SKIN RESEARCH Dec. 1968 Vol.10 No.5 577-583

COMPARATIVE STUDY ON THE DRUG-SENSITIVITY OF THREE STRAINS OF MUCORACEAE INCLUDING ABSIDIA RAMOSA ISOLATED FROM A CASE OF SKIN AND NAIL MUCORMYCOSIS PART 1: STUDIES ON THE IN-VITRO SENSITIVITY OF THREE STRAINS OF MUCO- RACEAE FOR 14 ANTIBIOTICS

KAZUE OHARA, M.D., KEIKO CHIBA, M.D., TAEKO OGURA, M.D. and SATOKO KITAMURA. M.D. Department of Dermatology, Kansai Medical School, Moriguchi, Osaka, Japan (Director: Prof. K. OHARA)

INTRODUCTION MATERIALS AND METHODS

The recent increase of incidence of mycotic I. The Strains infections has resulted in the development 1. Absidia ramosa (abbreviated as A.r.) isolated of many anti-fungal agents. Among them, from a case of skin and nail mucormycosis seen at the Department of Dermatology, Kansai Medical School griseofulvin for trichophytosis, nystatin for candidamycosis and amphotericin B for cryp- in 1959. 2. Rhizopus oryzae IFO No.5438 (abbreviated tococcosis or chromoblastomycosis, have been as R.o.) provided by Institute for Fermentation found very effective in clinical use. Osaka. For mucormycosis, amphotericin B and 3. Mucor pusillus (abbreviated as M.p.) provided nystatin seem to have been reported as effec- by the Institute of Microbiology, Osaka University. tive agents but no further report appears to have been found in the literature. This seems due to II. The Drugs a fact that cases of mucormycosis, being rather 1. Amphotericin B uncommon and rapidly fatal, are mostly dia- 2. Nystatin gnosed on autopsy and causative fungi are 3. Trichomycin, an antimycotic agent developed seldom isolated. from Streptomyces Hachijoensis isolated from a soil Chiba1), one of the authors, reported the of Hachijo island, Japan. 4. Naramycin, an agent developed from Strepto- sensitivity of four antibiotics and four chemi- myces Naraensis isolated from a soil of Nara, Japan. cals to Absidia ramosa isolated from a case of 5. Variotin, an antimycotic agent developed from primary skin and nail mucormycosis. Paecilomyces varioti Bainier variant antibioticus iso- The present study on sensitivity in vitro lated from a soil of Nagano, Japan. was made of three strains of mucoraceae, 6. Polymyxin B Absidia ramosa, Rhizopus oryzae and Mucor 7. Kanamycin pusillus for 14 antibiotics. 8. Dihydro-streptomycin

577 9. Erythromycin with the above mentioned 1% glucose liquid medium

10. Tetracyclin to 80 u/ml. Then four culture media, containing

11. Oxy-tetracyclin 32 u/ml, 16 u/ml, 8 u/ml and 4 u/ml of variotin were

12. Chlor-tetracyclin prepared. 13. Chloramphenicol b. Inoculation of Strains 14. Penicillin Two tenth cc of 0.85% NaCL solution of the spore

III. The Culture Media suspension (100-200/mm3) was put into a test tube A. For the Dilution Methods: which contained serial concentration of above men-

Sabouraud's liquid medium containing 1% glucose. tioned four anti-fungal agents. The m.i.c. of each antibiotic was examined after a

B. For the Disk Methods: seven day incubation at 25-28•Ž. The Nissan labeled medium consisting of the follow- ing B. Sensitivity Disk Methods

Kazamino acid Nissan (mixture of 13 amino acids) The disks employed were as follows: 16.5 g 1. Nystatin (Difco, U.S.A.)

Bovin heart extract 200.00 cc 2. Polymyxin B (Difco, U.S.A.) Water soluble starch 1.5 g 3. Kanamycin (Eiken, Japan)

Glucose 2.0 g 4. Dihydro-streptomycin (Eiken, Japan)

Powdered agar 15.0 g 5. Erythromycin (Eiken, Japan) Distilled water added to make 1,000.0 cc (pH 7.4•}0.1 6. Oxy-tetracyclin (Eiken, Japan) 7. Chlor-tetracyclin (Eiken, Japan)

IV. Experimental 8. Chloramphenicol (Eiken, Japan)

A. Dilution Methods 9. Tetracyclin (Eiken, Japan) 10. Penicillin (Eiken, Japan) a. Preparation of Solution of Drugs

1. Amphotericin B Each disk of three titers of each antibiotic, was put Fifty milligrams of amphotericin B was dissolved on the Nissan plate medium covered with 0.2 cc of in 5 ml of sterile distilled water. A 1: 100 ampho- a spore suspension at a 3 cm distance. After a 48- tericin B solution was diluted with a 1% glucose hours incubation at 37.0•Ž, the inhibitory zone thus liquid medium to 1: 1,000, 1: 10,000 and 1: 100,- developed was observed. 000, then from 1: 200,000 through 1: 800,000, serial Standerds of Evaluation: twofold dilution was performed. Very sensitive inhibitive zone posi- 2. Trichomycin tive for three disks.

To make a solution containing 1,000 u/ml of Sensitive inhibitive zone posi- trichomycin in a 1% glucose liquid medium, tricho- tive for medium and mycin, being water insoluble and labile to pH, had high titered disks. to be accurately adjusted to pH 7.6 with 0.1 N Relatively resistant inhibitive zone posi- NaOH and 1 N HCL. The solution was serially tive for only high diluted to 500 u/ml, 250 u/ml and 125 u/ml. titered disk.

Resistant inhibitive zone nega- 3. Naramycin tive for all three Naramycin being soluble in organic solvents except disks. for petroleum and ether, is water soluble within 2%. It was diluted to 1: 100 with the above mentioned 1% glucose liquid medium. The solution thus made RESULTS into a 1: 1,000 culture medium was subsequently diluted serially till 1: 32,000. A. The Dilution Methods (Table 1) 1. Amphotericin B 4. Variotin

A 1: 10 aqueous solution of variotin stock solution The minimum inhibitory concentration for

(8,000 u/ml) was prepared and then diluted serially M. P. was 2.5 mcg/ml (1: 400.000) and for A.r.

578 TABLE1 M.I.C. of 4 Antibioticsfor Three Strains of Mucoraceae (Dilution Method)

TABLE2 Sensitivity of Three Strains of Mucoraceaefor 10 Antibiotics (Disk Method)

™¿ very sensitive œO sensitive + relatively resistant -resistant

was 100 mcg/ml (1: 10,000). R.o. showed no containing 100 u. and 50 u., but never around inhibition even at a 1,000 mcg/ml (1: 1,000) the disk containing 25 u., proving sensitive. concentration. 2. Kanamycin 2. Trichomycin A.r. showed an inhibitive zone around the The m.i.c. was 500 u/ml for M.P., 1,000 u/ml disks containing 30 mcg and 10 mcg, but never for both A.r. and R.o., revealing to be relatively around a disk containing 5 mcg. It was evalu- resistant. ated as sensitive. The other two strains proved to be resistant developing no inhibitivive zone 3. Naramycin around three disks. The m.i.c. was 125 mcg/ml for M.p. and R.o., 3. Dihydro-streptomycin 250 mcg/Ml for A.r., proving relatively resis- A.r. showed an inhibitive zone around the tant. disk containing 50 mcg but never around te disks containing 10 mcg and 2 mcg. It proved 4. Variotin to be relatively resistant. Either R.o. or M.p. The m.i.c. was 4 u/ml for M.p., 16 u/ml for developed no zone of inhibition around the R.o. and 8 u/ml for A.r., revealing to be three titered disks ; both proved to be resistant. sensitive. 4. Polymyxin B, Tetracyclin, Oxytetracy- B. Sensitivity Disk Method (Table 2) clin, Chlortetracylcin, Chloramphenicol, Peni- 1. Nystatin cillin and Erythromycin. A.r. and M.p. developed a zone of inhibition The three strains developed no inhibitive around the disks containing 100 u., 50 u. and zone around the three titered disks of the 25 u. of nystatin, proving very sensitive. R.o. above mentioned 7 drugs. They proved to be developed a zone of inhibition around the disks resistant against these 7 antibiotics.

579 DISCUSSION 2. Nystatin Of all antibiotics, nystatin, next to am- A few reports2,5,9,12,13,14,15,8,16have been made photericin B has been favorably used for cases of the drug sensitivity of strains belonging to the of mucormycosis.5,6,7) family of Mucoraceae (Table 3). Bank et al.5)reported that nystatin showed an Three genera of Mucoraceae, Mucor, Rhi- m.i.c. of 0.5 u/ml against Rhizopus arrhizus zopus and Absidia which are pathogenic to which is isolated from a case of head and neck man and animals were found to be sensitive to mucormycosis. McCall et al.8)reported that the amphotericin B, nystatin, trichomycin and agent showed a marked inhibition for Rhizopus variotin by Watson et al.2), Chick et al.3), Ito et oryzae. Ito et al.9) reported that an m.i.c. of al.9),Sanbe et al.12),Yazima et al.13),Matsuda et nystatin is 3 u/ml for Rhizopus and 5 u/ml for al.14) and Takeuchi et al.15) Of these agents, Mucor, showing almost the same result for amphotericin B and nystatin have been favo- Candida albicans (3 to 10 u/ml). rably employed for cases of mucormycosis on a However, according to Watson et al.2) clinical and experimental basis. nystatin fails to show a marked anti-fungal activity for nine strains of Mucoraceae (four 1. Amphotericin B strains of Rhizopus stolonifer, four strains of The present results revealed that the m.i.c. Rhizopus microsporus and a strain of Mucor of amphotericin B was 2.5 mcg/ml for M.p., hiemalis) which have been isolated from cases 100 mcg/ml for A.r. and 1,000 mcg/ml for of gastrointestinal mucormycosis. The agent R.o. which proved to be almost non-sensitive. shows no growth inhibition at 25 u/ml to Watson et al.2)examined the in-vitro activity 1,000 u/ml for those nine strains for which of amphotericin B on nine strains of Muco amphotericin B shows a marked inhibition. raceae isolated from cases of gastro-intestinal In the present experimentation where the mucormycosis. As a result, the m.i.c. of am- disk method has been employed, M.p. and A.r. photericin B was found to be 30 to 60 mcg/ml show a growth inhibitory zone at 100 u., 50 u. for four strains of Rhizopus stolonifer and two and 25 u.(very sensitive) and R.o. at 100 u. and strains of Rhizopus microsporus, 2,500 mcg/ml 50 u.(sensitive). The results coincide with for one strain of Mucor hiemalis and 5,000 mcg/ those of other authors except for Watson et ml for two strains of Rhizopus microsporus al.2) respectively. Chick et al.3) reported that amphotericin B 3. Trichomycin protected a rabbit which had been given a Trichomycin, an antifungal agent developed lethal dose of Rhizopus spores. On the basis of in Japan showed antifungal activities against this experiment, they suggested that ampho- Candida albicans at m.i.c. of 0.15 mcg/ml tericin B might be useful in the treatment of (Danomae et al.10)),and 0.015 to 0.03 mcg/ml human cases of mucormycosis. (Hosoya et al.11)). So far as in-vitro sensitivity is concerned, It is also effective against strains of Mucora- amphotericin B has proved to be rather effec- ceae. Its m.i.c. was 62.5 mcg/ml for Absidia tive against some strains of Mucoraceae, but not ramosa (Sanbe et al.12)), 12,5 mcg/ml for so powerful as against Candida albicans or Absidia orchidis and 1.6 mcg/ml for Mucor Cryptococcus neoformans. According to Dario- pusillus (Yazima et al.13)).In spite of the above mae et al.4) an m.i.c. of amphotericin B for results, the present study has revealed that its Candida albicans and Cryptococcus neofor- m.i.c. was 500 u.(112 mcg)/ml for M.p. and mans proved to be 1.9 mcg/ml and 0.2 mcg/ml, 1,000 u.(224 mcg/ml) for A.r. and R.o., respectively. proving to be relatively resistant.

580 TABLE3 M.I.C. of Antibioticsfor Strains of Mucoraceae (Reported in the Literature and Presented by Authors in this Experiment)

4. Variotin chophyton at 0.5 u/ml) is reported to show its Variotin, a most efficient antifungal agent m.i.c. for Rhizopus nigricans to be at 16 u/ml developed in Japan, (its m.i.c. on Blastomyces (Matsuda et al.14)) or 160 u/ml (Takeuchi et dermatitidis being at 0.063 u/ml and on Tri- al.15))and for Rhizopus javanicus to be 0.8 u/ml

581 (Takeuchi et al.15)). raceae. The present results show that the m.i.c. is 4 u/ml for M.p., 8 u/ml for A.r. and 16 u/ml 6. Antibacterial agents for R.o., revealing a coincidence with Matsu- An attempt was made to evaluate if anti- da's results on Rhizopus nigricans. bacterial agents had any activity for Muco- raceae. 5. Naramycin In general, antibacterial agents have been Naramycin is accepted to be effective for reported as noneffective for fungi. But Mc- nonpathogenic or occasionally pathogenic Call and Strobos8) reported that polymyxin B yeasts and fungi, showing an m.i.c. of 0.31 mcg/ and chloramphenicol showed a moderate ml for Rhodotorula glutinis, 0.17 mcg/ml for inhibition (60 mcg) and slight inhibition Saccharomyces carlsbergensis, 1,000 mcg/ml (30 mcg) respectively for Rhizopus oryzae for Blastomyces dermatitidis, 1.0 mcg/ml for isolated from a case of mucormycosis of the Torula rubra, 125 mcg/ml for Alternaria tenuis, central nervous system. 250 mcg/ml for Aspergillus flavus, 500 mcg/ml The present results revealed that polymyxin for Aspergillus fumigatus, 7.5 mcg/ml for B and chloramphenicol failed to show any Candida albicans, 0.24 mcg/ml for Crypto- inhibitory effect for A.r., M.p. and R.o.. The coccus neoformans, 62.5 mcg/ml for Mucor other seven agents including kanamycin, mucedo and Rhizopus nigricans.16) dihydro-streptomycin, erythromycin, tetracy- In the present results, naramysin showed its clin, oxy-tetracyclin, chlor-tetracyclin and m.i.c. 125 mcg/ml for M.p. and R.o., and penicillin showed no activity for these three 250 mcg/ml for A.r., disclosing no marked strains except kanamycin and dihydrostrepto- activity against these three strains of Muco- mycin which proved slightly active to A.r..

SUMMARY The in-vitro sensitivity of strains of Mucorceae, Absidia ramosa, Rhizopus oryzae and Mucor pusillus was examined with 14 antibiotics, amphotericin B, nystatin, trichomycin, naramycin, variotin, polymyxin B, kanamycin, dihydro- stretomycin, erythromycin, tetracyclin, oxy-tetracyclin, chlor-tetracyclin, chloram- phenicol and penicillin. The results obtained were as follows : 1. All three strains are relatively resistant to trichomycin and naramycin, being resistant to polymyxin B, kanamycin, dihydro-streptomycin, erythromycin, tetracy- clin, oxy-tetracylin, chloramphenicol, chlor-tetracyclin and penicillin. 2. All three strains are sensitive to nystatin, variotin and amphotericin B. a. To nystatin, A.r. and M.p. are very sensitive, and R.o. is sensitive. b. To variotin, all three strains are sensitive. c. To amphotericin B, M.p. is very sensitive, while A.r. is slightly sensitive and R.o. is resistant. 3. In comparing the sensitivity of these three strains to the 14 antibiotics, M.p. proves to be most sensitive followed by A.r. and R.o. in descending order.

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