APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 600-601 Copyright © 1977 American Society for Microbiology
Vol. 34, No. 5 Printed in U.S.A.
Comparative Dose-Survival Curves of Representative Clostridium botulinum Type F Spores with Type A and B Spores ABE ANELLIS* AND D. BERKOWITZ Food Sciences Laboratory, U. S. Arny Natick Research and Development Command, Natick, Massachusetts 01 760
Received for publication 24 May 1977
Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with doseresponse data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters. Clostridium botulinum type F was unknown before 1960. Since then, several isolations (2, 4, 5, 7-9, 14) of the organism and encounters with its toxin (10, 12, 13) have been reported, mainly from aquatic sources. Due to the relative newness of this serotype, very little has been published on its radiation resistance. Using an aqueous spore suspension of the first reported isolate, the Langeland strain (9), Williams-Walls (15) observed approximately a 2-log reduction with a radiation dose of 0.3 Mrad of gamma rays (137Cs at 00C). In a more definitive irradiation study, she (11) obtained dose-survival curves (at 00°) for this strain and two of its laboratory mutants (OGB-69, OGB69R) in 0.067 M phosphate buffer (composition and pH not given). Interpolation of her doseresponse curve for the parent strain yielded an initial shoulder of 0.368 Mrad and a D value (the dose that reduced a spore population by 1-log cycle) of 0.171 Mrad. Although the incidence and distribution of type F, thus far, appear to be relatively uncommon compared to types A through E, it was of interest to compare the radioresistance of representative strains of type F spores with those of characteristic type A and B spores used by us to establish radappertization doses for prototype military foods. Spore crops of strains 33A, 40B, Eklund 83F (non-proteolytic), and Walls 8G-F (proteolytic) were produced and enumerated as cited previously (1), except that Sorensen phosphate buffer (0.067 M, pH 7.0) was used for washing and suspending of the spores. The washed cells were diluted in buffer to about 107 per ml, dispensed aseptically in 5.5-ml quantities in sterile Pyrex
tubes (16 by 150 mm), vacuum sealed, and frozen in solid C02-acetone (80°C). Duplicate tubes were irradiated in the dose range 0 to 1.2 Mrad in units of 0.1 Mrad with 'Co-gamma rays at -80 ± 2°C; the dose rate was 66.2 krad per min, and the transient dose was 42.0 krad. The tubes were thawed overnight (16 h) at 3 ± 10C, vortex mixed, diluted decimally with chilled sterile distilled water, and enumerated as indicated above. The semilog dose-survival curves in Fig. 1 represent average survival counts of 12-count tubes per dilution for each test organism. Table 1 shows the initial shoulder, or lag, and the D value for each test organism. The shoulders were obtained both graphically (3) and by ElBisi's (6) expression, s = D (log S + x/D), where s is the shoulder and S is the surviving spore fraction at dose x. The D values also were elicited graphically (by taking the reciprocals of the slopes of the linear regression curves) and computed by the relationship D = (s -x) logS, derived from El-Bisi's formula. The data required for solving the two equations were selected arbitrarily from the approximate midpoints of the exponential portions of survival curves as the best estimates. The non-proteolytic type F strain, Eklund 83F, was as radiation resistant as our indicator strain 33A, and was slightly less sensitive than our 40B indicator strain; the proteolytic type F strain, Walls 8G-F, was the most sensitive of the four tested (Table 1). Although the Langeland parent strain was examined under conditions somewhat different from ours (11), it appeared to be more, or at least not less, resistant than our strain 33A. If additional experience should indicate that 600
NOTES
VOL. 34, 1977
the incidence and/or distribution of type F C. botulinum are more widespread than currently known, then a more extensive investigation into the comparative radioresistance of this new type will be required for any furture radiation-inoculated pack studies. Furthermore, since type F cells and toxin apparently share the same aquatic environment with type E cells, it becomes essential that processes, used to prevent type E damage to seafood, should also be examined for protection against the type F hazard. The empirical equations used to compute the s and D values of our dose-survival curves agreed very closely with the graphic methods for obtaining these parameters (Table 1); thus, the two methods may be used interchangeably.
33A
-1
co
-2
-3
C=
-4
i
_
-5
-6
-7
0
0.2
0.4
0.6
0.8
1.0
1.2
MRAD
FIG. 1. Comparative dose-survival curves at -80' C of representative C. botulinum spores types A, B, and F suspended in phosphate buffer.
601
LITERATURE CITED 1. Anellis, A., D. Berkowitz, D. Kemper, and D. B. Rowley. 1972. Production of types A and B spores of Clostridium botulinum by the biphasic method: effect on spore population, radiation resistance, and toxigenicity. Appl. Microbiol. 23:734-739. 2. Craig, J. M., and K. S. Pilcher. 1966. Clostridium botulinum type F: isolation from salmon from the Columbia River. Science 153:311-312. 3. Durban, E., and N. Greez. 1969. Resistance of spores of Clostridium botulinum 33A to combinations of ultraviolet and gamma rays. Appl. Microbiol. 18:44-50. 4. Eklund, M. W., and F. Poysky. 1965. Clostridium botulinum type F from marine sediments. Science 149:306. 5. Eklund, M. W., F. Poysky, and D. Wieler. 1967. Demonstration and isolation of Cl. botulinum type F from the Pacific coast of the United States, p. 443-447. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall Ltd., London. 6. El-Bisi, H. M. 1967. Radiation death kinetics of Cl. botulinum spores at cryogenic temperatures, p. 89-107. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall, Ltd., London. 7. Gimenez, D. F., and A. S. Ciccareili. 1968. Clostridium botulinum type F in the soil of Argentina. Appl. Microbiol. 16:732-734. 8. Kautter, D. A., T. Lilly, Jr., A. J. Le Blanc, and R. A. Lynt. 1974. Incidence of Clostridium botulinum in crabmeat from the blue crab. Appl. Microbiol. 28:722. 9. Moller, V., and I. Scheibel. 1960. Preliminary report on the isolation of an apparently new type of Clostridium botulinum. Acta Pathol. Microbiol. Scand. 48:80. 10. U. S. Department of Health, Education, and Welfare. 1966. Botulism type F-Califomia. Morbid. Mortal. Weekly Rep. 15:359. 11. Walls, N. W. 1967. Physiological studies on Cl. botulinum, type F, p. 158-168. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall, Ltd., London. 12. Ward, B. Q., B. J. Carroll, E. S. Garrett, and G. B. Reese. 1967. Survey of the U. S. Gulf Coast for the presence of Clostridium botulinum. Appl. Microbiol. 15:629-636. 13. Wentz, M. W., R. A. Scott, and J. W. Vennes. 1967. Clostridium botulinum type F: seasonal inhibition by Bacillus licheniformis. Science 155:89-90. 14. Williams-Walls, N. J. 1968. Clostridium botulinum type F: isolation from crabs. Science 162:375-376. 15. WilliaMs-Walls, N. J. 1969. Effects on growth and toxin production of exposure of spores of Clostridium botulinum type F to sublethal doses of gamma irradiation. Appl. Microbiol. 17:128-134.
TABLE 1. Comparative resistancea to gamma irradiation at -80°C of representative C. botulinum spores types A, B, and F D value (Mrad)h Shoulder (Mrad) Strain
33Ac d 40Bc d
Graphic 0.335 0.425 0.305 0.365
Computed
Graphic
Computed
0.332 0.428 0.296 0.378
0.158 0.134 0.116 0.153
0.156
Walls 8G-Fd Eklund 83Fe aBased on survival counts. bIn Sorensen phosphate buffer, 0.067 M, pH 7.0. One of 10 strains used to establish a radiation 12 D dose for foods. d Proteolytic strain. eNon-proteolytic strain. c
0.134 0.114 0.157
Vol. 34, No. 5 Printed in U.S.A.
Comparative Dose-Survival Curves of Representative Clostridium botulinum Type F Spores with Type A and B Spores ABE ANELLIS* AND D. BERKOWITZ Food Sciences Laboratory, U. S. Arny Natick Research and Development Command, Natick, Massachusetts 01 760
Received for publication 24 May 1977
Radiation survival data of proteolytic (Walls 8G-F) and non-proteolytic (Eklund 83F) type F spores of Clostridium botulinum were compared with doseresponse data of radiation-resistant type A (33A) and B (40B) spores. Strain Eklund 83F was as resistant as strain 33A, whereas strain Walls 8G-F was the most sensitive of the four strains tested. The methods suggested for computing both an initial shoulder and a D value for the dose-survival curves yielded results comparable to the graphic techniques used to obtain these two parameters. Clostridium botulinum type F was unknown before 1960. Since then, several isolations (2, 4, 5, 7-9, 14) of the organism and encounters with its toxin (10, 12, 13) have been reported, mainly from aquatic sources. Due to the relative newness of this serotype, very little has been published on its radiation resistance. Using an aqueous spore suspension of the first reported isolate, the Langeland strain (9), Williams-Walls (15) observed approximately a 2-log reduction with a radiation dose of 0.3 Mrad of gamma rays (137Cs at 00C). In a more definitive irradiation study, she (11) obtained dose-survival curves (at 00°) for this strain and two of its laboratory mutants (OGB-69, OGB69R) in 0.067 M phosphate buffer (composition and pH not given). Interpolation of her doseresponse curve for the parent strain yielded an initial shoulder of 0.368 Mrad and a D value (the dose that reduced a spore population by 1-log cycle) of 0.171 Mrad. Although the incidence and distribution of type F, thus far, appear to be relatively uncommon compared to types A through E, it was of interest to compare the radioresistance of representative strains of type F spores with those of characteristic type A and B spores used by us to establish radappertization doses for prototype military foods. Spore crops of strains 33A, 40B, Eklund 83F (non-proteolytic), and Walls 8G-F (proteolytic) were produced and enumerated as cited previously (1), except that Sorensen phosphate buffer (0.067 M, pH 7.0) was used for washing and suspending of the spores. The washed cells were diluted in buffer to about 107 per ml, dispensed aseptically in 5.5-ml quantities in sterile Pyrex
tubes (16 by 150 mm), vacuum sealed, and frozen in solid C02-acetone (80°C). Duplicate tubes were irradiated in the dose range 0 to 1.2 Mrad in units of 0.1 Mrad with 'Co-gamma rays at -80 ± 2°C; the dose rate was 66.2 krad per min, and the transient dose was 42.0 krad. The tubes were thawed overnight (16 h) at 3 ± 10C, vortex mixed, diluted decimally with chilled sterile distilled water, and enumerated as indicated above. The semilog dose-survival curves in Fig. 1 represent average survival counts of 12-count tubes per dilution for each test organism. Table 1 shows the initial shoulder, or lag, and the D value for each test organism. The shoulders were obtained both graphically (3) and by ElBisi's (6) expression, s = D (log S + x/D), where s is the shoulder and S is the surviving spore fraction at dose x. The D values also were elicited graphically (by taking the reciprocals of the slopes of the linear regression curves) and computed by the relationship D = (s -x) logS, derived from El-Bisi's formula. The data required for solving the two equations were selected arbitrarily from the approximate midpoints of the exponential portions of survival curves as the best estimates. The non-proteolytic type F strain, Eklund 83F, was as radiation resistant as our indicator strain 33A, and was slightly less sensitive than our 40B indicator strain; the proteolytic type F strain, Walls 8G-F, was the most sensitive of the four tested (Table 1). Although the Langeland parent strain was examined under conditions somewhat different from ours (11), it appeared to be more, or at least not less, resistant than our strain 33A. If additional experience should indicate that 600
NOTES
VOL. 34, 1977
the incidence and/or distribution of type F C. botulinum are more widespread than currently known, then a more extensive investigation into the comparative radioresistance of this new type will be required for any furture radiation-inoculated pack studies. Furthermore, since type F cells and toxin apparently share the same aquatic environment with type E cells, it becomes essential that processes, used to prevent type E damage to seafood, should also be examined for protection against the type F hazard. The empirical equations used to compute the s and D values of our dose-survival curves agreed very closely with the graphic methods for obtaining these parameters (Table 1); thus, the two methods may be used interchangeably.
33A
-1
co
-2
-3
C=
-4
i
_
-5
-6
-7
0
0.2
0.4
0.6
0.8
1.0
1.2
MRAD
FIG. 1. Comparative dose-survival curves at -80' C of representative C. botulinum spores types A, B, and F suspended in phosphate buffer.
601
LITERATURE CITED 1. Anellis, A., D. Berkowitz, D. Kemper, and D. B. Rowley. 1972. Production of types A and B spores of Clostridium botulinum by the biphasic method: effect on spore population, radiation resistance, and toxigenicity. Appl. Microbiol. 23:734-739. 2. Craig, J. M., and K. S. Pilcher. 1966. Clostridium botulinum type F: isolation from salmon from the Columbia River. Science 153:311-312. 3. Durban, E., and N. Greez. 1969. Resistance of spores of Clostridium botulinum 33A to combinations of ultraviolet and gamma rays. Appl. Microbiol. 18:44-50. 4. Eklund, M. W., and F. Poysky. 1965. Clostridium botulinum type F from marine sediments. Science 149:306. 5. Eklund, M. W., F. Poysky, and D. Wieler. 1967. Demonstration and isolation of Cl. botulinum type F from the Pacific coast of the United States, p. 443-447. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall Ltd., London. 6. El-Bisi, H. M. 1967. Radiation death kinetics of Cl. botulinum spores at cryogenic temperatures, p. 89-107. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall, Ltd., London. 7. Gimenez, D. F., and A. S. Ciccareili. 1968. Clostridium botulinum type F in the soil of Argentina. Appl. Microbiol. 16:732-734. 8. Kautter, D. A., T. Lilly, Jr., A. J. Le Blanc, and R. A. Lynt. 1974. Incidence of Clostridium botulinum in crabmeat from the blue crab. Appl. Microbiol. 28:722. 9. Moller, V., and I. Scheibel. 1960. Preliminary report on the isolation of an apparently new type of Clostridium botulinum. Acta Pathol. Microbiol. Scand. 48:80. 10. U. S. Department of Health, Education, and Welfare. 1966. Botulism type F-Califomia. Morbid. Mortal. Weekly Rep. 15:359. 11. Walls, N. W. 1967. Physiological studies on Cl. botulinum, type F, p. 158-168. In M. Ingram and T. A. Roberts (ed.), Botulism 1966. Chapman and Hall, Ltd., London. 12. Ward, B. Q., B. J. Carroll, E. S. Garrett, and G. B. Reese. 1967. Survey of the U. S. Gulf Coast for the presence of Clostridium botulinum. Appl. Microbiol. 15:629-636. 13. Wentz, M. W., R. A. Scott, and J. W. Vennes. 1967. Clostridium botulinum type F: seasonal inhibition by Bacillus licheniformis. Science 155:89-90. 14. Williams-Walls, N. J. 1968. Clostridium botulinum type F: isolation from crabs. Science 162:375-376. 15. WilliaMs-Walls, N. J. 1969. Effects on growth and toxin production of exposure of spores of Clostridium botulinum type F to sublethal doses of gamma irradiation. Appl. Microbiol. 17:128-134.
TABLE 1. Comparative resistancea to gamma irradiation at -80°C of representative C. botulinum spores types A, B, and F D value (Mrad)h Shoulder (Mrad) Strain
33Ac d 40Bc d
Graphic 0.335 0.425 0.305 0.365
Computed
Graphic
Computed
0.332 0.428 0.296 0.378
0.158 0.134 0.116 0.153
0.156
Walls 8G-Fd Eklund 83Fe aBased on survival counts. bIn Sorensen phosphate buffer, 0.067 M, pH 7.0. One of 10 strains used to establish a radiation 12 D dose for foods. d Proteolytic strain. eNon-proteolytic strain. c
0.134 0.114 0.157
