Bacteriophages of Clostridium botulinum types A, B, E, and F and nontoxigenic strains resembling type E

Abstract

Recent investigations have shown cultures of Clostridium botulinum to be lysogenic. Vinet and Fredette (2) presented electron micrographs of bacteriophage obtained from C. botulinum type C. Inoue and Iida (1) induced the lysis of C. botulinum cultures with mitomycin C and ultraviolet light and observed bacteriophage in lysates of types A, C, D, and nonproteolytic type F. They also observed phage tail-like rods in lysates of type E and nonproteolytic type B. Proteolytic types B and F were not included in these studies. This paper presents (i) additional observations on the occurrence of lysogenicity in the different types of C. botulinum and (ii) the first observations of bacteriophage from type E, proteolytic and nonproteolytic types B, and proteolytic type F. C. botulinum type A and nonproteolytic types B and F were isolated at this laboratory from marine sediments. Other strains were obtained as follows: Beluga strain of type E from C. E. Dolman (Univ. of British Columbia, Vancouver, B.C.); strain 066B of type E and 066BNT (non-toxigenic variant of 066B) from D. A. Kautter (U.S. Food and Drug Administration, Washing-ton, D.C.); strain P4 (resembling type E except for the absence of toxinogenicity) from V. J. Cabelli (Northeast Marine Health Science Laboratory, Narragansett, R.I.); strain 8E of type E and proteolytic strain 169B of type B from C. F. Schmidt (Continental Can Co., Chicago, Ill.); and culture 8G, a proteolytic strain of type F from N W. Walls (Georgia Institute of Technology, Atlanta, Ga.). The cultures were maintained in cooked meat medium at 25 C. The nonparticulate medium used for induction of lysis experiments was TPGY (trypticase, 5%; peptone, 0.5%; glucose, 0.1 % yeast extract, 0.5%; and sodium thioglycolate, 0.1%; final pH, 7.0). Induction of lysis experiments were performed in screw-cap tubes (25 x 150 mm) containing 30 ml of TPGY medium. The tubes were inocu-lated with an 18-hr culture to give an optical density of 0.06 to 0.08 and incubated at 25 C until the optical density attained a value of 0.12. Then, different concentrations of mitomycin C (0.1 to 5.0 ,ug /ml) were added to the cultures. Optical density was measured in a Bausch & Lomb Spectronic-20 colorimeter at 525 nm. After lysis was complete, the cultures were clarified by centrifugation at 5,000 X g (Sorvall SS-34 rotor) for 15 min at 5 C. The pellet was discarded and the phage was sedimented from the supernatant fluid by centrifuging at 40,000 X g in an International model B-60 ultracentrifuge (rotor no. A-211) for 2 hr at 5 C. The pellet obtained was suspended in neutral 0.1 M am-monium acetate solution and again centrifuged at low and high speeds. The final pellet was resus-pended in 0.5 ml of 0.1 M ammonium acetate solution. Electron microscope specimen grids were dipped into the culture lysates and partially drained on filter paper. The grids were then dipped into a 2% solution of neutral potassium phosphotungstate solution, dried on filter paper, and examined in an RCA 3G electron microscope. Specimens were photographed at an initial magnification of X 21,000, and the negatives were further enlarged photographically to x258,000. Lysis occurred in all of the strains treated with mitomycin C except the nontoxigenic culture 066BNT (a nontoxigenic variant derived from toxigenic strain 066B). The concentration of mitomycin C yielding maximal lysis was (i) 1 ,ug/ml for type A, nonproteolytic and proteolytic types B, nontoxigenic strain P4, and proteolytic type F; (ii) 0.1 and 0.5 ,ug/ml for type E strain; and (iii) 0.5 and 1 ,ug/ml for nonproteolytic type F. Bacteriophage and phage tail-like structures from lysates of the different types of C. botulinum 270