B1 in an alcoholic extract of rice bran. This factor was stable

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1 EXPERIMENTS ON THE NUTRITION OF STREPTOCOCCI S. H. HUTNER Laboratory of Bacteriology, College of Agriculture, Cornell University, Ithaca, New York Received for publication September 24, 1937 The identification of the indispensable nutritional requirements of the streptococci has been the object of few investigations, despite the importance of the group and a number of clear-cut early advances. Hosoya and Kuroya (1923) discovered that a pyogenic streptococcus needed something accompanying vitamin B1 in an alcoholic extract of rice bran. This factor was stable to heat and acid; destroyed by drastic alkali treatment; adsorbed by fuller's earth; and precipitated by phosphotungstic acid. Freedman and Funk (1922), using the same hemolytic strain studied by Mueller, obtained similar data. Beef-heart infusion decolorized by norite charcoal allowed no growth, but was reactivated by the addition of peptone. Peptone alone did not permit growth. A streptococcus factor removed from autolyzed yeast by fuller's earth could be eluted with Ba(OH)2. Mueller (1922) found that norite-inactivated meat infusion was reactivated by the addition of acid-hydrolyzed casein as well as by peptone. One of the essential amino acids in the hydrolysate was identified as methionine. Another, found in the "monoamino" fraction of the Dakin butyl alcohol separation, and precipitated by HgCl2 in 5 per cent H2SO4 and by Ag2SO4, was not identified. Some additional properties of the hemolytic streptococcus factor were recorded: presence in spinach and blood; unextractibility by ether; stability toward oxidants and reductants. Orla-Jensen and associates (1936a, b) recently reported their extensive study of the nutrition of saprophytic streptococci. They found that the active material in skim milk, adsorbed onto 429

2 430 A. la. AtTrNE fuller's earth or norite, was eluted by a pyridine-methanol mixture. Deproteinized milk was made a better medium by the addition of casein. Riboflavin was viewed as essential. Several amino acids were found favorable, although ammonium salts sufficed as sole nitrogen source. Evaluation of these data is rendered difficult by the absence of details of the methods used for minimizing contamination of experimental media with material carried over from stock cultures. The present brief investigation developed from attempts to extend the work from Orla-Jensen's laboratory, and was as much aimed at finding the best species for experimental work as at identifying some of the growth essentials. CULTURAL TECHNIQUE The basic medium consisted of inorganic salts (NH4Cl 0.05 per cent; MgSO4.7H per cent; K2HPO per cent; KH2PO per cent; FeSO4 7H2O approximately per cent) plus Na citrate 11/2 H per cent and sucrose 2.0 per cent. The citrate served as buffer and to minimize heavy metal toxicity. Sucrose was chosen because it can be autoclaved without decomposition. Only sucrose-fermenting streptococci were used. The initial ph of all media was adjusted to 7.0 to 7.3. Cultures were maintained in 125 cc. Pyrex Erlenmeyer flasks containing 35 cc. of medium. Concentrations of nutrients were calculated, however, on a basis of a 25 cc. volume of medium so that all culture fluids were more dilute by the factor 5/7. Sterilization was by autoclaving for 10 minutes at 115'C. No evidence was found of destruction of growth essentials by this treatment. Growth was measured by the number of cubic centimeters of 0.1 N NaOH necessary to titrate a culture to pinkness with phenolphthalein after subtraction of the alkali expended on an uninoculated duplicate flask. Cultures were incubated at least 24 hours at 370C. - The stock broth used in most of this work had the following composition: Difco beef extract 0.3 per cent; Difco peptonized milk 0.5 per cent; Difco yeast extract 0.5 per cent; Difco tryptone 0.1 per cent; glucose 0.2 per cent; and CaCO3 in excess. The slight, or no, growth in

3 EXPERIMENTS ON NUTRITION OF STREPTOCOCCI control flasks made up of the basal solution plus adequate amino acids in the form of acid-hydrolyzed casein indicated that gross carry-over effects from the one-drop inocula were negligible. Eastman organic chemicals were used. CHOICE OF ORGANISMS Streptococcus liquefaciens was selected as our initial experimental object because of its good growth in peptone and exceptional resistance for a streptococcus to extreme physical conditions. Unfortunately the strain used, No. 805, produced so much alkali in deproteinized milk or yeast media that titration figures did not match growth. A strain of the closely related Streptococcus zymogenes (No. Inl) was found free of this objection but was studied only at the close of the investigation. The following vigorously-growing forms were selected: Streptococcus bovis No. 11. Isolated by Mrs. C. N. Stark from the mouth of cattle. Streptococcus inulinaceous. Ditto. Streptococcus asalignus (nina). Originally isolated by Prof. Frost. Streptococcus mastitidis No. ip1. Isolated by Dr. F. R. Smith from normal human feces and giving the precipitin test for Lancefield Group B. The Dochez "N. Y. 5" strain of "pyogenes" was chosen in preference to the typical "pyogenes" described by Evans (1936) because of its greater hardiness and more abundant growth both in skim milk and yeast media. Titration values for the forms enumerated were a satisfactory index of growth. AMINO ACID REQUIREMENTS The ease and thoroughness with which the protein can be removed from skim milk, leaving very little protein behind, made skim milk thus treated efficient in detecting amino acid requirements. The good growth most streptococci make in skim milk imply it to be a good source of their vitamins. Furthermore it is cheap and available, and its non-protein bulk consists largely of 431

4 432 S. H. HUTNER readily separable ash and lactose. Following Orla-Jensen's lead, skim milk fractions were made the basis of experimental work. Okuda and Zeller's (1921) directions for maximal protein removal were followed: a suspension of skim milk powder in water at ph 4.5 was steamed 10 minutes at 100'C. The protein coagulum was filtered off and washed with acidulated hot water. The combined filtrate and washings were brought to neutrality with NaOH and the calcium phosphate precipitate removed. The resulting deproteinized skim milk (PFM) was preserved with chloroform, as were all other putrescible solutions. No loss in activity was detected in the preparation after several months at room temperature in darkness. TABLE 1 Growth of S. pyogenes in deproteinized milk and hydrolyzed casein NaOH cc. PFM 1.0 per cent PFM 1.0 per cent plus AHC 0.5 per ceit PFM 2.0 per cent PFM 2.0 per cent plus AHC 0.5 per cent A sulfuric acid hydrolysate of casein (ARC) was prepared in the usual manner from Pfanstiehl's "vitamin-free" product. S. pyogenes makes a relatively poor growth in PFM plus ARC as compared with its growth in unaltered skim milk. A likely explanation is that in the process of protein removal serious amounts of growth factors are lost by adsorption on the protein. Freedman and Funk (1922) in fact noted that hydrolysates of ordinary unpurified proteins allowed growth while hydrolysates of purified proteins did not. Tests with PFM clearly showed dependence upon amino acids (table 1). The poor and somewhat irregular growth of this streptococcus in these media led to a comparison between PFM and yeast extract (Difco) as vitamin sources, all cultures being supplied with AHC at the 0.5 per cent level. The results are given in table 2.

5 EXPERIMENTS ON NUTRITION OF STREPTOCOCCI 433 There was clear evidence of complementary action-an indication of multiple factors. Other experiments proved that tryptophane was not a factor in the stimulation by yeast. Because of this complexity of growth factors, other streptococci growing far more vigorously in deproteinized milk were studied. It was always easy to elicit the casein stimulation (table 3). TABLE 2 Growth of S. pyogenes in deproteinized milk and yeast extract NaOH cc. AHC alone PFM 0.5 per cent PFM 1.0 per cent Yeast 0.1 per cent Yeast 0.5 per cent PFM 0.5 per cent plus yeast 0.1 per cent TABLE 3 Growth of streptococci in deproteinized milk, yeast, and hydrolyzed casein NaOH S. asa. S. bow. S. inu. S. mag. CC. CC. cc. Cc. PFM 2.0 per cent PFM 2.0 per cent plus AHC 0.5 per cent PFM 2.0 per cent plus yeast 0.25 per cent plus AHC 0.25 per cent Yeast 0.25 per cent plus AHC 0.25 per cent It will be noted that S. mastitidis and S. asalignus grow poorly in yeast. This is in marked contrast to S. bovis and the closely related S. inulinaceous, the first of which grows as luxuriantly as S. liquefaciens in milk or yeast media. A similar effect of hydrolyzed casein was found for Streptococcus liquefaciens, Streptococcus zymogenes, Streptococcus durans and Streptococcus salivarius. Attention was then directed to the identification of the essen-

6 434 S. H. HUTNER tial amino acids in the hydrolyzed casein. Cystine (furnished in slight excess) was found partly to replace hydrolyzed casein (table 4). Cystein hydrochloride (0.01 per cent), sterilized and added separately to avoid decomposition, was as effective. Trial of cystine was suggested by its sharing the property of Mueller's unidentified amino acid of being precipitated by acid mercuric reagents. The nature of the amino acid deficiencies coming into play after the satisfaction of the cystine requirement was the subject of a few expements. Supplements of methionine, tyrosine, leucine and phenylalanine together were entirely ineffective for the three streptococci tested: S. asalignus, S. bovis and S. mawstitidis. Occasionally growth with cystine would almost TABLE 4 Effect of replacement of casein hydrolysate by cystine... NaOH 8. a8a.. bow. S. ma.. _~~~~~~~C cc, c. cc PFM 1.0 per cent PFM 1.0 per cent pluscystine PFM 1.0 per cent plus AHC 0.25 per cent equal that with the casein hydrolysate but on serial subculture growth fell off almost entirely in the cystine flasks, but hardly at all in those with the hydrolysate. STREPTOCOCCUS VITAMINS Mueller (1922) reported disappointing results with heavy metal precipitation procedures for concentrating the S. pyogenes factor. It was felt that the complexity of the requirements of S. pyogenes might have been partly responsible, and that work with less exacting streptococci might yield clearer data. S. asalignus, S. bovis and S. mastitid8 were used. Heavy metal precipitation. One liter of PFM was evaporated on a steam bath under an electric fan to 250 cc., the temperature not rising above 750C. This concentrate was used for each

7 txpertments ON NUTRITION OF STREPTOCOCCI 435 precipitation. An untreated portion of concentrate served as control. (a) Ba(OH)2-ethanol. Fifty cubic centimeters of concentrate were chilled to 10'C. Hot saturated Ba(OH)2 was added in equal volume with vigorous stirring and, immediately after, an excess (400 cc.) of chilled 95 per cent ethanol. The curdy precipitate was placed in the refrigerator and shaken from time to time to prevent caking. After six hours the precipitate was filtered off with the aid of "filter-cel" (also used in the other precipitations) and washed with 95 per cent ethanol. Alcohol was removed from the filtrate by evaporation on a steam bath under electric fan. Barium was removed with H2SO4. On evaporation of an aliquot of the regenerated original barium precipitate abundant crystals of lactose formed. (b) Lead acetate-ba(oh)2. Fifty cubic centimeters of concentrate were treated with saturated basic lead acetate until no further precipitation occurred, then brought to ph 8.6 by the addition of cold saturated Ba(OH)2. After standing overnight the precipitate was filtered off and washed with water. It was then decomposed with a slight excess of H2SO4, the PbSO4 and BaSO4 filtered off, and the lead remaining in the filtrate removed with H2S. The PbS precipitate was washed with boiling water. H2S was removed from the PbS filtrate by bringing the solution to a boil. The original Pb-Ba filtrate was similarly treated. (c) HgSO4. Fifty cubic centimeters of chilled concentrate was treated with 25 cc. of West and Peterson's mercury reagent (a saturated solution of HgSO4 in 10 per cent H2SO4). Warm saturated Ba(OH)2 was added in small portions after successive chillings so that room temperature was not exceeded. When ph 7.0 was reached the flask was set overnight in the refrigerator, the combined mercury and BaSO4 precipitate filtered off and washed with small portions of ice water. The precipitate and filtrate were worked up as in the lead precipitation. (d) Copper-lime. Fifty cubic centimeters of concentrate were treated with 25 cc. of 25 per cent CuSO4. An excess of Ca(OH)2 suspension was added and the resulting bulky precipitate broken

8 436 S. H. HUTNER up into a fine suspension. The precipitate was filtered off and washed thoroughly with saturated Ca(OH)2 and was then decomposed with a slight excess of oxalic acid. H2S was passed in. The combined calcium oxalate and CuS precipitates were filtered off and washed with boiling water. The oxalate remaining in the filtrate was removed with CaCO3. The original filtrate was worked up in the same way as the precipitate. TABLE 5 Growth of streptococci in heavy metal fractions of deproteinized milk plus 0.6 per cent casein hydrolysate NaOH S. ata. S. bow. S. mae. cc. cc cc. AHC alone PFM 0.5 per cent Ba filtrate Ba precipitate Ba 0.5 per cent each together Pb filtrates Pb precipitate Pb 0.5 per cent together Hg filtrate Hg precipitate Hg 0.5 per cent each together Cu filtrate Cu precipitate Cu 0.5 per cent each together The growth tests were conducted with a concentration of each fraction corresponding to 0.5 per cent of the original deproteinized milk, considering only the organic matter content, as in all other experiments. This concentration of PFM allows about two-thirds maximal growth. The results are shown in table 5. The data are clearer for S. bovis than for S. asalignus and S. mastitidis. The mercury precipitation data point to multiple factors, one of them a base. Later adsorption experiments sup-

9 EXPERIMENTS ON NUTRITION OF STREPTOCOCCI port the belief that the principal loss of growth factors was by adsorption on the sulfide precipitates rather than by actual destruction. Fractionation with silver nitrate-ba(oh)2 was attempted but the very low activity of the fractions for S. bovis and S. mastitidis, the only strains tested, discouraged additional work. Fuller's earth adsorption Adsorption experiments with fuller's earth gave rise to the belief that all streptococci require at least one basic factor which may be characteristic for the genus. A large number of experiments were carried out with Eimer and Amend's fuller's earth and "Frankonite KL." Several other brands, in contrast, were quite inactive. The following is a TABLE 6 Growth in fuller'8-earth-treated deproteinized milk NaOH S. asa. S. bor. S. mas. CC. C. CC. PFM untreated Fuller's earth 0.4 per cent Fuller's earth 2.0 per cent Fuller's earth 10.0 per cent sample experiment: deproteinized milk was brought to ph 4.3 and treated with the following concentrations of Frankonite KL: 0.4, 2.0 and 10 per cent. The filtrates were brought back to neutrality and tested in concentrations corresponding, as in previous experiments, to 0.5 per cent of the original deproteinized milk. All cultures were with 0.25 per cent hydrolyzed casein. The results are shown in table 6. The lowest concentration of Frankonite removed virtually all the riboflavin judging by the disappearance of the pronounced yellowish-green color of the untreated deproteinized milk. S. liquefaciens also grows poorly in fuller's earth-treated PFM. Additions of pure vitamin B1 (gift of the Winthrop Chemical Company), riboflavin, guanine, uracil, and a hydrolysate of yeast nucleic acid-the last suggested by Richardson's (1936)

10 438 S. H. HBuNER findings for Staphylococcus aureu&-to the fuller's earth filtrates did not improve growth. It was hoped that differences in vitamin requirements might be a convenient supplementary mean for differentiating species of streptococci, particularly in the "t'ridans" group. Thirtyfive strains of S. bovi8 from Mrs. C. N. Stark's collection were inoculated into deproteinized milk plus casein hydrolysate, yeast extract, and both together. The widest diversity was revealed: some grew in either; others in one but not the other; others only in both together; and still others poorly even in both vitamin sources together. This behaviour was foreshadowed by their growth in litmus milk in which they were maintained where some grew so poorly as not to curdle the milk while others did so in a few hours. Similar results, though with fewer strains, were obtained with S. salivarius. DISCUSSION All streptococci investigated to date require at least one nonamn o-acid growth factor. Its adsorption on fuller's earth and precipitation by mercuric salts are properties of a base. If the theory of the close phylogenetic relation between staphylococci and streptococci be accepted, then Knight's (1937) discovery that Staphylococcus aureus needs nicotinic acid or its amide besides vitamin B1 or its cleavage products, is perhaps applicable to streptococci. The properties of nicotinic acid or of its amide are in keeping with those reported for the hypothetical streptococcus vitamin. Unfortunately, time did not permit trial of these compounds. The difficulty of concentrating vitamin preparations to the point where their amino-acid content is negligible precludes any sweeping statements as to the number of amino acids that might be required even should one succeed in replacing the casein hydrolysate with known amino acids. No streptococcus was found capable of growing with ammonium salts as a sole source of nitrogen; as previously mentioned growth would occasionally occur in deproteinized milk in the absence of the casein hydrolysate, but came to an abrupt end on serial subculture. Orla-Jensen's con-

11 EXPERIMENTS ON NUTRITION OF STREPTOCOCCI 439 clusions as to amino-acid requirements have been criticized on similar grounds by Wood, Anderson and Werkman (1937). Ehrismann and Dramburg (1937) in a study of a strain of S. pyogenes mention that cystine was supplied as sulfur source but experimental evidence in support of this was not given in their paper. A stimulating effect of riboflavin was not observed-a finding opposed to that of Orla-Jensen's. CONCLUSIONS By the use of deproteinized skim milk and a casein hydrolysate, amino acid requirements for several species of streptococci may be demonstrated. Strains of Streptococcus asalignus, Streptococcus bovis and Streptococcus mastitidis were found that required cystine (or cysteine) in addition to at least one more amino acid present in acid-hydrolyzed casein. Treatment of deproteinized milk with fuller's earth inactivated it for the above streptococci as well as for Streptococcus liquefaciens. Streptococcus bovis requires at least two non-amino-acid factors: one precipitated by mercuric sulfate, the other not. A great deal of variability in growth requirements was found within the species Streptococcus bovis and Streptococcus salivarius. The writer is indebted to the staff and graduate students of the Department of Dairy Industry and Bacteriology for their constant cooperation. REFERENCES EHRISMANN, O., AND DRAMBURG, K tmber die Verwertung von Aminosauren durch Streptokokken. I. tj'ber die Bedeutung von Aminosauren fur das Wachstum von Streptokokken. Zeit. Hyg., 119, EvANs, ALICE C Studies on hemolytic streptococci. II. Streptococcus pyogenes. Jour. Bact., 31, FREEDMAN, L., AND FUNK, C Nutritional factors in the growth of yeasts and bacteria. I. Vitamins. II. Protein hydrolysates. Jour. Metab. Res., 1, ; HosOYA, S., AND KUROYA, M Water-soluble vitamine and bacterial growth, with special reference to the chemical and physical properties

12 440 S. H. HUTNER of vitamin essential' for the growth of hemolytic streptococci. Gov. Inst. Infect. Dis. Tokyo Imp. Univ., 2, KNIGHT, B. C. J. G The nutrition of Staphylococcus aureus. The activities of nicotinamide, aneurin (vitamin B,) and related compounds. Biochem. Jour., 31, MUELLER, J. H Studies on cultural requirements of bacteria. Jour. Bact., 7, ; OKUDA, Y., AND ZOLLER, H. F The relations of hydrogen-ion concentration to the heat coagulation of proteins in Swiss cheese whey. Jour. Ind. Eng. Chem., 13, ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAER, A. 1936a Der Vitaminbedarf der Milchsiurebakterien. Centbl. Bakt., II Abt., 94, ORLA-JENSEN, S., OTTE, N. C., AND SNOG-KJAzR, A. 1936b Die StickstoffnAhrung der Milchaiurebakterien. Centbl. Bakt., II Abt., 94, RICHARDSON, G. M The nutrition of Staphylococcus aureus. Necessity of uracil for anaerobic growth. Biochem. Jour., 30, WOOD, H. G., ANDERSON, A. A., AND WERKMAN, C. H Growth-factors for propionic and lactic acid bacteria. Proc. Soc. Exptl. Biol. Med., Downloaded from on January 27, 2019 by guest