Download Nutritional Requirements of Streptococcus salivarius

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
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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-
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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
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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
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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
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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
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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).
The author gratefully acknowledges the technical assistance of Miss G. Matsson and
Mrs M. Claesson.
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