against phage B was prepared by intravenous inoculation of 5 pound rabbits CORYNEBACTERIUM DIPHTHERIAE1

Transcription

1 FURTHER OBSERVATIONS ON THE CHANGE TO VIRULENCE OF BACTERIOPHAGE-INFECTED AVIRULENT STRAINS OF CORYNEBACTERIUM DIPHTHERIAE1 VICTOR J. FREEMAN" AND I. UNA MORSE Department of Public Health and Preventive Medicine, University of Washington, School of Medicine, Seattle, Washington Received for publication September 4, 1951 It was recently reported from this laboratory (Freeman, 1951) that virulent strains of Corynebacterium diphtheriae were isolated from avirulent C. diphtheriae cultures which had been exposed to diphtheria bacteriophage. The studies described here establish the reproducibility of the original results, starting, however, from single cell cultures. Other observations suggest the immunologic identity of the original avirulent and derived virulent strains, and demonstrate the absence of preformed toxin in the avirulent strains, and of "true" lysogenicity in the derived virulent strains. MATERIALS AND METHODS Cultures. Avirulent C. diphtheriae strains from California listed as 770, 1174, 1180, 444, and 411 are the same as those described previously (Freeman, 1951). Strain 444I was isolated from one of the guinea pigs that died following inoculation with a bacteriophage B lysatel of strain 444. It was found to be virulent, lysogenic, and phage B-resistant. Strains 469 and 470 were representative avirulent and virulent strains, respectively, isolated from a bacteriophage lysate of strain 444 of the previous study (Freeman, 1951). Strains 123 and 124 were obtained from the Laboratory of the Seattle-King County Department of Public Health, where they were determined to be characteristic avirulent strains of C. diphtheriae. Whether strains 123 and 124 were cultures of cases or contacts is information which was not available. Bacteriophages and lysate preparation. The bacteriophages A and B used in these experiments, the methods of preparing lysates, the type of media employed, and the in vivo and in vitro methods of testing the toxigenicity of the lysates were described previously (Freeman, 1951), with the exception that broth was added as control instead of saline in the lysate experiments. Antisera and agglutination tests. Antisera were prepared against strains 469 and 470 by intravenous inoculation of 5 pound rabbits with 22 ml quantities of formalin-killed bacterial suspension, administered in graded doses. An antiserum against phage B was prepared by intravenous inoculation of 5 pound rabbits 1 Supported in part by grants from the National Institutes of Health, Public Health Service, and the State of Washington Biologic Research Fund. 2Present address: 3811 O'Hara Street, Pittsburgh 13, Pennsylvania. ' The term "lysate" here refers to the mixture of bacterial cells, bacteriophage, extracellular products, and products of cell lysis resulting from incubation of a susceptible bacterial culture with its appropriate bacteriophage. 407

2 408 vict'iyh,j. FRr, EMAN AND 1. UNA MOME [VOL. 63 with graded doses totaling 28 ml of a itz filtrate of a phage B broth lysate (titer approximately 1 X 105) of strain 444. One thousand units of antitoxin were administered in 250 A doses initially to the rabbits receiving the filtered lysate material to prevent death from toxin in the lysate. Absorption of agglutinating antisera and agglutination tests were conducted according to methods described elsewhere (Minzel and Freeman, 1950; Freeman and Minzel, 1950). Single cell isolation technique. Single cell isolations of strains 444, 411, 1180, and 444I were made with the De Fonbrune micromanipulator. Glass needles with points 2 to 5,u in diameter were drawn on the microforge. The needles were soaked in alcohol and washed in sterile distilled water prior to use. Sterile micropipettes were touched to the surface of 18-hour heart infusion broth cultures of the strains being isolated. The inocula thus obtained by capillary action were transferred to sterilized glass coverslips, to which had been added a very thin film of 1.4 per cent agar (Zelle, 1951). After replacing the coverslips in the moist chamber of the micromanipulator, single bacterial cells were isolated on the glass needles and transferred to Loeffler slants. Following overnight incubation at 37 C the single cell cultures were subcultured to broth media. Lyophilized cultures were prepared from the subcultures and transferred to other media made for the tests indicated hereafter. Cell disintegration technique. The final disintegration experiments were performed using a Mickle's tissue disintegrator (Mickle, 1948). The sediments of 4 centrifuge bottles, each containing 125 ml of inoculated heart infusion broth, were pooled following 18 hours of incubation at 37 C and a half hour centrifugation at 2,000 rpm. The combined sediment was resuspended in 5 to 7 ml of distilled water, transferred together with 10 g of glow beads to the disintegrator container, and then shaken in the cold room for 90 minutes. Shaken and unshaken samples were tested for bacterial viability by serial plate dilution counts and for toxigenicity by the guinea pig intradermal method. Temperature was recorded initially and finally. RESULTS Single cell cultures. Four cells each of strains 444 and 444I and 3 cells each of strains 1180 and 411 were isolated by micromanipulation and cultured. Cultural reactions were studied, and all strains were found to be characteristic of C. diphtheriae. The susceptibility pattern of the single cell cultures to bacteriophages A and B did not vary from that of the parent strains reported earlier (Freeman, 1951). All single cell cultures of strains 444 and 1180 were susceptible to both phages. The cultures of strain 411 were resistant to both, and those of 444I were resistant to phage B but susceptible to phage A. Bacteriophage B lysate preparations were made in broth media for each single cell culture (except single cell strains 444I). The results of the in vitro and in vivo toxigenicity tests on these lysates are recorded in table 1. From these results it can be seen that virulent subcultures developed from the combination of bacteriophage B with all subcultures of single cells isolated from strains 444 and None of the control cultures of these strains showed any evidence of

3 1952] CHANGE OF VIRULENCE OF C. DIPHTHERIAE 409 toxigenicity. Neither the controls nor the phage mixtures of the subcultures of strain 411 showed any toxigenicity. These results are consistent with the findings reported for the parent strains (Freeman, 1951). All of the 4 single cell cultures isolated from the virulent, lysogenic strain 444I of lysate origin proved to be toxigenic. The 4 cultures were all phage carriers, plaques being readily demonstrated when each culture was spotted on a plate culture of the parent strain 444. Because of this lysogenicity and the fact that the cultures were resistant to phage B, the lysate experiments were omitted in the case of the 444I strains. TABLE 1 Toxigenicity te8ts of single cell cultures and their bacteriophage B lysates I vito XEToD ITDEMAL IN VIVO MODt STRAINS Phage B Culture Phage B lysate Culture control lysage lysate culturol control Test Control: Test Controlt 444(1) + _ + _ - _ (2) (3) (4) (1) + O + O + - (2) _ (3) + O + O + _ 411 (1) (2) (3) I (1) (2) (3) (4) _ * Modified Elek plate toxigenicity test (see Freeman, 1951). t Guinea pig intradermal test (Fraser, 1937). t Antitoxin control. Not done. Bacteriophage A lysates were made for one of each set of the single cell cultures (excluding 444I). The lysates were prepared by the plate method (Freeman, 1951). All such lysates were negative when tested in vitro for toxigenicity. Agglutination tests. Table 2 shows the results of serologic comparison by slide agglutination of the parent strains and their single cell cultures and 2 virulent strains isolated from bacteriophage B lysates. The reactions recorded in part A of table 2 establish strains 469 and 470 as homologous. Both strains were derived from a phage lysate of the parent avirulent strain 444, but differed in that strain 469 was avirulent and phage-susceptible, whereas strain 470 was virulent and resistant to phage B. The results recorded in part B of table 2 show the parent avirulent strains, except strain 411, to be at least closely related, if not homologous, to strains

4 410 VICTOR J. FREEMAN AND I. UNA MORSE [VOL and 470. Although the parent strains originated from two sources, a case and a contact of diphtheria that were unrelated epidemiologically, both groups of cultures did come from the same state. Strain 411 originated from the same source as strains 444 and 1174 but did not agglutinate with either of the typing sera. It was the only strain of the parent group that was phage B-resistant and failed to yield virulent subcultures. Part C of table 2 gives the results obtained with the single cell cultures. Each single cell strain produced the same agglutination reactions with the typing TABLE 2 Comparison of parent and single cell avirulent strains and virulent lysate strains of Corynebacterium diphtheriae by slide agglutination test8 SERA* STRAINS Cross-absorbed sert Cnrol rabbtsrol rbi ei A B _ C 444SCt _ 1180SC S0C- 444ISC D * Final serum dilution was 1:80. t Sera 469 and 470 reciprocally absorbed with suspensions of strains 470 and 469, respectively. t SC = single cell cultures. sera as did its corresponding parent strain. The virulent single cell strains 444I produced the same reactions as the avirulent single cell strains 444 and Reproduction of virulence change in an unrelated strain. The reactions recorded in part D of table 2 show two strains that failed to agglutinate with either of the typing sera. Both of these strains were found to be avirulent by in vitro and in vivo tests. They were isolated in King County, Washington. Strain 123 was resistant to phage B, and strain 124 was susceptible. Toxigenicity tests, both in vitro and in vivo, repeatedly showed that the phage B lysates of strain 124 prepared in broth or on agar media were virulent, whereas strain 123 mixed with phage B remained avirulent.

5 19521 CHANGE OF VIRULENCE OF C. DIPHTHERIAE 411 Cell disintegration experiments. The possibility that the avirulent cultures contained some preformed toxin that was being released by cell lysis was considered. In 1942, Morton and Gonzalez demonstrated that diphtheria toxin could be freed from washed virulent cells of C. diphtheriae when the cells were ruptured by sonic vibration. They did not test avirulent cultures. Since three ultrasonic vibrating machines operating at different frequency ranges were available locally, they were used in an attempt to rupture cell suspensions of strain 444. No appreciable disintegration resulted. Possibly, diphtheria cells are insensitive to vibrations above the sonic range. A Mickle's tissue disintegrator was made available during the course of the foregoing experiments. Using strain 444, preliminary tests were conducted to determine the shaking time necessary for adequate cell rupture as measured by viability counts. It was found that the number of viable cells remaining after 90 minutes of shaking was negligible compared to the initial count, whereas at 60 minutes the viable count ran between 1 and 10 per cent of the original population. The temperature of the cell suspensions did not increase as a result of the long shaking. Actually, there was an average decrease in temperature of the TABLE 3 Per cent destruction and toxigenicity of avirulent and virulent strains of Corynebacterium diphtheriae subjected to disintegration CELL VIABILITY COUNTS PaR MlL TOXIGENICITY TESTS SUSPENSION PZ~~~~Rn CENT (INRADEEMAL) DISINTEGRATION Initial Final Ruptured Unruptured X X 10' >99 444I 4 X X 105 > samples of approximately 10 C from room temperature following 90 minutes of shaking. The results of the final disintegration experiments are recorded in table 3. The degree of cell destruction was very great, even though there was an appreciable final count. The disintegrated cell suspension of the initially avirulent culture 444 failed to produce any toxic reaction in the skin of the guinea pig. Evidence that toxin could withstand the shaking treatment was obtained by using the virulent strain 444I as a control. The characteristic intradermal reaction produced by toxigenic strains of C. diphtheriae was obtained following the disintegration of suspension 444I. Experiments with bacteriophage antiserum. If the lysogenicity of the derived virulent strains of C. diphtheriae was "apparent" rather than "true" (Delbrtick, 1946), phage-free cultures should be obtainable by growth in appropriate phage antisera. The antisera produced against phage B were of a low titer (1: 80 against undiluted phage4), but broth medium containing 10 per cent of the phage anti- 4 The highest dilution showing no lysis after equal parts of phage and antiserum were incubated at 37 C overnight and the resulting mixture was spotted on a susceptible base culture.

6 412 VICTOR J. FREEMAN AND I. UNA MORSE [VOL. 63 serum was found to prevent the demonstration of lysogenicity when the cultures grown in it were spotted directly on to an agar plate freshly inoculated with a known susceptible culture. The cultures from the serum broth medium were found to be still virulent. However, when the cultures from the serum broth were subcultured to broth media without antiserum, their lysogenicity was again manifest. Strain 444I was subcultured successively through five daily passages in 50 per cent phage antiseruin broth and finally into antiserum-free broth medium. Lysogenicity tests on all five antiserum pasages were negative, but the test on the final antiserum-free pasage was positive for phage. A control culture grown in 50 per cent normal rabbit serum showed no inhibition of lysogenicity. DISCUSSION The possibility that the original avirulent cultures of C. diphteriae, that showed the change in virulence when incubated with bacteriophage B, might have had a small number of virulent cells mixed in with them was suggested previously (Freeman, 1951). The results recorded herein with the single cell cultures eliminate such a consideration. Proved avirulent cultures that were isolated from single cells were demonstrated to yield virulent subcultures when incubated in the presence of bacteriophage B. The finding of all four of the single cell cultures of strain 444I to be virulent was consistent with the previous observation (Freeman, 1951) that 100 per cent of 72 single colony subcultures of one of the derived virulent cultures was virulent. Any comparison of avirulent and virulent cultures derived from the same parent strain of C. diphtheriae might be contributory to the ultimate explanation of the mechanism of toxigenicity of diphtheria cells. The serologic comparison reported herein showed a pair of commonly derived avirulent and virulent C. diphtheriae cultures to be antigenically homologous in so far as demonstrated by the agglutination technique. If one assumes that toxin formation is an intracellular phenomenon, then the observation that the two strains were antigenically homologous is not inconsistent. Serologically indistinguishable virulent and avirulent strains of C. diphtheriae have been reported elsewhere (Hewitt, 1948). It would be of value to know whether or not the virulent member of other such serologically homologous pairs was the carrier of a phage to which the avirulent member was susceptible. The readiness with which avirulent strains of C. diphtheriae can produce virulent subcultures in the presence of specific bacteriophage is very difficult to predict. That the phenomenon can occur in strains unrelated to the original California group is evidenced by the results recorded for the local strain 124. Strain 124 differed from the California strains both in epidemiologic origin and in serologic reaction. Over a dozen other avirulent cultures of C. diphtheriae were tested for susceptibility to phages A and B but were found to be resistant. Since the search for bacteriophages is a never-ending one, no definitive conclusion concerning the ability of resistant avirulent strains to show the virulenceconversion phenomenon can be drawn. It was pointed out previously (Freeman, 1951) that the hypothesis postulating

7 1952] CHANGE OF VIRULENCE OF C. DIPHTHERIAE 413 a preformed toxin, or a toxin precursor being released from avirulent cells by phage lysis had to be considered. The results of the experiment on cell disintegration described herein would seem to rule out such an hypothesis. The continued toxigenicity on subculture of the derived virulent culture is further evidence against the toxin release theory. Two principal hypotheses remain to be investigated, namely, the spontaneous development of toxigenic mutants with accompanying selection by phage lysis, and the complex phage-bacterium association per se. If the derived virulent subcultures could have been rendered phage-free, determination of the toxigenicity status of the resultant cultures probably would have indicated the mechanism of the phenomenon. But the use of bacteriophage antisera in the culture media in concentrations up to 50 per cent failed to yield stable phage-free cultures. It would seem logical that the simultaneous acquisition of lysogenicity and virulence in the same bacterial cell is a related, rather than coincidental phenomenon. The change to toxin production might well be interpreted as being due directly to the acquired lysogenicity. Conceivably, the bacteriophage may make possible the toxin production through some as yet undetermined association with the metabolic processes of the bacterial cell. SUMMARY AND CONCLUSIONS The ability to produce virulent Corynebacterium diphtheriae cultures from single cell subcultures of avirulent strains of C. diphtheriae when the avirulent strains were exposed to diphtheria bacteriophage, was demonstrated in all subcultures of two avirulent, phage-susceptible strains. The single cell subcultures of a phage-resistant avirulent C. diphtheriae strain remained avirulent when exposed to the bacteriophage. An avirulent and a virulent strain of C. diphtheriae that were both derived from the same phage lysate of an initially avirulent culture were shown to be antigenically homologous by slide agglutination technique. The parent phageresistant avirulent strain, together with its single cell subcultures, failed to agglutinate with either antiserum. The parent phage-susceptible strains and their single cell subcultuires agglutinated with both antisera, as did parent and single cell subcultures of a derived virulent strain. A phage-susceptible avirulent strain of C. diphtheriae that differed both in epidemiologic origin and in antigenicity was shown to yield virulent C. diphtheriae strains when exposed to diphtheria bacteriophage. Explanation of the mechanism of virulence change on the basis of release of preformed toxin by cell lysis was ruled out by the absence of toxicity when samples of the avirulent cultures were subjected to disintegration. Attempts to produce phage-free cultures of a derived virulent strain by subculturing in broth media containing up to 50 per cent of phage antiserum were unsuccessful. REFERENCES DELBRtrCK, M Bacterial viruses or bacteriophages. Biol. Revs., 21, FRASER, D. T The intracutaneous virulence test for Corynebacterium diphtheriae. Am. J. Pub. Health, 27, Supp. to no. 3,

8 414 VICTOR J. FREEMAN AND I. UNA MORSE [VOL. 63 FREEMAN, V. J Studies on the virulence of bacteriophage-infected strains of Corynebacterium diphtheriae. J. Bact., 61, FREEMAN, V. J., AND MINZEL, G. H Serologic studies of Corynebacterium diphtheriae. II. Antigenic relationship between small colony types of C. diphtheriae and C. diphtheriae-like bacilli as determined by the method of slide agglutination. Am. J. Hyg., 51, HEWITT, L. F Virulence and toxigenicity of different serological types of C. diphtheriae. Brit. J. Exptl. Path., 29, MICKLE, H Tissue disintegrator. J. Roy. Microscop. Soc., 68, MINZEL, G. H., AND FREEMAN, V. J Serologic studies of Corynebacterium diphtheriae. I. The use of a surface active agent in the preparation of uniform suspensions of C. diphtheriae for serologic typing. Am. J. Hyg., 51, MORTON, H. E., AND GONZALEZ, L. M On the site of formation of diphtherial toxin. J. Immunol., 45, ZELLE, M. R A simple single cell technique for genetic studies of bacteria. J. Bact., 61, 345.