surface of each plate and spread evenly with a sterile glass rod. Inoculated media were incubated The stock cultures of the C. perfringens strains

Transcription

1 STUDIES OF THE L-FORMS OF CLOSTRIDIUM PERFRINGENS I. RELATIONSHIP OF COLONY MORPHOLOGY AND REVERSIBILITY TOSHIO KAWATOMARI Department of Bacteriology, 406th Medical General Laboratory, APO 343, San Francisco, California Received for publication February 28, 1958 The L-forms of Clostridium perfringens were first reported by Dienes (1950). He described this form as minute, soft, pleomorphic colonies which developed when C. perfringens were grown on penicillin-treated horse serum agar plates. The reversion of L-colonies to bacterial forms has not been reported for C. perfringens. They have been observed in strains of Proteus (Dienes, 1949), Salmonellae (Weinberger et al., 1950), Hemophilus influenzae (Dienes and Weinberger, 1951), Bacteroides (Dienes, 1948), and Streptobacillus moniliformis (Dienes, 1943). The present study is the investigation of the transformation of known C. perfringens tvpes (A, B, C, D, E, and F), their respective L-forms, and an examination of the ability of the L-forms to revert to bacillary colonies. MATERIALS AND METHODS The strains of C. perfringens used in this study were obtained from Dr. L. S. McClung of Indiana University (type A, strain McC 146), and from the Wellcome Research Laboratories, Beckenham, England (types B, C, D, E, and F; strains CN-677, CN-882, CN-1635, CN-1241, and CN-2078, respectively). The media employed were a modified brainheart infusion agar developed by Barile and Yaguchi in this laboratory (1957, unpublished data), and the beef heart broth medium of Robertson (Wetzler et al., 1956). Brain-heart infusion agar was prepared by dissolving 37 g of dehydrated brain-heart infusion broth (Difco), 10 g of yeast extract (Oriental Yeast Company, Tokyo, Japan) and 12.5 g of agar in 850 ml of distilled water. The medium was adjusted to ph 7.8, autoclaved 15 min at 15 lb pressure, cooled to 50 C, and 150 ml of citrated human blood were added. The medium was distributed to sterile petri dishes in 25 ml aliquots. Two hours before streaking, 0.1 ml of a penicillin solution containing 500 units per ml was dropped on the 227 surface of each plate and spread evenly with a sterile glass rod. Inoculated media were incubated anaerobically in Brewer jars, using a hydrogen atmosphere. The stock cultures of the C. perfringens strains were maintained in the modified Robertson medium. Each C. perfringens culture used experimentally was purified by streaking to a chloral hydrate-sodium azide blood plate (Wetzler et al., 1956) and isolated colonies checked by the procedure routinely employed in this laboratory (Marshall et al., 1956). Pathogenicity tests were performed using guinea pigs. L-Forms of all six strains of C. perfringens (A to F) were induced by streaking a loopful of 6- to 8-hr bacillary growth from thioglycolate broth to penicillin-treated plates, which were then incubated anaerobically for 4 days at 37 C. After incubation, plates were examined macroscopically for the presence of minute, pin-point colonies with a slight zone of surrounding hemolysis. Plates showing such colonies were additionally inspected under a stereo-microscope. A Gram stain was made. If the minute colonies were observed to be partially submerged, gram-negative, and without microscopically demonstrable cellular morphology, they were considered to be L-forms. Serial transfers of such colonies to penicillin-free brain-heart infusion agar plates were made and observed for reversion to bacterial colonies. RESULTS Successive transfers, at 3 to 4 day intervals, over 6 months, showed no morphologic transformations or colony changes of the L-forms other than those considered as colony variations. The colonial morphology of each L-form of the six strains of C. perfringens examined are shown in figures 1 to 6. Young L-colonies often appeared dewdrop-like, viscous, and without submerged growth; however, after 48 hr all colonies took

2 228 KAWATOMARI [VOL. 76 Figure 1. In situ preparation of a cross-sectioned flat Clostridium perfringens type A L-colony. Magnification 14X.,,: :afa6.." A... Figure 2. Block of blood agar with type A Clostridium perfringens convex L-colonies. Magnification 8X.

3 * :.:..:,.0. : 1958] L-FORMS OF C. PERFRINGENS 229 6'a.!. A, Figure S. In situ preparation of Clostridium perfringens type B L-colony with daughter colonies. Magnification 25X :.E.g.S..*.._g,... _.. -.F,e,:..:.^.. :.:..R,.. R. _g:b. s *:F.F... w.... w..... Y & ws.;.. b c W A... Figure 4. Flat, radiating colonies of Clostridium perfringens type D on blood agar plate. Magnification 12X.

4 230 KAWATOMARI [VOL. 76 Figure 5. Blood agar plate with smooth, pin point, convex Clostridium perfringens type E L-colonies. Magnification 3X. Figure 6: In situ preparation of Clostridium perfringens type F L-colony. Magnification 25X.

5 1958] L-FORMS OF C. PERFRINGENS 231 on the appearance of mature L-forms. Mature L-colonies of C. perfringens types A and D occurred as two variants. One variant had a convex surface, with the submerged part of the colonies diffused into the agar without sharp demarkation (figure 1). The surface of these colonies became black upon prolonged incubation. The other colony type was flat with well-defined radiating branches into the agar below the surface of the plate (figure 2). The edges of these colonies also turned black upon prolonged incubation. C. perfringens types B and C produced a third L-colony type in addition to those observed with strains A and D. It appeared as a large central colony surrounded by an outcrop of numerous small daughter clones (figure 3). When this type occurred, it appeared to the exclusion of the types resembling A and D L-forms. During the first transfers, it often contained an agglomerated red pigment within the submerged part of the central colony. The L-colonies of strain E were usually smaller than those of the other five strains of C. perfringens. They were compact, domed, and glistening, macroscopically resembling a-hemolytic streptococci. L-Forms of strain F showed very flat, diffuse growth without discrete edges. This characteristic made them difficult to stain by the Dienes method (Dienes, 1945). All L-form colonies, except type E, softened the agar in their immediate vicinity. Some showed a well-defined zone of hemolysis. Hemolysis, however, was not always manifested. They propagated best in a hydrogen atmosphere, although scanty, submerged growth did occur when plates were incubated aerobically. Conversion of l-colonies to bacterial forms was accomplished bv inoculating tubes of the modified Robertson medium. After 24 to 72 hr incubation, gas formation, characteristic of C. perfringens, was noted. Smears prepared from these broth cultures showed gram-positive rods. Routine biochemical methods identified these bacteria as C. perfringens. Toxin production was demonstrated using guinea pigs injected intramuscularlv. At autopsy, the animals showed C. perfringens damage typical for each strain examined, and cultural recovery of the organism was accomplished. Tubes of brain-heart infusion broth (Difco), supplemented with 1 per cent yeast extract and 15 per cent human serum, inoculated with L- colonies also showed growth reverting to bacterial forms. Turbidity and cottony growth was observed after 3 to 4 days of incubation, and then gas formation after incubation for a week. As soon as gas was noted, a smear was prepared and examined for the presence of gram-positive rods. They were further identified as C. perfringens. Whereas the original reaction of the medium was about ph 7.8, the ph observed after reversion was found to be approximately 6.1. Five guinea pigs were inoculated with 0.5 ml of a saline suspension of C. perfringens strain A L-form. Two guinea pigs received 0.5 ml of a similar suspension of strain E. No reaction was observed in the animals. Six rabbits were inoculated each with an aliquot of the L-form of one of the strains of A to F of C. perfringens and no pathological response was elicited. DISCUSSION Dienes (1950) showed the effect of antimetabolites on the appearance of L-colonies. Penicillin was found to induce not only L- colonies, but also aberrant cell formation of C. perfringens. Several workers (Crofts and Evans, 1950; Johnstone et al., 1950; Ito et al., 1951; and Reimann, 1952) demonstrated rough bacterial colonies with long filamentous cells after such treatment. The Gram reaction and the pathogenicity of the rough forms are, however, always similar to those of the original smooth colonies. In the present experiments, rough colonies were not studied, but attention was focused solely on L-forms which had been successfully induced by penicillin from all six types of C. perfringens. When L-colonies were transferred from solid plates to liquid media, they reverted to the bacterial form. The reversion of L-forms to the bacterial colonies was not observed after continuous serial transfers on penicillin-free plates. Some of the L-strains were maintained for as long as six months without reversion by this method. When a single inoculum was put in the center of the plate, the colony grew to approximately 0.5 cm in diameter in a month, but did not revert to a bacillary form. This phenomenon has not been observed previously. On the contrary, it was observed that if L-forms were propagated in the absence of the antimetabolite which induced them, they promptly reverted to their respective bacterial forms (Dienes, 1949). The favorable

6 232 KAWATOMARI [VOL. 76 results obtained in the present study are probably due to the nutrients and metabolites present in the medium employed in these experiments, since the same plate has been used successfully for the cultivation of pleuropneumonialike organisms. The L-colonies progressed from surface-growing dewdrop forms to submerged types during incubation. 'T'hree forms of mature Lcolonies were observed, and the number and type of L- colonies appear to be characteristic for the strain of C. perfringens from which they originated. Unlike the original pathogenic bacterial cultures of C. perfringens, each L-form was found to be nonpathogenic for rabbits and guinea pigs. However, the bacterial cultures of the reverted L-forms were toxin producers. The L-forms of C. perfringens observed in these studies may be of diagnostic importance. Furthermore, they open a broad field of immunological studies. Inoculation of live L-forms into guinea pigs and rabbits did not evoke pathological reactions. Studies of immunization against C. perfringens and attempts to produce diagnostic sera against the bacillary types using the L-forms are being investigated. SUMMARY Six strains of Clostridium perfringens representing types A, B, C, D, E, and F were converted to L-forms with the aid of penicillin plate cultures. A reversion of the L-forms to their respective bacterial forms was not observed on plate cultures without penicillin. The L-forms reverted to bacterial forms in fluid media. Three types of mature Lcolonies of C. perfringens were described. The L-forms were practically nontoxic for guinea pigs and rabbits. REFERENCES CROFTS, J. E. AND EVANS, D. G The action of penicillin on Cl. welchii type A. Brit. J. Exptl. Pathol., 31, DIENES, L Reproduction of bacteria from the large bodies of Streptobacillus moniliformis. Proc. Soc. Exptl. Biol. Med., 53, DIENES, L Morphology and nature of pleuropneumonia group of organisms. J. Bacteriol., 50, DIENES, L The isolation of L type cultures from Bacteroides with the aid of penicillin and their reversion into the usual bacilli. J. Bacteriol., 56, DIENES, L The development of Proteus cultures in the presence of penicillin. J. Bacteriol., 57, DIENES, L Isolation of L type cultures from Clostridia. Proc. Soc. Exptl. Biol. Med., 75, DIENES, L. AND WEINBERGER, H. J The L-forms of bacteria. Bacteriol. Revs., 15, ITO, T., SAITO, Y., AND TSUKAHARA, T On the penicillin sensitivity of the Clostridium group. J. Antibiotics (Tokyo), 4, JOHNSTONE, K. I., CROFTS, J. E., AND EVANS, D. G Single cell culture of Cl. welchii type A morphologically changed by penicillin. Brit. J. Exptl. Pathol., 31, MARSHALL, J. D., JR., WETZLER, T. F., AND KAWATOMARI, T Differentiation of the Clostridia associated with medical specimens. U. S. Armed Forces Med. J., 7, REIMANN, B Wachstums-Beobachtungen an dem Bazillus Welch Typ F (B. enterotoxicus Zeissler) unter Penicillineinwirkung. Zentr. Bakteriol., Parasitenk., Abt. I. Orig., 159, WEINBERGER, H. J., MADOFF, S., AND DIENES, L The properties of L-forms isolated from Salmonella and the isolation of L-forms from Shigella. J. Bacteriol., 59, WETZLER, T. F., MARSHALL, J. D., JR., AND CARDELLA, M. A Rapid isolation of clostridiums by selective inhibition of aerobic flora. II. A systematic method as applied to surveys of clostridiums in Korea. Am. J. Clin. Pathol., 26,