Staphylococcus aureus S-6'

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1 APPLIED MICROBIOLOGY, July 1967, p Vol. 15, No American Society for Microbiology Printed in U.S.A Nutritional Requirements of Staphylococcus aureus S-6' ROBERT A. MAH, DANIEL Y. C. FUNG, AND STEPHEN A. MORSE Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, North Carolina Received for publication 20 February 1967 A synthetic medium was devised for growth of Staphylococcus aureus strain S-6. The growth yield in synthetic medium was compared to that in complex medium containing an equivalent amount of protein hydrolysate. Enterotoxin B formation in the two media was also compared. The defined medium was composed of inorganic salts, 11 amino acids (glycine, valine, leucine, threonine, phenylalanine, tyrosine, cysteine, methionine, proline, arginine, and histidine), and three vitamins (thiamine, nicotinic acid. and biotin). Biotin was a growth factor requirement of S-6 when glutamic acid but not glucose was used as a carbon source. The quantity of enterotoxin B produced in the defined medium was about one-seventh of that produced in complex medium, even though the growth yields were similar. Staphylococcus aureus strain S-6 has been thett" experiments, but was later modified as described in subject of many investigations concerned with the the text. All salts were chemically pure and conproduction of maximal yields of enterotoxin (3, 4, formed to either U.S.P. or A.C.S. standards. 18). Optimal conditions for enterotoxin forma- Vitamins. Identification by Gladstone (9) of the tion were determined in complex media such as acid unknown and thiamine growth factors led to the of first Fildes completely (5) as nicotinic defined casein caenhdoye hydrolysate or Brain Heart Infusion. rti. AAs a medium, consisting of 16 amino acids, vitamins, and result of such studies, sufficient quantities Of inorganic salts. Thiamine (as thiamine hydrochloride, enterotoxin B were purified (2, 15), and its amino Mallinckrodt Chemical Works, New York, N.Y.), acid composition was investigated in detail (1, 16). nicotinic acid (Merck and Co., Rahway, N.J.), and However, little is known about the actual factors later biotin (Calbiochem, Los Angeles, Calif.) were responsible for toxigenesis in these or other media. routinely incorporated into the defined medium. The Aside from Surgalla's work (17), defined media concentrations were: thiamine, Ag/ml; nicowere not used for studying factors affecting en- tinic acid, 1.2,ug/ml; and biotin, g/ml. terotoxin formation S. aureusalso, animal Inoculwn. S. aureus S-6 was obtained from M. S. terotn fnin S. abergdoll. This strain produces large amounts of assays were inadequate for precise quantitative enterotoxin B and small amounts of enterotoxin A. measurements of toxin. Although other synthetic Samples (3 ml) of a stationary-phase culture grown media have been employed for growing S. aureus, aerobically on 2% protein hydrolysate (Mead Johnindividual strain differences in amino acid require- son International, Evansville, Ind.) broth plus vitamins ments exist (9). The minimal amino acid and at 37 C were aseptically transferred to sterile culture growth requirements have not been reported for tubes, quick-frozen in a dry ice-acetone bath and kept S. aureus S-6. Development of a defined medium at - 15 C. The samples were thawed as needed, a 1% will facilitate evaluation of enterotoxigenic factors inoculum was introduced into protein hydrolysate bypermittinthe (PHP) broth, and the culture was incubated on a by permittig the controlled addition or deletion shaker at 37 C for 17 hr. It was then centrifuged and of possible toxigenic substrates. washed aseptically in sterile salts solution to remove soluble organic compounds, resuspended to the initial MATERIALS AND METHODS volume, and used as a 1% inoculum in the experi- Inorganic salts. Gladstone (9), Gale (6, 7), Surgalla mental vessels. Nephloflasks (300-ml; Bellco Glass (17), Ramsey and Padron (14), and others have re- Inc., Vineland, N.J.) containing a total of 50 ml of ported various salt solutions for culturing S. aureus. medium were the vessels of choice. The carbon source A comparison of the inorganic salts used by these was 1% glucose in all cases unless otherwise stated; it workers showed that Gale's mixture (6) supported was autoclaved and added separately. the best growth in media containing amino acids and Growth in defined media previously reported for vitamins. This solution was used in all the initial S. aureus was not evaluated in quantitative terms; relative degrees of turbidity were noted after daily Department publication no intervals, often up to 6 days (17). In the present in- 866

2 VOL. 15, 1967 NUTRITION OF S. A UREUS 867 vestigation, turbidity was measured by Klett-Summerson colorimetry (green filter at 540 m,u) after 24 hr of incubation at 37 C under aerobic conditions (shaken at 180 rev/min, Psycrotherm incubatorshaker, New Brunswick Scientific Co., Inc., New Brunswick, N.J.). On complex media, cells were in the maximal stationary phase after this length of incubation. Enterotoxin assay. Enterotoxin was assayed by the hemagglutination-inhibition method of Morse and Mah () and by the gel-diffusion method of Weirether et al. (19). RESULTS Amino acid requirements. Since PHP medium supported optimal growth of S-6 in the presence of nicotinic acid and thiamine, the initial composition of the defined medium was based on the constituent 18 amino acids present in PHP. These consisted of glycine, alanine, aspartic acid, valine, leucine, isoleucine, serine, threonine, phenylalanine, tyrosine, tryptophan, lysine, arginine, histidine, glutamic acid, cystine, methionine, and proline. All amino acids were obtained from commercial sources and were chromatographically pure. Each was added at a final concentration of 25 Ag/ml. From this mixture of amino acids, it was calculated that an equivalent amount of amino nitrogen was present in 0.08% PHP, and this concentration was used as an index of the maximal growth obtainable under the previously stated conditions. In the presence of glucose as a carbon source, growth in 0.08% PHP (270 Klett units) was similar to that in the defined medium with 18 amino acids (250 Klett units). To determine the essential amino acids for optimal growth of S-6, an arbitrary minimum yield of 200 Klett units in 24 hr was chosen. Media resulting in an extended lag phase may give rise to less than this arbitrary value even though the final cell yield may reach 200 Klett units. Such media would not be selected under these conditions. The following protocol was adopted for determining the amino acid requirement. Starting with the 18 amino acids in inorganic salts solution plus vitamins, each amino acid was singly deleted and the growth was recorded. Omission of some amino acids (aspartic, isoleucine, alanine, lysine, serine, tryptophan, methionine, and tyrosine) resulted in growth which satisfied the specified criteria (Table 1). Such amino acids were presumed unnecessary for optimal growth of S-6. This could be further tested by deletion of each of these nonessential amino acids in combination with the deletion of each of the 17 remaining amino acids. The resulting pair of amino acids which, upon omission, yielded acceptable growth could be tested further by the successive deletion of each of the remaining 16 amino acids. In the initial deletion experiments, any one of several amino acids could be omitted. However, Amino acid 1. Aspartic acid Isoleucine Alanine Lysine Tryptophan Glutamc acid. 7. Serine Methionine Histidine.... Tyrosine Threonine.... Leucine Glycine Phenylalanine Proline Arginine Valine Cysteine... None TABLE 1. I Growth in defined media 1, Amino acid combinations deleteda 1, 2,3 1,2,3,4 1,2, 3, 4, S6 1, 2, 3, 4, 5, , 2, 3, 1, 2, 3, 4,5,7 4,5,6,7 a The numbers correspond to the amino acids as listed on the left-hand margin. Growth is reported in Klett units after 24 hr of aerobic incubation at 37 C. Growth in the 18-amino acid control was 250 Klett units

3 868 MAH, FUNG, AND MORSE APPL. MICROBIOL. only one of the possibilities, usually the deletion yielding the highest growth, was tested. In the final experiments, deletion of an additional amino acid resulted in equivalent or better growth in only one or two cases. In such instances, both deletions were tested further. Aspartic acid was first omitted in addition to the successive omission of each of the remaining amino acids (Table 1, column 2). Equivalent or miproved growth resulted with the double omission of aspartic acid and any one of the following: isoleucine, lysine, glycine, serine, tyrosine, or tryptophan. Repetition of this procedure omitting the aspartic acid-isoleucine pair gave equivalent or improved growth with the additional deletion of several other amino acids (Table 1, column 3). Again eliminating the combination yielding the highest growth (aspartic acid, isoleucine, alanine; column 4), the remaining amino acids were reexamined. Finally, with omission of the combination, aspartic acid, isoleucine, alanine, lysine, and tryptophan, only glutamic acid, serine, and tyrosine deletions fulfilled the growth criteria (column 6). The combined deletion of aspartic acid, isoleucine, alanine, lysine, tryptophan, and glutamic acid permitted acceptable growth yields only for the serineless culture (column 7). The deletion of serine instead of glutamic acid with this same combination yielded similar results (column 8). Tyrosine was not tested further. The final amino acid mixture consisted of glycine, valine, leucine, threonine, phenylalanine, tyrosine, cysteine, methionine, proline, arginine, and histidine. Certain amino acids were essential for growth; their omission resulted in no growth at all (Table 1). A mixture composed of these apparently essential amino acids was not sufficient to satisfy the empirical growth requirements stated. Glutamic acid. Since S-6 grew well in PHP alone, it must use amino acids as a carbon source, because other organic compounds are not significant in this medium. Endogenous respiration of S. aureus can be attributed in part to the disappearance of glutamic acid from the free amino acid pool (11, 13). Furthermore, suspensions of S. aureus metabolize glutamic acid in the presence of either an endogenous (11) or exogenous (8, 11) energy source. This energy requirement is apparently necessary for transport of the amino acid across the cell membrane. Growth of S-6 was tested on glutamic acid (as monosodium glutamate, MSG) as the main carbon source in the defined medium. The yield was in the same range when grown on MSG (196 Klett units) or glucose (208 Klett units). Other amino acids were also tested as carbon sources in the defined medium. None of the following amino acids known to exhibit 02 uptake (11) was utilized: threonine, proline, glycine, alanine, arginine, cysteine, histidine, and serine. Each was tested by addition at a concentration of 0.2% and compared with the growth yield on 0.2% MSG. Proline gave the highest value (46 Klett units) compared with MSG (116 Klett units). Biotin. In early experiments, only thiamine and nicotinic acid were incorporated into the medium to satisfy the known vitamin requirements of S. aureus. Growth of S-6 in defined medium containing glucose as carbon source was virtually unaffected by the addition of yeast extract. However, MSG was not metabolized in defined medium in the presence of thiamine and nicotinic acid as the only added vitamins; with the addition of yeast extract, MSG was used as a carbon source. Biotin substituted for this growth factor requirement; cells incubated without biotin yielded ca. 47 Klett units. Addition of biotin to glucose-grown cells did not stimulate growth. Salts. The inorganic salts are reported as final concentration (per cent, w/v). The ammonium ion concentration was tested by deletion of the 0.4% (NH4)2HP04 in Gale's solution (6), replacement of the phosphate by 0.33% Na2HP04, and addition of 0.05% NH4Cl. The final concentrations of the other salts were: KH2PO4, 0.1%; MgSO4.7H20, 0.07%; NaCl, 0.1%; and FeSO4V 7H20, 0.001%; the final ph was 7.1. These modifications led to an increase in the growth yield when either glucose or glutamic acid served as carbon source. The results are shown in Table 2. This mixture of inorganic salts was adopted as the final combination. Enterotoxin formation. Growth in 0.5% PHP TABLE 2. Salt solution Comparison of salt solutions Growth (Klett units) Glutamic acid Glucose Gale (6) Modified TABLE 3. Enterotoxin formation in various media Growth units) Toxin Medium (Klett (j.ag/ml) 0.5% PHP % PHP + MSG % PHP + glucose Defined medium + glucose Defined medium + MSG

4 VOL. 15, 1967 NUTRITION OF S. AUREUS 869 without added glucose or MSG resulted in about seven times more toxin than in defined medium with glucose or MSG. This concentration of PHP supported a cell yield equivalent to the defined medium with added carbon source. Upon addition of glucose or MSG to the minimal PHP concentration (0.06%) necessary to support growth equivalent to the defined medium, toxigenesis was again very low (Table 3). DIScUSSION Determination of the 11 amino acids necessary for optimal growth of S-6 with glucose as a carbon source was based on the ability of cells to produce a minimum of 200 Klett units after incubation at 37 C for 24 hr under aerobic conditions. This turbidity corresponded to ca. 550 jug (dry weight) of cells per ml. Observation of positive growth under these conditions is more stringent than in previous reports. The final number and combination of amino acids might vary somewhat depending upon the order in which the deletions were tested. However, the present findings show that the initial single amino acid deletions yielding growth greater than 200 Klett units were almost all eventually deleted (tyrosine was the only exception). Thus, the sequence of deletions may not be important. Since S-6 grows on MSG, the cells either utilize endogenous energy reserves, at least for the initial transport of this amino acid, or the cells are permeable to MSG. The latter does not seem to be the case; preliminary manometric investigations show that cells grown on MSG do not oxidize it except in the presence of trace amounts of glucose. This contradiction has not yet been resolved. None of the 90 coagulase-positive strains studied by Gretler et al. () exhibited biotin dependency, although all 46 coagulase-negative strains studied showed complete (38 of 46) or partial (8 of 46) biotin dependency. The coagulase-positive S-6 exhibits a biotin requirement when grown on MSG but not glucose as a carbon source. This is not a general characteristic in other coagulasepositive strains of S. aureus so far investigated. The specific growth rate constants in 0.08 and 0.06% PHP with glucose as carbon source were 1.33 hr-' and 1.26 hr'-, respectively. In defined medium with glucose, the specific growth rate was hr-1. Thus, the medium is not as satisfactory as complex medium, but the specific growth rate is still quite rapid. In defined medium, all cultures were in the maximal stationary phase within hr of incubation when grown on glucose and 24 hr when grown on MSG. In complex PHPsupplemented medium, cultures were in the maximal stationary phase after hr, regardless of carbon source. ACKNOWLEDGMENTS We thank M. S. Bergdoll for the culture of S. aureus S-6 and for the samples of purified enterotoxin B and antiserum. In particular, we are grateful for the criticisms and suggestions of L. Regier and the able assistance of Lily Wyatt and Lena Meck. This investigation was supported by the U.S. Army Medical Research and Development Command, contract DA MD LITERATuRE CITED 1. BERGDOLL, M. S., F. S. CHu, I.-Y. HUANG, C. RowE, AND T. SHIH Staphylococcal enterotoxin B. III. The physicochemical properties and the N- and C-terminal amino acid sequences. Arch. Biochem. Biophys. 1: BERGDOLL, M. S., H. SUGIYAMA, AND G. M. DACK Staphylococcal enterotoxin. I. Purification. Arch. Biochem. Biophys. 85: CASMAN, E. P., AND R. W. BENNErr Culture medium for the production of staphylococcal enterotoxin A. J. Bacteriol. 86: FAvORITE, G. 0., AND W. McD. HAMMON The production of staphylococcus enterotoxin and alpha hemolysin in a simplified medium. J. Bacteriol. 41: FiLDES, P., G. M. RICHARDSON, B. C. J. G. KNIGHT, AN G. P. GLADSToNE Nutrient mixture suitable for the growth of Staphylococcus aureus. Brit. J. Exptl. Pathol. 17: GALE, E. F The arginine, omithine, and carbon dioxide requirements of streptococci (Lancefield Group D) and their relation to arginine dihydrolase activity. Brit. J. Exptl. Pathol. 26: GALE, E. F The assimilation of amino acids by bacteria. I. The passage of certain amino acids across the cell wall and their concentration in the internal environment of Streptococcusfaecalis. J. Gen. Microbiol. 1: GALE, E. F The assimilation of amino acids by Gram-positive bacteria and some actions of antibiotics thereon. Advan. Protein Chem. 8: GLADSTONE, G. P The nutrition of Staphylococcus aureus; nitrogen requirements. Brit. J. Exptl. Pathol. 18: GRETLER, A. C., P. MUCCIOLO, J. B. EVANS, AND C. F. NIVEN, JR Vitamin nutrition of the staphylococci with special reference to their biotin requirements. J. Bacteriol. 70: IVLER, D Comparative metabolism of virulent and avirulent staphylococci. Ann. N.Y. Acad. Sci. 8: MoRSE, S. A.,PAN R. A. MAH Microtiter hemagglutination-inhibition assay for staphylococcal enterotoxin B. Appl. Microbiol. 15: RAmsEY, H. H Endogenous respiration of Staphylococcus arueus. J. Bacteriol. 83: RAMSEY, H., AND J. L. PADRON Altered growth requirement accompanying chloram-

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