Microcapsule of Type III Strains of Group B Streptococcus: Production and Morphology

Transcription

1 INFECTION AND IMMUNITY, Jan. 1976, p Copyright ) 1976 American Society for Microbiology Vol. 13, No. 1 Printed in USA. Microcapsule of Type III Strains of Group B Streptococcus: Production and Morphology CAROL J. BAKERl* AND DENNIS L. KASPER The Channing Laboratory, Harvard Medical School, and Boston City Hospital, Boston, Massachusetts 2118 Received for publication 6 August 1975 The yield of purified type III polysaccharide of group B Streptococcus was significantly improved by modification of the growth medium. Culture of organisms in standard Todd-Hewitt broth resulted in acid accumulation during the exponential phase of growth and poor yield of type III polysaccharide when extracted from cells by washing with neutral buffer solution. By increasing the buffering capacity of the broth medium, acid accumulation was prevented, and the number of viable cells was increased at the stationary phase of growth. Further, by increasing the concentration of glucose in the buffered medium, the yield of type III polysaccharide was increased two to three times. Electron microscopic investigations of cells grown in the modified broth medium demonstrated a thicker microcapsule than was found in organisms grown in standard broth. The type-specific polysaccharides of group B Streptococcus extracted by treatment of whole cells with hot HCl have been shown to be chemically and immunologically distinct (4, 6, 11, 13, 14). These HCl polysaccharides are low-molecular-weight fragments of"native" antigens. However, for the isolation of high-molecularweight, antigenically complete substances, more gentle methods are needed. A recent investigation of purified type III polysaccharide extracted by washing cells with neutral buffer solution demonstrated the type III antigen in native or complete form to be a polymer, with sialic acid as a major chemical constituent (C. J. Baker and D. L. Kasper, J. Exp. Med., in press). After exposure to acid, this polysaccharide was readily degraded to an antigen immunologically identical to the type III-HCl polysaccharide. Preparation of type III antigen from organisms grown by methods used in previous investigations (6, 11, 13) of group B streptococcal antigens consistently resulted in low antigen yield. Because of the proved acid lability of native type III polysaccharide (Baker and Kasper, J. Exp. Med., in press) and the observation that the growth of organisms in standard conditions resulted in low antigen yield, experiments were conducted to modify the culture medium. A method was sought that would prevent exposure of cells to acid during the growth cycle and that would maximize the yield of type III polysaccharide. (Presented in part at the 14th Interscience Conference on Antimicrobial Agents and Chem- Present address: Department of Pediatrics, Baylor Col- ' lege of Medicine, Houston, Tex otherapy, San Francisco, California, September 1974.) MATERIALS AND METHODS Bacterial strains. Group B streptococcal strains M731 and M732 (type III) were originally isolated from infants with meningitis (Houston, 1973). Bacterial strains were stored in 1-ml aliquots of Todd- Hewitt broth (Difco Laboratories, Detroit, Mich.) at -7 C and thawed before use. Media and growth conditions. The liquid medium used in the experiments was Todd-Hewitt broth (Difco), which contained (per liter): 3 g of beef heart (infusion from 5 g), 2 g of neopeptone, 2 g of dextrose, 2 g of NaCl, 2.5 g of Na2CO3, and.4 g of Na2HPO4, at a final ph of 7.4. This broth will be referred to as the normal or standard medium. For certain experiments, this medium was modified by increasing the Na2HPO4 or glucose concentrations fourfold or eightfold. All ingredients were autoclaved together. Broth cultures were grown at 37 C in 25-ml Erlenmeyer flasks and stirred constantly. The broth media were inoculated with 1 ml of organisms, which had been grown for 1 to 12 h in Todd-Hewitt medium. The initial concentration was approximately 2 x 17 to 5 x 17 colony-forming units per ml, and subsequent growth was monitored by determining colony counts on plates containing 5% sheep blood in Trypticase soy agar (Difco). Under the above conditions, exponential growth was attained at 2 to 4 h; at this time a cell density of approximately 19 colony-forming units/ml had been attained. Chemical assays. The ph of the growth medium was measured by means of the radiometer ph meter 26 (Radiometer, Copenhagen, Denmark) with standard buffers. Glucose uptake was measured by determining the amount of glucose remaining in the medium after various intervals, using the oxidase re-

2 19 BAKER AND KASPER agent method (Worthington Biochemical Corp., Freehold, N.J.) with glucose standards. Electron microscopy. Suspensions ofwhole bacterial cells grown for 12 h in either standard or phosphate-glucose modified medium were prefixed to adjust the final concentration of fixative to.5% by the addition of 2.5% glutaraldehyde in.1 M sodium cacodylate buffer. CaCl2 was added to this suspension to achieve a concentration of.5%. After prefixation for 2 h at 4 C, the suspensions were centrifuged at 12, x g. The pellets obtained were fixed in 2.5% buffered glutaraldehyde for 2 h at 4 C, washed with buffer, and fixed again for 6 min in aqueous 1% OS4 (3). The pellets were then treated with.5% uranyl acetate in Michaelis buffer (ph 5.) for 2 h at 24 C. One-millimeter blocks were dehydrated in graded alcohols and embedded in Epon 812 according to the method of Luft (7). Thin sections were stained with lead citrate and examined in a Jem 1B electron microscope (Japan Electron Optics Laboratory, Tokyo, Japan). Polysaccharide components of bacterial cell walls are not seen readily with routine staining techniques; visualization of these antigens requires special stains, such as ruthenium red. Samples prepared for staining with ruthenium red were treated in a manner identical to that described above, except that the stain at a concentration of 7,g/ml was added to both the prefixation and fixation solutions (8). Preparation of native type III polysaccharide. Type III-specific polysaccharide antigen was isolated and purified in high-molecular-weight form by means of methods described previously (Baker and Kasper, J. Exp. Med., in press). Briefly, cells grown in 6- to 7-liter lots of standard or modified Todd-Hewitt broth media were washed with a neutral buffer solution. After ethanol fractionations, ultracentrifugation, and enzyme digestions for removal of protein and nucleic acid contaminants, separation of group B- from type III-specific polysaccharides was achieved by means of gel chromatography. The eluted fractions from the void volume of a Sepharose 4B K26, 1-cm column (Pharmacia Chemicals, Uppsala, Sweden) contained only type III serological activity. The fractions were pooled and concentrated on a PM3 filtration membrane, and 4 volumes of cold ethanol were added to precipitate the purified type III polysaccharide. Antigen yield was determined by weighing the lyophilized preparations. RESULTS The ph changes observed during the growth cycle of group B Streptococcus in the standard Todd-Hewitt broth are shown in Fig. 1, which compares the effects of fourfold and eightfold increases in concentration of Na2HPO4. Organisms were grown in 1 ml of broth, and 5-ml samples were removed at hourly intervals; these were filtered through membrane filters (diameter,.45,um; Millipore Corp., Bedford, Mass.) for ph determinations, and colony-form- I. INFECT. IMMUN. -o GROWTH (HOURS) FIG. 1. Hourly ph measurements of a culture medium for strain M731 grown in normal Todd-Hewitt broth modifiled with a fourfold or an eightfold increase in the Na2 HPO4 concentration. ing units per milliliter were determined for unfiltered aliquots. The ph of the standard Todd-Hewitt broth fell below 7. after 3 h of growth and decreased to 5.9 by 9 h. With use of the fourfold and eightfold Na2HPO4-buffered media, the ph was maintained above 6.2 and 6.5, respectively, during the entire growth cycle. Additionally, an average of a two-log increase in the number of viable organisms was observed after 12 h of growth in the eightfold buffered medium. The depletion of glucose in the medium by organisms growing in standard Todd-Hewitt broth is shown in Fig. 2. After 8 to 1 h of growth, the majority of the glucose was depleted from the standard medium. In contrast, glucose continued to be metabolized for several hours in the medium with a glucose excess. Similar results were observed when the medium was supplemented concurrently with Na2HPO4 and glucose. With the use of both an eightfold Na2HPO4 and glucose increase in the growth medium, the amount of purified, native type III polysaccharide extracted from a 6-liter lot of organisms was increased from 1 to 2 mg (standard conditions) to 3 to 4 mg. A decrease in viability of cells measured by colony-forming units per milliliter was correlated with onset of autolysis of organisms

3 VOL. 13, J I-. (9 -. LuJ C/) D ( Exl ts ~~~~~~I V - o-oo--o GROWTH (HOURS) FIG. 2. Glucose (mgll ml) depletion during growth of strain M732 in normal Todd-Hewitt broth and broth modified by increasing the glucose concentration fourfold or eightfold. grown in standard Todd-Hewitt medium after 8 to 1 h of growth. Initiation of autolysis was delayed beyond 12 h as both the Na2HPO4 and glucose concentrations were increased. By means of electron microscopy, the morphology of a type III strain of group B Streptococcus (M732) grown in standard medium was compared to that of this strain grown in modified liquid medium. In Fig. 3, an organism stained by standard techniques is shown. This organism demonstrated bilaminar morphology typical of other streptococci. The outer, moderately electron-dense layer is a microcapsule. This layer is morphologically similar to that of group A Streptococcus devoid of protein antigens (12). The thick, electron-dense, inner layer of the cell wall lies outside the indistinct cytoplasmic membrane. An organism stained with ruthenium red for demonstration of the capsular polysaccharide is seen in Fig. 4. The outer layer of microcapsule is better visualized with GROUP B STREPTOCOCCAL POLYSACCHARIDE (III) 191 \ 8X GLUCOSE_ BX_4X GLUCOSE NORMAL sx-~~~ this stain. This well-defined microcapsule is about two times the thickness of the inner cell wall. However, the thickness of the capsule is significantly increased by growing organisms in a medium modified with an eightfold concentration of Na2HPO4 and glucose, as demonstrated in the ruthenium red stain seen in Fig. 5. The capsular thickness of organisms grown in this modified medium was 1.5 to 2 times the thickness noted in organisms grown under standard culture conditions. More conventional techniques for the detection of capsules in microorganisms, such as staining of bacteria with India ink, the Quellung reaction, and slide agglutination with type III bacteria and homologous type-specific antisera, have been performed in conjunction with electron microscopy. Results obtained with these techniques have been consistent with the finding by electron microscopy of a microcapsule in a type III strain. This strain has a weakly positive Quellung reaction and a strongly positive slide agglutination. India ink preparation failed to demonstrate the presence of capsule. DISCUSSION Lancefield first suggested that the polysaccharide antigens of group B Streptococcus responsible for type serological specificity were capsular in nature (6). However, morphological studies for definition of the presence or absence of capsular material in group B streptococci have not been attempted previously. With use of a stain specific for polysaccharides (8), a microcapsule has been demonstrated in a type III strain of group B Streptococcus. Encapsulation of bacteria by polysaccharide has been suggested to be one factor responsible for the virulence of a microorganism; Rowley (1) believes that capsular thickness is a major factor. Although epidemiological studies have proved the enhanced invasiveness of type III strains among neonates with meningitis (1), other serotype strains of group B streptococci are postulated to have more capsular material (notably type Ia) (13). The type III polysaccharide isolated in native form, however, is a sialic acidcontaining polymer (Baker and Kasper, J. Exp. Med., in press; 2). Another encapsulated bacterium, Escherichia coli type K, has been shown to be associated with enhanced virulence among neonates (9). The K1 antigen is a polymer of sialic acid (5). The presence of sialic acid in certain bacterial antigens may be a more important factor than the capsular thickness in accounting for an organism's ability to invade the meninges of neonates.

4 FIG. 3. Electron micrograph ofgroup B Streptococcus (strain M732)grown in standard Todd-Hewitt broth and stained by standard techniques (x15,). Abbreviations: IL, inner layer ofcell wall; cap, microcapsule; bar,.1 pim. Downloaded from on May 12, 218 by guest FIG. 4. Electron micrograph ofgroup B Streptococcus (strain M732) grown in normal Todd-Hewitt broth and stained with ruthenium red (x15,). Abbreviations: IL, inner layer of cell wall; CM, cytoplasmic membrane; cap, microcapsule; bar,.1 pm. 192

5 VOL. 13, 1976 GROUP B STREPTOCOCCAL POLYSACCHARIDE (III) I L FIG. 5. Electron micrograph ofgroup B Streptococcus (strain M732) grown in Todd-Hewitt broth modified by the addition of excess Na2 HPO4 and glucose and stained with ruthenium red (x 15,). Abbreviations: IL, inner layer of cell wall; cap, microcapsule; bar,.1 pm. Previous investigation of the native type III polysaccharide has documented its acid lability (Baker and Kasper, J. Exp. Med., in press). The accumulation of acid in standard culture media during growth of organisms may produce acid-degraded antigenic substances. Because of the theoretical advantage of studying purified antigens isolated in native state from the bacterial cell surface, the buffering capacity of the Todd-Hewitt broth was increased to maintain a neutral ph environment. Furthermore, the yield oftype III polysaccharide could be substantially increased by additional modification of standard growth medium with glucose. This increased yield of polysaccharide can be accounted for by any one of a number of factors: (i) an increase in the number oforganisms available for extraction of polysaccharide; (ii) a change in the configuration of the surface polysaccharide (i.e., less density with buffering); or (iii) an increase in polysaccharide production by the organism. ACKNOWLEDGMENTS We wish to thank Diana Goroff for her excellent technical assistance and Claudia Ricci for her help in the preparation of this manuscript. This work was supported by Public Health Service research fellowship award 1 F22 AI235-1 from the National Institute of Health, Allergy, and Infectious Diseases. LITERATURE CITED 1. Baker, C. J., and F. F. Barrett Group B streptococcal infections in infants: the importance of various serotypes. J. Am. Med. Assoc. 23: Baker, C. J., and D. L. Kasper. Identification of sialic acid in polysaccharide antigens of group B Streptococcus. Infect. Immun. 13:-. 3. Devoe, L. W., and J. E. Gilchrist Release of endotoxin in the form of cell wall blebs during in vitro growth of Neisseria meningitidis. J. Exp. Med. 138: Freimer, E. H Type-specific polysaccharide antigens of group B streptococci. II. The chemical basis for serological specificity of the type II HCL antigen. J. Exp. Med. 125: Kasper, D. L., J. L. Winkelhake, W. D. Zollinger, B. L. Brandt, and M. S. Artenstein Immunochemical similarity between polysaccharide antigens of Escherichia coli 7:K, (L):NM and Group B Neisseria meningitidis. J. Immunol. 11: Lancefield, R. C., and E. H. Freimer Type-specific polysaccharide antigens of group B streptococci. J. Hyg. 64: Luft, J. H Improvements in epoxy resin embedding materials. J. Cell Biol. 9: Luft, J. H Fine structure ofcapillary and endocapillary layer as revealed by ruthenium red. Fed. Proc. Fed. Am. Soc. Exp. Biol. 25: Robbins, J. B., G. H. McCracken, Jr., E. C. Gotschlich, F. Orskov, I. Orskov, and L. A. Hanson Esche-

6 194 BAKER AND KASPER INFECT. IMMUN. richia coli K, capsular polysaccharide associated with 12. Swanson, J., and E. C. Gotschlich Electron microneonatal meningitis. N. Engl. J. Med. 29:1216- scopic studies on streptococci. II. Group A carbohy drate. J. Exp. Med. 138: Rowley, D. J Endotoxins and bacterial virulence. 13. Wilkinson, H. W Immunochemistry of purified J. Infect. Dis. 123: polysaccharide type antigens of group B streptococcal 11. Russell, H., and N. L. Norcross The isolation and types Ta, Ib, and Ic. Infect. Immun. 11: some physiochemical and biologic properties of the 14. Wilkinson, H. W., and R. G.Eagon Type-specific type III antigen of group B streptococci. J. Immunol. antigens of group B type Ic streptococci. Infect. Im- 19:9-96. mun. 4: