ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 1978, p. 465-469 0066-4804/78/0014-0445$02.00/0 Copyright © 1978 American Society for Microbiology
Vol. 14, No. 3
Printed in U.S.A.
Combined Activity of Minocycline and Amphotericin B In Vitro Against Medically Important Yeasts MICHAEL A. LEW,* KEVIN M. BECKETT, AND MYRON J. LEVIN
Department of Clinical Microbiology, Sidney Farber Cancer Institute, Boston, Massachusetts 02115 Received for publication 27 March 1978
The capacity of minocycline to enhance the activity of amphotericin B against Candida albicans, Torulopsis glabrata, Cryptococcus neoformans, and nonalbicans Candida was examined in vitro utilizing a time-killing curve technique. Synergism was apparent at 4 h with 5 of 5 strains of C. albicans, 8 of 8 strains of C. neoformans, and 1 of 12 strains of non-albicans Candida. Synergism was apparent at 24 h with the remaining 11 strains of non-albicans Candida and all 5 strains of T. glabrata. C. neoformans was the most susceptible of the yeasts to the minocycline-amphotericin B combination; seven strains showed a 3-log or greater reduction in colony count in 4 h and all strains showed this reduction in 24 h at amphotericin B concentrations of 0.4 ,tg/ml or less in the presence of minocycline.
Tetracycline has no useful activity against intact yeast cells, although it inhibits protein synthesis by cell-free yeast ribosomes (2). Kwan and co-workers noted that tetracycline, at a concentration of 100 ,tg/ml, acted synergistically with amphotericin B (AmB) against a strain of Saccharomyces cerevisiae in vitro (6), and tetracycline subsequently was shown to potentiate the effect of AmB in treating experimental coccidioidomycosis in mice (5). These findings prompted us to explore the possibility that other analogs of tetracycline would possess greater antifungal activity when combined with AmB. Initially, we tested four tetracycline analogs individually and in combination with AmB in vitro against 20 strains of Candida albicans (7). Minocycline, a relatively lipid-soluble analog, potentiated the activity of AmB against all strains at much lower concentrations than did the other analogs tested. Killing-curve studies with two Candida isolates indicated that minocycline, in the presence of subinhibitory levels of AmB, exerted fungicidal effects at concentrations that are attainable in human serum with oral therapy. We now describe the activity of minocycline and AmB in vitro against other species of medically important yeasts and against additional strains of C. albicans. (This work was presented in part at the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, October, 1977.) MATERIALS AND METHODS Organisms. A total of 30 strains of pathogenic
yeasts were studied. These included 5 strains of Candida albicans, 5 strains of Torulopsis glabrata, 8 465
strains of Cryptococcus neoformans, and 12 strains of
non-albicans species of Candida. Most isolates were obtained from clinical specimens processed in the Laboratory of Clinical Microbiology at the Sidney Farber Cancer Institute. Several strains of non-albicans Candida and C. neoformane were provided by Helen Buckley (Temple University School of Medicine, Philadelphia, Pa.). Organisms were identified by standard methods utilizing germ tube and chlamydospore formation, urease production, and assimilation of simple carbohydrates as identifying criteria (10). Antimicrobial agents. AmB, supplied as assay powder by E. R. Squibb & Sons (Princeton, N.J.), was dissolved in dimethylsulfoxide and stored at -70°C (7, 9). Working concentrations of AmB were prepared on the day of use by diluting thawed stock solution 1:10 in 0.1 M phosphate buffer (pH 8.0) containing 60% dimethylsulfoxide and making further dilutions in buffer and medium (7). The maximal concentration of dimethylsulfoxide was 0.04% at working concentrations of AmB. Minocycline, supplied as powdered reference standard by Lederle Laboratories (Pearl River, N.Y.), was dissolved in 0.1 M phosphate buffer (pH 6.0) at a concentration of 2,560 ,ug/ml and stored at -70°C (7). Working concentrations were prepared by making appropriate dilutions in buffer and medium. Synergism studies. All experiments were performed in yeast nitrogen base broth (YNB; Difco, Detroit, Mich.) supplemented with 0.5% glucose and L-asparagine (1.5 mg/ml). A time-killing curve technique was utilized, as described previously (7). Culture flasks were prepared containing AmB at concentrations ranging from 0.1 to 1.6 jLg/ml alone or in combination with minocycline at a concentration of 0.25 or 2.5 fLg/ml. Control flasks, containing no antimicrobial agent or minocycline alone at a concentration of 2.5 ,ug/ml plus dimethylsulfoxide at the highest working concentration, were included with each experiment. Inocula were adjusted with a spectrophotometer to yield a starting density of 5 x 105 colony-forming units
466
ANTIMICROB. AGENTS CHEMOTHER.
LEW, BECKE1T, AND LEVIN
reduction in colony-forming units. The reduction in colony-forming units for the strains of C. albicans and C. neoformans was rapid, with all or most of the decrease occurring within 4 h, followed by persistence or slight regrowth of surviving organisms between 4 and 24 h (Fig. 1 and 2). In contrast, the counts of viable T. glabrata cells appeared to diminish slowly and at a steady rate over 24 h (Fig. 3). Figure 4 summaizes the results of synergism studies against the strains discussed above and against 12 strains of non-albicans Candida spe-
per ml. Counts of viable organisms were determined in samples removed from flasks at 0, 4, and 24 h. Serial 10-fold dilutions of these samples were made in 0.9% NaCl containing Tween 80 (0.02%), and 0.1 ml of each dilution was incorporated into duplicate pour plates of Sabouraud dextrose agar. Antimicrobial synergism was defined as a reduction in the number of viable organisms by the antimicrobial combination which was 100-fold or greater than the reduction achieved by the most effective of the antimicrobial agents employed individually at the same concentration (8).
RESULTS Minocycline alone had no effect on the growth of the 30 strains of yeast at the maximum concentration tested (2.5 ,ug/ml). AmB had no individual antifungal effect at concentrations less than 0.4 ,ug/ml for most strains of C. neofornans (Fig. 1) or 0.8 ,Lg/ml for most strains of C. albicans and T. glabrata (Fig. 2 and 3). Even at these concentrations, the inhibitory effect of AmB was generally transient and not detectable at 24 h. The addition of minocycline to AmB produced a marked potentiating effect which was discernible for some strains of C. albicans and C. neoformans at an AmB concentration of 0.1 ,ug/ml (Fig. 1 and 2). Progressive increases in AmB concentration resulted in correspondingly greater reductions in cell counts with the addition of both the high (2.5 ,ug/ml) and the low (0.25 ,ug/ml) concentrations of minocycline. At AmB concentrations of -0.2 ,Lg/ml, the reductions in counts produced by the high minocychine concentration were measurably greater than those produced by the low concentration; at AmB concentrations of )0.4 ug/ml, both concentrations of minocycline produced an identical
CryptocoWcus neofor_ns
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AmBSQ4pg/ml
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4
4
24
Time Of Incubation (hr) FIG. 1. Effect of minocycline and AmB on the growth of eight strains of C. neoformans. Points and brackets indicate means and standard deviations. (A): (0) AmB, 0.1 pg/ml; (0) AmB, 0.1 pg/mi + minocycline, 0.25 pg/ml; (*) AmB, 0.1 pg/ml + mi-
nocycline, 2.5 pg/ml. (B): (A) AmB, 0.2 pg/ml; (A) AmB, 0.2 pg/ml + minocycline, 0.25 pg/mi; (A) AmB, 0.2 pg/ml + minocycline, 2.5 pg/ml. (C): (0) Growth control containing no antimicrobial agents; (O) AmB, 0.4 pg/ml; (OU) AmB, 0.4 pg/ml + minocycline, 0.25 pg/ml; (A) AmB, 0.4 pg/ml + minocycline, 2.5 pg/ml.
Condida albicons 8-
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Time Of Incubation (hr) FIG. 2. Effect of minocycline and AmB on the growth of five strains of C. albicans. Points and brackets indicate means and standard deviations for aii strains. (A): (0) AmB, 0.1 pg/ml; (0) AmB, 0.1 pg/ml + minocycline, 0.25 pg/mi; (0) AmB, 0.1 pg/mi + minocycline, 2.5 pg/ml. (B): (A) AmB, 0.2 pg/ml; (A) AmB, 0.2 pug/ml + minocycline, 0.25 pg/mi; (A) AmB, 0.2 pg/ml + minocycline, 2.5 pg/ml. (C): (0) Growth control containing no antimicrobial agents; (0) AmB, 0.4 pg/ml; (EO) AmB, 0.4 pg/ml + minocycline, 0.25 pg/ml; (M) AmB, 0.4 pg/ml + minocycline, 2.5 pg/ml. (D): (0) Growth control containing no antimicrobial agents; (V) AmB, 0.8 pg/ml; (7) AmB, 0.8 pg/ml + minocycline, 0.25 pg/ml; (V) AmB, 0.8 ptg/ml + minocycline, 2.5 pg/ml.
MINOCYCLINE AND AMPHOTERICIN B
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467
cies. Synergism was noted against eall organisms tested at an AmB concentration offs0.8 iLg/ml. As a group, the strains of C. albitcans and C. neoformans appeared to be the mosit susceptible to the combination of AmB and minocycline. Synergism was noted at 4 h agains;t all but one of these strains at an AmB concentrration of0.4 ,ug/ml and at 24 h against all strainS at an AmB concentration of 0.2 jg/ml. Syne,rgism at 4 h was noted against only one strain of non-albi-
cans Candida and against none of the strains of T. glabrata. Synergism at 24 h occurred against all strains of T. glabrata at an AmB concentration of s0.4 ,ug/ml. The strains of non-albicans Candida showed a wide range of susceptibility; however, synergism was noted against all strains at an AmnB concentration of 50.8 ,ug/ml. An analysis of the small number of isolates tested did not reveal any species-specific susceptibility patterns. As a group, the three strains of Candida tropicalis and four strains of Candida parapsilosis that were tested were comparable to Torukpss globrat the strains of T. glabrata in their susceptibility to AmB and minocycline (data not shown). A c Killing, defined as a 1,000-fold or greater re8duction in viable organisms (1), proved to be a more stringent criterion than synergism in as6sessing the susceptibility of the test strains to the combination of AmB and minocycline (Fig. 4. 4). By this measure, the strains of C. neoformans appeared most susceptible to the combination; 2in the presence of minocycline seven of eight AmS 04*g/ M AmBa1.6pg/ml strains were killed in 4 h at an AmB concentra4 24 tion of '0.4 ,ug/ml, and all strains were killed in 4 24 24 h at this concentration. An AmB concentraTim Of Incubotion(hr) tion of 0.8 1g/ml was required to kill the majority FIG. 3. Effect of minocycline and AmBon the of C. albicans isolates in 4 and 24 h. None of the growth of five strains of T. glabrata brackets indicate means and standard deviations for strains of T. glabrata was killed in 4 h; killing at all strains. (A): (5) AmB, 0.4 pg/ml; (El) AmB, 0.4 24 h required an AmB concentration of l1.6 pg/mi + minocycline, 0.25 pg/mi; (U) A?mB, 0.4 pg/ml Lg/ml. With killing as an end point, the strains + minocycline, 2.5 pg/ml. (B): (0) Girowth control of non-albicans Candida again showed a wide containing no antimicrobial agents; ((V) AmB, 0.8 range of susceptibility. Three strains (one each pg/ml; ('T) AmB, 0.8 pg/ml + min ocycline, 0.25 of C. guillermondii, C. parapsilosis, and C. pg/ml; (O) AmB, 0.8 pg/ml + minocycliine, 2.5 pg/ml. pseudotropicalis) were not killed at an AmB (C): (0) Growth control containing no oantimicrobial concentration of s1.6 ,ug/ml. agents; (K) AmB, 1.6 pg/mi; (O) AmA3, 1.6 pg/mi + DISCUSSION minocycline, 0.25 pg/ml. Curve for Am]B, 1.6 pg/ml + minocycline, 2.5 pg/mi, was superinpcDsed on curve Our previous studies indicated that minocyfor AmB, 1.6 pg/mi + minocycline, 0.25 pg/ml, and is not displayed. cline, among four tetracycline analogs tested, a
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C A B D C D A B FIG. 4. Concentration of AmB required to produce synergism or killing in the presence of minocycline. Open symbols, Synergism or killing at 4 h; closed symbols, synergism or killing at 24 h. (A) C. albicans; (B) C. neoformans; (C) T. glabrata; (D) non-albicans species of Candida.
468
ANTIMICROB. AGENTS CHEMOTHER.
LEW, BECKETT, AND LEVIN
was uniquely active in potentiating the fungicidal activity of AmB against C. albicans in vitro. Killing-curve studies with two strains of this organism indicated that minocycline had 30 times more activity, on a weight basis, than the next most potent analog (doxycycline) and that fungicidal activity was expressed at concentrations of both minocycline and AmB that are well within clinically attainable levels (7). In this report we show that minocycline possesses a comparable degree of activity against five additional strains of C. albicans and that its antifungal spectrum includes a number of species of yeasts that are important in producing human infections. When minocycline was used in combination with AmB, synergism was observed against all strains, and fungicidal activity (1,000fold or greater decrease in colony-forming units) was observed against 27 of 30 strains at antimicrobial concentrations that are attainable in the serum of patients receiving conventional doses of both agents (3, 11). Minocycline, when used by itself, had no antifungal activity at the concentrations employed against any of the yeast strains. AmnB alone showed little antifungal activity at concentrations of c0.4 ug/ml; even at this and higher concentrations, its activity was transient and small. This may be partially explained by the lability of AmB in standard culture media and by our use of an unbuffered medium which permits rapid decreases in pH as organic acids accumulate (4, 13). With all strains tested, however, synergism between AmB and minocycline was demonstrated at concentrations that were individually subinhibitory. The persistence of organisms between 4 and 24 h, following the rapid reduction in colony count observed during the first 4 h with most strains of C. albicans and C. neoformans, merits further investigation. A decline of AmB activity to subeffective levels, for reasons cited above, may have been a contributing factor. Other possible explanations include selection of organisms resistant to one or both members of the antimicrobial combination, and reversible injury of organisms which merely delayed their recovery in pour plates during the 48-h observation period. The latter seems unlikely, since plates held for longer periods of time failed to show increases in numbers of colonies after the first 48 h. The magnitude of the antifungal effect achieved by a given concentration of minocychine appeared dependent on the concentration of AmB that was present; this relationship appeared to hold even at concentrations at which AmB had no individual activity. In the presence of sufficiently high concentrations of AmB (gen-
erally 20.4 ,ug/ml), the reduction in colony-forming units produced by addition of minocychine was identical over a range of 0.25 to 2.5 ,ug of this compound per ml. At lower concentrations ofAmB, the higher concentration of minocycline had a measurably greater antifungal effect. This suggests that the concentration of AmB is the limiting factor in determining synergism between AmB and minocycline and is consistent with the hypothesis that AmB synergie with minocycline and other compounds by enhancing their uptake into the cell (6). Our observations suggest that minocycline may prove to be a useful agent for the treatment of human systemic fungal infections. Addition of minocycline to conventional AmB therapy may produce enhanced fungicidal activity in the central nervous system and other sites where the penetration of AmB is limited. Furthernore, the addition of minocycline may permit use of smaller total doses of AmB, thereby limiting the dose-related toxicity of this compound (3, 12). Further in vitro testing will be required to assess the activity of minocycline against filamentous and dimorphic fungi. Studies in animals to assess therapeutic efficacy and unanticipated toxicity should be conducted before the AmB-minocycline combination is considered for use in humans.
ACKNOWLEDGMENT This study was supported in part by Public Health Service Clinical Cancer Center grant 1-PO1-CA-19589-02 from the National Cancer Institute.
LITERATURE CID 1. Barry, A. L., and L D. Sabath. 1974. Special tests:
bactericidal activity and activity of antimicrobics in combination, p. 431-435. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology. American Society for Microbiology, Washington, D.C. 2. Battaner, E., and D. Vazquez. 1971. Inhibitors of protein synthesis by ribosomes of the 80-S type. Biochim. Biophys. Acta 246:316-330. 3. Bindschalder, D. D., and J. E. Bennett. 1969. A pharmacologic guide to the clinical use of amphotericin B. J. Infect. Dis. 120:427-436. 4. Cheung, S. C., G. Medoff, D. Schlesinger, and G. S. Kobayashi. 1975. Stability of amphotericin B in fungal 5.
6.
7.
8.
culture media. Antimicrob. Agents Chemother. 8:426-428. Huppert, M., S. H. Sun, and K. R. Vukovich. 1974. Combined amphotericin B-tetracycline therapy for experimental coccidioidomycosis. Antimicrob. Agents Chemother. 5:473-478. Kwan, C. N., G. Medoff, G. S. Kobayashi, D. Schlessinger, and H. J. Raskus. 1972. Potentiation of the antifungal effects of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 2:61-66. Lew, M A., K. M Beckett, and M. J. Levin. 1977. Antifungal activity of four tetracycline analogues against Candida albicans in vitro: potentiation by amphotericin B. J. Infect. Dis. 136:263-270. Moellering, R. C., Jr., C. Wennersten, and A. N.
VOL. 14, 1978 Weinberg. 1971. Studies on antibiotic synergism against enterococci. I. Bacteriologic studies. J. Lab. Clin. Med. 77:821-828. 9. Shadomy, S., J. A. McCay, and S. L. Schwartz. 1969. Bioassay for hamycin and amphotericin B in serum and other biological fluids. Appl. Microbiol. 17:497-503. 10. Silva-Hutner, M., and B. H. Cooper. 1974. Medically important yeasts, p. 491-507. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology. American Society for Microbiology, Washington, D.C.
MINOCYCLINE AND AMPHOTERICIN B
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11. Steigbigel, N. H, C. W. Reed, and M. Finland. 1968. Absorption and excretion of five tetracycline analogues in normal young men. Am. J. Med. Sci. 255:296-312. 12. Utz, J. P., J. E. Bennett, M. W. Brandriss, W. T. Butler, and G. J. Hill, U. 1964. Amphotericin B toxicity. Combined Clinical Staff Conference at the National Institutes of Health. Ann. Intern. Med. 61:334-354. 13. Utz, C. J., S. White, and S. Shadomy. 1976. New medium for in vitro susceptibility studies with amphotericin B. Antimicrob. Agents Chemother. 10:776-777.
Vol. 14, No. 3
Printed in U.S.A.
Combined Activity of Minocycline and Amphotericin B In Vitro Against Medically Important Yeasts MICHAEL A. LEW,* KEVIN M. BECKETT, AND MYRON J. LEVIN
Department of Clinical Microbiology, Sidney Farber Cancer Institute, Boston, Massachusetts 02115 Received for publication 27 March 1978
The capacity of minocycline to enhance the activity of amphotericin B against Candida albicans, Torulopsis glabrata, Cryptococcus neoformans, and nonalbicans Candida was examined in vitro utilizing a time-killing curve technique. Synergism was apparent at 4 h with 5 of 5 strains of C. albicans, 8 of 8 strains of C. neoformans, and 1 of 12 strains of non-albicans Candida. Synergism was apparent at 24 h with the remaining 11 strains of non-albicans Candida and all 5 strains of T. glabrata. C. neoformans was the most susceptible of the yeasts to the minocycline-amphotericin B combination; seven strains showed a 3-log or greater reduction in colony count in 4 h and all strains showed this reduction in 24 h at amphotericin B concentrations of 0.4 ,tg/ml or less in the presence of minocycline.
Tetracycline has no useful activity against intact yeast cells, although it inhibits protein synthesis by cell-free yeast ribosomes (2). Kwan and co-workers noted that tetracycline, at a concentration of 100 ,tg/ml, acted synergistically with amphotericin B (AmB) against a strain of Saccharomyces cerevisiae in vitro (6), and tetracycline subsequently was shown to potentiate the effect of AmB in treating experimental coccidioidomycosis in mice (5). These findings prompted us to explore the possibility that other analogs of tetracycline would possess greater antifungal activity when combined with AmB. Initially, we tested four tetracycline analogs individually and in combination with AmB in vitro against 20 strains of Candida albicans (7). Minocycline, a relatively lipid-soluble analog, potentiated the activity of AmB against all strains at much lower concentrations than did the other analogs tested. Killing-curve studies with two Candida isolates indicated that minocycline, in the presence of subinhibitory levels of AmB, exerted fungicidal effects at concentrations that are attainable in human serum with oral therapy. We now describe the activity of minocycline and AmB in vitro against other species of medically important yeasts and against additional strains of C. albicans. (This work was presented in part at the 17th Interscience Conference on Antimicrobial Agents and Chemotherapy, October, 1977.) MATERIALS AND METHODS Organisms. A total of 30 strains of pathogenic
yeasts were studied. These included 5 strains of Candida albicans, 5 strains of Torulopsis glabrata, 8 465
strains of Cryptococcus neoformans, and 12 strains of
non-albicans species of Candida. Most isolates were obtained from clinical specimens processed in the Laboratory of Clinical Microbiology at the Sidney Farber Cancer Institute. Several strains of non-albicans Candida and C. neoformane were provided by Helen Buckley (Temple University School of Medicine, Philadelphia, Pa.). Organisms were identified by standard methods utilizing germ tube and chlamydospore formation, urease production, and assimilation of simple carbohydrates as identifying criteria (10). Antimicrobial agents. AmB, supplied as assay powder by E. R. Squibb & Sons (Princeton, N.J.), was dissolved in dimethylsulfoxide and stored at -70°C (7, 9). Working concentrations of AmB were prepared on the day of use by diluting thawed stock solution 1:10 in 0.1 M phosphate buffer (pH 8.0) containing 60% dimethylsulfoxide and making further dilutions in buffer and medium (7). The maximal concentration of dimethylsulfoxide was 0.04% at working concentrations of AmB. Minocycline, supplied as powdered reference standard by Lederle Laboratories (Pearl River, N.Y.), was dissolved in 0.1 M phosphate buffer (pH 6.0) at a concentration of 2,560 ,ug/ml and stored at -70°C (7). Working concentrations were prepared by making appropriate dilutions in buffer and medium. Synergism studies. All experiments were performed in yeast nitrogen base broth (YNB; Difco, Detroit, Mich.) supplemented with 0.5% glucose and L-asparagine (1.5 mg/ml). A time-killing curve technique was utilized, as described previously (7). Culture flasks were prepared containing AmB at concentrations ranging from 0.1 to 1.6 jLg/ml alone or in combination with minocycline at a concentration of 0.25 or 2.5 fLg/ml. Control flasks, containing no antimicrobial agent or minocycline alone at a concentration of 2.5 ,ug/ml plus dimethylsulfoxide at the highest working concentration, were included with each experiment. Inocula were adjusted with a spectrophotometer to yield a starting density of 5 x 105 colony-forming units
466
ANTIMICROB. AGENTS CHEMOTHER.
LEW, BECKE1T, AND LEVIN
reduction in colony-forming units. The reduction in colony-forming units for the strains of C. albicans and C. neoformans was rapid, with all or most of the decrease occurring within 4 h, followed by persistence or slight regrowth of surviving organisms between 4 and 24 h (Fig. 1 and 2). In contrast, the counts of viable T. glabrata cells appeared to diminish slowly and at a steady rate over 24 h (Fig. 3). Figure 4 summaizes the results of synergism studies against the strains discussed above and against 12 strains of non-albicans Candida spe-
per ml. Counts of viable organisms were determined in samples removed from flasks at 0, 4, and 24 h. Serial 10-fold dilutions of these samples were made in 0.9% NaCl containing Tween 80 (0.02%), and 0.1 ml of each dilution was incorporated into duplicate pour plates of Sabouraud dextrose agar. Antimicrobial synergism was defined as a reduction in the number of viable organisms by the antimicrobial combination which was 100-fold or greater than the reduction achieved by the most effective of the antimicrobial agents employed individually at the same concentration (8).
RESULTS Minocycline alone had no effect on the growth of the 30 strains of yeast at the maximum concentration tested (2.5 ,ug/ml). AmB had no individual antifungal effect at concentrations less than 0.4 ,ug/ml for most strains of C. neofornans (Fig. 1) or 0.8 ,Lg/ml for most strains of C. albicans and T. glabrata (Fig. 2 and 3). Even at these concentrations, the inhibitory effect of AmB was generally transient and not detectable at 24 h. The addition of minocycline to AmB produced a marked potentiating effect which was discernible for some strains of C. albicans and C. neoformans at an AmB concentration of 0.1 ,ug/ml (Fig. 1 and 2). Progressive increases in AmB concentration resulted in correspondingly greater reductions in cell counts with the addition of both the high (2.5 ,ug/ml) and the low (0.25 ,ug/ml) concentrations of minocycline. At AmB concentrations of -0.2 ,Lg/ml, the reductions in counts produced by the high minocychine concentration were measurably greater than those produced by the low concentration; at AmB concentrations of )0.4 ug/ml, both concentrations of minocycline produced an identical
CryptocoWcus neofor_ns
AmBO0.2pag/ml
AmBaO.Ipig/ml 4
24
AmBSQ4pg/ml
24
4
4
24
Time Of Incubation (hr) FIG. 1. Effect of minocycline and AmB on the growth of eight strains of C. neoformans. Points and brackets indicate means and standard deviations. (A): (0) AmB, 0.1 pg/ml; (0) AmB, 0.1 pg/mi + minocycline, 0.25 pg/ml; (*) AmB, 0.1 pg/ml + mi-
nocycline, 2.5 pg/ml. (B): (A) AmB, 0.2 pg/ml; (A) AmB, 0.2 pg/ml + minocycline, 0.25 pg/mi; (A) AmB, 0.2 pg/ml + minocycline, 2.5 pg/ml. (C): (0) Growth control containing no antimicrobial agents; (O) AmB, 0.4 pg/ml; (OU) AmB, 0.4 pg/ml + minocycline, 0.25 pg/ml; (A) AmB, 0.4 pg/ml + minocycline, 2.5 pg/ml.
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B
AMB u0.1p~g/mI
AmB-0.2p~g/mI
2-
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24
4
24
AmB 0 .4pqg/mi 4
24
Am8s0.8p&gmt 4
24
Time Of Incubation (hr) FIG. 2. Effect of minocycline and AmB on the growth of five strains of C. albicans. Points and brackets indicate means and standard deviations for aii strains. (A): (0) AmB, 0.1 pg/ml; (0) AmB, 0.1 pg/ml + minocycline, 0.25 pg/mi; (0) AmB, 0.1 pg/mi + minocycline, 2.5 pg/ml. (B): (A) AmB, 0.2 pg/ml; (A) AmB, 0.2 pug/ml + minocycline, 0.25 pg/mi; (A) AmB, 0.2 pg/ml + minocycline, 2.5 pg/ml. (C): (0) Growth control containing no antimicrobial agents; (0) AmB, 0.4 pg/ml; (EO) AmB, 0.4 pg/ml + minocycline, 0.25 pg/ml; (M) AmB, 0.4 pg/ml + minocycline, 2.5 pg/ml. (D): (0) Growth control containing no antimicrobial agents; (V) AmB, 0.8 pg/ml; (7) AmB, 0.8 pg/ml + minocycline, 0.25 pg/ml; (V) AmB, 0.8 ptg/ml + minocycline, 2.5 pg/ml.
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467
cies. Synergism was noted against eall organisms tested at an AmB concentration offs0.8 iLg/ml. As a group, the strains of C. albitcans and C. neoformans appeared to be the mosit susceptible to the combination of AmB and minocycline. Synergism was noted at 4 h agains;t all but one of these strains at an AmB concentrration of0.4 ,ug/ml and at 24 h against all strainS at an AmB concentration of 0.2 jg/ml. Syne,rgism at 4 h was noted against only one strain of non-albi-
cans Candida and against none of the strains of T. glabrata. Synergism at 24 h occurred against all strains of T. glabrata at an AmB concentration of s0.4 ,ug/ml. The strains of non-albicans Candida showed a wide range of susceptibility; however, synergism was noted against all strains at an AmnB concentration of 50.8 ,ug/ml. An analysis of the small number of isolates tested did not reveal any species-specific susceptibility patterns. As a group, the three strains of Candida tropicalis and four strains of Candida parapsilosis that were tested were comparable to Torukpss globrat the strains of T. glabrata in their susceptibility to AmB and minocycline (data not shown). A c Killing, defined as a 1,000-fold or greater re8duction in viable organisms (1), proved to be a more stringent criterion than synergism in as6sessing the susceptibility of the test strains to the combination of AmB and minocycline (Fig. 4. 4). By this measure, the strains of C. neoformans appeared most susceptible to the combination; 2in the presence of minocycline seven of eight AmS 04*g/ M AmBa1.6pg/ml strains were killed in 4 h at an AmB concentra4 24 tion of '0.4 ,ug/ml, and all strains were killed in 4 24 24 h at this concentration. An AmB concentraTim Of Incubotion(hr) tion of 0.8 1g/ml was required to kill the majority FIG. 3. Effect of minocycline and AmBon the of C. albicans isolates in 4 and 24 h. None of the growth of five strains of T. glabrata brackets indicate means and standard deviations for strains of T. glabrata was killed in 4 h; killing at all strains. (A): (5) AmB, 0.4 pg/ml; (El) AmB, 0.4 24 h required an AmB concentration of l1.6 pg/mi + minocycline, 0.25 pg/mi; (U) A?mB, 0.4 pg/ml Lg/ml. With killing as an end point, the strains + minocycline, 2.5 pg/ml. (B): (0) Girowth control of non-albicans Candida again showed a wide containing no antimicrobial agents; ((V) AmB, 0.8 range of susceptibility. Three strains (one each pg/ml; ('T) AmB, 0.8 pg/ml + min ocycline, 0.25 of C. guillermondii, C. parapsilosis, and C. pg/ml; (O) AmB, 0.8 pg/ml + minocycliine, 2.5 pg/ml. pseudotropicalis) were not killed at an AmB (C): (0) Growth control containing no oantimicrobial concentration of s1.6 ,ug/ml. agents; (K) AmB, 1.6 pg/mi; (O) AmA3, 1.6 pg/mi + DISCUSSION minocycline, 0.25 pg/ml. Curve for Am]B, 1.6 pg/ml + minocycline, 2.5 pg/mi, was superinpcDsed on curve Our previous studies indicated that minocyfor AmB, 1.6 pg/mi + minocycline, 0.25 pg/ml, and is not displayed. cline, among four tetracycline analogs tested, a
AmEo nf tnd
Synergism
Kiiii ng >32-
.1.
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C A B D C D A B FIG. 4. Concentration of AmB required to produce synergism or killing in the presence of minocycline. Open symbols, Synergism or killing at 4 h; closed symbols, synergism or killing at 24 h. (A) C. albicans; (B) C. neoformans; (C) T. glabrata; (D) non-albicans species of Candida.
468
ANTIMICROB. AGENTS CHEMOTHER.
LEW, BECKETT, AND LEVIN
was uniquely active in potentiating the fungicidal activity of AmB against C. albicans in vitro. Killing-curve studies with two strains of this organism indicated that minocycline had 30 times more activity, on a weight basis, than the next most potent analog (doxycycline) and that fungicidal activity was expressed at concentrations of both minocycline and AmB that are well within clinically attainable levels (7). In this report we show that minocycline possesses a comparable degree of activity against five additional strains of C. albicans and that its antifungal spectrum includes a number of species of yeasts that are important in producing human infections. When minocycline was used in combination with AmB, synergism was observed against all strains, and fungicidal activity (1,000fold or greater decrease in colony-forming units) was observed against 27 of 30 strains at antimicrobial concentrations that are attainable in the serum of patients receiving conventional doses of both agents (3, 11). Minocycline, when used by itself, had no antifungal activity at the concentrations employed against any of the yeast strains. AmnB alone showed little antifungal activity at concentrations of c0.4 ug/ml; even at this and higher concentrations, its activity was transient and small. This may be partially explained by the lability of AmB in standard culture media and by our use of an unbuffered medium which permits rapid decreases in pH as organic acids accumulate (4, 13). With all strains tested, however, synergism between AmB and minocycline was demonstrated at concentrations that were individually subinhibitory. The persistence of organisms between 4 and 24 h, following the rapid reduction in colony count observed during the first 4 h with most strains of C. albicans and C. neoformans, merits further investigation. A decline of AmB activity to subeffective levels, for reasons cited above, may have been a contributing factor. Other possible explanations include selection of organisms resistant to one or both members of the antimicrobial combination, and reversible injury of organisms which merely delayed their recovery in pour plates during the 48-h observation period. The latter seems unlikely, since plates held for longer periods of time failed to show increases in numbers of colonies after the first 48 h. The magnitude of the antifungal effect achieved by a given concentration of minocychine appeared dependent on the concentration of AmB that was present; this relationship appeared to hold even at concentrations at which AmB had no individual activity. In the presence of sufficiently high concentrations of AmB (gen-
erally 20.4 ,ug/ml), the reduction in colony-forming units produced by addition of minocychine was identical over a range of 0.25 to 2.5 ,ug of this compound per ml. At lower concentrations ofAmB, the higher concentration of minocycline had a measurably greater antifungal effect. This suggests that the concentration of AmB is the limiting factor in determining synergism between AmB and minocycline and is consistent with the hypothesis that AmB synergie with minocycline and other compounds by enhancing their uptake into the cell (6). Our observations suggest that minocycline may prove to be a useful agent for the treatment of human systemic fungal infections. Addition of minocycline to conventional AmB therapy may produce enhanced fungicidal activity in the central nervous system and other sites where the penetration of AmB is limited. Furthernore, the addition of minocycline may permit use of smaller total doses of AmB, thereby limiting the dose-related toxicity of this compound (3, 12). Further in vitro testing will be required to assess the activity of minocycline against filamentous and dimorphic fungi. Studies in animals to assess therapeutic efficacy and unanticipated toxicity should be conducted before the AmB-minocycline combination is considered for use in humans.
ACKNOWLEDGMENT This study was supported in part by Public Health Service Clinical Cancer Center grant 1-PO1-CA-19589-02 from the National Cancer Institute.
LITERATURE CID 1. Barry, A. L., and L D. Sabath. 1974. Special tests:
bactericidal activity and activity of antimicrobics in combination, p. 431-435. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology. American Society for Microbiology, Washington, D.C. 2. Battaner, E., and D. Vazquez. 1971. Inhibitors of protein synthesis by ribosomes of the 80-S type. Biochim. Biophys. Acta 246:316-330. 3. Bindschalder, D. D., and J. E. Bennett. 1969. A pharmacologic guide to the clinical use of amphotericin B. J. Infect. Dis. 120:427-436. 4. Cheung, S. C., G. Medoff, D. Schlesinger, and G. S. Kobayashi. 1975. Stability of amphotericin B in fungal 5.
6.
7.
8.
culture media. Antimicrob. Agents Chemother. 8:426-428. Huppert, M., S. H. Sun, and K. R. Vukovich. 1974. Combined amphotericin B-tetracycline therapy for experimental coccidioidomycosis. Antimicrob. Agents Chemother. 5:473-478. Kwan, C. N., G. Medoff, G. S. Kobayashi, D. Schlessinger, and H. J. Raskus. 1972. Potentiation of the antifungal effects of antibiotics by amphotericin B. Antimicrob. Agents Chemother. 2:61-66. Lew, M A., K. M Beckett, and M. J. Levin. 1977. Antifungal activity of four tetracycline analogues against Candida albicans in vitro: potentiation by amphotericin B. J. Infect. Dis. 136:263-270. Moellering, R. C., Jr., C. Wennersten, and A. N.
VOL. 14, 1978 Weinberg. 1971. Studies on antibiotic synergism against enterococci. I. Bacteriologic studies. J. Lab. Clin. Med. 77:821-828. 9. Shadomy, S., J. A. McCay, and S. L. Schwartz. 1969. Bioassay for hamycin and amphotericin B in serum and other biological fluids. Appl. Microbiol. 17:497-503. 10. Silva-Hutner, M., and B. H. Cooper. 1974. Medically important yeasts, p. 491-507. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology. American Society for Microbiology, Washington, D.C.
MINOCYCLINE AND AMPHOTERICIN B
469
11. Steigbigel, N. H, C. W. Reed, and M. Finland. 1968. Absorption and excretion of five tetracycline analogues in normal young men. Am. J. Med. Sci. 255:296-312. 12. Utz, J. P., J. E. Bennett, M. W. Brandriss, W. T. Butler, and G. J. Hill, U. 1964. Amphotericin B toxicity. Combined Clinical Staff Conference at the National Institutes of Health. Ann. Intern. Med. 61:334-354. 13. Utz, C. J., S. White, and S. Shadomy. 1976. New medium for in vitro susceptibility studies with amphotericin B. Antimicrob. Agents Chemother. 10:776-777.
