Deposition and degradation of C3 on type III group B streptococci.

Antibody to the polysaccharide capsule of type III group B streptococci (GBS) and complement are essential to host defense against systemic infection in neonates. Interactions between C3 degradation products and specific neutrophil receptors mediate the attachment and ingestion of these organisms. To evaluate the influence of capsule on C3 disposition, we compared the C3 fragments released from a highly encapsulated clinical isolate (M861) with those from an unencapsulated mutant (COH 31-15) and an asialo mutant (COH 31-21) of type III GBS after opsonization with hypogammaglobulinemic serum. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis, the three strains displayed similar patterns of C3 degradation; both C3b and iC3b were detectable. However, as the duration of opsonization increased, C3 fragment bands became more prominent on the encapsulated strain. The capsule, and specifically sialylation of the capsular polysaccharide of type III GBS, promotes C3 fragment deposition. However, C3 was deposited and degraded to iC3b in the absence of capsule. Opsonization of strain M861 with serum containing antibody specific for the polysaccharide capsule facilitated C3 fragment deposition in the early phases of opsonization. Because iC3b is one of the C3 fragments on an encapsulated strain of type III GBS, the relative deficiency of neonatal neutrophil receptors for this ligand may contribute to the virulence of this organism. Sufficient concentrations of antibody may enhance opsonization by facilitating C3 deposition as well as by interacting with Fc receptors on neutrophils.     

Extracellular antigens of serotype III group B streptococci.

Two sialic acid-containing type III group B streptococcal antigens were obtained from a supernatant growth medium, purified by anion exchange or gel filtration, and found to be free of group B reactivity. Quantitation of the high-molecular-weight extracellular type III antigen indicated that approximately 20-fold more antigen was recoverable from the growth medium than could be obtained by neutral buffer extraction of whole cells.     

Phenotypic diversity in the alpha C protein of group B streptococci.

Group B streptococci (GBS) is the leading cause of neonatal sepsis and meningitis. C proteins are an immunologically important group of surface-associated antigens in GBS that remain incompletely characterized. Two C proteins have been designated alpha and beta on the basis of protease susceptibility. We recently used a monoclonal antibody to describe a protective epitope of the GBS alpha (or trypsin-resistant) C protein in the prototype Ia/c GBS strain. In the present study, we examined 51 GBS isolates for expression of C-protein alpha and beta antigens. The alpha antigen, as detected with monoclonal antibody in sodium dodecyl sulfate (SDS) extracts, appears as a heterogeneous series of proteins spaced 8 kDa apart on SDS-polyacrylamide gel electrophoresis, but has a maximum molecular mass that varies among strains from 62.5 to 167 kDa. By immunoblotting with human immunoglobulin A, polyclonal antiserum, or monoclonal antibody, the beta antigen, in contrast, appears as a single protein of molecular mass between 124 and 134 kDa. The amount of alpha antigen expressed by each strain was quantified by enzyme immunoassay inhibition and was found to vary markedly from strain to strain. The susceptibility of strains of GBS to opsonization and killing by human polymorphonuclear leukocytes in the presence of either complement alone or complement with alpha-specific monoclonal antibody was examined. Strains expressing the alpha antigen were less readily killed in the absence of specific antibody than were alpha-negative strains. Killing in the presence of alpha-specific monoclonal antibody was found to correlate directly with the maximum molecular mass of the alpha antigen and with the quantity of antigen on the bacterial cell surface. Isolates of GBS that express the alpha C protein vary widely in the quantity and molecular mass of the alpha antigen produced, and this heterogeneity appears to have biologic importance.     

Stimulation of complement component C3 synthesis in macrophagelike cell lines by group B streptococci.

Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses.     

Faecal carriage of group B streptococci.

Consecutive stool samples from 116 female and 98 male patients (both adults and children), and rectal and vaginal swabs from 28 and 53 cases respectively, were quantitatively cultured for group B streptococci using Islam's medium. Group B streptococcus was recovered from 5% and 2% of faeces in female and male patients respectively, and the colony counts ranged from 10(2) to 10(3)/g. In women, the faecal carriage rate was 6%, which was significantly lower than the rectal carriage rate (p 0.02), suggesting that the higher recovery rate (27%) from rectal specimens may be due to contamination of swabs by perianal skin flora. Type II group B streptococcus was the only faecal isolate in adults (numbers involved are small for statistical significance), and we suspect that this type strain may be the only resident gut flora in adults, and the gastrointestinal tract is unlikely to serve as the main reservoir of all group B streptococci.     

Detection of the C protein gene among group B streptococci using PCR.

AIM--To develop a polymerase chain reaction (PCR) for the specific detection of the C protein gene in strains of group B Streptococcus. METHODS--A single primer pair derived from the nucleotide sequence of the IgA binding beta antigen of the C protein complex permitted the specific amplification of a 592 base pair DNA fragment from the C protein gene. After 35 cycles of amplification this product could be detected by agarose gel electrophoresis. Southern blot hybridisation confirmed that this product was the C protein gene. RESULTS--PCR detected the C protein gene in 75 (63%) of 119 strains of group B streptococci analysed. The product was not detected in other Gram positive organisms, showing that this PCR assay was highly specific. The sensitivity of the assay was satisfactory to a dilution of 1 in 10,000 of extracted DNA. CONCLUSIONS--The C protein of group B streptococci is associated with neonatal sepsis. The specific detection of the C protein gene by PCR may help identify which strains are likely to be associated with infection by the organism.     

Use of phenotypic and molecular serotype identification methods to characterize previously nonserotypeable group B streptococci

Among 1,762 isolates of Streptococcus agalactiae (group B streptococcus [GBS]), 207 (12%) initially nonserotypeable isolates were tested by improved conventional serotyping methods (Lancefield antigen extraction with 0.1 and 0.2 N HCl, latex agglutination assays, and use of antisera against all known serotypes [Ia, Ib, and II to IX]) and a molecular serotype identification system (multiplex PCR-based reverse line blot [mPCR/RLB] assays targeting serotype-specific sites in the region spanning cpsH to cpsM). Serotypes were assigned to 71 (34%) of the 207 isolates by using antisera and to 204 (98.5%) of them by mPCR/RLB. Sequencing of a portion of the cpsE-cpsF-cpsG region of 141 persistently nonserotypeable isolates and 1 with discrepant conventional and molecular serotyping results was attempted. Major mutations were identified in 34 isolates (24%), including 11 (8%) from which no amplicons were obtained and 23 (16%) with sequence variation compared with published sequences; of the latter, 21 (15%) were associated with amino acid changes. By contrast, mutations were identified in only 12 (2.3%) of 516 serotypeable isolates for which this region has been sequenced previously. In summary, an improved serotyping scheme allowed serotype identification of more than one-third of the previously nonserotypeable GBS isolates. Molecular serotypes were assigned to almost all of the isolates by mPCR/RLB. Significant mutations (with no amplicons or with associated amino acid changes) were found in the cpsE-cpsF-cspG region of a higher proportion of nonserotypeable than of serotypeable isolates (32/141 versus 8/516; P < 0.001), but further investigation is needed to determine the genetic basis for most nonserotypeable GBS isolates.     

Primary carrier sites of group B streptococci in pregnant women correlated with serotype distributions and maternal parity.

Perianal, perineal, vulval, and vaginal swabs from women attending an antenatal clinic were quantitatively cultured for group B streptococci using Islam's medium. The isolation rates from the four sites were very similar with an overall carriage rate of 21%. The findings suggest that the type II strains, a faecal flora, probably colonise the perianal site from a faecal source, and that the type III strains colonise primarily the genital site and then spread to the perineoperianal region. The type I strains did not conform to any pattern, suggesting a possible secondary involvement of these sites from another source. The types I, R, X, and non-typable isolates were almost exclusively isolated from primigravidae and second gravidae; the type III strains were conspicuously absent in 47 primigravidae. The primigravidae and second gravidae women consistently had high colony counts.     

Cell-associated collagenolytic activity by group B streptococci.

Group B streptococci (GBS) are important pathogens in neonatal sepsis, pneumonia, and meningitis. The ability of GBS to invade the collagen-rich amniotic membrane of the placenta has been shown in vitro. In the presence of GBS, the collagen fibrils of the amnion appear disordered, suggesting a role for GBS in premature rupture of membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Sephadex G-200 column chromatography, and gelatin zymograms were used in this study to characterize cell-associated collagenolytic activities of GBS. The synthetic peptide 2-furanacryloyl-Leu-Gly-Pro-Ala (FALGPA), which mimics the primary structure of collagen, was degraded by GBS USF704, a clinical isolate from the placenta of a septic newborn. Cells of GBS USF704 (9 x 10(7) CFU/ml) hydrolyzed 902 nmol of FALGPA over a 24-h period. As reported for zinc metalloenzymes such as collagenase, the hydrolysis of FALGPA by GBS was inhibited by addition of EDTA or 1,10-phenanthroline. Boiling of the cells resulted in loss of activity, while higher activity was observed with crude GBS cell lysates (hydrolysis of 970 nmol of FALGPA in 1.5 h). Antiserum raised against collagenase from Clostridium histolyticum was found to cross-react with cell-associated proteins produced by GBS and to inhibit GBS FALGPA hydrolysis. Twenty-five additional GBS clinical isolates were screened and found to have various levels of FALGPA hydrolytic activity. These observations suggest a cell-associated collagenolytic activity by GBS which may be involved in premature rupture of membranes and neonatal disease.     

Extracellular neuraminidase production by group B streptococci.

Neuraminidase (sialidase) activity in concentrated culture filtrates of group B streptococci was measured with bovine submaxillary mucin as substrate. Group B streptococcal neuraminidase was not active on human alpha-1 acid glycoprotein and did not show increased activity on bovine submaxillary mucin that had been O-deacetylated by alkaline treatment. The enzyme was produced in a variety of media, including a chemically defined medium (FMC; Terleckyj et al., Infect. Immun. 11:649-655, 1975) supplemented with bovine serum albumin or human serum albumin. Maximal levels of activity were present in filtrates from cells grown in a dialyzable fraction of Todd-Hewitt broth harvested during the late exponential phase of growth. Dramatic decreases were seen when filtrates from the late stationary phase were assayed. The decrease in specific activity during the stationary phase was shown to be due to proteolytic digestion of neuraminidase and not to the elaboration of an extracellular neuraminic acid aldolase.     

Serious infection caused by group C streptococci.

Group C streptococci commonly cause infection in animals but only occasionally give rise to severe infection in man. We report here three cases of serious human infection due to this organism and discuss its pathogenicity in relation to the clinical manifestations of the disease.     

Lectins detecting group C streptococci

Lectin of Wisteria floribunda specifically agglutinates group C streptococci. The reaction is strongly inhibited by N-acetyl-d-galactosamine, slightly by lactose, and not at all by galactose. The utility of the simple test of the lectin with group C streptococci is discussed.     

Fatal infection associated with group C streptococci.

Serious infection caused by Lancefield group C streptococci is unusual in man. Two unrelated deaths associated with these organisms in a 55 year old woman who died after three days of diarrhoea and vomiting, and in a 65 old man who died after a week of non-specific symptoms, are presented. Post mortem examination showed septicaemia in the former and severe aortic stenosis with widespread septic emboli and probable meningitis in the latter. Lancefield group C streptococci were isolated from both cases. These organisms may be carried asymptomatically and usually cause disease in animals but cases of serious human infection have recently been described, mainly in elderly patients or those with other predisposing factors.     

Prevention of C3 deposition by capsular polysaccharide is a virulence mechanism of type III group B streptococci.

Strains of type III group B streptococci isolated from patients with neonatal sepsis are generally resistant to complement-mediated phagocytic killing in the absence of specific antibody. It has been suggested that the resistance of type III group B streptococci to phagocytosis results from inhibition of alternative-complement-pathway activation by sialic acid residues of the type III polysaccharide. To better define the relationship between structural features of the type III capsule and resistance of type III group B streptococci to complement-mediated phagocytic killing, we measured deposition of human C3 on group B streptococcal strains with altered capsule phenotypes. C3 binding was quantified by incubating bacteria with purified human 125I-C3 in 10% serum. Wild-type group B Streptococcus sp. strain COH1 bound eightfold fewer C3 molecules than did either of two isogenic mutant strains, one expressing a sialic acid-deficient capsule and the other lacking capsule completely. Similar results were obtained when the incubation with 125I-C3 was performed in serum chelated with Mg-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (MgEGTA), suggesting that the majority of C3 deposition occurred via the alternative pathway. In contrast to the wild-type strain, which was relatively resistant, both mutant strains were killed by human leukocytes in 10% serum with or without MgEGTA. We also measured C3 binding to 14 wild-type strains of type III group B streptococci expressing various amounts of capsule. Comparison of degree of encapsulation with C3 binding revealed a significant inverse correlation (r = -0.72; P less than 0.01). C3 fragments released by methylamine treatment of wild-type strain COH1 were predominantly in the form of C3bi, while those released from the acapsular mutant were predominantly C3b and those from the asialo mutant represented approximately equal amounts of C3b and C3bi. We conclude from these studies that the sialylated type III capsular polysaccharide inhibits alternative-pathway activation, prevents C3 deposition on group B streptococci, and protects the organisms from phagocytic killing.     

Rapid identification of group B streptococci by counterimmunoelectrophoresis.

Beta-hemolytic streptococcal isolates have been examined by counterimmunoelectrophoresis (CIE) with group B antiserum to determine whether this techinque is of value in the rapid identification of group B strains. Ninety stock cultures and 100 clinical isolates of beta-hemolytic streptococci including representatives of groups A, D, C, G, and B were inoculated into Todd-Hewitt broth; after incubation at 37 C for 1, 2, 3, and 4 h, aliquots of the whole broth cultures were removed and tested by CIE. Antigen was not regularly detected in the 1-, 2-, and 3-h samples, but after 4 h all 126 group B streptococcal strains identified by the capillary precipitin reaction gave CIE precipitin bands with group B antiserum. None of the 58 non-group B strains gave precipitin reactions with this antiserum. Cerebrospinal fluid from an infant with group B streptococcal meningitis and peritoneal fluid from a patient with group B streptococcal peritonitis had free group B antigen detected by the CIE technique. CIE of broth cultures and direct body fluids appears to be a rapid and sensitive method for the identification of group B streptococcal strains.     

Streptococcal group B type antigen X in group L streptococci.

Extracts from 19 of 29 group L streptococcal cultures reacted distinctly with antiserum against group B streptococcal type antigen X in coagglutination and immunodiffusion tests. The X antigen corresponded to those obtained by streptococci of serological groups B and G.     

Interaction of soluble fibronectin with group B streptococci.

Fibronectin binds to a variety of bacterial species, and we hypothesized that differential fibronectin binding might influence the invasive potential of group B streptococci (GBS). Human plasma fibronectin purified by a standard two-step chromatographic procedure was radiolabeled with 3H. Fifty GBS strains (invasive, colonizing, or bovine) representing serotypes Ia (10 strains), Ib (6 strains), Ia/c (6 strains), II (10 strains), III (11 strains), IV (1 strain), and nontypable (6 strains) were tested. No source or serotype variability was detected among GBS strains, and binding was uniformly less than 1.5% of available fibronectin. Lack of detectable binding occurred at both the log and stationary growth phases and persisted despite treatment with trypsin or neuraminidase or opsonization with immunoglobulin G containing high levels (greater than 40 micrograms/ml) of antibody specific for the Ia, II, or III GBS capsular polysaccharides. Incubation with GBS did not inhibit fibronectin binding to the Cowan 1 strain of Staphylococcus aureus. Strain COH 31-15, an isogenic, type III, capsule-deficient mutant of COH 31r/s, also failed to bind fibronectin. In contrast to other streptococci, GBS do not have readily detectable receptors for soluble fibronectin as part of their surface structures. If present, binding sites for soluble fibronectin are deep to surface structures, obscured from host defense systems, or require the presence of other factors to facilitate their recognition of fibronectin. The uniform ability of GBS to resist binding to soluble fibronectin could be a significant virulence factor in the pathogenesis of invasive infections of infants.     

Faecal carriage of group B streptococci

A study of 1,138 primarily healthy subjects of various ages and sex was conducted to determine the faecal isolation rate of group B streptococci. Five percent of 284 neonates (5 days old) and 4% of 267 healthy children (1–15 years old) were found to be faecal carriers. Adults were more frequently faecal carriers than children, group B streptococci being isolated in 15% of 361 adults and 11% of 226 pregnant patients. The isolation rate was independent of sex at all ages. Although group B streptococci were found more frequently in rectal than in faecal specimens from pregnant women (p0.001), the isolation rate for faecal specimens could be increased by using a more selective broth. Forty-four percent of strains isolated from faeces of 105 subjects belonged to serotype III, 27% to type Ia, 15% to type Ib, 11% to type II and 3% were nontypeable. The same serotype of group B streptococci was usually present at different sites in each subject.