INFZCTION AND IMMUNITY, Mar. 1976, p. 653-660Copyright © 1976 American Society for Microbiology

Vol. 13, No. 3Printed in U.S.A.

Chemical and Biological Properties of Extracellular SlimeProduced by Staphylococcus aureus Grown in High-

Carbohydrate, High-Salt MediumJ. H. BROCKI*AND B. REITER

National Institute for Research in Dairying, Shinfield Reading RG2 9AT, England

Received for publication 29 July 1975

Slime material produced by three strains of Staphylococcus aureus grown inthe high-carbohydrate, high-salt modified 110 medium contained ribitol teichoicacid and, in two of the three strains, a basic protein reacting with antisera to S.aureus whole cells and cell walls. The basic protein differed chemically andserologically from cell wall mucopeptide and protein A. Substances resemblingthe capsular antigen of the Smith diffuse strain of S. aureus were not detected,nor were any other uronic acid-containing components. When cell walls, slimematerial, and teichoic acid were injected intradermally into cows, only cell wallsproduced a skin reaction.

Two strains of Staphylococcus aureus grownin the high-carbohydrate, 'high-salt modified110 medum of Yoshida and Ekstedt (25) wereshown to possess enhanced virulence in bovineskin compared with organisms grown in con-ventional media (6). The staphylococci grown inmodified 110 medium produced large quantitiesof extracellular slime, and it is this slime whichis responsible for the increase in virulence (25).Ekstedt and Bernhard (12) have recently iso-

lated and purified the slime material producedby several strains of S. aureus grown on modi-fied 110 agar and found it to contain two immu-nologically distinct components. These werenot separated but were shown to consist mainlyof galactose, with smaller quantities of glucose,glucuronic and galacturonic acids, glycerolphosphate, and amino acids.Morse (22) reported that the capsular antigen

of the Smith diffuse strain ofS. aureus could beisolated from the supernatant fluid of station-ary-phase liquid cultures. It seemed likely thatthe same would apply to slime material pro-duced in modified 110 medium; in the presentwork studies on the slime were made on mate-rial isolated from the supernatant of station-ary-phase liquid cultures of two bovine mastitisstrains and one human strain of S. aureus, andthe composition and properties were comparedwith material isolated from the cocci by mildultrasonic treatment.

MATERIALS AND METHODSS. aureus strains Mexi and BB (both of bovine

mastitis origin), and 3528 (of human origin), and theI Present address: Fundacion F. Cuenca Villoro, Gascon

de Gotor 4, Zaragoza, Spain.

preparation of modified 110 medium were as previ-ously described (5, 6). The Smith strain of S. aureuswas obtained from the National Collection of TypeCultures (NCTC 10399), and the encapsulated dif-fuse variant was isolated from it by the serum softagar technique (14). For preparation of slime mate-rial, the organisms were grown in 10-liter batches ofmodified 110 medium without agitation for 22 h at37 C. Bacterial cells were separated from the culturesupernatant by centrifugation.

Preparation of slime material from culture su-pernatant (SS). The supernatant fluid was treatedwith formalin (final concentration 0.25%) for 6 h at37 C, concentrated to about 500 ml by rotary evapo-ration, dialyzed against distilled water, and lyophi-lized, yielding about 6 g of crude material. All sub-sequent operations were carried out at 4 C. Thecrude material was dissolved in 30 ml of distilledwater, and 30 ml of 60% (wt/vol) trichloroacetic acidwas added. A precipitate formed and was removedby centrifugation. To the supernatant, 180 ml ofabsolute alcohol and 30 ml of acetone were added,and the resulting precipitate, after being allowed toflocculate for 30 min, was collected by centrifuga-tion. This precipitate was washed successively inabsolute alcohol and ether, dissolved in 10 ml of 10%trichloroacetic acid, precipitated by 70 ml of absolutealcohol, and washed as before. This last precipita-tion and washing procedure was repeated, and thematerial was finally dried in a vacuum desiccator.Yield was normally about 150 mg for strains BB andMexi and about 100 mg for 3528.

Preparation of slime material from bacterialcells (SB). To avoid loss of slime material the cocciwere not washed, but were suspended in 15 ml of0.15 M NaCl and treated by ultrasonics using aDawe Soniprobe (Dawe Instruments Ltd, London) at75 W and 20 kc/s for 4 min. Under these conditionsno decrease in viable count occurred, indicating thatthe organisms were not being disrupted. The bacte-ria were immediately removed by centrifugation at

653

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

654 BROCK AND REITER

4 C. The supernatant was adjusted to pH 3.5 with 0.1N HCl and allowed to stand overnight at 4 C, and aslight precipitate was removed by centrifugation.Six volumes of absolute alcohol and 1 volume ofacetone were added, and the resulting precipitatewas treated subsequently in the manner describedabove for the material from the culture supernatant.The yield was normally about 30 mg.

Chemical methods. Total hexose was estimatedby the phenol-sulfuric acid method (10). Uronic acidswere estimated by the carbazole method (9). Phos-phorus was estimated by the molybdate method (8).Protein was estimated by the method of Lowry et al.(21). Amino sugars were estimated by the p-amino-benzaldehyde method (23), after hydrolysis of sam-ples in 2 N HCI for 2 h at 100 C. Amino acid analysiswas carried out on samples hydrolyzed in 6 N HCl inevacuated sealed tubes for 24 h at 100 C, using aJEOLCO amino acid analyzer. The specific colorreaction for the Smith surface antigen was carriedout as described by Haskell and Hanessian (16).

Treatment with Pronase. Samples (1 mg) in 1 mlof 0.05 M tris(hydroxymethyl)aminomethane per0.145 M NaCl, pH 7.4, were incubated with 0.1 mg ofPronase (Calbiochem) at 37 C for 18 h.Preparation of staphylococcal antigens. A sam-

ple of crude protein A from S. aureus strain Mexi,prepared by the Verwey method as described byLofkvist and Sjoquist (20), was kindly provided byM. W. Russell. Teichoic acid was prepared by ex-traction of cell walls with 10% trichloroacetic acid(3), modified as described by Brock and Reiter (5),and mucopeptide was obtained by treating the resi-due from this extraction with 0.5 M NaOH for 4 h atroom temperature (2) and then with trypsin (10 ,tg/ml) in 0.05 M tris (hydroxymethyl)aminomethaneper 0.145 M NaCl, pH 7.4. Mucopeptide was solubi-lized by treatment with lysozyme (10 ,tg/ml) in 0.1 Mphosphate buffer (pH 6.25) at 37 C for 24 h.The capsular antigen from the Smith diffuse

strain of S. aureus was prepared as described byMorse (22).

Immunological methods. Antisera to whole cellswere raised in rabbits by twice-weekly intravenousinjection of heat-killed (65 C, 2 h) cells, suspendedat an optical density of 0.55 (Hilger spectrophotom-eter, no. 58 filter). Antisera to cell walls were raisedby a similar protocol using a cell wall suspensioncontaining 3.5 mg/ml. Attempts to raise antisera tosemipurified slime material were performed in twodifferent ways. In the first attempt, rabbits receivedone intramuscular injection of 5 mg of slime in 1 mlof physiological saline emulsified with 1 ml ofFreund complete adjuvant (Difco), followed by asimilar injection 2 weeks later. In the second at-tempt, 2 mg of slime in 0.1 ml of physiological salinewas emulsified with 0.1 ml of the adjuvant andinjected into the footpad, followed by a second simi-lar injection 2 weeks later. Absorption of antisera,ring precipitin and agglutination tests, gel diffu-sion, and immunoelectrophoresis were carried outby standard methods.

Paper chromatography. Samples (1 to 2 mg) werehydrolyzed in 2 N HCl (0.2 ml) for 2 h at 100 C. Afterevaporation to dryness over potassium hydroxideand phosphorus pentoxide, hydrolysates were exam-

ined by descending paper chromatography using sol-vents and reagents of Baddiley et al. (4).

Skin reaction in cows. The material to be testedwas dissolved or suspended at 0.1 mg/ml in 0.15 MNaCl, and 0.1 ml was injected into the loose skin atthe neck of each of four Friesian cows. The skinthickness at the site of injection was determined bypinching a fold of skin together and measuring thethickness with calipers. The increase in skin thick-ness was obtained by subtracting a similarly ob-tained value for the thickness of adjacent normalskin.

RESULTSChemical composition of slime material. SS

of all three strains contained protein, phospho-rus, hexose, and hexosamine as major compo-nents (Table 1). The hexose content was veryvariable, accounting for 80% of the total of 3528SS and between 17 and 59% (varying withdifferent batches) of SS from strains BB andMexi. SB was qualitatively similar, except thathexose was present as only a minor component(

VOL. 13, 1976

Special effort was directed towards establish-ing the presence of uronic acids and amino-uronic acids, since aminoglucuronic acid is amajor component of the Smith surface antigen(16). However, the assay for uronic acids gavenegative results with each preparation, and nosubstances resembling aminoglucuronic acidwere detected by paper chromatography. Fur-thermore, in the specific color reaction for theSmith surface antigen, slime material from allthree strains failed to show the absorptionpeaks at 410 and 470 nm characteristic of theSmith surface antigen.

Solutions of the slime preparations gave onlyvery slight absorption at 260 nm, equivalent tono more than 2% of nucleic acids in the slimepreparations.Immunological reactions. Both SS and SB

from strains Mexi and BB gave similar immu-noelectrophoresis patterns with whole-cell anti-sera raised against either strain. Two arcs ap-peared, one strongly anodic and the otherweakly cathodic (Fig. 1, top). SS and SB fromstrain 3528 gave only the cathodic arc with anti-sera to all three strains (Fig. 1, bottom), and SSand SB from strains Mexi and BB gave only thecathodic arc against 3528 antiserum. The pres-ence of the anodic arc, together with glucosa-mine, phosphate, and ribitol derivatives in hy-drolysates, strongly suggested that teichoicacid was a component of the slime of all threestrains.

Rabbits inoculated with several differentslime material preparations either intramuscu-larly or via the footpads failed to produce anydetectable antibodies reacting with the slime.Skin reaction in cows. The skin reactions in

SLIME FROM S. AUREUS 655

cows injected with slime material, cell walls,and teichoic acid (Fig. 2) showed that only cellwalls gave a pronounced skin reaction, slimematerial and teichoic acid being largely with-out effect.

Separation and purification of the two im-munologically active components in SS fromstrain BB. SS from strain BB was chromato-graphed on diethylaminoethyl-cellulose, thecolumn being eluted initially with 0.02 M phos-phate (pH 6.85) followed by a linear 0 to 0.5 MNaCl gradient in the same phosphate buffer(Fig. 3). A serologically active protein-contain-ing peak (fraction I) was eluted with the initialbuffer (together with most of the mannose con-tent), and further serologically active materialwas eluted with the NaCl gradient (fraction II).Fraction I contained 28% protein and 0.1%phosphorus (with mannose accounting for mostofthe remainder), and fraction II contained 15%protein and 6.1% phosphorus. The reactions ofthese fractions in immunoelectrophoresis andgel diffusion (Fig. 4 and 5) showed that fractionI gave the anodic arc and fraction II the ca-thodic. Fraction I gave a stronger immunoelec-trophoresis arc against antiserum raised to or-ganisms grown in modified 110 medium thanagainst the antiserum to organisms grown in anormal medium (Fig. 4a). This might indicatethat fraction I was more readily produced in theformer medium. The electrophoretic mobility offraction II was similar to that of purified cellwall ribitol teichoic acid (Fig. 4b) from S. au-reus, and fraction II gave a line of identity withteichoic acid in gel diffusion (Fig. 5b). The iden-tity of fraction II as teichoic acid was confirmedwhen hydrolysis and paper chromatography re-

FIG. 1. Immunoelectrophoresis patterns of slime material against S. aureus antisera. (Top) Mexi SSagainst Mexi antiserum. (Bottom) 3528 SS against 3528 antiserum (upper trough) and Mexi antiserum(lower trough).

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

656 BROCK AND REITERIncreose In slkinthckness (mm)

COW 287

'A ,21- 'I' ,,^ ¢A**_

I t

0. t- taE0- 1

Cow 575

j)- -me

6 12 24 hor0

Cow 5392A,,II

l ..----------- -0; 11@.-O

:. *sLA '.

S...

6

Cow 265

06FIG. 2. Skin reactions in cows injected intrader-

mally with 10 Ag of S. aureus BB teichoic acid (0),SS (Q), and cell walls (E).

0 D 2Onm

vealed the presence of anhydroribitol, ribitolphosphates, inorganic phosphate, and glucosa-mine, these being typical of the ribitol cell wallteichoic acid of S. aureus.

Identification of fraction I. Fraction I wasfound to react with antisera to cell walls as wellas to whole cells, indicating that it was a cellwall component. Pronase destroyed all serologi-cal activity, indicating that protein was the ac-tive component. It seemed possible that itmight be related either to protein A or to thecell wall peptide structurally similar to muco-peptide described by Hisatsune et al. (18).However, its reaction in gel diffusion indicatedthat it was immunologically distinct from bothprotein A and mucopeptide (Fig. 5a).

Fraction I was further purified by chromatog-raphy on Sephadex G-200. The serologically ac-tive material was eluted in the void volume,and this procedure separated it from most of thecontaminating mannose. The amino acid com-position of purified fraction I was determinedand compared with that of mucopeptide fromstrain BB and with published figures for puri-fied protein A (Table 2). As expected, the onlyamino acids present in significant amount inthe mucopeptide were glutamic acid, lysine,alanine, and glycine in approximate ratios of1:1:2:4, but fraction I had a quite differentamino acid composition, which also differedfrom that of protein A, notably in respect tothreonine, which was the most abundant aminoacid in fraction I but was present in only traceamounts in protein A. This supports the sero-logical evidence that fraction I is a protein dis-

No C wo6w.t y

SwoIogcol Activty^,_ I I,..._0 10 20 30 g,o 40 50 60 70 80 90

-~W Froction number

FIG. 3. Elution ofSS from S. aureus BB from diethylaminoethyl-cellulose. Two hundred milligrams wasapplied to a column (30 by 0.9 cm) ofdiethylaminoethyl-cellulose and eluted with 0.02 M sodium phosphate(pH 6.85) followed by a NaCI gradient in the phosphate buffer. Fractions were 5 ml. Serological activity wasmonitored by ring precipitin test against S. aureus BB whole-cell antiserum. (O) Optical density at 280 nm.

INFECT. IMMUJN.

i12

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

SLIME FROM S. AUREUS 657

FIG. 4. Immunoelectrophoresis patterns ofpurified slime fractions from SS ofS. aureus BB. (a) fraction Iagainst antisera to strain Mexi grown in a conventional medium (upper trough) and in modified 110 medium(lower trough). (b) Fraction II (upper well) and teichoic acid from S. aureus BB (lower well) against Mexiantiserum.

tinct from both mucopeptide and protein A.Separation of immunologically active com-

ponents from other slime preparations. SSfrom strain Mexi and SB from Mexi and BBbehaved in the same way on diethylamino-ethyl-cellulose chromatography as describedabove. SS of strain 3528 yielded only a verysmall amount of material corresponding to frac-tion I, which was serologically inactive. Thisagrees with the absence ofan anodic componentin the slime material of this strain (see Fig. 1,bottom).

It appears therefore that only slime from thetwo strains Mexi and BB contains the immuno-logically active protein fraction I and not slimefrom strain 3528.

Reaction with antiserum to the Smith sur-face antigen. To ascertain whether the slimepreparation contained an antigen related to thecapsular antigen of the Smith diffuse strain ofS. aureus, the ability of the slime preparationfrom strain BB to remove agglutinating anti-bodies from antisera to the Smith diffuse strainwas investigated (Table 3). It can be seen thatwhereas 1 mg of Smith surface antigen re-moved all activity from 1 ml of antisera, up to10 mg of slime material reduced the activity byonly two dilutions. This indicates that theSmith surface antigen, or similar cross-react-ing antigens, were at most only very minorcomponents of the slime material. Since theSmith diffuse antiserum did not agglutinatenonencapsulated strains of S. aureus, it seems

reasonable to assume that the agglutinatingactivity was due to antibodies to the uniqueSmith surface antigen.

DISCUSSIONThe extracellular slime produced by S. au-

reus Mexi, BB, and 3528 contained cell wallteichoic acid and, in the case of Mexi and BB, aprotein antigen unrelated to either mucopep-tide or protein A. There was no evidence, eitherchemically or immunologically, for the pres-ence of the Smith surface antigen, and al-though the slime reacted with antisera againstkilled S. aureus, it was itself not immunogenicin rabbits and did not cause skin reactions incows. The findings differ in several respectsfrom those of Ekstedt and Bernhard (12), whoexamined slime material produced by severalstrains of S. aureus grown on modified 110agar. These workers reported the presence ofsignificant amounts of galactose and glucuronicand galacturonic acids, none of which were de-tected in slime from BB, Mexi, and 3528, butfound no ribitol teichoic acid, although they diddetect small amounts of glycerol phosphate. Aglycerol teichoic acid was also reported to bepresent in the slime of capsule produced by theWiley wound strain of S. aureus (17). None ofthese workers reported any ribitol teichoic acid,which is perhaps rather surprising in view ofthe large amount present in the slime from BB,Mexi, and 3528, and the fact that ribitol teichoicacid is a major cell wall component, whereas

VOL. 13, 1976

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

658 BROCK AND REITER

FIG. 5. Gel diffusion reactions ofpurified slime and cell wall components ofS. aureus BB. (a) Mucopeptide(MP), protein A (PA), and fraction I ofSS (SL) against BB antiserum (AS). (b) Teichoic acid (T) and frac-tion II of SS (F) against BB antiserum. (S)

glycerol teichoic acid occurs only in associationwith the cell membrane (1). As these workersdid not state that they had distinguished inhydrolysates between glycerol and ribitol phos-

phates, and between glycerol and anhydroribi-tol, it is possible that ribitol derivatives wereoverlooked. The extensive production of cellwall teichoic acid as a component of extracellu-

INFECT. IMMUN.

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/

SLIME FROM S. AUREUS 659

0.96" 1.06 0.53tr' -" 0.33tr

0.020.010.061.00tr

4.182.17trtrtrtr

0.060.06

1.01tr

0.401.000.840.840.650.39

0.50

0.671.551.221.000.331.441.110.89tr

0.220.33trtr

a Molar ratios calculated from results of Grov etal. (15) for protein A from S. aureus strain Cowan 1(NCTC 8530).

b Molar ratios: glutamic acid = 1.00.tr,

660 BROCK AND REITER

2. Archibald, A. R., H. E. Coapes, and G. H. Stafford.1969. The action of dilute alkali on bacterial cellwalls. Biochem. J. 113:899-900.

3. Armstrong, J. J., J. Baddiley, J. G. Buchanan, B.Carss, and G. R. Greenberg. 1958. Isolation andstructure of ribitol phosphate derivatives (teichoicacids) from bacterial cell walls. J. Chem. Soc. 4344-4354.

4. Baddiley, J., J. G. Buchanan, U. L. Rajbhandary, andA. R. Sanderson. 1962. Teichoic acid from the walls ofStaphylococcus aureus H. Structure of the N-acetyl-glucosaminylribitol residues. Biochem. J. 82:439-448.

5. Brock, J. H., and B. Reiter. 1971. Sensitisation of sheeperythrocytes by cell wall teichoic acid of Staphylococ-cus aureus. Immunochemistry 8:933-938.

6. Brock, J. H., A. Turvey, and B. Reiter. 1973. Virulenceof two mastitis strains of Staphylococcus aureus inbovine skin: enhancement by growth in high carbohy-drate-high salt medium or in raw milk. Infect. Im-mun. 7:865-872.

7. Bunting, M. E., C. F. Robinson, and H. Bunting. 1949.Factors affecting the elaboration of pigments andpolysaccharide by Serratia marcescens. J. Bacteriol.58:114-115.

8. Chen, P. S., T. Y. Toribara, and H. Warner. 1956.Microdetermination of phosphorus. Anal. Chem.28:1756-1758.

9. Dische, Z. 1947. A new specific color reaction of hexu-ronic acids. J. Biol. Chem. 167:189-198.

10. Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers,and F. Smith. 1956. Colorimetric method for determi-nation of sugars and related substances. Anal. Chem.28:350-356.

11. Duguid, J. P. 1948. The influence of cultural conditionson the morphology ofBacterium aerogenes with refer-ence to nuclear bodies and capsule size. J. Pathol.Bacteriol. 60:265-274.

12. Ekstedt, R. D., and J. M. Bernhard. 1973. Preparationand characterisation of a slime layer material pro-duced by Staphylococcus aureus. Proc. Soc. Exp. Biol.Med. 142:86-91.

13. Ekstedt, R. D., and K. Yoshida. 1969. Immunity tostaphylococcal infection in mice: effect of living ver-sus killed vaccine, role of circulating antibody, andinduction of protection-inducing antigen(s). J. Bacte-riol. 100:745-750.

14. Finkelstein, R. A., and S. E. Sulkin. 1958. Characteris-tics of coagulase positive and coagulase negativestaphylococci in serum soft agar. J. Bacteriol. 75:339-344.

15. Grov, A., B. Mykiestad, and P. Oeding. 1964. Immuno-chemical studies on antigen preparation from Staphy-lococcus aureus. I. Isolation and chemical characteri-sation of antigen A. Acta Pathol. Microbiol. Scand.61:588-596.

16. Haskell, T. H., and S. Hanessian. 1964. The purifica-tion and characterisation of a new immunising poly-saccharide prepared from Staphylococcus aureus.Biochim. Biophys. Acta 83:35-41.

17. Hisatsune, K., S. J. de Courcy, and S. Mudd. 1967.Studies on the carbohydrate-peptide fraction of thecentrifugal supernatants of Staphylococcus aureuscultures. I. Biochemistry 6:586-594.

18. Hisatsune, K., S. J. de Courcy, and S. Mudd. 1967.Studies on the carbohydrate-peptide fraction of thecentrifugal supernatants of Staphylococcus aureus.II. Biochemistry 6:595-603.

19. Karakawa, W. W., and J. A. Kaie. 1972. Characteriza-tion of the surface antigens of Staphylococcus aureus,strain K-93 M. J. Immunol. 108:1199-1208.

20. Lofkvist, T., and J. Sjoquist. 1962. Chemical and sero-logical analysis of antigen preparations from Staphy-lococcus aureus: a comparison between the productsobtained by Verwey's and Jensen's techniques. ActaPathol. Microbiol. Scand. 56:295-304.

21. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.

22. Morse, S. I. 1962. Isolation and properties of a surfaceantigen of Staphylococcus aureus. J. Exp. Med.115:295-311.

23. Rondle, C. J. M., and W. T. J. Morgan. 1955. Thedetermination of glucosamine and galactosamine.Biochem. J. 61:586-589.

24. Schwarzmann, S., and J. R. Boring. 1971. Antiphago-cytic effect of slime from a mucoid strain of Pseudo-monas aeruginosa. Infect. Immun. 3:762-767.

25. Yoshida, K., and R. D. Ekstedt. 1968. Relation of mu-coid growth of Staphylococcus aureus to clumpingfactor reaction, morphology in serum-soft agar, andvirulence. J. Bacteriol. 96:902-908.

INFECT. IMMUN.

on April 6, 2021 by guest

http://iai.asm.org/

Dow

nloaded from

http://iai.asm.org/