A streptomycin-sensitive revertant of a streptomycin-dependent strain of Vibrio cholerae*

BRIEF COMMUNICATIONS A streptomycin-sensitive revertant of a streptomycin-dependent strain of Vibrio cholerae * V. B. SINHA' & J. W. BHATTACHARJEE 2 Abstract A study was made of a streptomycin-sensitive revertant of a streptomycin-dependent strain of V. cholerae. The revertant, designated as strain KB599, appears to be avirulent in animal experimental models. However, no evidence of multiplication was found in infant mice. Earlier work has shown that live vaccines may be able to confer more effective immunity against chol- era than the killed vaccines used at present (1, 17, 18). Several attenuated strains of V. cholerae have been studied in order to evaluate their potential as immunizing agents. Naturally occurring attenuated strains of El Tor vibrios were isolated from water sources and de- scribed by Mukerjee (15). Treatment of V. cholerae with mutagenic agents in the laboratory has yielded a number of mutants that show attenuation of virulence in animal models (2, 9, 10). Streptomycin-dependent (sm-d) mutants of V. cholerae (K. Bhaskaran & V. B. Sinha, unpub- lished observations; 3, 16) and other bacterial species (12, 20, 21) are known to be avirulent. Felsenfeld et al. (8) compared the metabolic and immunological characteristics of sm-d V. cholerae with those of their parent strains. In the present study, we were interested to see if a streptomycin- independent revertant derived from sm-d mutants of V. cholerae would also show attenuation of viru- lence. Materials and methods Vibrio strains. Among the strains used, strain KB611 is a sm-d mutant isolated in this laboratory by Dr K. Bhaskaran and kindly made available to us. Strain KB599 is a streptomycin-independent rever- * Communication No. 2141 from the Department of Microbiology, Central Drug Research Institute, Lucknow 226001, India. I Scientist. 2 Holder of a Research Followship awarded by the Council of Scientific and Industrial Research, New Delhi, India. tant derived from KB611 and is streptomycin-sensi- tive. KB92 is a virulent Ogawa classical strain, which is the parent of KB611. Strain KB304 (Inaba 569B) was used as the positive control strain in infant mice. Culture media. The broth used was brain-heart infusion (Difco Laboratories). To prepare nutrient agar, the broth was solidified with 1% agar powder (Oxoid). Gelatin agar was prepared as described elsewhere (11). Isolation of revertants. Strain KB611 was grown in brain-heart infusion broth without streptomycin for 3 h. The spun deposit was seeded on nutrient agar. After 48 h the plates were examined for streptomy- cin-independent revertants. Animal experimental models. In ligated ileal loop studies (5), adult albino rabbits weighing 1-1.5 kg were starved for 48 h prior to challenge. Under anaesthesia 3 loops were prepared in each animal and inoculated with 1 ml of a 10-1 or 10-3 dilution of a 3-h brain-heart infusion culture of strains KB599 and KB92, respectively. After 18 h the animals were sacrificed and the loops examined for presence or absence of fluid. In virulence studies with the same strains in mice (2), 10-fold dilutions varying from 10-1 to 10-7 of a 3-h broth culture were prepared and 0.5 ml, suspended in 5% mucin (Wilson type 1701), was injected into Swiss mice weighing 14-16 g. Each dilution was inoculated into 10 mice, which were then observed for 48 h for any mortality. In a further experiment, 10-day-old rabbits (7) weighing 100-130 g were anaesthetized and 1 ml of a 3-h broth culture was introduced directly into the small intestine. The animals were observed and autopsied after 48 h. Multiplication of strain KB599 in vivo. In order to investigate the in vivo growth of strain KB599, viable counts were made 24 h after initiation of infection in the ligated ileal loops of rabbits and in infant mice. Each ileal loop was removed and homogenized in 100 ml of broth using a Kenwood liquidizer. The 3463 - 482 BULL. WORLD HEALTI ORGAN., Vol. 53, 1976 BRIEF COMMUNICATIONS 483 viable counts were obtained after incubation for 48 h at 37°C on gelatin agar plates. The infant mouse studies were made on 5-day-old sucklings starved overnight. A round-tipped 23-gauge needle attached to a 1-ml syringe was used to introduce 0.1 ml ofa 4-h broth culture into the stomach via the mouth. For tests using the control virulent strain KB304, 0.1 ml of a 10-1 dilution of a 3-h broth culture was intro- duced into the stomach. At 24 h the intestines were removed and homogenized, and viable counts were obtained as described above. Results Revertants. Streptomycin-independent revertants from the sm-d strain KB611 were rare; the frequency of reversion was 5 x 10-9. All the revertants were streptomycin-sensitive. One of them, designated strain KB599, was selected for further study. It resembled the parent strain KB611 in all respects except that it grew faster; however, it grew more slowly than KB92, the virulent parent strain of KB611. Animal models. The behaviour of strains KB599 and KB92 was compared in mice and rabbits. In each of 2 groups of 8 adult rabbits-I group inocu- lated with each strain-16 ileal loops were infected; however, all 16 loops in the KB92 group showed positive reactions, as against none in the KB599 group. In infant rabbits, all 5 of the animals infected with strain KB92 exhibited diarrhoea with fluid distension of the caecum, while of the 6 infected with strain KB599, 1 had diarrhoea and none had fluid distension of the caecum. In 2 tests of the virulence of the strains in mice, the LD50 as determined by the method of Reed & Muench (19) was <43 and <40 organisms for the control strain, and >2 x 107 and >5.6 x 106 organisms for strain KB599. Thus strain KB599 proved avirulent in these models. Over a 6- month period of study the attenuation of virulence remained a constant feature, which indicates the stability of the strain. In vivo recovery in mice and rabbits. In 7 infant mice injected with the control strain KB304, the average % survival a was 237.0% (standard error = 72.5 %), while it was only 3.070% (standard error = ± 1.31 %) in 8 injected with strain KB599 (number of organisms injected = 3 x 107). Average % survival of strain KB599 in 1 ileal loop in each of 8 adult a Average % survival = average number of bacteria recovered/number of bacteria injected x 100. rabbits was 4600% (standard error = ±2052%). There was no evidence of multiplication of strain KB599 in the infant mouse intestine, though it was able to multiply in the ileal loop, which can be regarded as a closed system. No multiplication of the strain was detectable in the gut of infant rabbits. Discussion Similar findings of avirulence in streptomycin- independent revertants derived from sm-d organisms have been reported in Brucella species (22) and in Shigella flexneri (9, 14). In the ligated ileal loop, strain KB599 is able to multiply but in the mouse intestine a considerable fall occurred in the number of viable organisms recoverable from the gut at 24 h. Sm-d mutants of Shigella, which are not assumed to multiply in the gut (12), have been successfully used as oral immu- nizing agents (13). Hence multiplication of the orga- nism in the gut may not be essential for induction of immunity, and this may even be true for vibrios unless they behave unlike shigellae. However, a streptomycin-independent revertant that is able to persist longer in the gut of infant mice than strain KB599 may prove to be more efficient in inducing immunity. ACKNOWLEDGEMENTS The authors are indebted to Dr K. Bhaskaran for suggesting the study, to Dr Nitya Nand for his interest and support, and to Dr B. S. Srivastava for critical reading of the manuscript. They thank Mr A. K. Sarkar for his skilful technical assistance. REFERENCES 1. BASU, S. ET AL. J. infect. Dis., 121, Suppl.: 556 (1970). 2. BHASKARAN, K. & SINHA, V. B. J. Hyg. (Camb.), 65: 135 (1967). 3. BHATTACHARJEE, J. W. & SINHA, V. B. Indian J. med. Res. (in press). 4. CHAICUMPA, W. & ROWLEY, D. J. infect. Dis., 125: 480 (1972). 5. DE, S. N. & CHATTERJI, D. N. J. Path. Bact., 66: 559 (1953). 6. DUPONT, H. L. ET AL. J. infect. Dis., 125: 5 (1972). 7. DUTTA, N. K. & HABBU, M. K. Brit. J. Pharmacol., 10: 153 (1955). 8. FELSENFELD, 0. ET AL., Appl. Microbiol., 19: 463 (1970). 9. FINKELSTEIN, R. A. ET AL. J. infect. Dis., 129: 117 (1974). 10. HOWARD, B. D. Nature (Lond.), 230: 97 (1971). 484 BRIEF COMMUNICATIONS 1 1. CRUICKSHANK, R., ed. Medical microbiology, 11th ed. Edinburgh, Livingstone, 1965, p. 764. 12. MEL, D. M. ET AL. Bull. Wld Hlth Org., 32: 633 (1965). 13. MEL, D. M. ET AL. Bull. Wld Hlth Org., 39: 375 (1968). 14. MIHAILOV, I. F. ET AL., Zh. Microbiol. (Mosk.), 45 (11): 10 (1968). 15. MUKERJEE, S. Bull. Wld Hlth Org., 29: 753 (1963). 16. OLITZKI, A. L. & OLrrZKI, Z. Exp. Med. Surg., 13: 332 (1955). 17. PANSE, M. V. & DUTTA, N. K. J. Immunol., 93: 243 (1954). 18. PANSE, M. V. ET AL. J. infect. Dis., 114: 26 (1964). 19. REED, L. J. & MUENCH, H. Amer. J. Hyg., 27: 493 (1938). 20. REITMAN, M. J. infect. Dis., 117: 701 (1967). 21. SERGEEV, V. V. ET AL. Zh. Microbiol. (Mosk.), 44: 56 (1967). 22. SIMON, E. M. & DAVID, T. B. J. Bact., 83: 1347 (1962).