Vol. 58, No. 4
INFECTION AND IMMUNITY, Apr. 1990, p. 1026-1029 0019-9567/90/041026-04$02.00/0 Copyright © 1990, American Society for Microbiology
Effect of Environmental Conditions on Production of Toxic Shock Syndrome Toxin 1 by Staphylococcus aureus AMY C. LEE WONG* AND MERLIN S. BERGDOLL Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 Received 24 July 1989/Accepted 17 January 1990
The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger. a
Mass.) and introduced into the medium via a glass tube (internal diameter, 1 mm) which was inserted through the headplate. When specified, a 12-mm-diameter coarse sintered glass sparger (Corning Glass Works, Corning, N.Y.) was used. Sterilized silicone antifoam (a 1:10 dilution of B emulsion; Dow Corning Corp., Midland, Mich.) was used when excessive foaming occurred. The gas flow rate was controlled by a flowmeter (Union Carbide Corp., Linde Division, Somerset, N.J.). Dissolved oxygen (DO) was detected with an autoclavable galvanic DO probe (Model M1016-0208; New Brunswick) which was connected to a DO analyzer (Model DO-50; New Brunswick). The pH of the culture was monitored with an autoclavable pH electrode (type 465-35 or 465-35-90-K9; Ingold Electrodes, Inc., Wilmington, Mass.). Automatic pH control was maintained by a pH controller (Model pH22; New Brunswick), using 1 M NaOH and HCI solutions. The DO probe, pH electrode, air filter, and connecting tubes were autoclaved separately and placed aseptically into their respective positions. The percent DO pH, and temperature of the medium were allowed to equilibrate to the appropriate controlled values before inoculation. Incubation conditions. The inoculum was 3.5 ml of an overnight culture of the specified strain. Unless stated otherwise, the strain used was FRI-1169, the initial staphylococcal population was about 107 CFU/ml, the incubation temperature was 37°C, and the culture was agitated at 280 rpm by a magnetic stirrer. Samples were removed at specified times. Methods of analysis. Bacterial growth was determined by surface plating the appropriate dilutions and culture fluids on plate count agar plates (Difco Laboratories, Detroit, Mich.). TSST-1, SEA, and SEC were quantitated by a single gel diffusion tube method (10) with a detection limit of 0.5 jig/ml. In addition, when the concentration was 95% purity by disc polyacrylamide gel electrophoresis [11]) were prepared by column chromatography (13). Results presented are the averages of duplicate experiments.
Production by Staphylococcus aureus of toxic shock syndrome toxin 1 (TSST-1) (2), a major causative agent of toxic shock syndrome (TSS), is affected by environmental conditions. Aerobic conditions have been shown to stimulate the production of TSST-1, while anaerobiosis is inhibitory (17; H. F. Pickrum, D. L. Lucas, and R. L. Stone, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B57, p. 27). However, high aeration rates in continuous cultures decrease toxin production (15). Cultures incubated aerobically make more TSST-1 with added CO2 than without (9, 18). Sarafian and Morse (15) noted that production of TSST-1 does not begin until after the exponential growth phase. Schlievert and Blomster (16) observed that toxin is produced during the logarithmic phase, and maximal production occurs just before the stationary phase. Taylor and Holland (17) showed that TSST-1 is produced maximally at a suboptimal specific growth rate. In this study, TSST-1 production was monitored throughout the growth cycle of S. aureus by using a bench top fermentor which allowed continuous measurement and control of aeration rates, atmospheric composition, pH, and temperature. MATERIALS AND METHODS Strains. S. aureus FRI-1123 (TSST-1), FRI-1169 (TSST-1), FRI-1183 (TSST-1 and enterotoxin C [SEC]), FRI-1187 (TSST-1, enterotoxin A [SEA], and SEC], and Harrisburg (TSST-1 and SEA), originally isolated from patients with TSS, were used. The Harrisburg strain was obtained originally from R. L. Stone, The Procter & Gamble Co., Cincinnati, Ohio. Strains were stored in the dried form on porcelain beads (8). Medium. The culture medium consisted of 3% NZ Amine NAK, a pancreatic digest of casein (Sheffield Products, Memphis, Tenn.), and 1% yeast extract (3+1 medium) (pH 6.5). Apparatus for controlled culture conditions. A New Brunswick Bioflo bench top fermentor (Model C30; New Brunswick Scientific Co., Inc., New Brunswick, N.J.) was used. The fermentor vessel was filled with 350 ml of 3 + 1 medium and sterilized by autoclaving. Gases were sterilized with an autoclavable bacteriological filter (Balston, Inc., Lexington, *
Corresponding author. 1026
PRODUCTION OF TSST-1
VOL. 58, 1990 11
TABLE 1. Effect of gas flow rate on growth of S. aureus FRI-1169 and on TSST-1 productiona Flow rate (cm3/min)
Air
CO2
(min) 0 50 100 100 100 100 200 400 0
0 0 0 5 10 20 0 0 5
N2 (100 cm3/min) a
34.5 25.7 26.1 23.1 25.3 27.8 26.2 22.7 39.2 46.0
10
TSST-1
Growth
Generation time
1027
CFU
100U
~ gX
(0m)
pgm
0.51 1.10 1.30 1.30
0.3 1.5 1.9 19.8
0.65 0.28 1.10 0.73 0.25 0.09
15.3 10.5 1.3 0.9 0.3 0.0
g11
CFU
10
F
0.6 1.4
1.5 15.2 23.5 37.5 1.2 1.2 1.4 0.0
108
le8
Incubation for 24 h at 37°C; stirrer speed, 280 rpm. IL
RESULTS AND DISCUSSION Effect of air flow rate. The exponential growth rates of FRI-1169 aerated at 50 to 400 cm3/min by the tube method were slightly higher than that of an unaerated culture, resulting in shorter generation times (Table 1). Cell yields after 24 h were 1.4 to 2.5 times higher with aeration. TSST-1 was detected at the mid- to late logarithmic phase, when production was at a maximal exponential rate. TSST-1 was produced at a higher rate relative to growth rate, as the productivity (defined as micrograms of TSST-1 per 1010 CFU) continued to increase. Although strain FRI-1169 grew to about 109 CFU/ml under completely anaerobic conditions, only 0.008 p.g of TSST-1 per ml was produced. Effect of DO. The exponential growth phase ended when the DO of aerated cultures (100 cm3 of air per min) dropped in 0%, for both the tube and sparger aerated cultures (Fig. 1 and 2), indicating that insufficient 02 was available to sustain exponential growth. However, the major differences in the tube and sparger aerations were as follows: (i) the exponential growth phase with the sparger was much longer, indicating an increased efficiency in providing oxygen to the culture, and (ii) when maximum growth of 6 x 1010 CFU/ml was reached (12 h) in the sparger aerated culture, the DO returned to above 0%, whereas in the tube aerated culture, growth was still taking place at 24 h and had reached only 1.3 x 1010 CFU/ml, with the DO remaining at 0%. The amount of toxin produced was about the same for both aeration methods (1.9 ,ug/ml) at the same growth level (1010 CFU/ml), but because of the increased growth with the sparger aeration, an additional 8.1 ,ug of toxin per ml was produced. Although more TSST-1 was produced aerobically than anaerobically, the fact that TSST-1 continued to be synthesized after the DO level decreased to 0% (Fig. 1 and 2) indicated that full aerobiosis was not essential. Decreasing the amount of air supplied with the sparger from 100 to 20 cm3/min resulted in reduced growth (2.2 x 1010 CFU/ml) but with a threefold increase in toxin production (30 p.g/ml). This could indicate that less 02 is required for toxin production than is required for growth. Alternatively, higher than optimal 0, levels could inhibit TSST-1 production. Excess 02 could be toxic by a variety of possible mechanisms: inhibition of enzyme systems, oxidation of 02-sensitive compounds (e.g., SH group-containing substances), or formation of free radicals (20). The degree of aeration and DO level have been shown to influence the production of other staphylococcal products. Carpenter and
0 9
107
s
106
a
HOURS
FIG. 1. Parameters for S. aureus FRI-1169 incubated at 370C for 24 h with a stirrer speed of 280 rpm and aeration with 100 cm3 of air per min. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
Silverman (5) observed that maximal enterotoxin B (SEB) production occurred within a narrow range of aeration rates (125 to 150 cm3/min). A constant DO of 100% resulted in profuse growth but no SEB or nuclease was produced, whereas 10% stimulated synthesis of both products. They hypothesized that production of SEB and nuclease was controlled by the overall energy balance of the cell. At high DO levels, being in a favorable energy state, the cells grew profusely and did not elaborate this enzyme or toxin. At low DO concentrations, an unfavorable energy state was accompanied by an accumulation of reduced NAD, which in turn led to the induction or depression of SEB and nuclease production. Protease (1) and coagulase (6) were also reported to be produced optimally by S. aureus strains under oxygen-limited growth conditions. Effect of carbon dioxide. Growth rates when the gases were supplied by the tube method were similar with or without CO2 (Fig. 3, Table 1); however, the exponential TSST-1 production rates were almost double with C02, resulting in a sharp initial increase in TSST-1, with 5 to 10 times higher toxin yields. Increasing the CO2 flow rates depressed total growth and toxin production, but TSST-1 production was more efficient, as the amount (micrograms) produced per 1010 CFU was higher (Table 1). Applying the same combination of gases by sparger resulted in a fivefold increase in growth (3.5 x 1010 CFU/ml) and a sixfold increase in toxin production (96 ,ug/ml). The most effective combination by sparger, 20 cm3 of air and 5 cm3 of CO2 per min, yielded 120 ,ug of TSST-1 per ml, with less than one-half the growth (1.6 x 1010 CFU/ml). It is possible that additional growth and toxin production would have occurred if the experiments were carried beyond 24 h.
1028
INFECT. IMMUN.
WONG AND BERGDOLL o'I
1011
10
1U
.
IL0
_1
0 cx
co x
109
I
0 I-
-1
.i
a N
0I-
11.
iL ci
a
o
0
108
0.1
10
0.01
L 610 -
0
4
S
12 HOURS
16
20
24
24
0.001
100
_
0 s
0
0
HOURS
FIG. 2. Parameters for S. aureus FRI-1169 incubated at 37°C for 24 h, with a stirrer speed of 280 rpm, and aerated with 100 cm3 of air per min with a sintered glass sparger. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
FIG. 3. Parameters for S. aureus FRI-1169 incubated at 37°C for 24 h with a stirrer speed of 280 rpm and aeration with 100 cm3 of air per min and 10 cm3 of CO2 per min. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
Production of TSST-1 by strains FRI-1123, FRI-1183, FRI-1187, and Harrisburg, which produce about 1/10 as much TSST-1 as strain FRI-1169 by the shake-flask method, was greatly enhanced by the sparger method from approximately 1 to >50 ,ug/ml for all strains. Production of SEA and SEC was affected only slightly for strains that produced
INFECTION AND IMMUNITY, Apr. 1990, p. 1026-1029 0019-9567/90/041026-04$02.00/0 Copyright © 1990, American Society for Microbiology
Effect of Environmental Conditions on Production of Toxic Shock Syndrome Toxin 1 by Staphylococcus aureus AMY C. LEE WONG* AND MERLIN S. BERGDOLL Food Research Institute, University of Wisconsin, Madison, Wisconsin 53706 Received 24 July 1989/Accepted 17 January 1990
The kinetics of toxic shock syndrome toxin 1 (TSST-1) production by Staphylococcus aureus was studied in fermentor in which aeration rate, atmospheric composition, pH, and temperature were controlled. The toxin was synthesized at a maximal rate during the exponential phase. High bacterial populations were not necessarily accompanied by high TSST-1 yields. Aerobiosis increased TSST-1 production, but excessive aeration had an adverse effect. Addition of CO2 enhanced TSST-1 yield by increasing toxin production rate and efficiency. Cultures with no pH control made more TSST-1 than those maintained at pH 5.5 to 7.5. Maximum TSST-1 yields were obtained when cultures were supplied with air (20 cm3/min) and CO2 (5 cm3/min) via a sintered glass sparger. a
Mass.) and introduced into the medium via a glass tube (internal diameter, 1 mm) which was inserted through the headplate. When specified, a 12-mm-diameter coarse sintered glass sparger (Corning Glass Works, Corning, N.Y.) was used. Sterilized silicone antifoam (a 1:10 dilution of B emulsion; Dow Corning Corp., Midland, Mich.) was used when excessive foaming occurred. The gas flow rate was controlled by a flowmeter (Union Carbide Corp., Linde Division, Somerset, N.J.). Dissolved oxygen (DO) was detected with an autoclavable galvanic DO probe (Model M1016-0208; New Brunswick) which was connected to a DO analyzer (Model DO-50; New Brunswick). The pH of the culture was monitored with an autoclavable pH electrode (type 465-35 or 465-35-90-K9; Ingold Electrodes, Inc., Wilmington, Mass.). Automatic pH control was maintained by a pH controller (Model pH22; New Brunswick), using 1 M NaOH and HCI solutions. The DO probe, pH electrode, air filter, and connecting tubes were autoclaved separately and placed aseptically into their respective positions. The percent DO pH, and temperature of the medium were allowed to equilibrate to the appropriate controlled values before inoculation. Incubation conditions. The inoculum was 3.5 ml of an overnight culture of the specified strain. Unless stated otherwise, the strain used was FRI-1169, the initial staphylococcal population was about 107 CFU/ml, the incubation temperature was 37°C, and the culture was agitated at 280 rpm by a magnetic stirrer. Samples were removed at specified times. Methods of analysis. Bacterial growth was determined by surface plating the appropriate dilutions and culture fluids on plate count agar plates (Difco Laboratories, Detroit, Mich.). TSST-1, SEA, and SEC were quantitated by a single gel diffusion tube method (10) with a detection limit of 0.5 jig/ml. In addition, when the concentration was 95% purity by disc polyacrylamide gel electrophoresis [11]) were prepared by column chromatography (13). Results presented are the averages of duplicate experiments.
Production by Staphylococcus aureus of toxic shock syndrome toxin 1 (TSST-1) (2), a major causative agent of toxic shock syndrome (TSS), is affected by environmental conditions. Aerobic conditions have been shown to stimulate the production of TSST-1, while anaerobiosis is inhibitory (17; H. F. Pickrum, D. L. Lucas, and R. L. Stone, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, B57, p. 27). However, high aeration rates in continuous cultures decrease toxin production (15). Cultures incubated aerobically make more TSST-1 with added CO2 than without (9, 18). Sarafian and Morse (15) noted that production of TSST-1 does not begin until after the exponential growth phase. Schlievert and Blomster (16) observed that toxin is produced during the logarithmic phase, and maximal production occurs just before the stationary phase. Taylor and Holland (17) showed that TSST-1 is produced maximally at a suboptimal specific growth rate. In this study, TSST-1 production was monitored throughout the growth cycle of S. aureus by using a bench top fermentor which allowed continuous measurement and control of aeration rates, atmospheric composition, pH, and temperature. MATERIALS AND METHODS Strains. S. aureus FRI-1123 (TSST-1), FRI-1169 (TSST-1), FRI-1183 (TSST-1 and enterotoxin C [SEC]), FRI-1187 (TSST-1, enterotoxin A [SEA], and SEC], and Harrisburg (TSST-1 and SEA), originally isolated from patients with TSS, were used. The Harrisburg strain was obtained originally from R. L. Stone, The Procter & Gamble Co., Cincinnati, Ohio. Strains were stored in the dried form on porcelain beads (8). Medium. The culture medium consisted of 3% NZ Amine NAK, a pancreatic digest of casein (Sheffield Products, Memphis, Tenn.), and 1% yeast extract (3+1 medium) (pH 6.5). Apparatus for controlled culture conditions. A New Brunswick Bioflo bench top fermentor (Model C30; New Brunswick Scientific Co., Inc., New Brunswick, N.J.) was used. The fermentor vessel was filled with 350 ml of 3 + 1 medium and sterilized by autoclaving. Gases were sterilized with an autoclavable bacteriological filter (Balston, Inc., Lexington, *
Corresponding author. 1026
PRODUCTION OF TSST-1
VOL. 58, 1990 11
TABLE 1. Effect of gas flow rate on growth of S. aureus FRI-1169 and on TSST-1 productiona Flow rate (cm3/min)
Air
CO2
(min) 0 50 100 100 100 100 200 400 0
0 0 0 5 10 20 0 0 5
N2 (100 cm3/min) a
34.5 25.7 26.1 23.1 25.3 27.8 26.2 22.7 39.2 46.0
10
TSST-1
Growth
Generation time
1027
CFU
100U
~ gX
(0m)
pgm
0.51 1.10 1.30 1.30
0.3 1.5 1.9 19.8
0.65 0.28 1.10 0.73 0.25 0.09
15.3 10.5 1.3 0.9 0.3 0.0
g11
CFU
10
F
0.6 1.4
1.5 15.2 23.5 37.5 1.2 1.2 1.4 0.0
108
le8
Incubation for 24 h at 37°C; stirrer speed, 280 rpm. IL
RESULTS AND DISCUSSION Effect of air flow rate. The exponential growth rates of FRI-1169 aerated at 50 to 400 cm3/min by the tube method were slightly higher than that of an unaerated culture, resulting in shorter generation times (Table 1). Cell yields after 24 h were 1.4 to 2.5 times higher with aeration. TSST-1 was detected at the mid- to late logarithmic phase, when production was at a maximal exponential rate. TSST-1 was produced at a higher rate relative to growth rate, as the productivity (defined as micrograms of TSST-1 per 1010 CFU) continued to increase. Although strain FRI-1169 grew to about 109 CFU/ml under completely anaerobic conditions, only 0.008 p.g of TSST-1 per ml was produced. Effect of DO. The exponential growth phase ended when the DO of aerated cultures (100 cm3 of air per min) dropped in 0%, for both the tube and sparger aerated cultures (Fig. 1 and 2), indicating that insufficient 02 was available to sustain exponential growth. However, the major differences in the tube and sparger aerations were as follows: (i) the exponential growth phase with the sparger was much longer, indicating an increased efficiency in providing oxygen to the culture, and (ii) when maximum growth of 6 x 1010 CFU/ml was reached (12 h) in the sparger aerated culture, the DO returned to above 0%, whereas in the tube aerated culture, growth was still taking place at 24 h and had reached only 1.3 x 1010 CFU/ml, with the DO remaining at 0%. The amount of toxin produced was about the same for both aeration methods (1.9 ,ug/ml) at the same growth level (1010 CFU/ml), but because of the increased growth with the sparger aeration, an additional 8.1 ,ug of toxin per ml was produced. Although more TSST-1 was produced aerobically than anaerobically, the fact that TSST-1 continued to be synthesized after the DO level decreased to 0% (Fig. 1 and 2) indicated that full aerobiosis was not essential. Decreasing the amount of air supplied with the sparger from 100 to 20 cm3/min resulted in reduced growth (2.2 x 1010 CFU/ml) but with a threefold increase in toxin production (30 p.g/ml). This could indicate that less 02 is required for toxin production than is required for growth. Alternatively, higher than optimal 0, levels could inhibit TSST-1 production. Excess 02 could be toxic by a variety of possible mechanisms: inhibition of enzyme systems, oxidation of 02-sensitive compounds (e.g., SH group-containing substances), or formation of free radicals (20). The degree of aeration and DO level have been shown to influence the production of other staphylococcal products. Carpenter and
0 9
107
s
106
a
HOURS
FIG. 1. Parameters for S. aureus FRI-1169 incubated at 370C for 24 h with a stirrer speed of 280 rpm and aeration with 100 cm3 of air per min. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
Silverman (5) observed that maximal enterotoxin B (SEB) production occurred within a narrow range of aeration rates (125 to 150 cm3/min). A constant DO of 100% resulted in profuse growth but no SEB or nuclease was produced, whereas 10% stimulated synthesis of both products. They hypothesized that production of SEB and nuclease was controlled by the overall energy balance of the cell. At high DO levels, being in a favorable energy state, the cells grew profusely and did not elaborate this enzyme or toxin. At low DO concentrations, an unfavorable energy state was accompanied by an accumulation of reduced NAD, which in turn led to the induction or depression of SEB and nuclease production. Protease (1) and coagulase (6) were also reported to be produced optimally by S. aureus strains under oxygen-limited growth conditions. Effect of carbon dioxide. Growth rates when the gases were supplied by the tube method were similar with or without CO2 (Fig. 3, Table 1); however, the exponential TSST-1 production rates were almost double with C02, resulting in a sharp initial increase in TSST-1, with 5 to 10 times higher toxin yields. Increasing the CO2 flow rates depressed total growth and toxin production, but TSST-1 production was more efficient, as the amount (micrograms) produced per 1010 CFU was higher (Table 1). Applying the same combination of gases by sparger resulted in a fivefold increase in growth (3.5 x 1010 CFU/ml) and a sixfold increase in toxin production (96 ,ug/ml). The most effective combination by sparger, 20 cm3 of air and 5 cm3 of CO2 per min, yielded 120 ,ug of TSST-1 per ml, with less than one-half the growth (1.6 x 1010 CFU/ml). It is possible that additional growth and toxin production would have occurred if the experiments were carried beyond 24 h.
1028
INFECT. IMMUN.
WONG AND BERGDOLL o'I
1011
10
1U
.
IL0
_1
0 cx
co x
109
I
0 I-
-1
.i
a N
0I-
11.
iL ci
a
o
0
108
0.1
10
0.01
L 610 -
0
4
S
12 HOURS
16
20
24
24
0.001
100
_
0 s
0
0
HOURS
FIG. 2. Parameters for S. aureus FRI-1169 incubated at 37°C for 24 h, with a stirrer speed of 280 rpm, and aerated with 100 cm3 of air per min with a sintered glass sparger. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
FIG. 3. Parameters for S. aureus FRI-1169 incubated at 37°C for 24 h with a stirrer speed of 280 rpm and aeration with 100 cm3 of air per min and 10 cm3 of CO2 per min. Symbols: 0, CFU per milliliter; *, micrograms of TSST-1 per milliliter; O, micrograms of TSST-1 per 1010 CFU; A, percent DO; A, pH.
Production of TSST-1 by strains FRI-1123, FRI-1183, FRI-1187, and Harrisburg, which produce about 1/10 as much TSST-1 as strain FRI-1169 by the shake-flask method, was greatly enhanced by the sparger method from approximately 1 to >50 ,ug/ml for all strains. Production of SEA and SEC was affected only slightly for strains that produced
