STUDIES ON STREPTOCOCCUS PYOGENES

Size: px

Start display at page:

Download "STUDIES ON STREPTOCOCCUS PYOGENES"

Easter Stevenson
6 years ago
Views:

STUDIES ON STREPTOCOCCUS PYOGENES II. OBSERVATIONS ON THE MICROSCOPICAL AND BIOLOGICAL ASPECTS OF THE DISINTEGRATION AND SOLUBILIZATION OF A TYPE 6 STRAIN BY SHAKING WITH GLASS BEADS' HUTTON D.

1 STUDIES ON STREPTOCOCCUS PYOGENES II. OBSERVATIONS ON THE MICROSCOPICAL AND BIOLOGICAL ASPECTS OF THE DISINTEGRATION AND SOLUBILIZATION OF A TYPE 6 STRAIN BY SHAKING WITH GLASS BEADS' HUTTON D. SLADE AND JEANETTE K. VETTER The Rheumatic Fever Research Institute, Chicago 8, Illinois Received for publication November 23, 1955 Slade and Vetter (1956) have shown that exposure to sonic oscillation of an aqueous suspension of a type 6 strain of Streptococcus pyogenes (Streptococcus haemolyticus) resulted in the solubilization of considerable quantities of the group-specific C-antigen, the type-specific M1- antigen, and the cell wall constituent rhamnose. Several hours of oscillation resulted in the release into solution of 85 per cent of the dry weight of the cell. Electron microscopic observations at various times during the oscillation process demonstrated that extensive disintegration of the structural elements of the cell occurred. Hess and Slade (1955) found that shaking the same strain of S. pyogenes with glass beads in a Mickle disintegrator resulted in the solubilization of only 60 per cent as much of the cell material as had been obtained by the sonic process. This result, plus the fact that cell walls of group A streptococci prepared by shaking with glass beads (Salton, 1953; Barkulis and Ekstedt, 1955) were found to contain M-antigen, indicated that the disintegration and solubilization of the cell by the latter process was not as extensive as that caused by sonic energy. It was thus of considerable interest to investigate further the shaking procedure. In addition, the previous results made it desirable to examine the possible advantages of MIickle extracts as compared to sonic extracts as a starting material for the preparation of purified MI-antigen. It will be shown that the extracts obtained by the two procedures differed sharply in several instances. In addition, microscopical observations of the process of cell destruction are presented. 1 Supported by a grant from the Chicago Heart Association and a contract (N7onr-1769) between The Rheumatic Fever Research Institute and the Office of Naval Research, Department of the Navy. METHODS The methods used for the culture of the organism and harvesting of the cells were the same as previously described (Hess and Slade, 1955). No capsules could be demonstrated on the cocci by india ink-stain methods. One batch of cells was used for all the work to be reported here. Living cells were enumerated by the pour plate method following 1 min of sonic oscillation. This treatment with strain S43 increased the plate count 2 to 3 times (Slade and Slamp, 1956). Preparation of extracts. The cells were shaken with glass beads (80,u diameter) in a Mickle instrument as previously described (Hess and Slade, 1955) except that the volume of cell suspension was increased to 20 ml and the weight of the beads to 20 g. The material was divided evenly between the two cups of the shaking machine. The fractional method of extraction (Slade and Vetter, 1956) was employed. At the conclusion of each shaking period, the cell-bead mixture was poured into stainless steel centrifuge tubes and centrifuged at 20,000 X G for 1 hr. The supernatant solution was decanted and the cell-bead mixture in each tube was resuspended in 10 ml of distilled water. The tube was stoppered and shaken lightly by hand. This washing step was essential in order to recover the soluble material admixed with the cell residue and glass beads. One washing was sufficient. At the conclusion of the fifth hr of shaking in water, the water was replaced with 0.15 M NaHCO3 and the cell-bead mixture shaken for 1 hr. Extraction with 0.15 M Na acetate then followed in a similar manner. All the above operations were carried out in a cold room at 2 C. The extracts after dialvsis for 17 hr were frozen and dried. All cell components not sedimented after ½ hr at 20,000 X G were considered as soluble. 27

2 28 SLADE AND VETTER [VOL. 72 TABLE I Chemical and biological data on soluble fractions released from group A streptococcus by shaking with glass beads Soluble Fractions Rhamnose Protein, Oscillation Time Living Cells per cent dry ineach Per cent Mg in each Per cent in Per cent of OclMg weight of dialyzable extract each extract original in extract each extract hr * _ 44.0t < < < _ Bicarbonate _ Acetate - _ Residue t Per cent recovered * Total dry weight of original cells in mg. t Total rhamnose present in original cells in mg. t Protein determined after hydrolysis in 2 N HCl. The residue remaining after the 5th hr of extraction in water was shaken for 1 hr in 0.15 M NaHC03. T The residue remaining after bicarbonate extraction was shaken for 1 hr in 0.15 M Na acetate S -rimc n hours Figure 1. Total cellular material and rhamnose solubilized by the shaking of Streptococcus pyogenes with glass beads. All other methods were the same as previously employed (Slade and Vetter, 1956). RESULTS Reduction in number of living cells. Column 2 of table 1 shows that in the first % hr of shaking the number of viable cells was reduced to 0.6 per cent of the original count. Within the next 15 min the count was found to be 0.2 per cent. No growth was obtained after 1 hr of shaking when tested in a dilution of Similar results were obtained in a dilution of 10-2 when tested after shaking for 2 and 3 hr. These results are expressed in table 1 as less than 0.1 per cent. Solubilization of the cell. Figure 1 shows that 67 per cent of the dry weight of the streptococcal cell was solubilized during the 5 hr of shaking. It is apparent that after the first j1 hr of shaking only 16 per cent additional cell material was rendered soluble. The most extensive release of cell material occurred in the first 15 min when 23 of the total solubilized in 5 hr was found in solution. The dry weight of the original cells and the weights of extracts obtained are given in column 3 of table 1. Total recovery was 97.0 per cent. Column 4 of table 1 shows the quantity (in per cent) of dialyzable material present in the extracts obtained between 0.25 and 5 hr of shaking. The quantity for each hourly interval between 0 and 4 hr ranged from 6-20 per cent. The first

1956] STUDIES ON STREPTOCOCCUS II 29 TABLE 2 Precipitin tests for group-specific and type-specific antigens in extracts obtained by shaking group A streptococcus with glass beads Treatment of Extract

3 1956] STUDIES ON STREPTOCOCCUS II 29 TABLE 2 Precipitin tests for group-specific and type-specific antigens in extracts obtained by shaking group A streptococcus with glass beads Treatment of Extract Antisera Employed Time of Shaking in Water (Hours) Carbonate Solubles Acetate Solubles Residue No acid extraction Acid extraction Group A Type 6 Group A Type 6 All materials were tested at a level of 1 mg (dry weight)/ml two extracts, which contained about 60 per cent of the total mateiial solubilized, possessed only 6-11 per cent of dialyzable material. An increase then occurred which reached a level at about 20 per cent for the 2-, 3-, and 4-hr extracts. The total quantity of dialyzable material in the extracts was equal to 10.8 per cent of the material solubilized in the complete experiment. After extraction in water for a total of 5 hr, the cellular residue was extracted by shaking for 1 hr each in 0.15 M NaHCO3 and 0.15 M Na acetate. The quantity of material solubilized in these two additional steps amounted to only 2.3 per cent of that extracted in water. The quantity of dialyzable materials in these two extracts was not determined. Solubilization of rhamnose. Determinations for the cell wall component, rhamnose, were made on all the extracts. It can be seen in column 5 of table 1 that small quantities were present in each case. It is apparent also that after the first two extracts were removed, the remaining extracts contained approximately the same quantity of rhamnose. The curve illustrating the rhamnose present in the soluble extracts is shown in the lower part of figure 1. The linear release of rhamnose following hr of extraction is evident. Column 6 of table 1 shows that the concentration of rhamnose in each aqueous extract increased from 0.3 per cent (0.25-hr extract) to 5.1 per cent in the 4-hr extract. After the fifth hr the concentration of rhamnose in the extract had increased to only 6.1 per cent. Hence, it is likely that continued shaking would not have significantly increased the concentiation of rhamnose in the extracts obtained. The 0.25-hr extract, although it showed the lowest concentration of rhamnose, contained the laigest quantity of that carbohy-drate (1.53 mg). Bicarbonate extracted a fraction containing 5.4 per cent rhaminose, and acetate extracted a fraction containing 7.1 per cent. The total weight of extract, however, in the latter case was only 1 mg. The rhamnose present in all the soluble fractions amounted to 14.3 per cent of the carbohydrate in the original cells. The bulk of the rhamnose remained in the residue (81.4 per cent), resulting in a rhamnose-rich material of 12.6 per cent carbohydrate. Protein content of the extracts. Column 8 of table 1 lists the protein values obtained on the extracts. The values, although not absolute, do indicate that the protein content of all the aqueous extracts was approximately the same. The bicarbonate extract is, however, somewhat higher. In the case of the whole cells and the residue, a preliminaryr mild hydrolysis in 2 N HCI was cariied out before the protein detertmination. The values indicate the protein content of the residue to be lower than that of the original cells. Antigens in the extracts. All the soluble fractions were tested for (1) the group-specific carbohydrate antigen andl (2) the type-specific protein antigen. The antisera were obtained fronm the U. S. Public Health Service2 and were labeled as "moderate" in titer. Table 2 presents the results obtained. It is evident that all the soluble extracts obtained by shakinig the cells in water, bicarbonate, and acetate, contained antigenic material which reacted with the specific, adsorbed homologous antisera. Precipitin tests w-ere made on the soluble fractions both with and without the acid digestion procedure of Swift et al. (1943). The results were similar in both cases. It is evident 2 Obtained through the courtesy of Dr. Elaine Updvke, Communicable Disease Center, U. S. Public Health Service, Chamblee, Georgia.

4 30 ;.:.,,' '... e '',.'. :..,. ;;: : :. :. *.: *.: ::..:.:. :... ::.. _r..y.... :.:...: :. SLADE AND VETTER A. --JV _t _Yh _S -Wk: KB.w. _F :...: :.: *: xt.. < r ; :.i :..... :. : ; 8.:t:. * i.: O. [VOI.. 72 ::.. Figutre 2. Cell residue after 15 min of shaking; X 11,503. Note presence of broken and unbroken cells. Shadowed at 110 with chromium. that the shaking method of solubilization of the cell did not greatly alter the stability of the antigens to digestion with 0.2 N HCl at 100 C. It can be seen in table 2 that the quantity of precipitin whichl formed was much less in those extracts obtained in the early stages of shaking than in those extracts obtained in the later stages. The soluble fraction obtained at the fifth hour gave the strongest precipitin reaction to both antisera of all the extracts obtained by shaking in water. The soluble fiactions obtained by shaking in bicarbonate an(l acetate also contained the groupspecific and the type-specific antigens. The reaction of the latter antigen with its homologous antibody was (onsiderably stronger in the carbonate extract than in the acetate extract. Acid extracts of the original cells and the insoluble residue were made according to the method of Swift et al. (1943). Table 2 shows that a positive reaction was obtained to both antigens in each case. Various extracts were also tested with types 3 and 17 antisera. No precipitin formed in any case. Observations with the electron microscope. Figure 2 shows several groups of cells after shaking for 15 min. It is evident that not all cells exhibit marked microscopic changes; however, only 6 per cent remain viable at this time (table 1). On the other hand, some cells have lost a part of their contents. In contrast to sonic energy (9-kc instrument), few "clean" cell walls have been seen at this stage. If shaking is continued it has been found that completely emptied cells are seen at about 1 hr; at 1 hr the cell wall material of these cells is seen to break up into pieces of varying size (figure 3). After 2 hr of shaking, further disintegration has taken place with a complete loss of the original structure of the cell (figure 4). Attention is called to the granular material in figures 3 and 4. Similar granules were noted in previous studies with sonic oscillation (Slade and Vetter, 1956).

5 19561 STUDIES ON STREPTOCOCCUS II 31 Figure 3. Cell residue after 1 hr of shaking; X 28,000. Note granular material. Shadowed at 11 with chromium. Figure 4. Cell residues after 2 hr of shaking; X 15,200. Note complete loss of cell structure. Shadowed at 110 with chromiumn.

32 SLADE AND VETTER [VO)L. 72 DISCUSSION It has been shown that, under the conditions used, the shaking procedure was highly effective in reducing the viable count to a low level within 15 min.

6 32 SLADE AND VETTER [VO)L. 72 DISCUSSION It has been shown that, under the conditions used, the shaking procedure was highly effective in reducing the viable count to a low level within 15 min. A much longer killing time (90 min) was noted by King and Alexander (1948) with Staphylococcus aureus when "ballotini" of twice the diameter and a cell suspension 50 times more dilute were used. The solubilization of one-half the total dry weight of cells in the first 15 min of shaking (table 1) coincided with the sharp drop in viable cells (96 per cent) which occurred during this period. On the other hand, Slade and Vetter (1956) have shown with the same organism and a suspension of equal density, the solubilization of 30 per cent of the cell weight by sonic oscillation coincided with a decrease of only 46 per cent in the number of viable cells. The faster rate of death in the Mlickle process can be attributed, in large measure, to the glass beads. The "pinching" of a cell between two beads would result in rupture of the cell wall and death of the cell. It is doubtful that sterility can be achieved by the shaking process within a reasonable period of time. After 3 hr living cells were still present. It seems likely that a few cells are given "protection" by debris from the cell wall, granular material, etc. The microscopic observations indicate that such conditions did exist in the present experiments. King and Alexander (1948) believe that a few resistant cells occur in each culture. The latter workers show that "resistance" was not a genetic property in S. aureus. The C-carbohydrate of group A streptococci has been shown by Schmidt (1952) and McCarty (1952) to consist, in part, of rhamnose and glucosamine. Two-thirds of the weight of the cell walls prepared by McCarty were found to be rhamnose. The immunological and chemical results presented here demonstrate that a part of the group-specific C-carbohydrate has been solubilized. The steady increase in the concentration of rhamnose in each extract indicates also that the pentose is a part of the less soluble components of the cell. A marked difference between the oscillation and shaking methods of solubilizing the streptococcus is indicated by the rhamnose content of the soluble extracts. The present work shows that 15 per cent of the total rhamnose recovered was in a soluble form whereas Slade and Vetter (1956) found that sonic oscillation solubilized 85 per cent of the carbohydrate. Thus rhamnose accounts for a part of the approximately 20 per cent additional cell material solubilized by the sonic method as compared to the shaking procedure. The precipitin tests presented here show that extracts prepared by both methods contain the group-specific and type-specific antigens. Conroy and Updyke (1954) found that extracts prepared in a paint shaker with glow beads gave positive precipitin tests for the group-specific antigen. No information was given as to the presence of the type-specific antigen in the same extracts. Little indication is obtained by precipitin tests as to the quantity of antigenic material present. The rhamnose values (table 1) strongly indicate, however, that the sonic procedure has solubilized several times as much C-carbohydrate as the shaking procedure. Thus it is likely that the M-protein antigen, also considered to be near the surface of the cell (Lancefield, 1954), will be partly solubilized. For these reasons extracts prepared by sonic oscillation are to be preferred as starting material for the fractionation and purification of M-antigen. The selection of the proper duration of oscillation and a suitable solvent have been shown to aid greatly in the concentration of certain cell components in a single extract (Hess and Slade, 1956). The disintegration of streptococci by shaking with "ballotini" beads can be characterized by a phase of rapid release of the cytoplasmic components. These components are readily soluble and are in part dialyzable. During this period (½1 hr), 99 per cent of the cells lost their viability. Following this, a phase of slow solubilization occurs which results in part in the release of components which are a part of, or associated with, the cell wall. These components are rhamnose, C-carbohydrate, and M-protein. ACKNOWLEDGMENT The authors wish to express their appreciation to Dr. J. P. Marbarger, University of Illinois School of Medicine, for having the electron microscope work done at that institution, and to Miss Elizabeth Bush for preparing the mounts and taking the pictures. SUMMARY The disintegration and solubilization of Streptococcus pyogenes (Streptococcus haemolyticus) type

7 1956] STUDIES ON STREPTOCOCCUS II 33 6 stain S43 by shaking with tiny glass spheres has been studied. All cellular components not sedimented after centrifugation for / hr at 20,000 X G were considered as soluble. Fifty-eight per cent of the dry weight of the cell was solubilized in water in 1 hr. The viable count during this period was reduced to less than 1 per cent. The maximum quantity of cell material solubilized (68 per cent) was reached in approximately 3 hr. Bicarbonate extracted an additional 1.5 per cent; acetate was negligible. The cell wall constituent rhamnose was found in all soluble fractions. The concentration of rhamnose in each fraction increased gradually until a concentration of 6 per cent was present in the fraction obtained between the 4th and 5th hr. The largest quantity of rhamnose was solubilized in the first 15 min of shaking. Eighty-one per cent of the rhamnose remained in an insoluble state in the final residue. Positive precipitin tests for the group-specific C-carbohydrate and type-specific M-protein were obtained on all the extracts. The strongest reactions were obtained on fractions released in the later stages of shaking. Electron microscopic observations of disintegrated cells at various stages of shaking are presented. Extensive destruction of cell structures occurred after several hours. Granular particles of about 100 m,u diameter are seen upon rupture of the cell. The disintegration and solubilization of streptococci by shaking with "ballotini" can be divided into 2 phases: (1) rupture of the cell wall and release of cytoplasmic constituents, and (2) partial solubilization of structures assoeiated with or a part of the cell wall. Differences between the sonic oscillation and shaking methods of solubilizing streptococci are discussed. REFERENCES BARKULIS, S. S. AND EKSTEDT, R. D The composition and antigenic properties of the cell walls of group A hemolytic streptococci. Bacteriol. Proc., 1955, p. 33. CONROY, E. AND UPDYKE, E. L The use of a fraction of mechanically disrupted cells for production of group A streptococcus typing antisera. Science, 119, HESS, E. L. AND SLADE, H. D An electrophoretic examination of cell-free extracts from various serological types of group A hemolytic streptococci. Biochim. et Biophys. Acta, 16, HESS, E. L. AND SLADE, H. D Effect of time of exposure to sonic energy on the composition of cell-free extracts from a type 6 strain of Streptococcus pyogenes. Arch. Biochem. and Biophys., acceptedfor publication. KING, H. K. AND ALEXANDER, H The mechanical destruction of bacteria. J. Gen. Microbiol., 2, LANCEFIELD, R. C Streptococcal Infections, p. 6, The Columbia University Press, New York. MCCARTY, M The lysis of group A hemolytic streptococci by extracellular enzymes of Streptomyces albus II. Nature of the cellular substrate attacked by the lytic enzymes. J. Exptl. Med., 96, SALTON, M. R. J Studies of the bacterial cell wall IV. The composition of the cell walls of some gram-positive and gram-negative bacteria. Biochim. et Biophys. Acta, 10, SCHMIDT, W. C Group A streptococcus polysaccharide: Studies on its preparation, chemical composition, and cellular localization after intravenous injection into mice. J. Exptl. Med., 95, SLADE, H. D. AND SLAMP, W. C Sonic oscillation as an aid in the counting of group A streptococci by the pour-plate method. J. Bacteriol., 71, SLADE, H. D. AND VETTER, J. K Studies on Streptococcus pyogenes I. Observations on the microscopical and biological aspects of the disintegration and solubilization of a type 6 strain by sonic oscillation. J. Bacteriol., 71, SWIFT, H. F., WILSON, A. T., AND LANCEFIELD, R. C Typing group A hemolytic streptococci by M precipitin reactions in capillary pipettes. J. Exptl. Med., 78,

TYPING OF GROUP A STREPTOCOCCI BY IMMUNOFLUORESCENCE

JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp. 1377-1382 June, 1964 Copyright 1964 by the Anmerican Society for Microbiology Printed in U.S.A. TYPING OF GROUP A STREPTOCOCCI BY IMMUNOFLUORESCENCE I. PREPARATION