Journal of Antimicrobial Chemotherapy (1977) 3, 95-100

In vitro growth inhibitory studies of phacidin against some dermatophytes, opportunistic and systemic fungi, and yeasts pathogenic on man

Awatar S. Sekhon and A. Funk

Phacidin, an antibiotic produced by Potebniamyces balsamicola var. boycei, was tested for activity against some species of dermatophytes, yeasts pathogenic to man, opportunistic and systemic dimorphic fungi, using standard tube double dilution procedure. Results indicated that among dermatophytes, Trichophyton mentagrophytes, T. rubrum, and Epidermophytonfloccosumwere very susceptible (MFCs 1-56 ug/ml of the medium); the MFCs for the species of Microsporum ranged from 3-125 to 25 ug/ml. Scopulariopsis brevicaulis and Aspergillus nidulans gave the highest MFCs (400 ug/ml). For other species of opportunistic fungi, i.e. A fumigatus, A. niger, A.flavus, and A. terreus, the MFCs recorded were from 25 to 100 ug/ml. In the group of yeasts, which included six species of Candida, two species of Cryptococcus, one species of Saccharomyces, Torulopsis, and Trichosporon, the MFCs were in the range of 1-56 to 50 ug/ml. Surprisingly, the incitants of Blastomycosis, Coccidioidomycosis, Histoplasmosis, and Sporotrichosis were found to be very susceptible to phacidin (MFCs, 0-195 to 12-5 ug/ml). Introduction

Preliminary studies have shown that phacidin, y-pyrone antibiotic produced by the ascomycete Potebniamyces balsamicola var. boycei (Poulton, Williams & McMullan, 1974), inhibited the growth of a wide variety of saprophytic and plant pathogenic fungi, along with a few isolates of Candida albicans and another species of Candida (Funk & McMullan, 1974). However, the effect of this antibiotic on the fungi and yeasts that cause superficial and deep seated mycoses in man has not been determined. The present study was conducted to determine the spectrum of phacidin against the medically important fungi and yeasts. Materials and methods

Organisms The strains of dermatophytes, opportunistic and systemic fungi, and yeasts pathogenic on man and their sources are listed in Table I. Stock cultures of the opportunistic and dermatophytes, yeasts, and systemic dimorphic fungi were maintained on the Pablum cereal agar (Sekhon & Carmichael, 1972), phytone-yeast extract (BBL), and brain heart infusion agar (BHI, Difco) slants, respectively at 25° or 37°C. 95

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Provincial Laboratory of Public Health, The University of Alberta, Edmonton, Alberta, T6G 2J2, and Pacific Forest Research Centre, 506 West Burnside Road, Victoria, British Columbia, Canada

353 354 345 350 297 298 299 355 344 346 303 356 341 342 220 343 357 339 340 305 306 307 352 328 348 349 326 331 332 327 328 244

Yeasts Candida albicans Candida albicans Candida guilliermondii Candida guilliermondii Candida krusei Candida krusei Candida parapsilosis Candida parapsilosis Candida pseudotropicalis Candida pseudotropicalis Candida tropicalis Candida tropicalis Cryptococcus albidus Cryptococcus albidus Cryptococcus neoformans Saccharomyces cerevisiae Saccharomyces cerevisiae Trichosporon cutaneum Trichosporon cutaneum Torulopsis glabrata Torulopsis glabrata Torulopsis glabrata Torulopsis glabrata

Dermatophytes Epidermophyton floccosum Epidermophyton floccosum Epidermophyton floccosum Microsporum canis Microsporum canis Microsporum canis 'Nannizzia gypsea (—) 'Nannizzia gypsea (+) Scopulariopsis brevicaulis

Organism

1-58 1-58 0-39-0-78 250 12-5-25-0 12-5 6-25 3-125 400.0

6.25 1-56-3-125 250 6-25-12-5 250 12-5-25 250 250 12-5 6-25-12-5 500 500 6-25 1-56-3-125 3-125 3.125 3-125-6-25 12-5-25-0 12-5-25-0 3-125-6-25 12-5 500 12-5

Phacidin" PLMt No. MFC (ng/ml)

1-56 0-39-07-8 0-78

12-5-25-0

(0-78-1-56)

6-25-1-25

0-78-1-56

(12-5)

(3-125-6-25)"

Antibiotics myxin" MFC (ng/ml)

(1-5-1-7)

(1-4-1-6)

(2-1-2-3)

Griseofulvinc MIC (ng/ml) after 100 h

Human, Provincial Lab. , Edmonton Human, Provincial Lab. , Edmonton Human, Provincial Lab, , Edmonton Human, Provincial Lab. , Edmonton Unknown Human, Provincial Lab, , Edmonton UAMHft 1486(—) UAMH 1485 (+) Human, Provincial Lab, , Edmonton

As No. 5, N.Y. State Dept. of Health, Albany, N.Y. Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton

Human, isolated and identified by the Division of Mycology, Provincial Laboratory of Public Health, University of Alberta, Edmonton, Alberta, according to the method stated previously (Sekhon & Hargesheimer, 1975)

Source

Table I. Sources and results of the sensitivity tests on some human pathogenic yeasts, dermatophytes, and opportunistic and systemic dimorphic fungi to phacidin

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318 358 253

Histoplasma capsulatum

Histoplasma capsulatum

Sporothrix schenckii 6-25-12-5

0195

0-39-0-78

1-56-3-125 0-78 6-25 1-56

250-500 4000 500 500

1000 500 50-0-1000 500

4000 1-56 3-125 3-125-6-25 0-78-1-58 500 500-1000

6-25

0195 0195 0-78-1-56

(1-3-1-5)

(1-9-2-1)

Griseofulvinc MIC (ug/ml) after 100-h

As 49B, N.Y. State Dept. of Health, Albany, N.Y. As 49A, N.Y. State Dept. of Health, Albany, N.Y. Human Provincial Lab., Edmonton UAMH 3537, as B 1393 from the Centre for Disease Control, Atlanta, Georgia, U.S.A. As 811, from Dr Leo Kaufman, Centre for Disease Control, Atlanta, Georgia, U.S.A. As G8, Walter Reed Army Institute of Research, Washington, D.C., U.S.A. As B959, from Dr Leo Kaufman, Centre for Disease Control, Atlanta, Georgia, U.S.A.

Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton As PC7 from Dr K. E. Papas, Dept. of Plant Pathology, University of Georgia, Athens, Georgia, U.S.A. As No. 31, N.Y. State Dept. of Health, Albany, N.Y. As 31B, N.Y. State Dept. of Health, Albany, N.Y. Unknown As 35A, N.Y. State Dept. of Health, Albany, N.Y.

Human, Provincial Lab., Edmonton UAMH2822(+) UAMH2823(-) Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton Human, Provincial Lab., Edmonton As 1050, from Dr L. Ajello, Centre for Disease Control, Atlanta, Georgia, U.S.A.

Source

c

° The MFCs are from duplicate determinations. " The values given for myxin are from a recent investigation of Sekhon & Hargesheimer [Journal of Clinical Pathology 28: 547-9 (1975]. The MICs given in brackets are for the different strains of the same species. For details, please refer to work of Korzybski et al. (1967]. ' The values given in brackets are for the different strains of the same species. • M. gypseum, imperfect state of N. gypsea. f Imperfect state of Arthroderma benhamiae. t Provincial Laboratory Mycology Number. t t University of Alberta Mold Herbarium and Culture Collection.

217 218 288 314

221 213 163 212

Aspergillus niger Aspergillus nidulans Aspergillus terreus Aspergillus terreus

Systemic dimorphic fungi Blastomyces dermatitidis Blastomyces dermatitidis Coccidioides immitis Histoplasma capsulatum

320 325 347 252

245 335 336 334 351 167 190

Opportunistic Fungi Aspergillus fumigatus Aspergillus fumigatus Aspergillus fumigatus Aspergillus flavus

Scopulariopsis brevicaulis 'Trichophyton mentagrophytes f Trichophyton mentagrophytes Trichophyton rubrum Trichophyton rubrum Trichophyton terrestre Trichophyton terrestre

Organism

PLMt Phacidin" No. MFC (ug/ml)

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98

A. S. Sekhon and A. Funk

Inoculum preparation Strains of the opportunistic fungi and dermatophytes were grown in petriplates containing pablum cereal agar (15 ml/plate) for 7 days at 25°C. Yeasts were cultured on phytoneyeast extract agar and the systemic dimorphic fungi on BHI agar slants for 48 to 240 h at 25 or 37°C. Inoculum suspensions for the yeasts and systemic dimorphic fungi were prepared according to Sekhon & Hargesheimer (1975); for opportunistic and dermatophytic fungi as described elsewhere (Sekhon, Padhye & Carmichael, 1974). The spectrophotometric readings of these suspensions were in the range of 85 to 95 %T at 520 nm.

Antibiotic test medium and susceptibility tests The yeast nitrogen base (lx) broth was used to perform susceptibility tests as described in detail by Sekhon & Hargesheimer (1975). Concentrations of the antibiotic ranged from 400 to 0-139 ug/ml of the test medium in tubes. Appropriate solvent controls were included and the observations indicated that, except for the first two dilutions for the majority of the strains and fourth dilution for one strain only, solvent did not inhibit the growth of any strain when present in the amounts corresponding to the tubes that contained up to 100 ug of phacidin per ml of the medium. Thus, in accordance with the previous study (Sekhon & Hargesheimer, 1975), this investigation has also demonstrated that whatever the minimal fungicidal concentrations (MFCs) have been obtained are only due to the presence of phacidin. The MFCs were recorded after an initial incubation of tubes containing broth, inoculum suspension, and the antibiotic at 30°C for 48 h, and then inoculating the Sabouraud dextrose agar plates (for dermatophytes and opportunistic fungi) and BHI slants (for yeasts and systemic dimorphic fungi) from the inoculum taken from the tube which showed visible growth and from the next two tubes which contained higher concentrations. These plates and slants were incubated at 30°C for 48 to 96 h and then the MFCs were recorded. Results

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Antibiotic Phacidin used in this study was produced according to the method of Funk & McMullan (1974). Stock solution of the antibiotic was prepared in A^.A^.dimethylformamide (1 mg/ml).

The MFCs of phacidin obtained for the organisms tested are presented in Table I. Also given for comparison are previously publishedfindingsof tests (Table I) with myxin (Sekhon & Hargesheimer, 1975) and griseofulvin (Korzybski, Kowszyk-Gindifer & Kurylowicz 1967). Among yeasts, strains of six Candida species, Cryptococcus neoformans, C. albidus, Saccharomyces cerevisiae, Trichosporon cutaneum, and Torulopsis glabrata, the MFCs ranged 1-58 to 50|ig/ml. Three strains of Epidermophytonfloccosumwere very susceptible to phacidin (MFC, 0-39 to 1-56 ug/ml). For other strains of dermatophytes, the highest value recorded was for Scopulariopsis brevicaulis (400 ug/ml); whereas, the lowest MFC was for Trichophyton mentagrophytes and T. rubrum. However, T. terrestre was somewhat resistant to phacidin. The fungi that generally cause opportunistic infection in human beings, their MFCs with the exception of Aspergillus nidulans (400 ug/jil), varied from 12-5 to 100 ug/ml. On the other hand, the response of systemic dimorphic fungi, causing blastomycosis, coccidioidomycosis, histoplasmosis and

Activity of phacidin against fungi and yeasts

99

sporotrichosis, was much better (MFCs, 0-139 to 12-5 ug/ml) when their response is compared with the strains belonging to the fungi of the 'opportunistic' group. Discussion

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The results of this study have demonstrated that phacidin, a newly discovered antibiotic, is effective in vitro against the fungi and yeasts of medical importance. Most mycotic infections are currently treated with either mycostatin, griseofulvin, 5-fluorocytosine, or amphotericin B. All of these antibiotics are either inhibitory to only a limited number of fungi and/or yeasts or, because of their side effects, the dose given is reduced to a point at which it probably has no therapeutic value; consequently, new antifungal compounds are badly needed to cure mycotic infections in human. Our studies with phacidin when compared to those reported for myxin (Sekhon & Hargesheimer, 1975) indicate that both these antibiotics may be of value in human infections. Since there are no data available as to the toxicity of these two potent, wide spectrum antibiotics, it is impossible to draw any conclusion with regard to the application of phacidin in clinical use. One thing which is clear, however, that one may not have any difficulties in obtaining or producing the phacidin as it can readily be made available. This point is emphasized because there were limitations associated in the availability of myxin (Sekhon, 1975). In vitro effectiveness of phacidin for dermatophytes indicates that MFCs of this antibiotic were very similar to the minimal inhibitory concentrations reported for griseofulvin (Korzybski et al, 1967). With the exception of Microsporum canis, the MFC for phacidin and MICs for griseofulvin are very close to one another. For other dermatophytes, e.g. E. floccosum, and T. mentagrophytes, the MFCs for phacidin were lower than the MICs of griseofulvin (see Table II). These results are certainly significant, since griseofulvin is being used with much success in the treatment of dermatophytoses; further, this antifungal therapeutic agent has been reported to be less effective in cases of toenail infections, as only 56-9 and 16-7 % success was achieved in fingernail and toenail infections, respectively (Anderson, 1965). Thus, it is clear that there remains a need for more effective antifungal antibiotic not only to treat the cases of deep seated mycoses, but also of superficial infections caused by fungi or yeasts. This point is further stressed because the most commonly used and available antibiotics, amphotericin B and 5-fluorocytosine, in Canada proved ineffective in a recently reported case of deep scopulariopsosis and endophthalamitis in vitro and in vivo, respectively (Sekhon, Willans & Harvey, 1974; Skidmore & Cockcroft, 1974). The MFCs of this study for the group of opportunistic fungi (i.e. Aspergillus species) are higher than those reported by Shadomy & Espinel-Ingroff (1974); however, the resistance of these fungi to phacidin and amphotericin B shows a proportional trend. That is, the organism that required greater concentration of phacidin also needed the higher concentration of amphotericin B. Similarly, the strains that were highly sensitive to phacidin were also sensitive to amphotericin B (Shadomy & Espinel-Ingroff, 1974). Phacidin, as indicated by the results, proved very effective against species of systemic dimorphic fungi (H. capsulation, S. schenckii, B. dermatitidis, and C. immitis) and yeasts except C. tropicalis and T. glabrata, which are otherwise either completely resistant or less susceptible to 5-fluorocytosine. The findings also suggest that in vitro response of strains tested in this investigation is, certainly, better than the latter antifungal compound (5-fluorocytosine). It has also been noted that the results of this study for C. albicans are in agreement with the previously reported data by Funk & McMullan (1974). However, a direct

100

A. S. Sekhon and A. Funk

correlation between the methods used in the present and the former study cannot be established. Acknowledgements

References

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The authors express their gratefulness to Dr J. M. S. Dixon, Director, Provincial Laboratory of Public Health, The University of Alberta, Edmonton, for his advice, suggestions, and criticisms of the manuscript. We thank Eleanor E. McMullan for her valuable help in purification of phacidin, Miss Erika Hargesheimer for technical assistance, and Dr J. W. Carmichael for his suggestion to take up the investigation. The work reported herein was made possible by a research grant, MA 5216, from the Medical Research Council of Canada, Ottawa, Canada.

Anderson, D. W. Griseofulvin: Biology and clinical usefulness. Annals of Allergy 23:103 (1965). Funk, A. & McMullan, E. E. Phacidin, a fungal growth inhibitor from Potebniamyces balsamicola var. boycei. Canadian Journal of Microbiology 20: 422-5 (1974). Korzybski, T., Kowzyk-Gindifer, Z. & Kurylowicz, W. Antibiotics, Volume II. Origin, Nature & Properties (1967), pp. 1235^*8. Poulton, G. A., Williams, M. E. & McMullan, E. E. Phacidin, a novel y-pyrone fungal growth inhibitor from Potebniamyces balsamicola var. boycei. Tetrahedron Letters 30: 2611-4 (1974). Sekhon, A. S. Sensitivity of Scopulariopsis brevicaulis to some antimicrobial compounds. Mycopathologia 56: 177-9 (1975). Sekhon, A. S. & Carmichael, J. W. Pyrolysis-gas-liquid chromatography of some dermatophytes. Canadian Journal of Microbiology 18: 1593-1601 (1972). Sekhon, A. S. & Harhesheimer, E. Sensitivity of some human pathogenic yeasts and systemic fungi to myxin. Journal of Clinical Pathology 28: 547-9 (1975). Sekhon, A. S., Padhye, A. A. & Carmichael, J. W. Disc-gel electrophoresis of + and — strains and of the gymnothecial cultures of Arthroderma tuberculatum. Sabouraudia 12: 12-17 (1974). Sekhon, A. S., Willans, D. J. & Harvey, J. H. Deep scopulariopsosis: a case report and sensitivity studies. Journal of Clinical Pathology 27: 837-43 (1974). Shadomy, S. & Espinel-Ingroff. Susceptibility testing of antifungal agents. In Manual of Clinical Microbiology. Second Edition, American Society for Microbiology, Washington, D.C., Ed. Lennette, E. H., Spaulding, E. H. & Truant, J. P. (1974), pp. 569-74. Skidmore, A. & Cockcroft, W. H. Intravenous feeding and endophthalmitis. The Forty-second Annual Meeting, The Laboratory Division of the Canadian Public Health Association, Ottawa, 27-30 November, p. 69. {Manuscript accepted 26 January 1976)

In vitro growth inhibitory studies of phacidin against some dermatophytes, opportunistic and systemic fungi, and yeasts pathogenic on man.

Journal of Antimicrobial Chemotherapy (1977) 3, 95-100 In vitro growth inhibitory studies of phacidin against some dermatophytes, opportunistic and s...
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