CHEMICALLY DEFINED MEDIUM FOR GROWTH OF

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 88, No. 1, p July, 1964 Copyright 1964 American Society for Microbiology Printed in U.S.A. CHEMICALLY DEFINED MEDIUM FOR GROWTH OF STREPTOCOCCUS PYOGENES M. N. MICKELSON National Animal Disease Laboratory, U.S. Department of Agriculture, Ames, Iowa Agricultural Research Service, Received for publication 6 March 1964 ABSTRACT MICKELSON, M. N. (National Animal Disease Laboratory, Ames, Iowa). Chemically defined medium for growth of Streptococcus pyogenes. J. Bacteriol. 88: Three strains of group A hemolytic streptococci representing three serological types were serially subcultured for an extended period (100 or more transfers) in a peptide-free and protein-free culture medium. An amino acid assay medium, modified by addition of small amounts of glutamine, ammonium acetate, and 0.1 M phosphate (ph 7), was used. In this medium, high concentrations of glutamic acid or glutamine were required, and biotin was stimulatory to growth. Biotin could be partially replaced with NaHCO3. Maximal growth was obtained with NaHCO3 when biotin was present, and aspartic acid and asparagine were omitted from the medium. In 24 to 48 hr of incubation at 37 C, luxuriant growth was obtained with complete removal and quantitative fermentation of 1% glucose to lactic acid. Optical densities of cultures were equivalent to those obtained with the best infusion media. The group A streptococci are generally regarded as being fastidious in their nutritional requirements. In addition to amino acids, B vitamins, purines, pyrimidines, and inorganic ions, they have usually been reported to need whole blood, serum, or other tissue fluids for growth. No fully defined medium has been found which would permit luxuriant growth of group A hemolytic streptococci upon continuous culture. The availability of such a medium would be of great value in studies on the nature and synthesis of cellular components and the pathogenicity of these microorganisms. Various workers have reported that proteins or peptides are required for growth or to stimulate growth of the group A hemolytic streptococci in an otherwise chemically defined medium. Woolley (1941) described a water-soluble nondialyzable material from liver; Sprince and Woolley (1945), strepogenin; Slade, Knox, and Slamp (1951), a nondialyzable peptide from pancreatic digest of casein; and Slade and Slamp (1955), heated ovalbumin. Ogburn, Harris, and Harris (1958) studied the production of proteinase by two cultures of group A streptococci in a chemically defined medium. They added small quantities of casein hydrolysate to the medium, and, by gradually reducing the amount in serial cultures until it was eliminated, they were able to obtain growth with optical densities as high as 0.24 at 540 to 590 mp. They did not report on continued cultivation of the organisms in the chemically defined medium. It is the purpose of this report to show that group A streptococci will grow luxuriantly in a protein- and peptide-free medium. MATERIALS AND METHODS Three strains of Streptococcus pyogenes (Richards, N19, and S43) were obtained from the collection of H. D. Slade. They belonged to serological types 3, 19, and 6, respectively (Slade and Slamp, 1955). All produced,b-hemolysis on bovine blood-agar. The stock cultures were transferred biweekly in serum broth and stored at 2 C. The serum broth (ph 7.0) was a beef infusion broth to which were added sterile 1% horse serum, 10 g of peptone (Difco), and 5 g of NaCl per liter. The medium for preparing the inoculum for growth studies was Brain Heart Infusion (BBL) broth to which were added 0.1 M glucose and 0.1 M phosphate buffer (ph 7.0). The buffer was sterilized separately and added after the medium was cooled. Portions (1 ml) of an 18- to 20-hr culture in Brain Heart Infusion broth were frozen in small culture tubes and held at -60 C for use as needed. To prepare the inoculum for growth 158

2 VOL. 88 X 1964 NUTRITION OF S. PYOGENES 159 studies, 0.8 ml of the thawed frozen culture was transferred to 8 ml of Brain Heart Infusion broth, and after 18 to 20 hr of incubation the cells were sedimented by centrifugation, washed once with an equal volume of sterile distilled water, and resuspended to give an optical density of 0.40 (#55 filter) in a Klett-Summerson colorimeter. A 0.1-ml amount of this suspension was used to inoculate 5 ml of the chemically defined culture medium. For a number of transfers, or until the cultures began to grow well, a 10% (v/v) inoculum was transferred; thereafter, a 1% inoculum was usually sufficient. All cultures were incubated at 37 C. The basic synthetic medium was that of Williams (1955), modified by the addition of 20,ug of L-glutamine and 40,ug of ammonium acetate per ml. The ph of the medium was adjusted to 7, and the buffer content of the medium was increased by adding 0.1 M phosphate (ph 7.0). A 1.0 M potassium phosphate solution was autoclaved separately, and 0.5 ml was added to each 5-ml final volume of the medium after it had been sterilized and cooled. The complete medium, exclusive of the phosphate, was autoclaved for 15 min. Darkening of the medium by improper sterilization impaired growth of the three strains of S. pyogenes. This medium is referred to as the modified Williams medium. Spray-dried whole egg white (Swift and Co., Chicago, Ill.) was used in some of the media. This sample had a good reducing value for 2, 6-dichlorophenolindophenol after heating for 10 min at 100 C (Slade and Slamp, 1955). Growth of the cultures was measured by titration of the acid formed with 0.05 N NaOH by use of bromothymol blue indicator or by optical density measurements in a Klett-Summerson colorimeter with a S55 filter (range 520 to 600 my). Glucose and lactic acid were determined by the anthrone procedure and Barker and Summerson procedure, respectively, as described by Umbreit, Burris, and Stauffer (1957). RESULTS Growth in the basic synthetic medium. Growth response of three strains of group A streptococci to heated ovalbumin was greater in the unmodified medium of Williams (1955) than in that of Slade and Slamp (1955) (Fig. 1). Appreciable growth and acid formation occurred in Z OVALBUMIN Mg/Ml FIG. 1. Growth response of Streptococcus pyogenes Richards to heated ovalbumin in two di grent chemically defined media. Cultures were titrated after 48 hr of incubation at 37 C. the unmodified Williams medium without egg albumin; this growth was transferable. After five to ten daily transfers, good growth of each of the three strains was obtained in the proteinand peptide-free medium. Increasing the buffer capacity of the medium with 0.1 M phosphate (ph 7.0) resulted in luxuriant growth and the quantitative fermentation of 1 NO glucose to lactic acid with a final ph of 5.1 to 5.2 (Table 1). A further increase in phosphate concentration to TABLE, 1. Growth and fermentation in the modified Williams medium with and without 0.1 phosphate of three strains of Streptococcus pyogenes* GlucseCarbon Medium Culture densityt Optical mfer- as lactic mented uoerecovered acid With phosphate Richards N S Without phos- Richards phate N S * Glucose, 1%. t Optical density measured after 48 hr of incubation at 37 C.

3 160 MICKELSON J. BACTERIOL. TABLE 2. Effect of size of inoculum on growth of Streptococcus pyogenes in the modified Williams medium Inoculum (1%, v/v) Optical density after Dilution Optical 24 hr 48 hr density : : : M resulted in some growth inhibition. In the original Williams medium, the ph dropped to 4.8 to 4.9 with only one-third of the glucose consumed. Though a rather large inoculum, 10% by volume, was used during the early subcultures, a 1% inoculum was adequate after five to ten successive daily transfers in the synthetic medium. The results of subculturing with various quantities of inoculum are shown in Table 2. Amino acid requirements for growth in Slade and Slamp's and Wtilliams' media. The three strains of S. pyogenes did not grow in Slade and Slamp's chemically defined medium without ovalbumin, whereas moderate growth occurred in the original Williams medium. When the latter was modified, as indicated, good growth resulted (Table 1). The composition of the two media is compared in Table 3. The three strains have been subcultured more than 100 times in the modified Williams medium. The amino acids which were present in the Williams medium but not in Slade and Slamp's medium were glutamic acid, aspartic acid, asparagine, norleucine, hydroxyproline, and cysteine. Tests to determine which of these amino acids were critical for growth were done with the three strains of S. pyogenes which had been transferred 60 times in the modified Williams medium. The amino acids in the above list were omitted from the modified Williams medium one at a time, except for the simultaneous omission of cystine and cysteine. The effect of their omission on the growth of three strains of S. pyogenes is shown in Table 4. When the two sulfur-containing amino acids were not present in the medium, growth did not occur. The presence of either cystine or cysteine corrected the deficiency. Only with the Richards strain was there evidence that the presence of both was beneficial. Glutamic acid was essential for the growth of all three strains of S. pyogenes. Aspartic acid, asparagine, norleucine, or hydroxyproline did not affect growth under these conditions. In the original Williams medium, glutamic acid was present in a concentration of 0.5 mg/ml, whereas Slade and Slamp's medium contained no glutamic acid but did contain 0.1 mg/ml of glutamine. When L-glutamic acid or -glutamine were used in equivalent concentrations (0.5 mg/ml), the growth of the three strains of S. pyogenes was equal. The glutamine, in this case, was sterilized by filtration. In addition to the six amino acids mentioned, there were four B vitamins in the Williams medium which were not present in Slade and Slamp's medium. The growth response to these amino acids and vitamins by S. pyogenes Richards is shown in Fig. 2. The other two strains responded similarly. The amino acid and vitamin mixture of Slade and Slamp's medium was used. The concentrations of amino acids, salts, vitamins, and buffers were the same as in the modified Williams medium. Each culture was transferred six times in each medium with a 1 % inoculum. The first tubes were inoculated with 1 % inoculum from a 24-hr culture in modified Williams medium. The growth of S. pyogenes Richards after 48 hr of incubation in each of the four media is shown in Fig. 2. The growth of all strains was similar. The addition of glutamic acid to the basal medium of Slade and Slamp (1955) permitted minimal growth, after dilution of the nutrients carried over in the first subcultures. Further supplementation with aspartic acid, asparagine, hydroxyproline, and norleucine caused no appreciable improvement in the growth-promoting properties of the medium. Addition of biotin, folic acid, p-aminobenzoic acid, and pyridoxamine caused nearly a twofold increase in growth over the basal medium and about 70% of the maximal growth that was obtained when both the extra vitamins and amino acids were added to the basal medium. The four amino acids stimulated growth only when the four "extra" vitamins were present in the medium (Fig. 2, curve 4). Effect of biotin, folic acid, pyridoxamine, and p-aminobenzoic acid. Experiments were performed to determine which of the vitamins was responsible for the stimulation of growth in the presence

4 VOL. 88, 1964 NUTRITION OF S. PYOGENES 161 TABLE 3. Composition of the Williams medium, the modified Williams medium, and the medium of Slade and Slamp* Williams Slade and Williams Slade and Component mediumt Slamp Component mediumt Slamp meu, mediuml e,m mediumt Amino acids B vitamins L-Alanine Folic acid L-Arginine... O Biotin L-Asparagine p-aminobenzoic acid L-Aspartic acid Thiamine L-Cysteine HCl Riboflavine L-Cystine Pyridoxal HCI L-Glutamic acid Pyridoxamine Glycine Ca-pentothenate L-Histidine HC Niacin L-Hydroxyproline L-Isoleucine Salts L-Leucine K2HP L-Lysine HCl KH2PO L-Methionine Na2HPO L-Norleucine gmgso7h2o L-Phenylalanine FeSO4-7H L-Proline MnSO4-4H L-Serine ZnSO4*7H L-Threonine... O NaCi L-Tryptophan Na(C2H302)2-3H L-Tyrosine... O Glucose L-Valine L-Glutamine ph Adenine Guanine Uracil * Amounts are expressed as final concentration in mg/ml. t Williams (1955). Modified Williams medium contained, in addition to the listed components, L- glutamine, 0.05; NH4C2H302, 0.10; and 0.1 M phosphate (ph 7.0). t Slade and Slamp (1955). of aspartic acid, asparagine, norleucine, and hydroxyproline. The basic medium was changed by elimination of biotin, folic acid, pyridoxamine, and p-aminobenzoic acid singly. A 1% inoculum from a 24-hr culture in the modified Williams medium was used to inoculate the test medium, and six daily transfers were made. Growth of each strain was measured after 48 hr of incubation at 37 C. Elimination of biotin caused about a 50% decrease in the amount of growth with each of the strains of S. pyogenes (Table 5). No decrease in growth was noted when folic acid, pyridoxamine, or p-aminobenzoic acid was eliminated from the medium. The presently known reactions of biotin enzymes are all concerned with carbon dioxide TABLE 4. Effect of certain amino acids on the growth of three strains of Streptococcus pyogenes in a chemically defined medium Amino acid or amide omitted Optical density Richards N19 S43 Glutamic acid Cystine and cysteine Asparagine Aspartic acid Norleucine Cystine Cysteine Hydroxyproline Complete medium

5 162 MICKELSON J. BACTERIOL. z -J 4 0 I SERIAL SUBCULTURE FIG. 2. Effect of certain amino acids and B vitamins on growth of Streptococcus pyogenes Richards in a chemically defined medium. Serial subculture: (1) Slade and Slamp's amino acid mixture plus glutamic acid; (2) medium 1 + aspartic acid, asparagine, hydroxyproline, norleucine; (3) medium 1 + biotin, folic acid, p-aminobenzoic acid, pyridoxamine; (4) medium 1 + supplements of 2 and S. Turbidities were read in a Klett-Summerson colorimeter with a #5 filter after 72 hr of incubation. fixation and fatty acid biosynthesis (Ochoa and Kaziro, 1961). To determine whether NaHCO3 would replace all or part of the biotin stimulation of the growth of S. pyogenes, 1 mg/ml of NaHCO3 was added to the modified Williams medium from which biotin was omitted. Growth was compared, during six transfers, with TABLE 5. _ 4 that obtained in the complete medium and in complete medium with biotin omitted. The omission of biotin reduced growth of S. pyogenes Richards more than one-half (Table 6). Growth in the biotin-free medium was increased twofold by addition of NaHCO3; however, growth was not restored to the level obtained in the complete medium. Omission of aspartic acid, asparagine, and biotin resulted in the same effect on growth as did biotin omission. When NaHCO3 was added to this medium, growth was erratic on successive transfers and no better than when biotin alone was omitted. However, when NaHCO3 was added to media containing biotin but not aspartic acid and asparagine, growth after four transfers was maximal or equal to that in the modified Williams medium. DISCUSSION The data show that it is possible to obtain luxuriant growth of the three strains of group A hemolytic streptococci, through repeated subculture, in a chemically defined medium. Though it was necessary to use a large inoculum for the first five to ten subcultures, a 1% inoculum was sufficient when the organisms became established in the medium. The ability of the cultures to grow in the chemically defined medium may be the result of adaptation and selection from the original culture of those cells which had the highest biochemical capabilities. When the cells were able to synthesize their growth essentials at an adequate rate from the simple chemicals present, luxuriant growth ensued. Cultures transferred from the chemically defined medium to one supplemented with Casitone, then back to the Effect of biotin, pyridoxamine, p-aminobenzoic acid, asparagine, and aspartic acid on the growth of Streptococcus pyogenes Richards* Complete medium minus Serial subculture Complete medium Biotin, p-amino asparagine, and Biotin Folic acid Pyridoxamine benzoic aspartic acid acid * Results expresed as optical density measured after 48 hr of incubation at 37 C.

6 VOL. 88, 1964 NUTRITION OF S. PYOGENES 163 chemically defined medium again, required a short period of adaptation (two to three subcultures) before they would grow luxuriantly in the chemically defined medium. The group A streptococci are more sensitive to acid than are certain other lactic acid bacteria, and it was necessary to increase the buffer capacity of the medium. Glutamic acid or glutamine in a high concentration was required for growth, and biotin was stimulatory under our conditions. The need of glutamine for growth of the hemolytic streptococci was established by Mclllwain (1946). He also demonstrated the deamination of glutamine by the hemolytic streptococci during glucolysis and that glutamic acid did not replace glutamine. Glutamic acid was as satisfactory as glutamine for growth under our conditions with the strains studied. Since we were interested in a maximal rather than a minimal medium, small amounts of glutamine and ammonium ion were added. Although biotin was present in the medium used by Slade and Knox (1950) for the growth of S. pyogenes Richards, they reported only a small improvement in growth due to biotin, folic acid, and p-aminobenzoic acid. Biotin alone of these three vitamins produced a pronounced stimulation of growth. That NaHCO3 partially replaced biotin as a growth stimulant indicates that biotin was probably involved in CO2 fixation by these organisms. In the presence of biotin and NaHCO3, and in the absence of asparagine and aspartic acid, growth was maximal after several transfers; in the presence of asparagine and asparatic acid, growth was inferior. This suggests, as shown with Lactobacillus arabinosus by Lardy, Potter, and Burris (1949), that aspartic acid and asparagine interfere with CO2 utilization. A major change in cells grown in the chemically defined medium was their loss of M protein (Mickelson and Slade, 1964). Fox (1961) noted that peptides were necessary for M protein synthesis by cell suspensions of S. pyogenes in an amino acid mixture. The effect on synthesis of other proteins associated with S. pyogenes is not known. A chemically defined growth medium may be of value in studying the synthesis of proteins associated with the group A streptococci. Although more strains of S. pyogenes need to be examined for their ability to grow in this medium, the results of these studies indicate that the group A streptococci probably have TABLE 6. Effect of NaHCO3 on growth of Streptococcus pyogenes Richards in a biotin-free synthetic medium* Serial subculture Mediumt * Results expressed as optical density measured after 48 hr of incubation at 37 C. t Composition of media: (1) modified Williams medium; (2) modified Williams medium, no biotin; (3) modified Williams medium, no aspartic acid, or asparagine; (4) modified Williams medium, no biotin, aspartic acid, or asparagine; (5) medium 2 plus 1 mg/ml of NaHCO3; (6) medium 3 plus 1 mg/ml of NaHCO3 ; (7) medium 4 plus 1 mg/ml of NaHCO3. nutritional needs similar to those of other lactic acid bacteria. ACKNOWLEDGMENT Grateful acknowledgment is expressed to H. D. Slade for supplying the cultures used in this work. ADDENDUM Four additional strains of S. pyogenes representing serological types 1, 5, 25, and 30 have recently been serially cultured for a period of 6 weeks in the chemically defined medium described in this report. LITERATURE CITED Fox, E. N Peptide requirements for the synthesis of streptococcal proteins. J. Biol. Chem. 236: LARDY, H. A., R. L. POTTER, AND R. H. BURRIS Metabolic functions of biotin. I. The role of biotin in NaHCO3 utilization by Lactobacillus arabinosus studied with C14. J. Biol. Chem. 179: MCILLWAIN, H Ammonia formation from glutamine by hemolytic streptococci; its reciprocal relationship with glycolysis. Biochem. J. 40: MICKELSON, M. N., AND H. D. SLADE Absence of type-specific M antigen from group A

7 164 MICKELSON J. BACTERIOL. streptococci grown in a chemically defined medium. J. Bacteriol. 87:1251. OCHOA, S., AND Y. KAZIRO Biotin enzymes. Federation Proc. 20: OGBURN, C. A., T. N. HARRIS, AND S. HARRIS Extracellular antigens in steady-state cultures of the hemolytic streptococcus: production of proteinase at low ph. J. Bacteriol. 76: SLADE, H. D., AND G. A. KNOX Nutrition and the role of reducing agents in the formation of streptolysin 0 by a group A hemolytic streptococcus. J. Bacteriol. 60: SLADE, H. D., G. KNOX, AND W. C. SLAMP The amino acid nutrition of group A hemolytic streptococci with reference to the effect of glutathione on the cysteine requirement. J. Bacteriol. 62: SLADE, H. D., AND W. C. SLAMP The requirement of ovalbumin for the growth of group A hemolytic streptococcus in a synthetic medium. J. Exptl. Med. 102: SPRINCE, H., AND D. W. WOOLLEY The occurrence of the growth factor strepogenin in purified proteins. J. Am. Chem. Soc. 67: UMBREIT, W. W., R. H. BURRIS, AND J. F. STAUF- FER Manometric techniques, 3rd ed. Burgess Publishing Co., Minneapolis. WILLIAMS, H "Essential" amino acid content of animal feeds. Cornell Univ. Agri. Expt. Sta. Mem WOOLLEY, D. W A new growth factor required by certain hemolytic streptococci. J. Exptl. Med. 73: Downloaded from on April 10, 2018 by guest