Characterization of CMP-N-acetylneuraminic acid synthetase of group B streptococci.

The capsular polysaccharide is a critical virulence factor for group B streptococci associated with human infections, yet little is known about capsule biosynthesis. We detected CMP-Neu5Ac synthetase, the enzyme which activates N-acetylneuraminic acid (Neu5Ac, or sialic acid) for transfer to the nascent capsular polysaccharide, in multiple group B streptococcus serotypes, all of which elaborate capsules containing Neu5Ac. CMP-Neu5Ac synthetase isolated from a high-producing type Ib strain was purified 87-fold. The enzyme had apparent Km values of 7.6 for Neu5Ac and 1.4 for CTP and a pH optimum of 8.3 to 9.4, required magnesium, and was stimulated by dithiothreitol. This is the first characterization of an enzyme involved in group B streptococcus capsular polysaccharide biosynthesis.     

Platelet aggregation by group B streptococci

Forty-six strains of group B streptococci (GBS), including various serotypes and non-serotypable strains, were tested for their ability to induce platelet aggregation in human platelet-rich plasma; four strains, all belonging to type III, showed a positive reaction. The characteristics of the reaction were investigated in these four positive strains. Aggregation was dependent on the ratio of bacteria to platelets, being maximal at a ratio of 4.3. Platelet aggregation was inhibited by EDTA (100% inhibition at 3.1 mM), indomethacin (100% inhibition at 10 mM), acetylsalicylic acid (93-100% inhibition at 5.0 mM) and quinacrine (100% inhibition at 0.25 mM). Thus the reaction was cation-dependent and required cyclooxygenase activity. Assays for cytosolic lactate dehydrogenase did not indicate platelet lysis. GBS induced the release of [3H]serotonin, which was maximal (68-78%) at 10 min after the reaction was started. Experiments with gel-filtered platelets suggested that GBS-induced platelet aggregation required both fibrinogen and heat-resistant (56 degrees C, 30 min) serum factors. Type-specific antisera prevented the platelet aggregation activity of heat-killed bacteria, but not of live bacteria. Trypsin digestion of the bacterial cells caused an almost complete loss of the platelet aggregation activity.     

Colonisation of babies and their families by group B streptococci.

A high incidence of group B streptococcal disease of the newborn in West Berkshire led to a prospective study of the condition. Cultures taken from 1090 babies shortly after birth showed that 65 (6%) were colonised with the streptococcus. Thirty of these babies were assigned to group 1. Bacteriological samples were taken from babies and mothers at birth and at four, eight, and 12 weeks, and also from fathers and siblings. Fifty uncolonised babies and their families were similarly studied and served as controls (group 2). In group 1,28 of the 30 mothers and 14 of the 28 fathers examined were colonised by group B streptococci. In group 2 the streptococci were isolated from three babies, 12 mothers, and 11 out of 45 fathers during follow-up. These findings suggest that group B streptococci are carried predominantly in the lower gastrointestinal and genitourinary tracts. Most families are lightly colonised, but in others maternal colonisation is stable and heavy and the incidence of paternal colonisation high. Results of serotyping suggest that sexual transmission occurs, which may explain the difficulty in eradicating the organism during pregnancy.     

Selective broth for isolation of group B streptococci

[This corrects the article on p. 884 in vol. 26.].     

Degradation of amniotic collagen fibrils by group B streptococci

Amniotic membranes collected after both cesarean and vaginal deliveries were inoculated with group B streptococci (GBS) in this in vitro study. Transmission electron microscopic examination of segments of uninoculated control amniotic membranes revealed compact, wellordered, clearly defined layers of collagen fibrils. Examination by both scanning and transmission electron microscopy of amniotic membrane segments inoculated in vitro with group B streptococci revealed bacterial attachment to the membrane surface and migration through the membrane accompanied by disordered collagen fibril layers. Degradation of the collagen fibrils during bacterial invasion may cause weakening of the amniotic membranes and thus be a contributing factor in cases of premature rupture of membranes associated with group B streptococcal colonization of the mother.     

Findings of serotypes of group B streptococci in human and bovine sources.

Five hundred and fifty-five strains of S. agalactiae of human or bovine origin were serologically typed. In human strains, serotype Ia was the most frequent irrespective of the source and kind of cultivation material, but serotype R was very frequent in urine. In bovine strains, one serotype was found as a rule in one stable both in small private and large socialist farms. The reason for such uniformity of serotypes is not known. Monocolonisation is one of the alternatives, but it seems more reasonable to assume that, the most resistant or more invasive strain will predominate in the herd in the course of time.     

Adhesion of group B streptococci to vaginal epithelial cells

The work presents the results of studies on the optimum and standard conditions for the in vitro determination of the adhesiveness of group B streptococci with epithelial cell suspensions. Vaginal epithelium has proved to be the most convenient and adequate system for studying the adhesiveness of group B streptococci. The optimum infective dose of these bacteria has been found to range from 50 to 200 cocci per cell. The characteristics of the adhesion of group B streptococci to vaginal epithelium are highly reproducible and exhibit low dependence on the time of the incubation of the bacteria with epithelial cells; fluctuations in the adhesiveness of the cultures in the definite range of pH shifts are seemingly determined by the serotype of the strains.     

Antigenic specificity of opsonophagocytic antibodies in rabbit anti-sera to group B streptococci.

An opsonophagocytic assay has been developed which requires human polymorphonuclear leukocytes, immune serum, and complement for optimal killing of Group B streptococci. Only with all three of these components was killing of greater than 1.0 log10 of the initial inoculum achieved, using rabbit antisera directed to homologous strains of each of the five known serotypes of Group B streptococci. Titers of specific antisera which opsonized the strains and resulted in greater than 1 log 10 reduction of colony-forming units, ranged from 1:100 (serotype Ib) to 1:3200 (serotype Ia). Cross-reactions between serotype-specific sera and heterologous strains were seen in certain instances. Type Ic strain and serotype Ic antiserum demonstrated cross-reactions with types Ia and Ib which were explainable by known shared antigens among these types. The only other cross-reaction which resulted in greater than 1 log 10 reduction in colony-forming units was when unabsorbed antiserum to strain Ia was used to opsonize a strain of serotype III. Opsonization of 10 serotype III strains was demonstrated with a single type III antiserum. Killing of nine of these strains required polymorphonuclear leukocytes, complement, and antiserum, but one strain, D136C, the reference strain, could be killed (greater than 1 log 10 reduction in colony-forming units) without either complement or specific antiserum. Inhibition studies were performed utilizing large m.w. polysaccharide antigens extracted from each serotype. These antigens inhibited opsonization of homologous strains by homologous antisera with 50% inhibition points ranging between 0.5 and 4 mug.     

A comparison of four methods for the serotyping of group B streptococci.

Group B streptococci are implicated in a wide range of clinical conditions in human adults and neonates. The Group is subdivided into five serotypes Ia, Ib, Ic, II and III, which are differentiated on the basis of capsular polysaccharides. In the interests of epidemiology and efficiency a cheap, rapid method which is easily interpreted would be advantageous. In this study four methods of serotyping, namely, counter-immunoelectrophoresis (CIEP), microimmunodiffusion (MID), coagglutination (COA), and the Lancefield capillary precipitin (CP) test were compared in terms of ease of operation and interpretation, accuracy and rapidity. Todd Hewitt Broth (THB) cultures and acid extracts of the group B streptococcal strains were used as antigens for these methods. It was concluded that COA using THB cultures allows cheap and rapid screening for presumptive serotyping, having a 93-96% correlation with the CP test. MID gives an accurate (100% correlation with the CP test) and unambiguous confirmatory diagnosis of serotype.     

Protective properties of certain external proteins of group B streptococci

On the basis of genes, which control synthesis of externally localized proteins of group B streptococci (bac and scaAB), recombinant polypeptides P6 and ScaAB were obtained. Data on protective activity of these polypeptides during experimental infection of immunized mice as well as in opsonophagocytic test on cultivated peritoneal macrophages are presented. It has been shown that protective effect of specific antibodies to P6 was dependent from intensity of immune response. Titer of specific IgG to P6 equal 1:25000 was protective for mice during challenge with LD50. During sublethal challenge level of humoral immunity determined both rate of microorganism elimination and degree of decrease of concentration of streptococci in the spleen. Recombinant polypeptide ScaAB also had marked protective activity and protective titer ScaAB-specific IgG was significantly lower compared with the first polypeptide (1:1600). It has been established that both types of antibodies have opsonizing activity against different strains of group B streptococci. Opsonizing properties of antibodies to P6 were restricted to Bac protein-producing streptococci whereas specificity of antibodies to ScaAB was not restricted by type and group borders. Opsonization of both group B and group A streptococci was revealed. It has been established that protective efficacy mediated by antibodies was dependent not only from their opsonizing characteristics but also from availability of protein antigens, which under certain conditions can be shielded by capsular polysaccharide. It has been assumed that vaccine preparation developed on the basis of polypeptides P6 and ScaAB is promising for further research.     

Influence of capsular neuraminic acid on properties of streptococci of serological group B.

Neuraminic acid is thought to be a critical virulence factor of group B streptococci. The present study was designed to further characterize a previously described type III group B streptococcus and its transposon-mutagenized asialo capsular mutant. The wild-type group B streptococcus grew as short chains with a uniform turbidity and had diffuse colonies in soft agar media. In contrast, the asialo mutant grew in fluid media as a granular sediment, formed significantly longer chains and had compact colonies in soft agar. These differences, possibly related to the surface charge of the bacteria, could also be demonstrated in salt aggregation tests and hexadecane adherence studies. The wild-type group B streptococcus showed hydrophilic, and the asialo mutant hydrophobic surface properties. Removal of neuraminic acid from the wild-type strain changed the surface properties from hydrophilic to hydrophobic. A similar masking effect of capsular neuraminic acid could be observed in adherence and phagocytosis experiments. In contrast to the wild-type strain, the asialo mutant adhered significantly more to buccal epithelial cells and was phagocytosed more by polymorphonuclear leucocytes. These altered properties might possibly be of importance for group B streptococcal pathogenicity.