Growth of Allescheria boydii in Antibiotic- Containing Media

JOURNAL OF BATERIOLOGY, Nov., 1965 opyright 1965 American Society for Microbiology Vol. 90, No. 5 Printed in U.S.A. Growth of Allescheria boydii in Antibiotic- ontaining Media JOHN AZIN, JR., AND DAVID W. DEKER Department of Microbiology, University of Iowa, Iowa ity, Iowa Received for publication 8 July 1965 ABSTRAT AZIN, JOHN, JR. (University of Iowa, Iowa ity), AND DAVID W. DEKER. Growth of Allescheria boydii in antibiotic-containing media. J. Bacteriol. 90:1308-1313. 1965. -Thirteen isolates of Allescheria boydii were surveyed for their ability to grow and sporulate on media containing cycloheximide and chloramphenicol. The fungus grew well in the presence of 4 to 8 mg/ml of cycloheximide, whereas ascocarps and coremia were always inhibited at a concentration of the drug lower than that required to inhibit growth and conidial production. In certain strains, ascocarp production was inhibited by concentrations of cycloheximide (0.4 mg/ml) normally used in selective media; however, two strains produced mature ascocarps at a concentration of 1 mg/ml of the antibiotic, and two strains produced immature ascocarps at a concentration of 4 mg/ml. Growth inhibition was not observed with concentrations of chloramphenicol as high as 1,000 mg per liter in any of eight strains tested. Laboratory media containing cycloheximide and chloramphenicol are used widely for routine culture studies and for isolation of pathogenic fungi from clinical specimens. hloramphenicol functions in these media to inhibit the growth of certain bacterial contaminants, whereas cycloheximide serves to inhibit the growth of many saprophytic fungi. Georg, Ajello, and Papageorge (1954) demonstrated that ryptococcus neoformans, Allescheria boydii, and Aspergillus fumigatus are sensitive to the concentration of cycloheximide commonly employed in laboratory media. They suggested that these organisms would not be recovered from clinical materials and other substrates with the use of a medium containing cycloheximide in amounts which would effectively inhibit saprophytic molds. During a study of fungi isolated from soil at a suspected endemic site of histoplasmosis in Iowa (azin, Mculloch, and Braun, 1962), A. boydii was recovered from one of the specimens even though cycloheximide-containing medium was being used to culture the organs of mice inoculated with soil suspensions. The isolate of A. boydii recovered from mouse tissues was typical except for its failure to produce ascocarps on cycloheximide-containing medium. The typical ascigerous stage of the fungus was observed, however, when the strain was transferred to Sabouraud medium or to zapek Dox medium. Repeated subcultures failed to elicit ascocarp production when the fungus was grown on antibiotic-containing medium. The present investigations were designed to determine the concentrations of cycloheximide and chloramphenicol necessary to inhibit growth of the fungus, and to confirm our observation that development of mature ascocarps is inhibited in a medium containing cycloheximide and chloramphenicol. MATERIALS AND METHODS ultures of A. boydii used in this study were those maintained in the culture collection of the Department of Microbiology at the University of Iowa (azin and Decker, 1964) and five additional strains (D-1, N-20, N-34, N-35, and N-50) obtained from Paul W. Kirk, Jr., Department of Botany, Duke University. The Kirk isolates were originally isolated from wood that had been submerged in seawater for 6 weeks or more. To determine whether some constituent of the medium other than the antibiotic agents was responsible for inhibition of ascocarp production, two commercially available antibiotic-containing media were tested. Mycosel Agar (BBL) and Mycobiotic Agar (Difco), each containing the same concentration of antibiotics but differing in the nature of peptone used in the medium, were compared for their ability to suppress the production of sexual fruiting structures by A. boydii. Specific experiments were also designed to test individually each of the two antibiotics (cycloheximide and chloramphenicol) often incorporated into selective diagnostic media used 1308

VOL. 90, 1965 A. BOYDII IN ANTIBIOTI-ONTAINING MEDIA 1309 for isolation of fungi. zapek Dox Agar was selected as a basal medium that contained no organic nitrogen, and Sabouraud Dextrose Agar (ph 6.5) was selected as a basal medium of high organic nitrogen content. Antibiotics were added to the media in the same concentrations found in the commercially prepared media (50 mg/liter of chloramphenicol and 0.4 mg/ml of cycloheximide). hloramphenicol preparations (Parke, Davis & o., Detroit, Mich.) used in this study included: hloromycetin Ophthalmic (each 25 mg of hloromycetin contained borate buffer equivalent to 100 mg of boric acid); hloromycetin Succinate (each 1-g vial of chloramphenicol Sodium Succinate contains an equivalent of 1 g of hloromycetin); hloromycetin Intramuscular (each 2.5 ml of suspension prepared as directed contains 1 g of hloromycetin and 10 mg of sodium carboxymethyl cellulose in 0.9% sodium chloride solution containing 1:10,000 Phemerol chloride); and hloromycetin Solution (each 1 ml contains 250 mg of hloromycetin dissolved in a 50% aqueous solution of N,N-dimethylacetamide with tartaric acid and sodium tartrate equivalent to 5 mg of tartaric acid.) Acti-Dione (The Upjohn o., Kalamazoo, Mich.) consisting of cycloheximide, 85 to 100%, w/w (B-[2-(3,5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-glutarimide), was prepared in solution by first dissolving it in the minimal amount of methanol. Antibiotics were added to media before sterilization in an autoclave at 121 for 10 min. The test media, prepared as agar slants, were inoculated with a suspension of the fungus consisting of 500 to 1,000 viable spores and mycelial fragments, and were incubated at room temperature (azin and Decker, 1964). omparative growth readings were made visually at weekly intervals for 4 weeks and again at the end of 8 weeks. Lactophenol cotton blue slide mounts were prepared from each tube and examined microscopically for the presence of characteristic morphological structures. TABLE 1. Growth and sporulation of Allescheria boydii on zapek Dox Agar containing cycloheximide and chloramphenicol Antibiotic A. boydii strain 800, 804, 801 802 803, 807 805 806 D-1, N-20, N-34 N-so N-35 hloramphenicol * (succinate), 50 mg/ t AS AS AS K AS K liter hloramphenicol (intravenous), 50 AS AS K AS K mg/liter ycloheximide, 400 mg/liter K A AS K AS K ycloheximide, 400 mg/liter and hloramphenicol A AS K AS K (succinate), 50 mg/ liter ycloheximide, 3+ 400 mg/liter and hloramphenicol A A K AS K (intravenous), 50 mg/liter None AS K AS AS K AS K * Growth readings relative to control zapek Dox medium without antibiotics. t = conidia; A = ascocarps; S = ascospores; K = coremia.

1310 AZIN AND DEKER J. BAITERIOL. RESULTS The different peptones used in Mycosel and Mycobiotic Agar did not appear to influence inhibition by cycloheximide or chloramphenicol of growth or sporulation of the organisms. All isolates tested grew equally well on the two media; results obtained with Sabouraud Agar (ph 6.5) were essentially the same. Ascocarps were less numerous and slower in maturing on the organic nitrogen-containing media than they were on the zapek Dox Agar. To determine whether the inhibitory effect produced by Mycosel or Mycobiotic Agar on A. boydii was a constant feature of the genus, or whether it represented a unique strain sensitivity, 13 different isolates of A. boydii were inoculated onto zapek Dox Agar slants containing combinations of cycloheximide and chloramphenicol (Table 1). At 1 week, a slightly slower rate of growth occurred on the antibiotic-containing media as compared with the controls. By 2 weeks, growth on the antibiotic-containing media was equal to that on the control media. Ascocarp production was retarded in the media containing antibiotics, and where fructifications did appear many of them were immature and lacked ascospores. To evaluate whether the inhibitory effect of the antibiotics on A. boydii was caused by chlor- TABLE 2. Growth and sporulation of Allescheria boydii strains 803 and 807 on zapek Dox Agar containing chloramphenicol and borate hloramphenicol prepn hloram- Boric phenicol acid concn concn Ophthalmic* Intravenous Intramuscular Succinate None 803 807 803 807 803 807 803 807 803 807 mg/liter mg/litr 1,000 t t A 50 A 100 - AS AS 50 AS AS A AS 10 - ASK AS AS AS AS AS 1,000 4,000 - - - - - - - - 500 2,000 - - - - - - - 100 400 3+ AS 50 200 A AS AS 10 40 AS AS AS AS AS AS AS AS - 4,000-2,000-400 - 200 AS - 40 AS AS -- AS AS AS AS AS AS AS AS AS AS * Additional boric acid was not added to this medium; the concentrations shown represent that amount supplied by the commercial product. t Growth readings relative to control zapek Dox medium without antibiotics. $ = conidia; A = ascocarps; S = ascospores; K = coremia.

VOL. 90, 1965 A. BOYDII IN ANTIBIOTI-ONTAINING MEDIA 1311 amphenicol or by cycloheximide, the fungus was grown in the presence of serial dilutions of each of the two antibiotics. In the presence of the ophthalmic preparation of chloramphenicol, growth of the fungus was inhibited, in most cases, at the concentration of chloramphenicol approximating that which is normally contained in selective culture media (50 mg per liter). Of the eight strains of A. boydii tested, however, one grew sparsely in the presence of 1,000 mg per liter, and in the presence of 500 mg per liter of the antibiotic preparation growth of the fungus was equal to that of the control. Sporulation of the fungus did not occur at 50 mg per liter of chloramphenicol but did occur at concentrations of 10 mg per liter and below. Further examination of these findings revealed that borate ion contained in the ophthalmic preparation of chloramphenicol was responsible for the inhibitory effects of the commercial product. Inhibition studies with three other commercial preparations of chloramphenicol revealed that growth of A. boydii is not inhibited by 1,000 mg per liter of chloramphenicol incorporated in the medium. But the data did suggest that sporulation of the fungus may be influenced bv concentrations of chloramphenicol used in selective culture media. A summary of effects produced by TABLE 3. Growth and sporulation of Allescheria boydii on zapek Dox Agar containing cycloheximide A. boydii 800 801 802 803 804 805 806 807 D-1 N-20 N-34 N-35 N-50 ycloheximide concn (mg/ml) 16 8 4 2 1 0.5 0.25 0.125 0.062 0.031 None 2+ * t 3+ 3+ 2+ A A 3+ A A A AS K A A AS K AS K A A AS AS K AS K AS AS AS AS K AS K AS AS AS AS K AS K AS AS AS AS * Growth readings relative to control zapek Dox medium without antibiotics. t = conidia; A = ascocarps; S = ascospores; K = coremia. AS K AS K AS AS AS AS AS K AS K AS AS AS AS

1312 AZIN AND DEKER J. BATERIOL. chloramphenicol and borate ion concentrations on growth and sporulation of two of eight strains of A. boydii tested is presented in Table 2. Thirteen strains of A. boydii were grown in the presence of serial dilutions of cycloheximide contained in zapek Dox Agar to determine the inhibitory concentration of the drug for the organism. Results shown in Table 3 indicate that three strains of A. boydii could grow in the presence of no more than 4 mg/ml of cycloheximide, nine strains could grow in the presence of no more than 8 mg/ml of cycloheximide, and one strain could grow sparsely in the presence of 16 mg/ml of the drug. Sporulation of the fungus was inhibited by concentrations of the drug lower than that required to inhibit growth. Sporulation was inhibited in strain 801 by 0.062 mg/ml of the antibiotic, a concentration much lower than the 0.4 mg/ml concentration used in selective media. Sporulation was also inhibited in two other strains (807 and D-1) by a concentration of 0.5 mg/ml, but not by 0.25 mg/ml of the antibiotic. DISUSSION Several investigators have studied the effects of cycloheximide on the growth of pathogenic fungi (Whiffen, 1948; Georg et al., 1954; Huppert and Walker, 1958; McDonough et al., 1960a, b; Negroni and Daglio, 1962; Kuehn and Orr, 1962). As the result of studies by Georg et al. (1954), it is generally accepted that. neoformans, A. boydii, and A. fumigatus are sensitive to concentrations of cycloheximide normally used in selective media. These investigators stated that the fungus would not be expected to be recovered from clinical materials and other substrates with medium containing cycloheximide in amounts which would effectively inhibit saprophytic molds. Since we were successful in isolating A. boydii on cycloheximide-containing medium and have had no difficulty in maintaining the fungus on the medium, it appeared that the existing discrepancy should be resolved. Georg et al. (1954) added serial dilutions of cycloheximide to Sabouraud dextrose broth at ph 6.5 and used very small inocula (25 cells or mycelial fragments by hemocytometer count) in testing the sensitivity of fungi to the drug. Growth was compared with that of fungi growing in medium without cycloheximide after 14 and 21 days of incubation at 30. Our experimental design provided for a much larger inoculum, the use of a solid medium, and a longer incubation period than that used by Georg et al. (1954). These factors may account for some of the differences observed in our findings. Growth readings were made over a period of 8 weeks in the present investigation. Although a slight growth inhibition of A. boydii by 0.4 mg/ml of cycloheximide was noted at the end of 1 week, no inhibition was obvious after 2 weeks. At the end of 4 weeks of incubation, the fungi were usually slightly inhibited by one twofold concentration of cycloheximide lower than that sufficient to inhibit growth at 8 weeks. These inhibitory concentrations of cycloheximide, however, were 10 to 20 times higher than concentrations of the antibiotic normally used in selective culture media. By using a relatively large inoculum, good growth is provided in all media containing insufficient cycloheximide to inhibit growth. That this factor may be of importance is suggested by the results of Georg et al. (1954) in which cycloheximide dilutions over a wide range are shown to be partially inhibitory. They observed growth over a range of six twofold dilutions of cycloheximide, whereas in the present study the range over which partial inhibition was observed was never more than one twofold dilution. The observation by Whiffen (1948) that 25,ug/ml of cycloheximide contained in an agar medium were sufficient to inhibit the growth of M. apiospermum for 72 hr is difficult to evaluate in light of the present study. We found that all 13 strains tested could grow in the presence of 160 times as much cycloheximide (4 mg/ml); moreover, 9 of 13 A. boydii strains could grow well in the presence of 8 mg/ml of cycloheximide, and one strain grew sparsely in the presence of 16 mg/ml of the antibiotic. Whereas our results compare somewhat more favorably with those of Georg et al. (1954), they contrast sharply with the earlier findings of Whiffen (1948). The two strains of A. boydii tested by Georg et al. (1954) grew moderately well in the presence of 0.032 mg/ml of cycloheximide and sparsely in the presence of 4 mg/ml of the antibiotic. The short observation period used by Whiffen (1948) or an inadvertent selection of an unusually sensitive strain of M. apiospermum might possibly account for the wide variation in results. In our study, strain 806 never exhibited more than growth in the presence of any concentration of cycloheximide tested. This does not reflect growth inhibition produced by the antibiotic, however, since growth also occurred in the control zapek Dox Agar as well. When grown on Sabouraud Dextrose Agar, A. boydii 806 reacted similarly to all other strains. This strain, described in an earlier report (azin and

VTOL. 90, 1965 A. BOYDII IN ANTIBIOTI-ONTAINING MEDIA 1313 Decker, 1964), was found to be unable to assimilate sucrose. An interesting effect observed in this study is that coremial production and the ascigerous stage of A. boydii are inhibited at a lower concentration of cycloheximide than is the conidial stage. onidia were produced by the fungus in the presence of all concentrations of cycloheximide where growth occurred. In those strains which produced ascocarps or coremia in the presence of c cloheximide, the structures were always inhibited at a concentration of the antibiotic lower than that required to inhibit growth. Sensitivity of the ascigerous stage of the organism varied from strain to strain. Those strains of A. boydii most recently isolated appeared to be most consistent in their response to cycloheximide. Apparently these same strains may be slightly more sensitive to the antibiotic than strains maintained in artificial culture for some time. Growth of A. boydii does not appear to be inhibited to any great extent by a concentration of 0.4 mg/ml of cycloheximide, which is normally incorporated in selective media for pathogenic fungi. In media such as Mycosel Agar or Mycobiotic Agar, the organism could be expected to grow; however, sporulation may be inhibited to the extent that the ascigerous stage of the organism could be absent or present only in an immature state. Results also suggest that the effects produced on A. boydii by chloramphenicol at concentrations of the antibiotic normally used in selective media are negligible. Although there was some evidence to show that ascocarp production in some strains of A. boydii may be influenced by concentrations of chloramphenicol used in selective culture media, evaluation of our results does not suggest that this effect is additive to the inhibitory effects produced by cycloheximide alone. Of the eight strains of A. boydii tested, none was inhibited by chloramphenicol in a manner similar to that observed by Tong et al. (1958). They demonstrated, in a single isolate of A. boydii, 75% growth inhibition by 20,g/ml of chloramphenicol; 100% growth inhibition occurred in the presence of 100 Ag/ml of the drug. They also found that when the organism was isolated again from their patient, who had undergone 3 weeks of chloramphenicol therapy, the fungus had rapidly developed resistance to the drug and was able to grow luxuriantly in the presence of 100,ug/ml of chloramphenicol. The type of chloramphenicol preparation used in their sensitivity studies was not mentioned. None of our strains was inhibited by any of three different chloramphenicol preparations at a concentration of 1,000,Ag/ml. An ophthalmic preparation of chloramphenicol produced a growth-inhibiting effect on the organism, but this effect was shown to be caused by borate used as a buffer in the antibiotic preparation. LITERATURE ITED AZIN, J., JR., AND D. W. DEKER. 1964. arbohydrate nutrition and sporulation of Allescheria boydii. J. Bacteriol. 88:1624-1628. AZIN, J., JR., W. F. MULLOH, AND J. L. BRAUN. 1962. Isolation of Histoplasma capsulatum, Allescheria boydii and Microsporum gypseum from Iowa soil in an attempt to determine the probable point source of a case of histoplasmosis. J. Iowa Med. Soc. 52:348-351. GEORG, L. K., L. AJELLO, AND. PAPAGEORGE. 1954. Use of cycloheximide in the selective isolation of fungi pathogenic to man. J. Lab. lin. Med. 44:422-428. HUPPERT, M., AND L. J. WALKER. 1958. The selective and differential effects of cycloheximide on many strains of occidioides immitis. Am. J. lin. Pathol. 29:291-295. KUEHN, H. H., AND G. F. ORR. 1962. Tolerance of certain fungi to actidione and its use in isolation of gymnoascaceae. Sabouraudia 1:220-229. MDONOUGH, E. S., L. AJELLO, L. K. GEORG, AND S. BRINKMAN. 1960a. In vitro effects of antibiotics on yeast phase of Blastomyces dermatitidis and other fungi. J. Lab. lin. Med. 55:116-119. McDONOUGH, E. S., L. K. GEORG, L. AJELLO, AND S. BRINKMAN. 1960b. Growth of dimorphic human pathogenic fungi on media containing cycloheximide and chloramphenicol. Mycopathol. Mycol. Appl. 13:113-120. NEGRONI, P., AND. A. N. DAGLIO. 1962. Sensibilidad de andida y Torulopsis a la icloheximide (Actidione). An. Soc. ient. Arg. 173 :69-72. TONG, J. L., E. H. VALENTINE, J. R. DURRANE, G. M. WILSON, AND D. A. FISHER. 1958. Pulmonary infection with Allescheria boydii. Report of a fatal case. Am. Rev. Tuberc. Pulmonary Diseases 78:604-609. WHIFFEN, A. J. 1948. The production, assay, and antibiotic activity of actidione, an antibiotic from Streptomyces griseus. J. Bacteriol. 56:283-291.