INFECTION AND IMMUNITY, Jan. 1976, p. 189-194Copyright ©) 1976 American Society for Microbiology

Vol. 13, No. 1Printed in USA.

Microcapsule of Type III Strains of Group B Streptococcus:Production and MorphologyCAROL J. BAKERl* AND DENNIS L. KASPER

The Channing Laboratory, Harvard Medical School, and Boston City Hospital, Boston, Massachusetts 02118

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 orga-nisms in standard Todd-Hewitt broth resulted in acid accumulation during theexponential phase of growth and poor yield of type III polysaccharide whenextracted from cells by washing with neutral buffer solution. By increasing thebuffering capacity of the broth medium, acid accumulation was prevented, andthe number of viable cells was increased at the stationary phase of growth.Further, by increasing the concentration of glucose in the buffered medium, theyield of type III polysaccharide was increased two to three times. Electronmicroscopic investigations of cells grown in the modified broth medium demon-strated a thicker microcapsule than was found in organisms grown in standardbroth.

The type-specific polysaccharides of group BStreptococcus extracted by treatment of wholecells with hot HCl have been shown to be chemi-cally and immunologically distinct (4, 6, 11, 13,14). These HCl polysaccharides are low-molecu-lar-weight fragments of"native" antigens. How-ever, for the isolation of high-molecular-weight, antigenically complete substances,more gentle methods are needed. A recent in-vestigation of purified type III polysaccharideextracted by washing cells with neutral buffersolution demonstrated the type III antigen innative or complete form to be a polymer, withsialic acid as a major chemical constituent (C.J. Baker and D. L. Kasper, J. Exp. Med., inpress). After exposure to acid, this polysaccha-ride was readily degraded to an antigen immu-nologically identical to the type III-HCl polysac-charide. Preparation of type III antigen fromorganisms grown by methods used in previousinvestigations (6, 11, 13) of group B streptococ-cal antigens consistently resulted in low anti-gen yield. Because of the proved acid lability ofnative type III polysaccharide (Baker and Kas-per, J. Exp. Med., in press) and the observa-tion that the growth of organisms in standardconditions resulted in low antigen yield, experi-ments were conducted to modify the culturemedium. A method was sought that would pre-vent exposure of cells to acid during the growthcycle and that would maximize the yield of typeIII polysaccharide.

(Presented in part at the 14th InterscienceConference on Antimicrobial Agents and Chem-

' Present address: Department of Pediatrics, Baylor Col-lege of Medicine, Houston, Tex. 77025.

otherapy, San Francisco, California, Septem-ber 1974.)

MATERIALS AND METHODSBacterial strains. Group B streptococcal strains

M731 and M732 (type III) were originally isolatedfrom infants with meningitis (Houston, 1973). Bacte-rial strains were stored in 1-ml aliquots of Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) at-70 C and thawed before use.Media and growth conditions. The liquid me-

dium used in the experiments was Todd-Hewittbroth (Difco), which contained (per liter): 3 g of beefheart (infusion from 500 g), 20 g of neopeptone, 2 g ofdextrose, 2 g of NaCl, 2.5 g of Na2CO3, and 0.4 g ofNa2HPO4, at a final pH of 7.4. This broth will bereferred to as the normal or standard medium. Forcertain experiments, this medium was modified byincreasing the Na2HPO4 or glucose concentrationsfourfold or eightfold. All ingredients were auto-claved together.

Broth cultures were grown at 37 C in 250-ml Er-lenmeyer flasks and stirred constantly. The brothmedia were inoculated with 1 ml of organisms,which had been grown for 10 to 12 h in Todd-Hewittmedium. The initial concentration was approxi-mately 2 x 107 to 5 x 107 colony-forming units perml, and subsequent growth was monitored by deter-mining colony counts on plates containing 5% sheepblood in Trypticase soy agar (Difco). Under theabove conditions, exponential growth was attainedat 2 to 4 h; at this time a cell density of approxi-mately 109 colony-forming units/ml had been at-tained.

Chemical assays. The pH of the growth mediumwas measured by means of the radiometer pH meter26 (Radiometer, Copenhagen, Denmark) with stand-ard buffers. Glucose uptake was measured by deter-mining the amount of glucose remaining in the me-dium after various intervals, using the oxidase re-

189

190 BAKER AND KASPER

agent method (Worthington Biochemical Corp.,Freehold, N.J.) with glucose standards.

Electron microscopy. Suspensions ofwhole bacte-rial cells grown for 12 h in either standard or phos-phate-glucose modified medium were prefixed to ad-just the final concentration of fixative to 0.5% by theaddition of 2.5% glutaraldehyde in 0.1 M sodiumcacodylate buffer. CaCl2 was added to this suspen-sion to achieve a concentration of 0.05%. After prefix-ation for 2 h at 4 C, the suspensions were centri-fuged at 12,000 x g. The pellets obtained were fixedin 2.5% buffered glutaraldehyde for 2 h at 4 C,washed with buffer, and fixed again for 60 min inaqueous 1% OS04 (3).The pellets were then treated with 0.5% uranyl

acetate in Michaelis buffer (pH 5.0) for 2 h at 24 C.One-millimeter blocks were dehydrated in gradedalcohols and embedded in Epon 812 according to themethod of Luft (7). Thin sections were stained withlead citrate and examined in a Jem 100B electronmicroscope (Japan Electron Optics Laboratory, To-kyo, Japan).

Polysaccharide components of bacterial cell wallsare not seen readily with routine staining tech-niques; visualization of these antigens requires spe-cial stains, such as ruthenium red. Samples pre-pared for staining with ruthenium red were treatedin a manner identical to that described above, ex-cept that the stain at a concentration of 700 ,g/mlwas added to both the prefixation and fixation solu-tions (8).

Preparation of native type III polysaccharide.Type III-specific polysaccharide antigen was iso-lated and purified in high-molecular-weight form bymeans of methods described previously (Baker andKasper, J. Exp. Med., in press). Briefly, cellsgrown in 60- to 70-liter lots of standard or modifiedTodd-Hewitt broth media were washed with a neu-tral buffer solution. After ethanol fractionations,ultracentrifugation, and enzyme digestions for re-moval of protein and nucleic acid contaminants,separation of group B- from type III-specific polysac-charides was achieved by means of gel chromatogra-phy. The eluted fractions from the void volume of aSepharose 4B K26, 100-cm column (PharmaciaChemicals, Uppsala, Sweden) contained only typeIII serological activity. The fractions were pooledand concentrated on a PM30 filtration membrane,and 4 volumes of cold ethanol were added to precipi-tate the purified type III polysaccharide. Antigenyield was determined by weighing the lyophilizedpreparations.

RESULTSThe pH changes observed during the growth

cycle of group B Streptococcus in the standardTodd-Hewitt broth are shown in Fig. 1, whichcompares the effects of fourfold and eightfoldincreases in concentration of Na2HPO4. Orga-nisms were grown in 100 ml of broth, and 5-mlsamples were removed at hourly intervals;these were filtered through membrane filters(diameter, 0.45 ,um; Millipore Corp., Bedford,Mass.) for pH determinations, and colony-form-

INFECT. IMMUN.

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-o0 2 4 6 8 10 12GROWTH (HOURS)

FIG. 1. Hourly pH measurements ofa culture me-dium for strain M731 grown in normal Todd-Hewittbroth modifiled with a fourfold or an eightfold in-crease in the Na2 HPO4 concentration.

ing units per milliliter were determined forunfiltered aliquots. The pH of the standardTodd-Hewitt broth fell below 7.0 after 3 h ofgrowth and decreased to 5.9 by 9 h. With use ofthe fourfold and eightfold Na2HPO4-bufferedmedia, the pH was maintained above 6.2 and6.5, respectively, during the entire growth cy-cle. Additionally, an average of a two-log in-crease in the number of viable organisms wasobserved after 12 h of growth in the eightfoldbuffered medium.The depletion of glucose in the medium by

organisms growing in standard Todd-Hewittbroth is shown in Fig. 2. After 8 to 10 h ofgrowth, the majority of the glucose was de-pleted from the standard medium. In contrast,glucose continued to be metabolized for severalhours in the medium with a glucose excess.Similar results were observed when the me-dium was supplemented concurrently withNa2HPO4 and glucose. With the use of both aneightfold Na2HPO4 and glucose increase in thegrowth medium, the amount of purified, nativetype III polysaccharide extracted from a 60-literlot 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 corre-lated with onset of autolysis of organisms

GROUP B STREPTOCOCCAL POLYSACCHARIDE (III) 191

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1200

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1000

800

600

400

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FIG. 2. Glucose (mgll00 ml) depletion duringgrowth of strain M732 in normal Todd-Hewitt brothand broth modified by increasing the glucose concen-tration fourfold or eightfold.

grown in standard Todd-Hewitt medium after 8to 10 h of growth. Initiation of autolysis was

delayed beyond 12 h as both the Na2HPO4 andglucose concentrations were increased.By means of electron microscopy, the mor-

phology of a type III strain of group B Strepto-coccus (M732) grown in standard medium wascompared to that of this strain grown in modi-fied liquid medium. In Fig. 3, an organismstained by standard techniques is shown. Thisorganism demonstrated bilaminar morphologytypical of other streptococci. The outer, moder-ately electron-dense layer is a microcapsule.This layer is morphologically similar to that ofgroup A Streptococcus devoid of protein anti-gens (12). The thick, electron-dense, inner layerof the cell wall lies outside the indistinct cyto-plasmic membrane. An organism stained withruthenium red for demonstration of the capsu-lar polysaccharide is seen in Fig. 4. The outerlayer of microcapsule is better visualized with

this stain. This well-defined microcapsule isabout two times the thickness of the inner cellwall. However, the thickness of the capsule issignificantly increased by growing organismsin a medium modified with an eightfold concen-tration of Na2HPO4 and glucose, as demon-strated in the ruthenium red stain seen in Fig.5. The capsular thickness of organisms grownin this modified medium was 1.5 to 2 times thethickness noted in organisms grown understandard culture conditions.More conventional techniques for the detec-

tion of capsules in microorganisms, such asstaining of bacteria with India ink, the Quel-lung reaction, and slide agglutination withtype III bacteria and homologous type-specificantisera, have been performed in conjunctionwith electron microscopy. Results obtainedwith these techniques have been consistentwith the finding by electron microscopy of amicrocapsule in a type III strain. This strainhas a weakly positive Quellung reaction and astrongly positive slide agglutination. India inkpreparation failed to demonstrate the presenceof capsule.

DISCUSSION

Lancefield first suggested that the polysac-charide antigens of group B Streptococcus re-sponsible for type serological specificity werecapsular in nature (6). However, morphologicalstudies for definition of the presence or absenceof capsular material in group B streptococcihave not been attempted previously. With useof a stain specific for polysaccharides (8), amicrocapsule has been demonstrated in a typeIII strain of group B Streptococcus. Encapsula-tion of bacteria by polysaccharide has been sug-gested to be one factor responsible for the viru-lence of a microorganism; Rowley (10) believesthat capsular thickness is a major factor. Al-though epidemiological studies have proved theenhanced invasiveness of type III strainsamong neonates with meningitis (1), other sero-type strains of group B streptococci are postu-lated to have more capsular material (notablytype Ia) (13). The type III polysaccharide iso-lated in native form, however, is a sialic acid-containing polymer (Baker and Kasper, J. Exp.Med., in press; 2). Another encapsulated bac-terium, Escherichia coli type K, has beenshown to be associated with enhanced virulenceamong neonates (9). The K1 antigen is a poly-mer of sialic acid (5). The presence of sialic acidin certain bacterial antigens may be a moreimportant factor than the capsular thickness inaccounting for an organism's ability to invadethe meninges of neonates.

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VOL. 13, 1976

FIG. 3. Electron micrograph ofgroup B Streptococcus (strain M732)grown in standard Todd-Hewitt brothand stained by standard techniques (x150,000). Abbreviations: IL, inner layer ofcell wall; cap, microcapsule;bar, 0.1 pim.

FIG. 4. Electron micrograph ofgroup B Streptococcus (strain M732) grown in normal Todd-Hewitt brothand stained with ruthenium red (x150,000). Abbreviations: IL, inner layer of cell wall; CM, cytoplasmicmembrane; cap, microcapsule; bar, 0.1 pm.

192

GROUP B STREPTOCOCCAL POLYSACCHARIDE (III) 193

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FIG. 5. Electron micrograph ofgroup B Streptococcus (strain M732) grown in Todd-Hewitt broth modifiedby the addition of excess Na2HPO4 and glucose and stained with ruthenium red (x 150,000). Abbreviations:IL, inner layer of cell wall; cap, microcapsule; bar, 0.1 pm.

Previous investigation of the native type IIIpolysaccharide has documented its acid lability(Baker and Kasper, J. Exp. Med., in press).The accumulation of acid in standard culturemedia during growth oforganisms may produceacid-degraded antigenic substances. Because ofthe theoretical advantage of studying purifiedantigens isolated in native state from the bacte-rial cell surface, the buffering capacity of theTodd-Hewitt broth was increased to maintain aneutral pH environment. Furthermore, theyield oftype III polysaccharide could be substan-tially increased by additional modification ofstandard growth medium with glucose. Thisincreased yield of polysaccharide can be ac-counted for by any one of a number of factors:(i) an increase in the number oforganisms avail-able for extraction of polysaccharide; (ii) achange in the configuration of the surface poly-saccharide (i.e., less density with buffering); or(iii) an increase in polysaccharide production bythe organism.

ACKNOWLEDGMENTS

We wish to thank Diana Goroff for her excellent techni-cal assistance and Claudia Ricci for her help in the prepara-tion of this manuscript.

This work was supported by Public Health Service re-search fellowship award 1 F22 AI02305-1 from the NationalInstitute of Health, Allergy, and Infectious Diseases.

LITERATURE CITED1. Baker, C. J., and F. F. Barrett. 1974. Group B strepto-

coccal infections in infants: the importance of variousserotypes. J. Am. Med. Assoc. 230:1158-1160.

2. Baker, C. J., and D. L. Kasper. Identification of sialicacid in polysaccharide antigens of group B Streptococ-cus. Infect. Immun. 13:000-000.

3. Devoe, L. W., and J. E. Gilchrist. 1973. Release ofendotoxin in the form of cell wall blebs during in vitrogrowth of Neisseria meningitidis. J. Exp. Med.138:1156-1167.

4. Freimer, E. H. 1967. Type-specific polysaccharide anti-gens of group B streptococci. II. The chemical basisfor serological specificity of the type II HCL antigen.J. Exp. Med. 125:381-392.

5. Kasper, D. L., J. L. Winkelhake, W. D. Zollinger, B.L. Brandt, and M. S. Artenstein. 1972. Immunochemi-cal similarity between polysaccharide antigens ofEscherichia coli 07:K, (L):NM and Group B Neisseriameningitidis. J. Immunol. 110:262-268.

6. Lancefield, R. C., and E. H. Freimer. 1966. Type-spe-cific polysaccharide antigens of group B streptococci.J. Hyg. 64:191-203.

7. Luft, J. H. 1961. Improvements in epoxy resin embed-ding materials. J. Cell Biol. 9:409-417.

8. Luft, J. H. 1966. Fine structure ofcapillary and endocap-illary layer as revealed by ruthenium red. Fed. Proc.Fed. Am. Soc. Exp. Biol. 25:1773-1789.

9. Robbins, J. B., G. H. McCracken, Jr., E. C. Gotschlich,F. Orskov, I. Orskov, and L. A. Hanson. 1974. Esche-

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richia coli K, capsular polysaccharide associated with 12. Swanson, J., and E. C. Gotschlich. 1973. Electron micro-neonatal meningitis. N. Engl. J. Med. 290:1216- scopic studies on streptococci. II. Group A carbohy-1220. drate. J. Exp. Med. 138:245-258.

10. Rowley, D. J. 1971. Endotoxins and bacterial virulence. 13. Wilkinson, H. W. 1975. Immunochemistry of purifiedJ. Infect. Dis. 123:317-327. polysaccharide type antigens of group B streptococcal

11. Russell, H., and N. L. Norcross. 1972. The isolation and types Ta, Ib, and Ic. Infect. Immun. 11:845-852.some physiochemical and biologic properties of the 14. Wilkinson, H. W., and R. G.Eagon. 1971. Type-specifictype III antigen of group B streptococci. J. Immunol. antigens of group B type Ic streptococci. Infect. Im-109:90-96. mun. 4:596-604.