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The Action of Culture Filtrates of Aspergillus Clavatus on Dermatophytes1
THE ACTION OF CULTURE FILTRATES OF ASPERGILLTJS CLAVATUS ON DERMATOPHYTES' IN VITRO EXPRRThIENTS
KURT LOEWENTHAL, M.D.
Clavacin is an antibiotic substance derived from Aspergillus clavatus and from a number of related molds (1). It was shown to be active, in vitro, against some
dermatophytes including Trichophyton gypseum, Trichophyton purpureum, Microsporon audouini and also against Monilia albicans and Cryptococcus neoformans (2). The present study extends these investigations and discusses the mechanism of the action of clavacin upon the test organisms.
The test fungi were Trichophyton violaceum, Trichophyton suiphureum, Trichophyton schoenleini, Microsporon lanosum, Epidermophyton floccosum, and also Sporotrichum scheneki as a representative of the fungi producing deep
mycoses. Control organisms which had already proven their sensitivity to clavacin were also employed, including Monilia albicans, Trichophyton gypseum, Trichophyton purpureum and Microsporon audouini.
Since crystalline clavacin could not be obtained a culture filtrate was obtained from a strain of A. clavatus of our own stock cultures. The fungus was grown in a liquid medium which contained 40 Gm. dextrose and 10 Gm. peptone in 1,000 cc. of distilled water. The inocula were kept at 28 C. for 7 days and the viable material separated by passage through a Seitz filter. The amber colored filtrate was kept at ice box temperature. To determine the influence, if any, of the hydrogen ion concentration upon the activity of the filtrate the latter was divided into two parts; one part was adjusted to a pH of 4.0 and the other part to a pH of 5.8. A second batch of culture filtrate was prepared which was inferior in potency to the former. It also was employed at pH values of 4.0 and 5.8 respectively. The absolute potency of both batches, expressed in content of clavacin, was not known. METhOD
Fragments from the test fungous cultures were placed into test tubes containing 10 cc. of the various culture filtrates. Two inoculations were made for each hydrogen ion concentration and for each batch of filtrate, amounting to 8 inocula for each fungus and assay. The inoculated tubes were kept at room temperature. Two, 7 and 9 days later, particles of the inocula were removed from the filtrates and, after thorough rinsing in sterile distilled water, transplanted upon dextrosepeptone agar slants. Only one tube of each pH and potency was used for transplantations while the other remained undisturbed. The inocula in the filtrates 1
From the Long Island College Hospital and the Department of Dermatology and
Syphilology, Long Island College of Medicine, Brooklyn, N. Y. Received for Publication Jan. 6, 1947. 41
42
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
and the transplants on the agar slants were observed for evidence of fungous growth, at intervals of 3 to 5 days, for at least 21 days. RESULTS
The investigation of T. gypseum, T. violaceum, M. audouini, M. lanosum, and E. fioccoswn is complete while S. schencki, T. schoenleini and T. suiphureum demand some amplification. The results of 26 assays are summarized in table 1. S. schencki showed no appreciable inhibition by the weak filtrate at both PH; M. albicans was not influenced by the potent filtrate at p115.8. T. gypseum was inhibited and killed by the potent filtrate at a pH of 4.0; the potent filtrate of pH 5.8 and the weak filtrate of both pH did not produce fungicidal or fungistatic effect.
M. lanosum was inhibited and killed by both the strong and weak filtrate at pH 4.0. At 5.8 no growth was visible in the potent filtrate, but the first transplant revealed evidence of growth after 19 days of incubation. The later transplants did not grow. In the weak filtrate of pH 5.8 some inocula developed growth. Particles from the apparently sterile inocula revealed vigorous growth when the transplantation was done not later than 2 days after exposure to the filtrate. T. schoenleini and T. sulphureum were inhibited and killed by the potent ifitrate at pH 4.0. T. purpureum was inhibited and killed by the potent filtrate at both pH. It was inhibited by the weak filtrate at both pH, but transplants from pH 4.0 after 7 days' exposure, and from pH 5.8 after 9 days' exposure, revealed fungous growth. M. audouini, T. violaceum and E. fioccosum were inhibited and killed by the potent and weak filtrate at both pH. COMMENT
These experiments reveal that culture filtrates of A. clavatus possess a wider range of action than previously reported. All dermatophytes tested were shown to be susceptible. M. albicans and S. seheneki were not demonstrably influenced; the former, however, was shown in earlier reports to react to some extent to higher concentrations of clavacin and long exposures (2). The effect was determined by the following factors: the type of fungus, the potency of the filtrate (presumably identical with the clavacin concentration in
the filtrate), the time of contact of fungus and filtrate, and the hydrogen ion concentration of the filtrate. (See photographs 1 and 2.) In the table the lungi were arranged in their order of sensitivity. The highest degree of resistance was shown by M. albicans and S. scheneki. T. gypseum possessed a higher degree of sensitivity; it was killed by the potent filtrate at a low pH. M. lanosuin, more susceptible, was killed by both filtrates at pH 4.0, but showed some growth at pH 5.8. The position of T. schoenleini and T. suiphureum is tentative until more information will be available; both were killed
43
ACTION OF ASPERGILLUS CLAVATUS ON DERMATOPHYTES
TABLE 1 WEAL FILTRATE
pH 4.0
POTENT PILTEATE
pH 5.8
pH 4.0
pH 5.8
E. fioccosum
0
0
0
0 0 0
0 0 0
0 0
0 0 0
0
0
0 0 0 0
0 0
0
0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
+ + +
0
Transplant III Transplant II Transplant I Inoculum
T. violaceum
Transplant III Transplant II Transplant I Inoculum
0
0 0 0
M. audouini
Transplant III Transplant II Transplant I Inoculum
T. purpureum
Transplant III Transplant II Transplant I Inoculum
+ + 0
0
0 0 0
0
0 0 0 0 0 0
T. suiphureum 0 0 0 0
Transplant III Transplant II Transplant I Inoculum
T. schoenleini
0
Transplant III Transplant II Transplant I
0 0 0
Inoculum
M. lanosum
Transplant III Transplant II Transplant I Inoculum
0 0 0 0
Transplant I = after 2 days' incubation Transplant II = after 7 days' incubation Transplant III = after 9 days' incubation
+ = fungous growth
o = no fungous growth • = no information available
0 0
+ +
0 0 0 0
0 0
+ 0
44
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE 1—Concluded WEAK EILTLATE
T. gypeuin Transplant III Transplant II Transplant I Inoculum S. scheneki
Transplant III Transplant II Transplant I Inoculum M. albicans
Transplant III Transplant II Transplant I Inoculum
POTEN'X PILTRATE
pH 4.0
pH 5.8
pH 4.0
pH 5.8
+ + + +
+ +
0 0 0 0
+ + + +
+ + + +
. . . .
.
+ +
+ + . . . .
+ +
. . . .
. .
. .
+ + + +
by the potent filtrate at pH 4.0. Both filtrates were fungistatic for T. purpureum the more potent also killed the fungus at both hydrogen ion concentrations. The highest degree of sensitivity was revealed by M. audouini, T. violaceum and E. floccosum. That the more potent filtrate, presumably containing clavacin in higher concen-
tration, exerts a greater antifungal power was exemplified by T. purpureum and T. gypseum. The potent filtrate inhibited and killed the former while the action of the less potent filtrate was mainly fungistatic. The latter was not influenced by the weak filtrate but it was killed by the potent filtrate at pH 4.0. The influence of length of contact between filtrate and fungus was illustrated
by M. lanosum and T. purpureum. Transplants of the former, from inoctda exposed for 2 days showed growth upon culture medium while later transplants remained sterile. Transplants of T. purpureum from inocula exposed for 7 days grew while after 9 days no growth took place. The effect of the hydrogen ion concentration upon the antifungal action of the filtrates could be best illustrated by fungi with medium sensitivity. (See photograph 3.) Fungi highest in the scale of sensitivity were killed, and fungi lowest in the scale remained alive, independent of more or less acid pH. (S. schencki, T. purpureum, M. audouini, T. violaceum, and E. inguinale). T. gypseum, for instance, was not influenced by the potent filtrate of pH 5.8; a change of the pH to 4.0 however, produced both fungistatic and fungicidal effect. M. lanosum grew in the weak filtrate at pH 5.8 while the same filtrate at pH 4.0 inhibited and killed the fungus. This phenomenon could be observed to some extent also with T.
purpureum. It may be assumed that it operated as a general principle but
45
ACTION OF ASPERGILLUS CLAVATUS ON DERMATOPHYTES
FIG.
1
F. •0
PMqd Pqo 'Vt __j___jfltfrlLU •WTT19
Ji.%41k, ••..ft !"fl' Psey I SN
'---
5t
—
ii -J
rn
-.
FIG. 2
FIGs. 1 AND 2. First test tube of each group of four contains the inoculum in the potent filtrate. The following tubes contain transplants on solid media, made at intervals of 2, 7, and 9 days. 1) Responses to the filtrate vary with type of fungus. M. lanosum is of medium sensitivity; E. fioccosum is highly susceptible. 2) Less acid pH is less effective than more acid pH, demonstrated on M. lanosum. 3) Effect depends also on time of exposure. Two days of exposure of M. lanosum at pH 5.8 permits growth; seven and 9 days inhibit growth.
46
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
escaped demonstration by the overwhelming influence of the other determining factors; that is, sensitivity of fungus, potency of filtrate and time of exposure. That the high degree of acidity alone was not responsible for the antifungal effect can be shown on T. gypseum and T. purpureum. The former grew well in the weak filtrate at pH 4.0 but was inhibited and killed by the potent filtrate of the same p1-I. T. purpureum, kept for seven days in the weak filtrate at pH 4.0, grew upon transplantation, but none of the transplants from the potent filtrate of the same pH showed evidence of growth.
FIG. 3. It shows the influence of a weak filtrate at pH 4.0 and 5.8 resp., upon fungi of various degrees of susceptibility. T. gypseum, relatively little susceptible, was not influenced at both pH. T. purpureum, of medium sensitivity, grew at pH 5.8, but was killed at pH 4.0. M. audouini, very sensitive, was killed at both pH.
It is noteworthy that the data relating to pH and antifungal power are in agree-
ment with previous reports. Peck and his coworkers demonstrated that the antifungal power of human sweat increased with an increase in the hydrcgen ion
concentration. They also showed that the fungistatic and fungicidal action of sodium propionate solutions decreased above pH 6.0. Similar results with the latter were obtained by Keeney (3). SUMMARY
Culture filtrates of Aspergillus clavatus revealed fungistatic and fungicidal
tction, in vitro, upon a variety of dermatophytes including Trichophyton
ACTION OF ASPERGILLTJS CLAVATUS ON DERMATOPHYTES
47
gypseum, T. purpureum, T. violaceum, T. schoenleini, M. audouini, M. lanosum, and E. floccosum. M. albicans and S. schencki were not appreciably influenced. The different fungi vary in their susceptibility to the filtrates. The degree of
susceptibility increases in the following order: T. gypseum, M. lanosum, T. purpureum, M. audouini, T. violaceum, E. floccosum. Three other factors determined the effect of the filtrates, namely, the potency, the time of contact with the test fungi, and the hydrogen ion concentration. BIBLIOGRAPHY 1. a. WIESNEB, B. B.: Bactericidal effect of Aspergillus clavatus. Nature, 149: 356, 1942 b. CHAIN, E., FLOREY, H. W., AND JENNINGS, M. A.: An antibacterial substance produced by Penicillium claviforme. Brit. J. Exper. Path. 23: 202, 1942. c. WAKSMAN, S. A., HoRNING, E. S., ur SPENCER, E. L.: Two antigonistic fungi, Asper-
gillus fumigatus and Aspergillus clavatus, and their antibiotic substances. J. Bact. 45: 233, 1943. d. WAKSMAN, S. A., AND BuGlE, E.: Action of antibiotic substances upon Ceratostomella ulmi. Proc. Soc. Exper. Biol. & Med. 54: 79—82, 1943. e. WAKSMAN, S. A., HORNING, E. S., AND SPENCER, E. L. L. The production of two antibacterial substances, fumigacin and clavacin. Science, 96: 202—203, 1942. f. Rismici, H., BIRRENSHAW, J., MICHAEL S. E., BRACKEN, A., Grs, W. E., AND Hop-
KINS, W. A.: Patulin in the common cold; collaborative research on a derivative of Penicillium patulin Bainier. Lancet, 2: 625—635; comment p. 684, 1943. g. CHAIN, E., FLOREY, H. W., AND JENNINGS, M. A.: Identity of patulin and claviformin. Lancet, 1: 112—114, 1944.
h. KAROW, E. 0., AND FOSTER, J. W.: An antibiotic substance from species of Gymnoascus and Penicillium. Science, 99:265—266, 1944. a. REILLY, H. C., SCHATZ, A., AND WAKSMAN, S. A.: Antifungal properties of antibiotic
substances. J. Bact. 49: 585, 1945. b. HERRICK, J. A.: Antifungal properties of clavacin. Proc. Soc. Exper. Biol. & Med., 59:41,1945. c. TOLMACH, J. A., AND LOEWENTEAL, K.: Action of clavacin on some dermatophytes.
Preliminary report. J. Invest. Dermat;, 7:207—208, 1946. e. HOPKINS, J. G., Fisirsa, J. K., HILLEGAS, A. B., LEDIN, R. B., REBELL, G. C., AND
CAMP, E.: Fungistatic agents for treatment of dermatophytosis. J. Invest. Dermat. 7:239—253, 1946.
3. a. PECK, S. M., AND ROSENFELD, H.: The effects of hydrogen ion concentration, fatty acids and vitamin Con the growth of fungi. J. Invest. Dermat. 1: 237—265, 1938. b. PECK, S. M., ROSENFELD, H., LEIFER, W., AND BIEBMAN, W.: Role of sweat as a fungi-
cide. Arch. Dermat. & Syph. 39: 126—148, 1939.
c. KEENEY, E. L.: Fungistatic and fungicidal effect of sodium propionate on common pathogens. Bull. Johns Hopkins Hosp. 73:379—390, 1943.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
KURT LOEWENTHAL, M.D.
Clavacin is an antibiotic substance derived from Aspergillus clavatus and from a number of related molds (1). It was shown to be active, in vitro, against some
dermatophytes including Trichophyton gypseum, Trichophyton purpureum, Microsporon audouini and also against Monilia albicans and Cryptococcus neoformans (2). The present study extends these investigations and discusses the mechanism of the action of clavacin upon the test organisms.
The test fungi were Trichophyton violaceum, Trichophyton suiphureum, Trichophyton schoenleini, Microsporon lanosum, Epidermophyton floccosum, and also Sporotrichum scheneki as a representative of the fungi producing deep
mycoses. Control organisms which had already proven their sensitivity to clavacin were also employed, including Monilia albicans, Trichophyton gypseum, Trichophyton purpureum and Microsporon audouini.
Since crystalline clavacin could not be obtained a culture filtrate was obtained from a strain of A. clavatus of our own stock cultures. The fungus was grown in a liquid medium which contained 40 Gm. dextrose and 10 Gm. peptone in 1,000 cc. of distilled water. The inocula were kept at 28 C. for 7 days and the viable material separated by passage through a Seitz filter. The amber colored filtrate was kept at ice box temperature. To determine the influence, if any, of the hydrogen ion concentration upon the activity of the filtrate the latter was divided into two parts; one part was adjusted to a pH of 4.0 and the other part to a pH of 5.8. A second batch of culture filtrate was prepared which was inferior in potency to the former. It also was employed at pH values of 4.0 and 5.8 respectively. The absolute potency of both batches, expressed in content of clavacin, was not known. METhOD
Fragments from the test fungous cultures were placed into test tubes containing 10 cc. of the various culture filtrates. Two inoculations were made for each hydrogen ion concentration and for each batch of filtrate, amounting to 8 inocula for each fungus and assay. The inoculated tubes were kept at room temperature. Two, 7 and 9 days later, particles of the inocula were removed from the filtrates and, after thorough rinsing in sterile distilled water, transplanted upon dextrosepeptone agar slants. Only one tube of each pH and potency was used for transplantations while the other remained undisturbed. The inocula in the filtrates 1
From the Long Island College Hospital and the Department of Dermatology and
Syphilology, Long Island College of Medicine, Brooklyn, N. Y. Received for Publication Jan. 6, 1947. 41
42
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
and the transplants on the agar slants were observed for evidence of fungous growth, at intervals of 3 to 5 days, for at least 21 days. RESULTS
The investigation of T. gypseum, T. violaceum, M. audouini, M. lanosum, and E. fioccoswn is complete while S. schencki, T. schoenleini and T. suiphureum demand some amplification. The results of 26 assays are summarized in table 1. S. schencki showed no appreciable inhibition by the weak filtrate at both PH; M. albicans was not influenced by the potent filtrate at p115.8. T. gypseum was inhibited and killed by the potent filtrate at a pH of 4.0; the potent filtrate of pH 5.8 and the weak filtrate of both pH did not produce fungicidal or fungistatic effect.
M. lanosum was inhibited and killed by both the strong and weak filtrate at pH 4.0. At 5.8 no growth was visible in the potent filtrate, but the first transplant revealed evidence of growth after 19 days of incubation. The later transplants did not grow. In the weak filtrate of pH 5.8 some inocula developed growth. Particles from the apparently sterile inocula revealed vigorous growth when the transplantation was done not later than 2 days after exposure to the filtrate. T. schoenleini and T. sulphureum were inhibited and killed by the potent ifitrate at pH 4.0. T. purpureum was inhibited and killed by the potent filtrate at both pH. It was inhibited by the weak filtrate at both pH, but transplants from pH 4.0 after 7 days' exposure, and from pH 5.8 after 9 days' exposure, revealed fungous growth. M. audouini, T. violaceum and E. fioccosum were inhibited and killed by the potent and weak filtrate at both pH. COMMENT
These experiments reveal that culture filtrates of A. clavatus possess a wider range of action than previously reported. All dermatophytes tested were shown to be susceptible. M. albicans and S. seheneki were not demonstrably influenced; the former, however, was shown in earlier reports to react to some extent to higher concentrations of clavacin and long exposures (2). The effect was determined by the following factors: the type of fungus, the potency of the filtrate (presumably identical with the clavacin concentration in
the filtrate), the time of contact of fungus and filtrate, and the hydrogen ion concentration of the filtrate. (See photographs 1 and 2.) In the table the lungi were arranged in their order of sensitivity. The highest degree of resistance was shown by M. albicans and S. scheneki. T. gypseum possessed a higher degree of sensitivity; it was killed by the potent filtrate at a low pH. M. lanosuin, more susceptible, was killed by both filtrates at pH 4.0, but showed some growth at pH 5.8. The position of T. schoenleini and T. suiphureum is tentative until more information will be available; both were killed
43
ACTION OF ASPERGILLUS CLAVATUS ON DERMATOPHYTES
TABLE 1 WEAL FILTRATE
pH 4.0
POTENT PILTEATE
pH 5.8
pH 4.0
pH 5.8
E. fioccosum
0
0
0
0 0 0
0 0 0
0 0
0 0 0
0
0
0 0 0 0
0 0
0
0
0 0 0 0
0 0 0 0
0 0 0 0
0 0 0 0
0
+ + +
0
Transplant III Transplant II Transplant I Inoculum
T. violaceum
Transplant III Transplant II Transplant I Inoculum
0
0 0 0
M. audouini
Transplant III Transplant II Transplant I Inoculum
T. purpureum
Transplant III Transplant II Transplant I Inoculum
+ + 0
0
0 0 0
0
0 0 0 0 0 0
T. suiphureum 0 0 0 0
Transplant III Transplant II Transplant I Inoculum
T. schoenleini
0
Transplant III Transplant II Transplant I
0 0 0
Inoculum
M. lanosum
Transplant III Transplant II Transplant I Inoculum
0 0 0 0
Transplant I = after 2 days' incubation Transplant II = after 7 days' incubation Transplant III = after 9 days' incubation
+ = fungous growth
o = no fungous growth • = no information available
0 0
+ +
0 0 0 0
0 0
+ 0
44
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
TABLE 1—Concluded WEAK EILTLATE
T. gypeuin Transplant III Transplant II Transplant I Inoculum S. scheneki
Transplant III Transplant II Transplant I Inoculum M. albicans
Transplant III Transplant II Transplant I Inoculum
POTEN'X PILTRATE
pH 4.0
pH 5.8
pH 4.0
pH 5.8
+ + + +
+ +
0 0 0 0
+ + + +
+ + + +
. . . .
.
+ +
+ + . . . .
+ +
. . . .
. .
. .
+ + + +
by the potent filtrate at pH 4.0. Both filtrates were fungistatic for T. purpureum the more potent also killed the fungus at both hydrogen ion concentrations. The highest degree of sensitivity was revealed by M. audouini, T. violaceum and E. floccosum. That the more potent filtrate, presumably containing clavacin in higher concen-
tration, exerts a greater antifungal power was exemplified by T. purpureum and T. gypseum. The potent filtrate inhibited and killed the former while the action of the less potent filtrate was mainly fungistatic. The latter was not influenced by the weak filtrate but it was killed by the potent filtrate at pH 4.0. The influence of length of contact between filtrate and fungus was illustrated
by M. lanosum and T. purpureum. Transplants of the former, from inoctda exposed for 2 days showed growth upon culture medium while later transplants remained sterile. Transplants of T. purpureum from inocula exposed for 7 days grew while after 9 days no growth took place. The effect of the hydrogen ion concentration upon the antifungal action of the filtrates could be best illustrated by fungi with medium sensitivity. (See photograph 3.) Fungi highest in the scale of sensitivity were killed, and fungi lowest in the scale remained alive, independent of more or less acid pH. (S. schencki, T. purpureum, M. audouini, T. violaceum, and E. inguinale). T. gypseum, for instance, was not influenced by the potent filtrate of pH 5.8; a change of the pH to 4.0 however, produced both fungistatic and fungicidal effect. M. lanosum grew in the weak filtrate at pH 5.8 while the same filtrate at pH 4.0 inhibited and killed the fungus. This phenomenon could be observed to some extent also with T.
purpureum. It may be assumed that it operated as a general principle but
45
ACTION OF ASPERGILLUS CLAVATUS ON DERMATOPHYTES
FIG.
1
F. •0
PMqd Pqo 'Vt __j___jfltfrlLU •WTT19
Ji.%41k, ••..ft !"fl' Psey I SN
'---
5t
—
ii -J
rn
-.
FIG. 2
FIGs. 1 AND 2. First test tube of each group of four contains the inoculum in the potent filtrate. The following tubes contain transplants on solid media, made at intervals of 2, 7, and 9 days. 1) Responses to the filtrate vary with type of fungus. M. lanosum is of medium sensitivity; E. fioccosum is highly susceptible. 2) Less acid pH is less effective than more acid pH, demonstrated on M. lanosum. 3) Effect depends also on time of exposure. Two days of exposure of M. lanosum at pH 5.8 permits growth; seven and 9 days inhibit growth.
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escaped demonstration by the overwhelming influence of the other determining factors; that is, sensitivity of fungus, potency of filtrate and time of exposure. That the high degree of acidity alone was not responsible for the antifungal effect can be shown on T. gypseum and T. purpureum. The former grew well in the weak filtrate at pH 4.0 but was inhibited and killed by the potent filtrate of the same p1-I. T. purpureum, kept for seven days in the weak filtrate at pH 4.0, grew upon transplantation, but none of the transplants from the potent filtrate of the same pH showed evidence of growth.
FIG. 3. It shows the influence of a weak filtrate at pH 4.0 and 5.8 resp., upon fungi of various degrees of susceptibility. T. gypseum, relatively little susceptible, was not influenced at both pH. T. purpureum, of medium sensitivity, grew at pH 5.8, but was killed at pH 4.0. M. audouini, very sensitive, was killed at both pH.
It is noteworthy that the data relating to pH and antifungal power are in agree-
ment with previous reports. Peck and his coworkers demonstrated that the antifungal power of human sweat increased with an increase in the hydrcgen ion
concentration. They also showed that the fungistatic and fungicidal action of sodium propionate solutions decreased above pH 6.0. Similar results with the latter were obtained by Keeney (3). SUMMARY
Culture filtrates of Aspergillus clavatus revealed fungistatic and fungicidal
tction, in vitro, upon a variety of dermatophytes including Trichophyton
ACTION OF ASPERGILLTJS CLAVATUS ON DERMATOPHYTES
47
gypseum, T. purpureum, T. violaceum, T. schoenleini, M. audouini, M. lanosum, and E. floccosum. M. albicans and S. schencki were not appreciably influenced. The different fungi vary in their susceptibility to the filtrates. The degree of
susceptibility increases in the following order: T. gypseum, M. lanosum, T. purpureum, M. audouini, T. violaceum, E. floccosum. Three other factors determined the effect of the filtrates, namely, the potency, the time of contact with the test fungi, and the hydrogen ion concentration. BIBLIOGRAPHY 1. a. WIESNEB, B. B.: Bactericidal effect of Aspergillus clavatus. Nature, 149: 356, 1942 b. CHAIN, E., FLOREY, H. W., AND JENNINGS, M. A.: An antibacterial substance produced by Penicillium claviforme. Brit. J. Exper. Path. 23: 202, 1942. c. WAKSMAN, S. A., HoRNING, E. S., ur SPENCER, E. L.: Two antigonistic fungi, Asper-
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