APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1976, p. 145-158 Copyright © 1976 American Society for Microbiology
Vol. 32, No. 1 Printed in U.S.A.
Observations on Bacteriophages of Clostridium botulinum Type C Isolates from Different Sources and the Role of Certain Phages in Toxigenicity H. HARIHARAN* AND W. R. MITCHELL
Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario NIG 2W1, Canada Received for publication 19 February 1976
Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.
Though publications to date (10, 11, 17, 18, 20) on phage conversion in type C and D organisms have considerably helped to elucidate the mechanism of conversion of nontoxigenic forms to toxigenic form, and vice versa, the factors responsible for varying toxigenic properties and varying degrees of toxigenic stability of different strains have not been adequately explained. It has been postulated that highly toxigenic strains have a better chance to initiate the disease in the host. Since in nature birds are the main victims of type C botulism, it was considered necessary to characterize and study the phages of strains connected to avian botulism. In a liquid culture, whether phage-bacterium interaction finally results in a highly toxigenic culture or a weakly toxigenic culture is important, but this difference has not been emphasized in the phage-conversion experiments dealing with Clostridium botulinum type C. There was a ineed to go a step further from the basic laboratory studies on toxigenic conver-
sions towards a natural system where the disease occurs only when a minimum lethal dose of toxin is available to the host. MATERIALS AND METHODS Cultures. Strains NS1, NS2, NS3, and NS4 were isolated from soil specimens obtained from Nicholson's Island (NI), an area in Ontario endemic for avian botulism. Strains NPG1, NPG2, and NPG3 were from digestive tracts, and strain NPL1 was from the livers of healthy pheasants from Nicholson's Island. Isolates TML1, TML2, and TMG were obtained from the tissue of mallards that died in a type C botulism outbreak in Sunnyside Beach, Toronto, Ontario. Strains 6810 and 6811 were of terrestrial origin and strains 6812, 6813, and 6814 of marine origin, all of which were from W. P. Segner, Continental Can Co., Chicago, Ill. Strain Stockholm C was from K. Inoue, Hokkaido Institute of Public Health, Hokkaido, Japan, and strain X220B2 was from W. I. Jensen, Bear River Research Station, Utah. Strains CaC and CI3C were from Connaught Laboratories, and strains A028 (nontoxigenic) and A028 were reinfected with phages 1CIx+ 145
146
APPL. ENVIRON. MICROBIOL.
HARIHARAN AND MITCHELL
and 2C1x- (CE,B and CEa prepared according to the method of Eklund et al. 111] were from M. W. Eklund, National Marine and Fisheries Service, Seattle, Wash.). Toxigenic potencies of different strains were determined on mice. From freshly grown cultures, 0.2ml quantities of sediments were inoculated into 20 ml of air-exhausted fortified egg-meat medium (FEM) (11) and incubated for 5 days at 33°C. The mean lethal doses (LD.,,,) of culture supernatants were determined according to the method of Reed and Muench (21). Strains NS3, NS4, NPG3, and NPL1 produced less than 450 mouse LD;, per ml of toxin and were designated as hypotoxigenic. All other strains except X220B2 produced 37,500 to 425,000 mouse LD-,5 of toxin per ml and were designated as highly toxigenic. Strain X220B2 was classified as intermediate. Preparation of phage suspensions. For electron microscopic examination, phage suspensions were prepared according to the method of Eklund et al. (9), except that cultures were grown on TYG medium (8). Specimens were examined in a Phillips EM 200 microscope. For phage-conversion experiments, toxin-free phage suspensions were prepared. Culture lysates were prepared and concentrated as for electron microscopy, except that TYG broth instead of ammonium acetate solution was used as a suspending medium for phages. Before carrying out high-speed centrifugation, the lysates were filtered through a Millipore membrane of 0.45-,um size. The phage pellet obtained was suspended in TYG and centrifuged at high speed twice further to remove all the free toxin. The suspensions were treated with 40 Ag of crystalline deoxyribonuclease per ml for 3 h at 30°C, filter sterilized, and checked for freedom from toxin by injecting 0.4 ml intraperitoneally into two 20- to 25-g white mice. Assay of phage suspension. All the phage concentrates were tested on strain A028 as host to determine whether or not phages carrying lysis were present in the concentrate and to find out the amount of such phages, by employing the lysis of broth culture method (1). Conversion testing. Phage suspensions of all strains were tested for their ability in toxigenic conversion of strain A028. Salient steps of the procedure are shown in Fig. 1. A 0.1-ml portion of the culture was incubated into 10 ml of freshly prepared TYG broth filled in screw-capped tubes having the same optical density and incubated at 33°C until the optical density reached a value of 0.25 to 0.3 (Spectronic 20 at 525 nm; Bausch and Lomb, Inc., Rochester, N.Y.). Fresh TYG tubes were inoculated with 1 ml of the 0.3-optical density culture. Phage suspensions were added at 0.01-, 0.1-, and 1-ml quantities, and the tubes were incubated for 4 h. A 1-ml quantity of the culture thus treated with phage suspension was inoculated into 20 ml of FEM in duplicate and incubated at 33°C for 5 days. The cultures were centrifuged to sediment the bacteria, and the supernatants were injected in 0.4-ml quantities into two mice. The mice were observed for 4 days, and deaths were recorded. Approximate LD5, values were calculated. Culture supernatants containing less than
500 mouse LD.,,, of toxin per ml were considered as hypotoxigenic, and those containing more than 30,000 LD.5, toxin per ml were considered as highly toxigenic. Further phage characterization procedures. (i) Preparation of anti-phage sera. Concentrated phage suspensions of phage 1CIx',+ with a titer of 10-"' (as determined by lysis of broth culture method) were prepared, and free toxin was removed by washing the phage several times with phosphate buffer, pH 7.00. Two rabbits were injected intramuscularly with 2 ml of a mixture (1:1) of complete Freund adjuvant and the phage stock. Along with this injection, 3 ml of a phage suspension was given intravenously. After the first day, the rabbits received 1 ml of phage suspension intramuscularly twice weekly for 2 weeks. Two weeks after the last inoculation all the rabbits were bled by cardiac puncture, and the serum sample was pooled. The "k" value (1) of 1CIx'+ antiserum was determined against the homologous phage. The procedure was similar to that described by Adams (1). (ii) Plaque-forming ability on host strain A028. The phage suspensions from various strains were tested on strain A028 by an agar overlay procedure similar to that described by Eklund and Poysky (8). (iii) Phage identification by determination of antigenic similarity. For the determination of antigenic similarity of plaque-forming phages with phage 1CI"X+, dilutions of phage suspensions were mixed with 1CI'x+ antiserum and plated at the end of a 15-min incubation at 37°C by the agar overlay procedure on A028. Identification of phages that did not produce plaques on A028 but at the same time converted nontoxigenic A028 to toxigenic form in broth was attempted by the determination of ability of anti-phage 1C'l'x+ serum to inhibit toxigenic conversion. Phage suspension in 0.3-mi quantities was mixed with equal quantities of anti-1C0x+ serum and incubated for 15 min. Immediately after the incubation period, 0.2-ml quantities of the phageantiserum and 1 ml of actively growing A028 culture at 0.25 to 0.3 optical density units were inoculated
Nontoxigenic A028
(Actively growing) OD = 0.3 1 ml
Phage concentrate (sterile, toxin-free, deoxyribonuclease-treated) 0.1 ml
TYG broth 4-h incubation 1 ml
FEM (20 ml) t 5-day incubation Culture supernatant FIG. 1. Assay of toxicity in mice, using the broth method ofphage conversion. OD, Optical density.
VOL. 32, 1976
TOXIGENIC CONVERSION OF C. BOTULINUM TYPE C
into fresh TYG medium, and the conversion testing procedure was carried out as described earlier. Phage suspensions incubated with normal rabbit serum were tested as controls. Additional experiments for elucidating phagemediated variation in toxigenicity. (i) Determination of amount of conversion. The percentage of toxigenic cells was determined on two converted cultures. The first one was a highly toxigenic culture resulting from infection of A028 by phages of strain S-6810. The other was a hypotoxigenic culture resulting from infection of A028 by phages of strain Stockholm. A three-layer plate method (Fig. 2) was used for plating of the converted cultures to avoid injury to cells by oxygen. The culture under study was streaked on a reduced blood agar plate. The first overlay (melted and cooked to 45°C) was poured immediately on the surface of the plate, and as soon as these solidified the second overlay was added on the top. When the overlay was set, the plates were incubated in anaerobic jars with silica gel under the GasPak anaerobic system. After 18 h of incubation at 33°C, the plates were removed and 10 colonies were cultured individually in FEM. The FEM cultures were incubated for 5 days, and mouse LD.5,( of the supernatants was determined. (ii) Determination of lysogenic characteristics of cells from converted cultures. The FEM cultures of the above experiment, originating from different colonies, were tested for their lysogenic properties by determining their sensitivity or resistance to further phage infection. The FEM cultures were subcultured into fresh TYG broth, and agar layer procedures for detection of plaques were carried out on
147
these TYG cultures using the phage suspension of strain Stockholm.
RESULTS Electron microscopy. All toxigenic strains produced bacteriophages. Based on size alone, mainly two morphologically distinct types of phages were found to be present: a large type, similar to phage 1CItx (Fig. 3 and 4 show phage 1Ct"N+ and the large phage of strain NS2), and a small type (Fig. 5, 6, and 7 show small phages of strains 2C1x-, NS1, and 6811). A suitable criterion for the classification of Streak (converted culture)
F
3% TYG agar with sodium thioglycolate 2% TYG agar withJcysteine-hydrochloride Blood
agar t 18-h incubation
10 colonies cultured individually in FEM l 5-day incubation
Mouse LD5,, of supernatant determined FIG. 2. Procedure for determination of amount of
conversion.
FIG. 3. Large phage 1C"'+t exhibiting hexagonal head and partially sheathed tail. x115,500.
148
HARIHARAN AND MITCHELL
APPL. ENVIRON. MICROBIOL.
FIG. 4. Large phage of strain NS2 with hexagonal head and partially sheathed tail. x115,500.
FIG. 5. Small phage 2C'x-r with hexagonal head and a sheathed tail. x115,500.
VOL. 32, 1976
TOXIGENIC CONVERSION OF C. BOTULINUM TYPE C
149
FIG. 6. Small phage of strain NS1 with hexagonal head and a sheath. x115,500.
FIG. 7. Small phage of strain 6811 with hexagonal head and sheathless tail. x115,500.
phages was the tail length. The phages hav- were considered as the "small" phage. A third ing tail lengths in the range of 180 to 390 nm group was the "intermediate" type. (Figure 8 (usually >200 nm) were considered as the shows one of this type of phage of strain 6812.) "large" phage, and phages having tail lengths Though in general the head of the large phage in the range of 60 to 165 nm (usually
Vol. 32, No. 1 Printed in U.S.A.
Observations on Bacteriophages of Clostridium botulinum Type C Isolates from Different Sources and the Role of Certain Phages in Toxigenicity H. HARIHARAN* AND W. R. MITCHELL
Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario NIG 2W1, Canada Received for publication 19 February 1976
Twenty strains of Clostridium botulinum type C, including 12 isolates from avian sources with varying toxigenic properties, were examined by electron microscope for the presence of bacteriophages. All toxigenic strains were infected with one or two types of phages. Three types of phages designated large, small, and intermediate were observed. Most of the strains carried the large and small phage, with the large phage being present in much greater numbers. Since there is evidence that highly toxigenic strains of C. botulinum type C are responsible for large outbreaks of botulism in wild birds, the phenomenon of toxigenic variation among the type C strains was investigated. Experiments were carried out employing a broth medium on a phagefree nontoxigenic strain for elucidating the role of bacteriophages in toxigenicity. All phage suspensions contained large phages, with the exception of one that caused conversion. The exception was a preparation containing an intermediate type of phage. Phages from different strains produced cultures of varying toxigenic characteristics. By employing a tube-lytic test and an agar-overlay-phage assay technique, it was determined that whenever the phage-bacterium relationship resulted in an initial high degree of lysis, the potency of toxin in the culture was weak. It appeared that in highly toxigenic strains, the phage-bacterium relationship is characterized by a stable lysogenic type of association. It was also found that in a highly toxigenic converted culture the percentage of toxigenic cells was 100, whereas in hypotoxigenic culture the percentage was only 20.
Though publications to date (10, 11, 17, 18, 20) on phage conversion in type C and D organisms have considerably helped to elucidate the mechanism of conversion of nontoxigenic forms to toxigenic form, and vice versa, the factors responsible for varying toxigenic properties and varying degrees of toxigenic stability of different strains have not been adequately explained. It has been postulated that highly toxigenic strains have a better chance to initiate the disease in the host. Since in nature birds are the main victims of type C botulism, it was considered necessary to characterize and study the phages of strains connected to avian botulism. In a liquid culture, whether phage-bacterium interaction finally results in a highly toxigenic culture or a weakly toxigenic culture is important, but this difference has not been emphasized in the phage-conversion experiments dealing with Clostridium botulinum type C. There was a ineed to go a step further from the basic laboratory studies on toxigenic conver-
sions towards a natural system where the disease occurs only when a minimum lethal dose of toxin is available to the host. MATERIALS AND METHODS Cultures. Strains NS1, NS2, NS3, and NS4 were isolated from soil specimens obtained from Nicholson's Island (NI), an area in Ontario endemic for avian botulism. Strains NPG1, NPG2, and NPG3 were from digestive tracts, and strain NPL1 was from the livers of healthy pheasants from Nicholson's Island. Isolates TML1, TML2, and TMG were obtained from the tissue of mallards that died in a type C botulism outbreak in Sunnyside Beach, Toronto, Ontario. Strains 6810 and 6811 were of terrestrial origin and strains 6812, 6813, and 6814 of marine origin, all of which were from W. P. Segner, Continental Can Co., Chicago, Ill. Strain Stockholm C was from K. Inoue, Hokkaido Institute of Public Health, Hokkaido, Japan, and strain X220B2 was from W. I. Jensen, Bear River Research Station, Utah. Strains CaC and CI3C were from Connaught Laboratories, and strains A028 (nontoxigenic) and A028 were reinfected with phages 1CIx+ 145
146
APPL. ENVIRON. MICROBIOL.
HARIHARAN AND MITCHELL
and 2C1x- (CE,B and CEa prepared according to the method of Eklund et al. 111] were from M. W. Eklund, National Marine and Fisheries Service, Seattle, Wash.). Toxigenic potencies of different strains were determined on mice. From freshly grown cultures, 0.2ml quantities of sediments were inoculated into 20 ml of air-exhausted fortified egg-meat medium (FEM) (11) and incubated for 5 days at 33°C. The mean lethal doses (LD.,,,) of culture supernatants were determined according to the method of Reed and Muench (21). Strains NS3, NS4, NPG3, and NPL1 produced less than 450 mouse LD;, per ml of toxin and were designated as hypotoxigenic. All other strains except X220B2 produced 37,500 to 425,000 mouse LD-,5 of toxin per ml and were designated as highly toxigenic. Strain X220B2 was classified as intermediate. Preparation of phage suspensions. For electron microscopic examination, phage suspensions were prepared according to the method of Eklund et al. (9), except that cultures were grown on TYG medium (8). Specimens were examined in a Phillips EM 200 microscope. For phage-conversion experiments, toxin-free phage suspensions were prepared. Culture lysates were prepared and concentrated as for electron microscopy, except that TYG broth instead of ammonium acetate solution was used as a suspending medium for phages. Before carrying out high-speed centrifugation, the lysates were filtered through a Millipore membrane of 0.45-,um size. The phage pellet obtained was suspended in TYG and centrifuged at high speed twice further to remove all the free toxin. The suspensions were treated with 40 Ag of crystalline deoxyribonuclease per ml for 3 h at 30°C, filter sterilized, and checked for freedom from toxin by injecting 0.4 ml intraperitoneally into two 20- to 25-g white mice. Assay of phage suspension. All the phage concentrates were tested on strain A028 as host to determine whether or not phages carrying lysis were present in the concentrate and to find out the amount of such phages, by employing the lysis of broth culture method (1). Conversion testing. Phage suspensions of all strains were tested for their ability in toxigenic conversion of strain A028. Salient steps of the procedure are shown in Fig. 1. A 0.1-ml portion of the culture was incubated into 10 ml of freshly prepared TYG broth filled in screw-capped tubes having the same optical density and incubated at 33°C until the optical density reached a value of 0.25 to 0.3 (Spectronic 20 at 525 nm; Bausch and Lomb, Inc., Rochester, N.Y.). Fresh TYG tubes were inoculated with 1 ml of the 0.3-optical density culture. Phage suspensions were added at 0.01-, 0.1-, and 1-ml quantities, and the tubes were incubated for 4 h. A 1-ml quantity of the culture thus treated with phage suspension was inoculated into 20 ml of FEM in duplicate and incubated at 33°C for 5 days. The cultures were centrifuged to sediment the bacteria, and the supernatants were injected in 0.4-ml quantities into two mice. The mice were observed for 4 days, and deaths were recorded. Approximate LD5, values were calculated. Culture supernatants containing less than
500 mouse LD.,,, of toxin per ml were considered as hypotoxigenic, and those containing more than 30,000 LD.5, toxin per ml were considered as highly toxigenic. Further phage characterization procedures. (i) Preparation of anti-phage sera. Concentrated phage suspensions of phage 1CIx',+ with a titer of 10-"' (as determined by lysis of broth culture method) were prepared, and free toxin was removed by washing the phage several times with phosphate buffer, pH 7.00. Two rabbits were injected intramuscularly with 2 ml of a mixture (1:1) of complete Freund adjuvant and the phage stock. Along with this injection, 3 ml of a phage suspension was given intravenously. After the first day, the rabbits received 1 ml of phage suspension intramuscularly twice weekly for 2 weeks. Two weeks after the last inoculation all the rabbits were bled by cardiac puncture, and the serum sample was pooled. The "k" value (1) of 1CIx'+ antiserum was determined against the homologous phage. The procedure was similar to that described by Adams (1). (ii) Plaque-forming ability on host strain A028. The phage suspensions from various strains were tested on strain A028 by an agar overlay procedure similar to that described by Eklund and Poysky (8). (iii) Phage identification by determination of antigenic similarity. For the determination of antigenic similarity of plaque-forming phages with phage 1CI"X+, dilutions of phage suspensions were mixed with 1CI'x+ antiserum and plated at the end of a 15-min incubation at 37°C by the agar overlay procedure on A028. Identification of phages that did not produce plaques on A028 but at the same time converted nontoxigenic A028 to toxigenic form in broth was attempted by the determination of ability of anti-phage 1C'l'x+ serum to inhibit toxigenic conversion. Phage suspension in 0.3-mi quantities was mixed with equal quantities of anti-1C0x+ serum and incubated for 15 min. Immediately after the incubation period, 0.2-ml quantities of the phageantiserum and 1 ml of actively growing A028 culture at 0.25 to 0.3 optical density units were inoculated
Nontoxigenic A028
(Actively growing) OD = 0.3 1 ml
Phage concentrate (sterile, toxin-free, deoxyribonuclease-treated) 0.1 ml
TYG broth 4-h incubation 1 ml
FEM (20 ml) t 5-day incubation Culture supernatant FIG. 1. Assay of toxicity in mice, using the broth method ofphage conversion. OD, Optical density.
VOL. 32, 1976
TOXIGENIC CONVERSION OF C. BOTULINUM TYPE C
into fresh TYG medium, and the conversion testing procedure was carried out as described earlier. Phage suspensions incubated with normal rabbit serum were tested as controls. Additional experiments for elucidating phagemediated variation in toxigenicity. (i) Determination of amount of conversion. The percentage of toxigenic cells was determined on two converted cultures. The first one was a highly toxigenic culture resulting from infection of A028 by phages of strain S-6810. The other was a hypotoxigenic culture resulting from infection of A028 by phages of strain Stockholm. A three-layer plate method (Fig. 2) was used for plating of the converted cultures to avoid injury to cells by oxygen. The culture under study was streaked on a reduced blood agar plate. The first overlay (melted and cooked to 45°C) was poured immediately on the surface of the plate, and as soon as these solidified the second overlay was added on the top. When the overlay was set, the plates were incubated in anaerobic jars with silica gel under the GasPak anaerobic system. After 18 h of incubation at 33°C, the plates were removed and 10 colonies were cultured individually in FEM. The FEM cultures were incubated for 5 days, and mouse LD.5,( of the supernatants was determined. (ii) Determination of lysogenic characteristics of cells from converted cultures. The FEM cultures of the above experiment, originating from different colonies, were tested for their lysogenic properties by determining their sensitivity or resistance to further phage infection. The FEM cultures were subcultured into fresh TYG broth, and agar layer procedures for detection of plaques were carried out on
147
these TYG cultures using the phage suspension of strain Stockholm.
RESULTS Electron microscopy. All toxigenic strains produced bacteriophages. Based on size alone, mainly two morphologically distinct types of phages were found to be present: a large type, similar to phage 1CItx (Fig. 3 and 4 show phage 1Ct"N+ and the large phage of strain NS2), and a small type (Fig. 5, 6, and 7 show small phages of strains 2C1x-, NS1, and 6811). A suitable criterion for the classification of Streak (converted culture)
F
3% TYG agar with sodium thioglycolate 2% TYG agar withJcysteine-hydrochloride Blood
agar t 18-h incubation
10 colonies cultured individually in FEM l 5-day incubation
Mouse LD5,, of supernatant determined FIG. 2. Procedure for determination of amount of
conversion.
FIG. 3. Large phage 1C"'+t exhibiting hexagonal head and partially sheathed tail. x115,500.
148
HARIHARAN AND MITCHELL
APPL. ENVIRON. MICROBIOL.
FIG. 4. Large phage of strain NS2 with hexagonal head and partially sheathed tail. x115,500.
FIG. 5. Small phage 2C'x-r with hexagonal head and a sheathed tail. x115,500.
VOL. 32, 1976
TOXIGENIC CONVERSION OF C. BOTULINUM TYPE C
149
FIG. 6. Small phage of strain NS1 with hexagonal head and a sheath. x115,500.
FIG. 7. Small phage of strain 6811 with hexagonal head and sheathless tail. x115,500.
phages was the tail length. The phages hav- were considered as the "small" phage. A third ing tail lengths in the range of 180 to 390 nm group was the "intermediate" type. (Figure 8 (usually >200 nm) were considered as the shows one of this type of phage of strain 6812.) "large" phage, and phages having tail lengths Though in general the head of the large phage in the range of 60 to 165 nm (usually
