrophoresis, and electroelution. Purified beta C protein antigen (25 micrograms) with Freund's adjuvant was used to immunize rabbits. Rabbits developed enzyme-linked immunosorbent assay titers of > 1:1.6 x 10(6), and sera from immunized rabbits were administered to pregnant mice. Their neonatal pups were then challenged with a strain of GBS expressing beta C protein; 68% of these pups were protected by immune antiserum, whereas no controls were protected (P < 0.001). The immune serum (diluted 1:100) facilitated opsonophagocytic killing of GBS strains expressing the beta C protein but not those that do not express the antigen (mean log kill +/- standard deviation = 0.71 +/- 0.8 log10 CFU for beta+ strains and 0.09 +/- 0.2 for beta- strains; P = 0.02). In subsequent experiments, adult female mice were actively immunized with two doses of 2, 5, or 10 micrograms of beta C protein 2 months prior to mating. One- to two-day-old offspring of these dams were challenged with GBS and were protected in a dose-dependent manner, with 96% survival in the high-dose (10-micrograms) group and 20% survival in a sham-immunized control group (P < 0.001). Thus, active immunization of mice with the GBS beta C protein confers protection against lethal infection with beta+ GBS to their offspring.

Influence of serotype of group B streptococci on C3 degradation.

Serotype III strains of group B streptococci (GBS) are isolated from the majority of young infants with bacteremia or meningitis. We hypothesized that serotype-associated differences in structure of the type-specific capsular polysaccharide or the presence of c protein would influence the extent to which C3 degradation occurs on GBS and that type-specific antibody would alter C3 deposition or degradation patterns. When clinical isolates of GBS representing serotypes Ia, Ib/c, II (with or without c protein) and III were employed with hypogammaglobulinemic serum as an opsonic source, a remarkable similarity was observed in patterns of C3 deposition and degradation for each of the four GBS serotypes and between strains with or without c protein. Both C3b and iC3b were detected by 5 min and throughout a 90-min opsonization interval. Less deposition occurred at 5 min on serotypes Ia and Ib/c than on types II and III GBS. Minimal degradation to C3d or smaller fragments was observed. Type-specific antibody facilitated C3b deposition on GBS and C3b degradation to iC3b early in opsonization. Possibly, accessibility of C3 fragments to neutrophil receptors, rather than the extent to which the surface permits C3 degradation, accounts for differential virulence among GBS serotypes.

Adherence of Vaginal and Pharyngeal Strains of Group B Streptococci to Human Vaginal and Pharyngeal Epithelial Cells

In vitro tests for adherence to human vaginal and pharyngeal epithelial cells were used to study the problem of tissue-specific tropism in group B streptococci (GBS). Twenty-two vaginal or pharyngeal clinical isolates of GBS (serotypes Ia, Ib, II, and III) were used. No significant differences in adherence to vaginal and pharyngeal epithelial cells were found between GBS from both sources: statistical analysis furnished no evidence for tissue-specific tropism. Serotype III vaginal GBS adhered better to vaginal and pharyngeal epithelial cells than did serotype III GBS strains isolated from the pharynx. However, pronounced differences in the level of adherence were found among strains of the same serotypes and from the same sources. Thus, the results obtained suggest that differences in adherence may rather be strain-dependent than type-dependent.

A search for new group-B streptococcal serotypes.

New serotypes were sought among 165 clinical isolates of group-B streptococci that were untypable by antisera for the conventional types Ia, Ib, II and III. The strains were tested for sialic acid, an integral component of the group-B streptococcal type-polysaccharides; trypsin-treated bacteria were tested by slide-agglutination with the sialic-acid-specific lectin from the snail Cepaea hortensis. Sialic acid was detected in 96 of the strains; in 95 of these, new type antigens were identified serologically (type IV, 52; provisional type V, 34; candidate type NT6, seven; provisional type V and candidate type NT6, one; candidate type 7271, one); the remaining strain was found to possess a small amount of Ia antigen. Sialic acid was not detected in 69 strains, and none of these possessed a polysaccharide type-antigen. Chemical measurement of the sialic-acid content of cultures by Aminoff's method gave results in conformity with the lectin-agglutination test and the presence of polysaccharide type-antigens.

Beta-lactam antibiotic susceptibility of bacteria responsible for neonatal meningitis.

Neonatal meningitis is caused by group B streptococci (GBS), Escherichia coli, and Listeria monocytogenes, in order of frequency. Newly developed cephalosporins with a broad spectrum of activity have altered the therapy of meningitis due to gram-negative bacilli. However, we found that clinical isolates of GBS and L. monocytogenes did not demonstrate uniform susceptibility to beta-lactam antibiotics. Antibiotic potencies for GBS tested were: cefotaxime, penicillin, ceftriaxone, amoxicillin, cefamandole, cephalothin, and moxalactam. N-formimidoyl thienamycin was most active for L. monocytogenes followed by penicillin, cephalothin and chloramphenicol; broad-spectrum cephalosporins were not active against Listeria organisms that were tested. These agents should not be utilized as solitary therapy of meningitis until the organism has been characterized with antibiotic susceptibilities.