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Journal of Geiieral Microbiology ( I 97 I), 67, 69-76 Printed in Great Britain Nutritional Requirements of Streptococcus salivarius By J. C A R L S S O N Department of Oral Microbiology, Uiziversity of Umed, S-901 87 Unied, Sweden (Acceptedfor publication 28 April 1971) SUMMARY The nutritional requirements of Streptococcus salivarius ATCC I 34 r 9 were studied. Ammonia could serve as the major nitrogen source in a medium containing glucose, cysteine, nicotinic acid, biotin, thiamin, riboflavin, pantothenic acid and inorganic salts. Streptococcus sakvarius NCTC 861 8 and I I oral isolates of S. salii7ariusgrew in this medium. For growth of S. salivarius ATCC9759 the medium had to be supplemented with glutamic acid. The cysteine requirement of S. saliitarius ATCC 13419 could be replaced by cystine, homocysteine, homocystine or thiosulphate. Urea could be used as nitrogen source by S. salivarius ~ ~ ~ ~ 1 3 4 1 9 , S. sahYarius ATCC9759 and by five of the I I oral isolates of S. salii7arius. INTRODUCTION The predominant streptococcus in ruminants, Streptococcus bovis, has been considered as the only species of the genus Streptococcus which is capable of using ammonium salts as the major source of nitrogen for growth (Prescott, Williams & Ragland, 1959; Wolin, Manning & Nelson, 1959). Recently, however, it was found that when growing anaerobically the human oral streptococci S. sangiris and S. inutans may also utilize ammonia as major source of nitrogen (Carlsson, 197oa, b, 1971). This paper reports similar simple nutritional requirements of the predominant streptococcus of human saliva, Streptococcus salivariirs. METHODS Micro-organisms. Streprococcus salii*arius, American Type Culture Collection (ATCC) strain 13419 was used for the development of the media. Subsequently, the following strains 5 9 , National Collection of Type Cultures were tested : S. sulivarius ~ ~ ~ ~ 9 S.7 salivarius (~cTC)8618and 11 strains isolated from the oral cavity in man and considered to be S. salivarius in a numerical taxonomic study (Carlsson, 1968). Chemicals. DL-Homoserine, DL-homocystine and DL-homocysteine thiolactone HC1 were from Sigma Chemical Co., St Louis, Missouri, U.S.A. Thiouracil was from Nutritional Biochemicals Corporation, Cleveland, Ohio, U.S.A. Sodium thioglycollate as well as ingredients for Mitis Salivarius agar and Brain Heart Infusion agar were from Difco Laboratories, Detroit, Michigan, U S A . All other ingredients in the chemically defined media and other chemicals were of the purest grade available from the British Drug Houses Ltd, Poole, Dorset. Distilled water was used after filtration through mixed-bed ion-exchange resins (Crystalab Deeminizer, Crystal Research Laboratories Inc., Hartford, Connecticut, U.S.A.). Culture media. Amino acids and salts were dissolved according to Williams (1955) and vitamins according to Leslie (1961). All ingredients were sterilized by filtration (membrane filter, type GS, Millipore Corporation, Bedford, Massachussetts, U.S.A.). The media were Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 10 J. C A R L S S O N distributed in 5 ml. amounts into screw-capped tubes (16 x I 10mm.) and were kept frozen (- 20') until used. Assays. Bacterial growth yield was estimated by determining the extinction ( E )at 420 nm. in a double-beam spectrophotometer with the uninoculated medium as control. The culture was diluted in water to give an extinction less than 0.8. The dry weight of the three reference strains was determined using two independent cultures of each strain. The bacteria were harvested by centrifugation, suspended in 10 ml. of water, recentrifuged and then dried for 2 days at 110' on pre-weighed pieces of aluminium foil. The dried bacteria were weighed on an electrobalance (model G, Gram electrobalance, Cahn Instrument Co., Paramount, California, U.S.A.). The dry weight of bacteria in cultures with an extinction of 1-0at 420 nm. was 0.131 to 0.136 mg./ml. Table I. Composition of media for Streptococcus salivarius 0.1 M Potassium phosphate buffer (pH 7.0) in all media. Concentration of components (mg./l.) in medium Component Glucose L-Cysteine HCI Pyridoxine HCI Nicotinic acid Biotin p-Aminobenzoic acid Thiamin HCl Riboflavin Ca-pant othenate (NH,),SO, NH,HCO, Salts B MgSO,. 7Hz0 FeSO, .7Hz0 MnSO,. 4 H z 0 NaCl I c4 10,000 50 I2 2'3 0.006 0'0I 0.05 0'2 1'2 c 31 10,000 79 6.1 0.024 0.67 0.38 2.4 1,320 - 1,320 200 I0 I0 I0 200 I0 I0 I0 - c 39 10,000 79 6.1 0.024 0.67 0.38 2.4 1,975 200 I0 I0 I0 Experimental procedure. Streptococcus saharius ATCC 13419 grew in a medium (C4, Table I) previously developed for S. mutans (Carlsson, I970b). After 15 daily transfers, 0-1 ml. of a 24 h. culture was inoculated into a number of media each of which lacked one of the ingredients of medium C4. However, none of the salts (B) were omitted. The organism was subcultured daily for 10days with 0.1ml. inocula in each of these media. If the growth had ceased before that time, the procedure was repeated with 0.5 ml. inocula. The growth in each tube was followed for 5 days by scoring -, + or + + and finally the extinction of the culture at 420 nm. was measured on the fifth day. When the qualitative composition of a minimal medium for growth had been elaborated, the concentration of each component necessary for optimal yield was determined. Strain ATCC 13419 was subcultured in media with one component in such a low concentration that the yield was 20 to 50 % of maximal. A series of media with various concentrations of this component was inoculated with 0.1ml. from such a limited culture grown for 24 h. The growth yield in these media was determined after 72 h. by measuring the extinction at 420 nm. When a minimal medium with optimal concentration of each component had been developed for strain ~ ~ ~ ~ 1 3the 4 1growth 9 , yields of the other strains were tested in that medium. If any strain failed to grow, the medium was supplemented with various compon- Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 71 Nutrition of Streptococcus salivarius ents until transferable growth was obtained. A strain was considered to be able to grow in a medium if it could be subcultured ten times in the medium with 2 % (v/v) inocula. Preparation of media, daily transfers and sampling from culture tubes were done in a sterile bench with horizontal laminar air flow (Munktell, Gryksbo, Sweden). The purity of the cultures were checked at least once a week by streaking one drop of the culture on the surface of a Mitis Salivarius agar plate and a Brain Heart Infusion agar plate. All media were incubated in the dark at 37". For incubations in atmospheres other than air, the cuIture tubes were placed in a jar, the air evacuated to a pressure of - I -0 kg./cm.2 and replaced by the required gas. When the gas contained hydrogen the jar was provided with a palladium-asbestos catalyst in brass net. If not otherwise stated, anaerobic growth refers to growth in an atmosphere of 95 % H2 and 5 % C 0 2 . -6 -5 -3 -3 Logl,, iiiolarity of component -4 -1 0 Log,, molarity of component Fig. I. Growth yield of Streptococcus salivavius ATCC 13419in media containing various concentrations of one of the components. The complete medium contained (per ml.): glucose, 10nig.; (NH&304, 1.32 mg. ;L-cysteine HCJ, 50 pg; biotin, 2.4 ng. ; Capantothenate, 4-8pg. ;nicotinic acid, 12.3 pg. ;riboflavin, 0.19pg. ; thiamin HCI, 0.34 pg. ; MgS04.7Hz0, 0.2 mg. ; FeSO,. 7H20, 10pg. ; MnSO,. 4H20, 10pg. ;NaC1, 10pg., and potassium phosphate buffer (pH 7.0) 0.I mmole. Cultures grown in 95&: H2+ 5 "/b CO,. The mean extinction of three cultures ( S.U.) in each medium after 72 h. is given. RESULTS Growth of Streptococcus salivarius ATCC 13419. The organism grew in medium C 4 (Table I) when incubated in an atmosphere containing 95 % H2 and 5 % CO,. Only pyridoxine and p-aminobenzoic acid could be omitted from this medium without interfering with the growth yield. The effect of omitting each of the salts (B) was not tested. The growth yield at various concentrations of the components in a minimal medium is Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 72 J. CARLSSON shown in Fig. I. From these results medium C 31 (Table I) was composed. In medium C 31 Streptococcus salivarius ATCC 13419 grew luxuriantly with a yield of about 0.8 mg. dry wt of bacterialml. of medium, when incubated in an atmosphere containing 95 % H,+ 5 % CO, or 95 % N, + 5 % CO,. The organism failed to grow in this medium when incubated aerobically or in an atmosphere containing only H, or N,. When (NH& SO, in medium C 31 was replaced by urea or NH,HC03 the organism grew anaerobically (Fig. 2 ) as well as aerobically. The carbon dioxide requirement of S. sulivarius ATCC 13419 in aerobic culture in medium C 31 could be replaced by 10 mwglutamic acid or oxaloacetic acid. The addition of aspartic acid, a-oxoglutaric acid or succinic acid to medium C 31 gave inconsistent growth aerobically. 7 6 -4 -3 -2 Log,, rnolarity of nitrogen compound -I Fig. 2. Growth yield of Streptococcus salivarim ATCC 13419 in medium C 31 (Table I ) containing different concentrations of ammonium sulphate (0)or different concentration of urea (0) or ammonium bicarbonate).( substituting for the ammonium sulphate. Cultures grown in 95 yo H, + 5 76 CO,. The mean extinction of three cultures (2s.D.) in each medium after 72 h. is given. The cysteine requirement of Streptococcus sdlivarius ATCC 13419 in anaerobic culture in medium C 39 could be replaced by 0.5 mM L-cystine, DL-homocystine, DL-homocysteine thiolactone or sodium thiosulphate (Fig. 3), but not with 0.5 mMDL-homoserine, L-methionine, thioglycollic acid, mercaptoethanol, thiourea, thiouracil, sodium sulphite or sodium sulphide. Only L-cysteine supported growth aerobically. Growth of other strains of Streptococcus salivarius. Streptococcus salivarius NCTC 8618 grew in medium C 39. Streptococcus salivarius ATCC 9759 did not grow in medium C 3 I nor C 39 unless these media were supplemented with glutamic acid. The growth yield when medium C 37 was supplemented with different concentrations of glutamic acid is shown in Fig. 4. Succinic acid, oxaloacetic acid and a-oxoglutaric acid in a concentration of 10mM could not substitute for the glutamic acid requirement of S. salivarius A T C C ~ ? All ~ ~ .I I oral strains of S. salivarius grew anaerobically in medium C 39. Three of the I I strains also grew Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 Nutrition of Streptococcus salivarius 73 aerobically in this medium. When medium C 39 was supplemented with 10mM glutamic acid an additional two strains grew aerobically. Five of the 1 1 oral strains grew anaerobically in medium C 39 when NH,HCO, was replaced by 20 m M urea. Streptococcus salivurius ATCCg759 could also use urea instead of NH,HCO, in medium C 39, when this medium was supplemented with 10mM g1utamic:acid. Streptococcus salivarius N C T C ~ 8~ could I not utilize urea. When L-cysteine in medium C 39 was replaced by 0.5 mM sodium thiosulphate, S. salivarius ~ c ~ C 8 6 1and 8 six of the oral strains grew in this medium. Streptococcus salivarius ATCC9-759 could also use sodium thiosulphate instead of L-cysteine in medium C 39, when this medium was supplemented with 10mM-glutamic acid. I -6 -5 -4 Log,, molarity of sulphur compound -3 I I I 1 1 1 1 1 I I I I I I l l 1 -2 -3 -3 Log,, molarity of glutamic and/or (,NH,),SO, Fig. 4 Fig. 3 Fig. 3. Growth yield of Streptococcus salivarius A T C C I ~in~ medium I ~ C 31 containing different concentrations of L-cysteine (0)and with different concentrations of sodium thiosulphate (0) substituting for L-cysteine. Cultures grown in 95 "'0 H, 5 7 : COz. The mean extinction of three cultures (_+ s.D.) in each medium after 72 h. is given. + Fig. 4. Growth yield of Streptococcus salivarius ~ ~ c c g 7 5when 9 medium C 31 was supplemented with different concentrations of glutamic acid (m) and when medium 31 10mM glutamic acid contained different concentrations of ammonium sulphate (0)Cultures . grown in 95 yo H, 5 %, CO,. The mean extinction of three cultures (_+s.D.) in each medium after 72 h. is given. + + DISCUSSION Streptococcus salivarius was among the first streptococci in which the nutritive requirements were studied (Smiley, Niven & Sherman, 1943). A medium for transferable growth of I 4 of 2 I strains of S. salivarius contained inorganic salts, glucose, sodium thioglycollate, glutamic acid, leucine, arginine, isoleucine, lysine, methionine, tyrosine, riboflavin, nicotinic Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 J 74 J. C A R L S S O N acid, pantothenic acid, biotin, thiamin and uracil. Two oral strains of S. salivarius studied by Paul (1961) required glutamic acid, cystine, alanine, and lysine, and were stimulated by aspartic acid or asparagine and by histidine or isoleucine or tyrosine. The present study shows that the nutritional requirements of S. salivarius are much simpler, if the oxidationreduction potential of the medium is lowered and if the culture is provided with carbon dioxide. In the presence of glucose and five B-group vitamins, ammonia could serve as sole nitrogen source. Assimilation of carbon dioxide has been demonstrated in other streptococci. When grown on complex nitrogen sources Streptococcus bovis (Wright, 1960), S. angiosus (Martin & Njven, 1960) and S. faecium var. durans (Lachica & Hartman, 1968) incorporate carbon dioxide mainly as the P-carboxyl group of aspartic acid. In S.faecalis var. liquefuciens carbon dioxide is fixed in oxaloacetic acid by a pyruvate carboxylase, and oxaloacetic acid is transaminated to aspartic acid (Hartman, 1970). In S. bovis grown in an ammonium salt medium exogenous carbon dioxide is the sole source of the a-carboxyl group of glutamic acid (Burchall, Niederman & Wolin, 1964) and the carboxyl and guanidine carbons of arginine (Niederman & Wolin, 1967). In addition S. bovis incorporates acetate mainly into glutamic acid (Prescott, Ragland & Hurley, 1965). These results indicate that a tricarboxylic acid pathway to a-oxoglutaric acid may be present in streptococci. The primary site of incorporation of ammonia into amino acids in Streptococcus bovis is a nicotinamide adenine dinucleotide phosphate (NADP)-linked glutamate dehydrogenase (Burchall, Niederman & Wolin, 1964). An asparagine synthetase has also been demonstrated in this organism (Burchall, Reichelt & Wolin, 1964) and ammonia as well as carbon dioxide may also be fixed by enzymes involved in carbamoyl phosphate synthesis (Niederman & Wolin, 1967). The close overall similarity between Streptococcus bovis and S. salivarius (Carlsson, 1968) and the similarity in nutritional requirements of these species suggest that ammonia and carbon dioxide are assimilated in similar ways in these streptococci. Streptococcus salivarius could utilize cysteine, cystine, homocysteine, homocystine and thiosulphate as sulphur source. This is in accordance with findings in S. bovis (Prescott, 196I). Unlike S. bovis, S. salivarius could not utilize thioglycollic acid, thiourea, thiouracil and sulphide. Both organisms failed to utilize methionine, sulphate and sulphite. It is not known how streptococci synthesize their sulphur-containing amino acids. An interesting question is how Streptococcus salivarius satisfies its nutritional requirements in the natural habitat, the surface of the tongue (Krasse, 1954). Although a luxuriant growth of this organism in the mouth requires the presence of readily fermentable carbohydrates in the diet (Carlsson, 1969, S. salivarius can establish itself in the mouth of infants before they have consumed any milk (Carlsson, Grahnkn, Jonsson & Wikner, 1970). The requirements for ammonia, carbon dioxide and vitamins may be provided by saliva or by the microbiota inhabiting the crypts of the tongue. Saliva contains 3 to 6 pmoles ammonia/ml. (Battistone & Burnett, 1961) and 0.1 to 5 ml. carbon dioxide/ml. of fluid (Jenkins, 1966). All the B-group vitamins have been demonstrated in saliva, but there are wide differences in the amounts reported by various workers (Makila, 1968). Urea may also be a valuable nitrogen source for Streptococcus salivarius. The concentration of urea in saliva is closely related to its plasma level (Nikiforuk, Jackson, Cox & Grainger, 1956). When urea is supplied in the diet the ammonia production in the mouth is greater than the rate of ammonia utilization (Stephan, 1943; Frostell, 1960; Kleinberg, 1967). Urea has been included in sweets in order to prevent the drop of pH in microbial aggregations on the teeth, thereby preventing dental caries (Frostell, 1967, 1970). Such Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 05 May 2017 12:45:59 Nutrition of Streptococcus salivarius 7s sweets will be adequate carbon and nitrogen sources for S. salivarius as well as for the cariesconducive streptococci S. mutam and S. sanguis (Carlsson, rg7oa, by 1971). From the results of the present study these sweets cannot be recommended as caries-preventing agents. This work was supported by the Swedish Medical Research Council (B 70-24X-2793-01). 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