Vol. 220, No. 1
INFECrION AND IMMUNrrY, Apr. 1978, p. 158-160
0019-9567/78/0020-0158$02.00/0 Copyright i 1978 American Society for Microbiology
Printed un U.S.A.
Effect of Minerals on Staphylococcal Enterotoxin B Production GABRIELA M. KELLER, RICHARD S. HANSON AND MERLIN S. BERGDOLL* Food Research Institute and'Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 21 September 1977
Raising the magnesium level from 0.4 to 1.5 mM in a medium containing only amino acids (2.08%), salts, and vitamins increased enterotoxin B production by Staphylococcus aureus S-6 by about 80%. The level of phosphates in the medium was lowered to one-tenth (to 2.87 mM) the original amount without adversely affecting growth and enterotoxin production. The optimum level of potassium was between 15 and 45 mM. Ammonium salts were not essential in the amino acid medium. Sufficient quantities of trace metals were already present. Revising the salt mixture according to the information obtained resulted in a doubling of the enterotoxin B production. Several investigators have studied the production of enterotoxin B by Staphylococcus aureus S-6 in defined media. Mah et al. (4) and Miller and Fung (5) attempted to determine the minimal requirements for production of enterotoxin B, whereas Surgalla (6) did this for enterotoxin A. Wu and Bergdoll (8) attempted to develop a defined medium that would give optimum production of enterotoxin B. The salt mixtures used in the various investigations were modifications of the one proposed by Surgalla (6). Because optimum growth was not the aim of the defined medium studies, changes in the mineral mixture appeared to have little effect on growth and enterotoxin production in these media. Even though the situation is different when maximal growth is desired, Wu and Bergdoll (8) did not investigate the effect of the mineral mixture on growth and enterotoxin production. This paper presents data on the effect of changes in the mineral composition on the growth and enterotoxin B production of S. aureus S-6 in a defined medium. MATERIALS AND METHODS Organism. S. aureus S-6 was used (7).
Inoculum and incubation. The inoculum was prepared and the incubation was carried out as described by Wu and Bergdoll (8). Cell yield determination. The turbidity was measured as absorbance at 525 nm with a Beckman spectrophotometer. The cell yields were determined directly as dry weights with the cells harvested by centrifugation of 40 ml of culture. A factor relating wet weight to dry weight was obtained by drying weighed portions of the wet cells. A factor of 0.25 was calculated from the wet and dry weights. Enterotoxin assay. Enterotoxin was quantitated on single gel diffusion tubes as described by Kato et al. (3).
RESULTS AND DISCUSSION Magnesium. Jarvis (Ph.D. thesis, Massey University, Palmerston North, New Zealand, 1974) reported a linear relationship between staphylococcal enterotoxin production and magnesium concentration in the range of 0.05 to 0.4 mM. A further increase in toxin production might be expected for concentrations above 0.4 mM, the level used by Wu and Bergdoll (8). The results of adding magnesium to casein hydrolysate and defined media are given in Table 1. The added magnesium had no effect on the casein hydrolysate medium, indicating that a sufficient amount of the element was present in this medium, which would be expected, as casein contains considerable quantities of magnesium. An increase in the magnesium content in the defined medium to 1.5 mM resulted in an increase in viable cell count and enterotoxin production, but caused no change in cell dry weight. At the lower level of magnesium (0.4 mM), the cells formed larger clusters and the sizes of cells varied greatly. Some cells had diameters two to three
Media. The defined medium (AAWB medium) used in these studies was medium 4 of Wu and Bergdoll (8), containing 2.08% amino acids, and is identical to that described in the preceding paper except that it contained 48.8 mg of MgSO4 per liter. The salts in the medium were varied to determine the quantities necessary for optimum production of enterotoxin B by strain S-6. The casein hydrolysate medium contained 3% each of Protein Hydrolysate Powder (PHP; Mead Johnson International, Evansville, Ind.) and N-Z Amine NAK (NAK; Humko-Sheffield Chemical Co., Norwich, N.Y.). 158
VOL. 20, 1978
MINERALS AND ENTEROTOXIN B PRODUCTION
TABLE 1. Effect of magnesium concentration on staphylococcal cell yield and enterotoxin B production by strain S-6 Enterotoxin yield Medium MeimMgSO4 (MM) (mM)
3% PHP + 3% NAK
ob
0.5b
Viable count
CellWtdr
per
vol per dry
(CFUa/ml) (g/liter) (mg/
10'0
3x 3 x 10'°
5.22 5.12
wt
liter)
(mg/g)
349 312
67
61
0.4 6 x 109 20 94 4.70 1.5 3.1 x 10'° 4.55 167 37 a CFU, Colony-forming units. 'Amount of MgSO4 added to the medium that contains an undetermined amount of magnesium.
159
Trace elements. Trace-element deficiencies might be encountered in the defined medium, since high cell densities of 3.5 to 4.0 (dry weight) per ml were reached. The addition of various amounts of a trace-element solution to the medium (Table 5) indicated a small improvement TABLE 2. Effect ofphosphate concentration on staphylococcal growth and enterotoxin B production by strain S-6 (28-h cultures)
AAWB
Enterotoxin yield
P04 j (mM)
p P
Turbidity
(A525)b
per vol (mg/liter)
per absorbance
(mg/A525 unit)
8.4 20.8 28.7 5.74 8.5 20.0 2.87 8.6 18.6 1.44 8.5 7.7 0.72 8.5 4.3 0.36 8.4 3.8 3.7 0.29 8.4 a After growth. b A525, Absorbance at 525
153 135 180 90 60 42 42
7.4
times larger than normal. Abnormal cell mor6.8 phology disappeared at magnesium concentra9.7 tions of 1.5 mM or higher. 11.7 14.0 Phosphate. Higher concentrations of phos11.1 phate salts (about double) have been used in 11.4 defined media than are present in more complex media. In an attempt to determine what levels of phosphate were needed, the sodium and ponm. tassium phosphates were reduced by an equal amount with potassium acetate added to mainTABLE 3. Effect ofpotassium concentration on tain the potassium content at the original level growth and enterotoxin production (35-h cultures) (14.7 mM). Addition of 14.7 mM potassium aceTurbidToxin tate to the casein hydrolysate medium had no pH Medium (A ) (mg/liter) (mM) effect on growth or enterotoxin production of strain S-6. The results show that the original AAWBb 12.9 105 8.5 1.5 level of phosphate was 10 times that needed for 2.87 mM 2.5 8.5 16.8 155 optimal growth and enterotoxin production (TaP0438.5 155 15.3 5.0 ble 2). 8.5 155 10.0 16.6 Potassium. Potassium levels of less than 5 8.5 17.5 210 15.0 mM were reported to limit growth of S. aureus 8.5 16.3 202 30.0 (2), and levels of 35 mM had resulted in inhibi240 8.6 16.9 45.0 tion of enterotoxin synthesis by strain S-6 in a 173 8.4 17.0 60.0 complex (1) but not in a defined medium (Jarvis, 168 8.4 18.5 75.0 Ph.D. thesis). The results of varying the potas210 8.4 15.2 100.0 sium level from 1.5 to 100 mM in the AAWB 177 20.6 14.7 8.6 medium with low phosphate (2.87 mM) by the AAWBb 0 addition of potassium acetate (Table 3) showed A525, Absorbance at 525 nm. that high toxin yields were reached with potash Mg content = 1.5 mM. sium levels above 15 mM. Since the lag phase was prolonged substantially at potassium levels TABLE 4. Effect of ammonium concentration on growth and enterotoxin production in the AA WB of 45 mM and higher, a potassium concentration medium (35-h cultures) of 30 mM was chosen as a compromise between maximum toxin production and rapid growth. Toxin per Cell dry wt Toxin Vol per pH dry wt Ammonium. In the defined media, a nitrogen (NH0)2SO4 glie) (g/ite) (mM) (mg/g) pH (mg/liter) source other than the amino acids should not be 46 159 3.47 needed. To test this, the (NH4)SO4 content of 8.3 7.6 44 170 3.85 8.4 3.8 the medium was varied from 0 to 7.6 mM. The 46 164 8.3 3.60 1.9 results (Table 4) show that leaving out the am40 140 3.50 8.3 0.76 monium salts had little effect on the growth and 43 8.3 3.49 151 0 enterotoxin production. Because a small amount might be beneficial, it was decided to reduce the "Phosphate reduced 10-fold, potassium increased to 30 mM, and magnesium increased to 1.5 mM. amount to one-fourth the original amount.
(MM)S0
160
INFECT. IMMUN.
KELLER, HANSON, AND BERGDOLL
TABLE 5. Effect of trace elements on growth and enterotoxin production in the AA WB medium
(35-h cultures) Toxin yield
TESb (ml/liter)
Cell yield (dry wt)
per vol (mg/liter)
ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by Public Health Service grant Al 07615 from the National Institute of Allergy and Infectious Diseases.
per dry wt
(mg/g) 47 160 157 44 148 38 4 152 39 a Modified salt mixture; see text. 'ZnSO4 7H20, 0.5 mM; CuSO., 5H20, 0.5 mM; MnSO4 H20, 1.0 mM; Na2MoSO,12HAO, 0.004 mM; CoC12 6H20, 0.005 mM; CaC12 2H20, 20 mM.
1. Friedman, M. E. 1966. Inhibition of staphylococcal enterotoxin B formation in broth cultures. J. Bacteriol. 92:277-278. 2. Haynes, W. C., R. W. Huehne, and L. J. Rhodes. 1954. The effect of potassium upon the growth ofMicrococcus pyogenes. Apple. Microbiol. 2:339-344. 3. Kato, E., M. Khan, L. Kujovich, and M. S. Bergdoll. 1966. Production of enterotoxin A. Appl. Microbiol.
in cell yield, but toxin production was not stimulated. No toxic effects were observed when the same levels of the trace-element solution were added to the casein hydrolysate medium (data not shown). Modified salt mixture. The information from the tests outlined above was used in revising the salt mixture of Wu and Bergdoll (8). The results with the modified salt mixture (mg/liter) [(NH4)2S04, 250; Na3 citrate- 2H20, 500; KH2PO4, 200; Na2HPO4, 200; K acetate, 2,800; MgSO4, 180; FeSO4 7H20, 10] showed that enterotoxin production was increased from 85 to about 175 mg/liter (average of two experiments).
4. Mah, R. A., D. Y. C. Fung, and S. A. Morse. 1967. Nutritional requirements of Staphylococcus aureus S-6. Appl. Microbiol. 15:866-870. 5. Miller, R. D., and D. Y. C. Fung. 1973. Amino acid requirements for the production of enterotoxin B by Staphylococcus aureus S-6 in a chemically defined medium. Apple. Microbiol. 25:800-806. 6. Surgalla, M. J. 1947. A study of the production of staphylococcal enterotoxin in chemically defined medium. J. Infect. Dis. 81:97-111. 7. Wu, C. H., and M. S. Bergdoli. 1971. Stimulation of staphylococcal enterotoxin B production. I. Stimulation by fractions from a pancreatic digest of casein. Infect. Immun. 3:777-793. 8. Wu, C. H., and M. S. Bergdoli. 1971. Stimulation of enterotoxin B production. II. Synthetic medium for staphylococcal growth and enterotoxin B production. Infect. Immun. 3:784-792.
0 1 2
3.39 3.55 3.87 3.88
LITERATURE CITED
14:966-972.
INFECrION AND IMMUNrrY, Apr. 1978, p. 158-160
0019-9567/78/0020-0158$02.00/0 Copyright i 1978 American Society for Microbiology
Printed un U.S.A.
Effect of Minerals on Staphylococcal Enterotoxin B Production GABRIELA M. KELLER, RICHARD S. HANSON AND MERLIN S. BERGDOLL* Food Research Institute and'Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706 Received for publication 21 September 1977
Raising the magnesium level from 0.4 to 1.5 mM in a medium containing only amino acids (2.08%), salts, and vitamins increased enterotoxin B production by Staphylococcus aureus S-6 by about 80%. The level of phosphates in the medium was lowered to one-tenth (to 2.87 mM) the original amount without adversely affecting growth and enterotoxin production. The optimum level of potassium was between 15 and 45 mM. Ammonium salts were not essential in the amino acid medium. Sufficient quantities of trace metals were already present. Revising the salt mixture according to the information obtained resulted in a doubling of the enterotoxin B production. Several investigators have studied the production of enterotoxin B by Staphylococcus aureus S-6 in defined media. Mah et al. (4) and Miller and Fung (5) attempted to determine the minimal requirements for production of enterotoxin B, whereas Surgalla (6) did this for enterotoxin A. Wu and Bergdoll (8) attempted to develop a defined medium that would give optimum production of enterotoxin B. The salt mixtures used in the various investigations were modifications of the one proposed by Surgalla (6). Because optimum growth was not the aim of the defined medium studies, changes in the mineral mixture appeared to have little effect on growth and enterotoxin production in these media. Even though the situation is different when maximal growth is desired, Wu and Bergdoll (8) did not investigate the effect of the mineral mixture on growth and enterotoxin production. This paper presents data on the effect of changes in the mineral composition on the growth and enterotoxin B production of S. aureus S-6 in a defined medium. MATERIALS AND METHODS Organism. S. aureus S-6 was used (7).
Inoculum and incubation. The inoculum was prepared and the incubation was carried out as described by Wu and Bergdoll (8). Cell yield determination. The turbidity was measured as absorbance at 525 nm with a Beckman spectrophotometer. The cell yields were determined directly as dry weights with the cells harvested by centrifugation of 40 ml of culture. A factor relating wet weight to dry weight was obtained by drying weighed portions of the wet cells. A factor of 0.25 was calculated from the wet and dry weights. Enterotoxin assay. Enterotoxin was quantitated on single gel diffusion tubes as described by Kato et al. (3).
RESULTS AND DISCUSSION Magnesium. Jarvis (Ph.D. thesis, Massey University, Palmerston North, New Zealand, 1974) reported a linear relationship between staphylococcal enterotoxin production and magnesium concentration in the range of 0.05 to 0.4 mM. A further increase in toxin production might be expected for concentrations above 0.4 mM, the level used by Wu and Bergdoll (8). The results of adding magnesium to casein hydrolysate and defined media are given in Table 1. The added magnesium had no effect on the casein hydrolysate medium, indicating that a sufficient amount of the element was present in this medium, which would be expected, as casein contains considerable quantities of magnesium. An increase in the magnesium content in the defined medium to 1.5 mM resulted in an increase in viable cell count and enterotoxin production, but caused no change in cell dry weight. At the lower level of magnesium (0.4 mM), the cells formed larger clusters and the sizes of cells varied greatly. Some cells had diameters two to three
Media. The defined medium (AAWB medium) used in these studies was medium 4 of Wu and Bergdoll (8), containing 2.08% amino acids, and is identical to that described in the preceding paper except that it contained 48.8 mg of MgSO4 per liter. The salts in the medium were varied to determine the quantities necessary for optimum production of enterotoxin B by strain S-6. The casein hydrolysate medium contained 3% each of Protein Hydrolysate Powder (PHP; Mead Johnson International, Evansville, Ind.) and N-Z Amine NAK (NAK; Humko-Sheffield Chemical Co., Norwich, N.Y.). 158
VOL. 20, 1978
MINERALS AND ENTEROTOXIN B PRODUCTION
TABLE 1. Effect of magnesium concentration on staphylococcal cell yield and enterotoxin B production by strain S-6 Enterotoxin yield Medium MeimMgSO4 (MM) (mM)
3% PHP + 3% NAK
ob
0.5b
Viable count
CellWtdr
per
vol per dry
(CFUa/ml) (g/liter) (mg/
10'0
3x 3 x 10'°
5.22 5.12
wt
liter)
(mg/g)
349 312
67
61
0.4 6 x 109 20 94 4.70 1.5 3.1 x 10'° 4.55 167 37 a CFU, Colony-forming units. 'Amount of MgSO4 added to the medium that contains an undetermined amount of magnesium.
159
Trace elements. Trace-element deficiencies might be encountered in the defined medium, since high cell densities of 3.5 to 4.0 (dry weight) per ml were reached. The addition of various amounts of a trace-element solution to the medium (Table 5) indicated a small improvement TABLE 2. Effect ofphosphate concentration on staphylococcal growth and enterotoxin B production by strain S-6 (28-h cultures)
AAWB
Enterotoxin yield
P04 j (mM)
p P
Turbidity
(A525)b
per vol (mg/liter)
per absorbance
(mg/A525 unit)
8.4 20.8 28.7 5.74 8.5 20.0 2.87 8.6 18.6 1.44 8.5 7.7 0.72 8.5 4.3 0.36 8.4 3.8 3.7 0.29 8.4 a After growth. b A525, Absorbance at 525
153 135 180 90 60 42 42
7.4
times larger than normal. Abnormal cell mor6.8 phology disappeared at magnesium concentra9.7 tions of 1.5 mM or higher. 11.7 14.0 Phosphate. Higher concentrations of phos11.1 phate salts (about double) have been used in 11.4 defined media than are present in more complex media. In an attempt to determine what levels of phosphate were needed, the sodium and ponm. tassium phosphates were reduced by an equal amount with potassium acetate added to mainTABLE 3. Effect ofpotassium concentration on tain the potassium content at the original level growth and enterotoxin production (35-h cultures) (14.7 mM). Addition of 14.7 mM potassium aceTurbidToxin tate to the casein hydrolysate medium had no pH Medium (A ) (mg/liter) (mM) effect on growth or enterotoxin production of strain S-6. The results show that the original AAWBb 12.9 105 8.5 1.5 level of phosphate was 10 times that needed for 2.87 mM 2.5 8.5 16.8 155 optimal growth and enterotoxin production (TaP0438.5 155 15.3 5.0 ble 2). 8.5 155 10.0 16.6 Potassium. Potassium levels of less than 5 8.5 17.5 210 15.0 mM were reported to limit growth of S. aureus 8.5 16.3 202 30.0 (2), and levels of 35 mM had resulted in inhibi240 8.6 16.9 45.0 tion of enterotoxin synthesis by strain S-6 in a 173 8.4 17.0 60.0 complex (1) but not in a defined medium (Jarvis, 168 8.4 18.5 75.0 Ph.D. thesis). The results of varying the potas210 8.4 15.2 100.0 sium level from 1.5 to 100 mM in the AAWB 177 20.6 14.7 8.6 medium with low phosphate (2.87 mM) by the AAWBb 0 addition of potassium acetate (Table 3) showed A525, Absorbance at 525 nm. that high toxin yields were reached with potash Mg content = 1.5 mM. sium levels above 15 mM. Since the lag phase was prolonged substantially at potassium levels TABLE 4. Effect of ammonium concentration on growth and enterotoxin production in the AA WB of 45 mM and higher, a potassium concentration medium (35-h cultures) of 30 mM was chosen as a compromise between maximum toxin production and rapid growth. Toxin per Cell dry wt Toxin Vol per pH dry wt Ammonium. In the defined media, a nitrogen (NH0)2SO4 glie) (g/ite) (mM) (mg/g) pH (mg/liter) source other than the amino acids should not be 46 159 3.47 needed. To test this, the (NH4)SO4 content of 8.3 7.6 44 170 3.85 8.4 3.8 the medium was varied from 0 to 7.6 mM. The 46 164 8.3 3.60 1.9 results (Table 4) show that leaving out the am40 140 3.50 8.3 0.76 monium salts had little effect on the growth and 43 8.3 3.49 151 0 enterotoxin production. Because a small amount might be beneficial, it was decided to reduce the "Phosphate reduced 10-fold, potassium increased to 30 mM, and magnesium increased to 1.5 mM. amount to one-fourth the original amount.
(MM)S0
160
INFECT. IMMUN.
KELLER, HANSON, AND BERGDOLL
TABLE 5. Effect of trace elements on growth and enterotoxin production in the AA WB medium
(35-h cultures) Toxin yield
TESb (ml/liter)
Cell yield (dry wt)
per vol (mg/liter)
ACKNOWLEDGMENTS This research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, and by Public Health Service grant Al 07615 from the National Institute of Allergy and Infectious Diseases.
per dry wt
(mg/g) 47 160 157 44 148 38 4 152 39 a Modified salt mixture; see text. 'ZnSO4 7H20, 0.5 mM; CuSO., 5H20, 0.5 mM; MnSO4 H20, 1.0 mM; Na2MoSO,12HAO, 0.004 mM; CoC12 6H20, 0.005 mM; CaC12 2H20, 20 mM.
1. Friedman, M. E. 1966. Inhibition of staphylococcal enterotoxin B formation in broth cultures. J. Bacteriol. 92:277-278. 2. Haynes, W. C., R. W. Huehne, and L. J. Rhodes. 1954. The effect of potassium upon the growth ofMicrococcus pyogenes. Apple. Microbiol. 2:339-344. 3. Kato, E., M. Khan, L. Kujovich, and M. S. Bergdoll. 1966. Production of enterotoxin A. Appl. Microbiol.
in cell yield, but toxin production was not stimulated. No toxic effects were observed when the same levels of the trace-element solution were added to the casein hydrolysate medium (data not shown). Modified salt mixture. The information from the tests outlined above was used in revising the salt mixture of Wu and Bergdoll (8). The results with the modified salt mixture (mg/liter) [(NH4)2S04, 250; Na3 citrate- 2H20, 500; KH2PO4, 200; Na2HPO4, 200; K acetate, 2,800; MgSO4, 180; FeSO4 7H20, 10] showed that enterotoxin production was increased from 85 to about 175 mg/liter (average of two experiments).
4. Mah, R. A., D. Y. C. Fung, and S. A. Morse. 1967. Nutritional requirements of Staphylococcus aureus S-6. Appl. Microbiol. 15:866-870. 5. Miller, R. D., and D. Y. C. Fung. 1973. Amino acid requirements for the production of enterotoxin B by Staphylococcus aureus S-6 in a chemically defined medium. Apple. Microbiol. 25:800-806. 6. Surgalla, M. J. 1947. A study of the production of staphylococcal enterotoxin in chemically defined medium. J. Infect. Dis. 81:97-111. 7. Wu, C. H., and M. S. Bergdoli. 1971. Stimulation of staphylococcal enterotoxin B production. I. Stimulation by fractions from a pancreatic digest of casein. Infect. Immun. 3:777-793. 8. Wu, C. H., and M. S. Bergdoli. 1971. Stimulation of enterotoxin B production. II. Synthetic medium for staphylococcal growth and enterotoxin B production. Infect. Immun. 3:784-792.
0 1 2
3.39 3.55 3.87 3.88
LITERATURE CITED
14:966-972.
