Group B streptococcal colonization patterns in mothers and their infants.

Maternity patients and their newborn infants were cultured for group B streptococci (GBS) at Tampa General Hospital, Tampa, Fla., from September 1982 to May 1983. Culture swabs were placed into Lim Group B Strep Broth (GIBCO Laboratories, Madison, Wis.) and quantitated for GBS. A strong correlation was found between the numbers of GBS in the maternal vagina and the infant rectum. Infants symptomatic for early-onset GBS disease were delivered by mothers heavily colonized (greater than or equal to 3 X 10(4) GBS per swab) at the vagina. Such mothers were identified as GBS carriers by slide coagglutination and latex agglutination after their broth cultures had been incubated for 5 h. These data indicate that maternity patients at high risk of delivering infants heavily colonized with GBS and potentially symptomatic for early-onset GBS disease can be rapidly and selectively identified.     

Cytomegalovirus infection in Gambian mothers and their babies.

A 15 month longitudinal study of cytomegalovirus (CMV) infection in 178 Gambian mothers and their babies was undertaken. Twenty five (14%) of the babies were congenitally infected despite the fact that 87% of their mothers were antibody positive to the virus. Two of the 25 congenitally infected infants had evidence of severe neurological damage; skin sepsis was also a prominent feature in congenitally infected infants. The other children soon became infected. At 6 months of age, 53% of the infants were shedding virus either in urine or saliva. By the age of 12 months 86% of the infants had serological evidence of CMV infection. Preliminary evidence suggests that sibling to sibling infection in crowded compounds might be a major route of transmission.     

Enzyme-linked immunosorbent assay for group B streptococcal antibodies.

We report here on the development of enzyme-linked immunosorbent assays (ELISAs) for antibodies to types II and III group B streptococci. Streptococcal antigens were prepared by trichloroacetic acid extraction and fractional alcohol precipitation. Microtiter wells were coated with antigen in 0.1 M carbonate buffer at pH 9.6. Lyophilization was found to be an essential step for efficient binding of the streptococcal antigens. After incubation with antibody-containing rabbit serum, bound antibody was detected with peroxidase-labeled goat anti-rabbit immunoglobulin G. Optimal antigen concentrations were 200 micrograms/ml for type II and 100 micrograms/ml for type III. An ELISA is also described that uses intact bacteria as antigen. Hyperimmune rabbit serum reacted at a titer in excess of 512 against trichloroacetic acid-soluble antigen and 4,096 against whole bacteria. Sera from human subjects were also tested. Most human sera contained antibody which bound to intact bacteria but not to trichloroacetic acid-solubilized streptococcal antigens. Antibody titers in human sera against intact bacteria correlated very well with opsonic antibody activity measured in a chemiluminescence assay.     

Seroprevalence of bactericidal and anti-outer membrane vesicle antibodies to Neisseria meningitidis group B in England

Outer membrane vesicle (OMV) and recombinant protein-based vaccines targeted against multiple strains of group B meningococci are under development. The serum bactericidal antibody (SBA) assay has been designated the surrogate of protection, but the exact cutoff has not been determined. We measured the SBA titers in 2,415 serum samples and the anti-OMV IgG antibody concentrations in 2,672 serum samples representative of the English population to establish a baseline of natural immunity. SBA and anti-OMV IgG antibody titers are high in infants in the first 3 months of life, declining thereafter, presumably as maternal immunity wanes. About 6% of the subjects in the 1- to 11-year-old age group had SBA titers >or=4. During the teenage years, there was a marked increase in the percentage of subjects with SBA titers >or=4, rising to over 50% in 19-year-olds, with about 20% of older adults achieving this titer. The peak in SBA and anti-OMV IgG titers coincided with the peak in meningococcal carriage. Simple mathematical models confirm that the relationship between observed seroprevalence and carriage by age is consistent with carriage inducing SBA and that following an episode of carriage, SBA levels may remain elevated for many months. With the exception of children aged 3 to 11 months, there was no clear relationship between disease incidence and seroprevalence.     

Deficiency of IgG subclasses in mothers of infants with group B streptococcal septicemia

Serum IgG subclasses were studied in 19 mothers of infants with serious infections caused by group B streptococci (GBS) and compared with a control group of 20 mothers of healthy infants. 13 of 19 mothers showed decreased subclass levels: 10 of 19 low IgG2, 9 of 19 low IgG1 and 4 of 19 low IgG3. The levels of IgG1, IgG2 and IgG3 were significantly lower among mothers of GBS-infected infants than among the controls. Thus, there is indirect evidence that the infants were immunodeficient at birth.     

Interaction of group B streptococcal type-specific polysaccharides with wheat germ agglutinin and other lectins

The group B streptococci (GBS) are known to have type-specific polysaccharides rich in N-acetylneuraminic acid end groups, which are thought to be important immunological determinants. Wheat germ agglutinin (WGA) has affinity for N-acetylneuraminic acid as well as N-acetylglucosamine, and readily precipitates the type but not the group polysaccharide. A WGA-Sepharose affinity column was used to isolate complete type polysaccharides of representative strains of the 4 major GBS types. WGA, other lectins, and rabbit antisera were then used to characterize the products of various extraction procedures and chemical degradations, including mild acid hydrolysis and treatment with neuraminidase. Results of lectin binding studies were consistent with proposed chemical structures of types Ia, Ib and II. Differences were noted, however, between the cross-reactive antigens of pneumococcus type 14 and the desialated GBS type III polysaccharide. Although structurally similar, indirect evidence from lectin binding studies suggest that these antigens may not be identical.     

Detection of group B streptococcal antibodies in human sera by radioimmunoassay: concentrations of type-specific antibodies in sera of adults and infants infected with group B streptococci.

Current interest in determining the possible protective role of antibodies against group B streptococcal disease prompted this study of the feasibility of using a radioimmunoassay to measure type-specific immunity in humans. The radioimmunoassay was standardized as a quantitative test for antibodies against the carbohydrate (CHO) antigens of all five group B types. The data showed that the CHO antigens extracted by a cold trichloroacetic acid-sonification method measure more antibodies than do the corresponding CHO antigens extracted by hot hydrochloric acid; that the Ia CHOs extracted from two different types, Ia and Ic, measure the same quantity of Ia antibodies; and that human sera contain antibodies reactive with all five type-specific CHOs. No evidence of "protective" antibody was found in the serum samples studied, although there was evidence of and antibody response in adults to prolonged colonization by group B streptococci. The wide ranges of antibody concentration in a serum bank collection, the broad reactivity of all human sera tested, and the mixed populations of antibodies in human sera that react with different determinants on the same type-specific CHO antigen (type III) indicate that further studies must be done to better define normal and susceptible populations and to determine antigenic components important in protection.     

Detection of group B streptococcal antigen in early-onset and late-onset group B streptococcal disease with the Wellcogen Strep B latex agglutination test.

The Wellcogen Strep B latex agglutination test (Wellcome Diagnostics, Dartford, England) was evaluated as a method of detecting group B streptococcal antigen in urine, cerebrospinal fluid, and serum from neonates with early-onset (less than or equal to 7 days of age) and late-onset group B streptococcal disease. Urine was the best source of antigen, which was detected in 100% of six neonates with early-onset group B streptococcal disease who had urine available in the first 12 h of illness and in 88% of 17 group B streptococcus-infected neonates with urine available in the first 48 h of illness. Antigen was not detected in any samples from patients without group B streptococcal disease except in the urine of one patient with Proteus mirabilis meningitis. The Wellcogen Strep B latex test of the lot tested compares favorably with a noncommercially available latex agglutination test.     

The hemolytic and cytolytic activity of group B streptococcal hemolysin and its possible role in early onset group B streptococcal disease.

The in vitro and cytolytic properties of the hemolysin of group B streptococcus (GBS) were investigated using sheep erythrocytes and McCoy cells adapted for growth in a serum-deficient medium. The relationship between the hemolysin, various carrier molecules and phospholipids was examined. Starch-based carriers interfered with the inhibitory activity of phospholipids and solvents for the phospholipids reduced the activity of the hemolysin. These technical problems were resolved by use of an albumin-based carrier, a strain producing large amounts of hemolysin and sonication of the phospholipid. The hemolysin was cytolytic for McCoy cells and this activity and its hemolytic action on sheep erythrocytes were inhibited by a number of phospholipid components of surfactant. It is possible that GBS hemolysin has a direct or indirect role in the pathogenesis of the pneumonitis of early onset GBS infection.     

Kinetics of phagocyte response to group B streptococcal infections in newborn rats.

To test the hypothesis that inadequate in vivo mobilization of leukocytes may contribute to the unique susceptibility of neonates to infection, we studied the kinetics of phagocyte response to neonatal and adult rats to intraperitoneal infection with group B streptococcus, type Ia. The 50% lethal dose was considerably greater for adults than for neonates (1.1 x 10(7) colony-forming units per g versus 2.7 x 10(2) colony-forming units per g). After challenge with group B streptococcus, type Ia, the number of neonatal peritoneal leukocytes increased more slowly than did those of adult rats. For example, at 4 h, the adult neutrophil count was 41 times greater than that of the neonate, but at 24 h, neonatal peritoneal neutrophils had not yet reached the adult 4-h level. Peritoneal macrophages also increased more rapidly in adults than in neonates. After intraperitoneal infection, both adults and neonates developed bacteremia, but adults cleared the bacteria with greater efficiency. Adult blood neutrophils increased 247% by 12 h and then decreased; neonatal neutrophils steadily decreased to a 57% reduction by 24 h. These data suggest that the neonatal neutrophil response to group B streptococcus, type Ia, infection is inadequate and may contribute to the high mortality associated with this infection.     

A latex agglutination test for the measurement of antibodies to group-specific streptococcal polysaccharides

Group-specific polysaccharides of streptococci, extracted with formamide and purified by various methods, were adsorbed onto polystyrene latex particles, causing them to be agglutinated specifically by homologous antisera. This method was used to determine the group-specific antibody titres of hyperimmune rabbit sera.     

Guanine-plus-cytosine composition of five group B streptococcal serotypes.

The percent guanine-plus-cytosine content of deoxyribonucleic acid of each of the five serotypes of group B streptococci was determined by thermal denaturation. The range of guanine-plus-cytosine content was 35.1 to 36.9%, with a mean value of 35.9%. These values suggest a genetic homogeneity to the serotypes of the group B streptococci.     

Production of interleukin-1 by mononuclear cells of newborns and their mothers.

We have examined neutrophil phagocytosis and intracellular killing of Staphylococcus aureus in patients with primary biliary cirrhosis, alcoholic liver disease and chronic active hepatitis in comparison with age and sex matched controls. Significant decrease in neutrophil phagocytosis was found in both early and advanced primary biliary cirrhosis while impaired phagocytosis was seen in alcoholic cirrhosis but not in alcoholic fatty liver. In both disorders the effect appeared to be mediated by the patient's serum as there was no difference between patient and control neutrophil function when the test was performed in AB serum. Although total bacterial killing was reduced in chronic active hepatitis, phagocytosis was not impaired. Intracellular killing was not affected in any of the liver disease groups. These results support the view that serum factors exist in patients with liver disease which inhibit neutrophil phagocytosis. While these studies confirm earlier finding in alcoholic liver disease, this is the first demonstration of such a defect in primary biliary cirrhosis.     

Transfer of antirotaviral antibodies from mothers to their infants

Levels of rotavirus-specific immunoglobulin G (IgG), IgA, IgM, and secretory immunoglobulin in maternal and cord serum, colostrum and milk, and infants' stools were measured by enzyme-linked immunosorbent assay in 92 mothers and their infants. Although antirotaviral IgG, IgA, and secretory immunoglobulin were present in most maternal sera, only IgG crossed the placenta. All samples of colostrum and milk tested contained antirotaviral secretory immunoglobulin and IgA except those of two women in whom IgA deficiency was subsequently described. Specific IgM and IgG were also detected in many colostral samples. Antirotaviral IgA was detected in many colostral samples. Antirotaviral IgA was detected in stools of breast-fed but not bottle-fed neonates. Apparently the human infant receives rotaviral antibodies both transplacentally and via maternal colostrum and milk.     

Role of C5a-ase in group B streptococcal resistance to opsonophagocytic killing.

Type III group B streptococci (GBS) can be subdivided into three subtypes, RDP III-1, III-2, and III-3, on the basis of numerical analysis of HindIII restriction endonuclease digestion patterns (HindIII RDP) with their chromosomal DNAs. In the present study, the effect of C5a on opsonophagocytic killing of a representative strain from each RDP type was investigated by using a novel optical method for determining opsonophagocytic killing, and the effect of C5a-ase treatment of C5a on opsonophagocytic killing was also investigated. Pre-stimulation of polymorphonuclear leukocytes (PMNs) with C5a significantly increased opsonophagocytic killing of all three strains. The increase in killing was abolished by pretreating the C5a with GBS that express C5a-ase, a treatment that also destroyed the chemoattractant activity of the C5a. The kinetics of killing of the RDP III-2 strain differed from those of the other two strains. The survival of the RDP III-2 bacteria continued to decline over the entire 60-min incubation of the opsonophagocytic assay when PMNs were prestimulated with C5a or with C5a that had been inactivated with GBS C5a-ase (dC5a). In contrast, killing of the RDP III-1 and III-3 strains almost ceased after 20 or 60 min when PMNs were prestimulated with dC5a or C5a, respectively. A difference in bacterial killing between the III-2 strain and the III-1 and III-3 strains therefore became increasingly apparent with prolonged incubation time. The percentage of bacteria surviving in the extracellular fluid was approximately the same as the percentages of bacteria surviving in both intracellular and extracellular locations when PMNs were prestimulated with either C5a or dC5a. These data imply that the majority of bacterial killing occurred following phagocytosis and suggest that the enhanced killing of GBS following prestimulation of PMNs with C5a resulted from increased ingestion of the bacteria.     

Binding of group B streptococcal phosphoglycerate kinase to plasminogen and actin

The glycolytic enzyme, phosphoglycerate kinase (PGK) of group B streptococci (GBS), has previously been identified as expressed on the GBS cell surface. The data presented describes the ability of group B streptococcal phosphoglycerate kinase (GBS-PGK) to bind to plasminogen and to bind actin. GBS-PGK binding to plasminogen was inhibited by the lysine analogue, 6-aminocaproic acid, suggesting plasminogen binding is achieved through GBS-PGK lysine residues. In addition to GBS-PGK surface expression, GBS-PGK was also found to be released from the bacterial cell suggesting GBS-PGK may affect its environment independent of GBS. To determine the effect of GBS-PGK on the actin cytoskeleton within a host cell, GBS-PGK attached to green fluorescent protein was transfected into and expressed in HeLa cells. Transfected GBS-PGK disrupted the actin cytoskeleton resulting in a compact or ovoid shaped HeLa cell rather than a typical epithelioid appearance.In conclusion, we have shown GBS-PGK binds to plasminogen and actin. We have also shown that GBS-PGK can be released from the bacterial cell and that transfected GBS-PGK can alter the epithelial cell cytoskeleton.     

Heterogeneity of group A streptococcal pyrogenic exotoxin type B.

Streptococcal pyrogenic exotoxin type B purified from culture filtrates of either the NY-5 or T-19 strain of group A streptococcus was found to be heterogeneous in charge. Three protein fractions with isoelectric points of 8.0, 8.4, and 9.0 were isolated by differential solubility in ethanol and acetate-buffered saline followed by isoelectric focusing and shown to be antigenically identical to streptococcal pyrogenic exotoxin type B. The molecular weights of all three fractions were approximately 17,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with aggregates forming in the presence of hyaluronic acid. Only the pI 8.4 fraction showed the characteristic activities of streptococcal pyrogenic exotoxin in rabbits: pyrogenicity and ability to enhance susceptibility to lethal endotoxin shock. The pI 8.0 and pI 9.0 fractions were not pyrogenic, but could be used to immunize against pyrogenicity. These two fractions failed either to enhance lethal endotoxin shock or to immunize against enhancement activity. When the isolated fractions were electrofocused again they appeared heterogeneous, suggesting an instability of the B toxin molecular forms.     

Characterization of PepB, a group B streptococcal oligopeptidase.

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.