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Yeast-Like Fungi B Postgrad Med J: first published as 10.1136/pgmj.55.647.683 on 1 September 1979. Downloaded from Postgraduate Medical Journal (September 1979) 55, 683-686 In vitro synergy and antagonism of antifungal agents against yeast-like fungi B. DUPONT E. DROUHET M.D. M.D. Institut Pasteur, Service de Mycologie, 75015, Paris Summary ever, with 3 antifungal agents, such a study is very Some antifungal agents have been tested qualitatively long and cannot be used routinely for clinical in various associations against species of Candida, application. Cryptococcus and Torulopsis. The method used was A rapid and simple technique was proposed to the channel test and the results are confirmed by com- determine whether combination of antifungal agents parison with the serial dilution test in agar. is synergistic or not (Dupont and Drouhet, 1978). The antagonism in vitro with the combination of This method is derived from a technique using paper amphotericin B imidazole suggests that caution must strips impregnated with antibiotics employed in be exercised before prescribing the antifungal drugs bacteriology for the study of antibacterial combi- Protected by copyright. in combination for man. The frequent synergy of nations (Chabbert, 1973). flucytosine with econazole is, however, encouraging because of the low toxicity of the 2 drugs. Under the Materials and methods limited conditions described, the combination of Twenty-six strains of Candida albicans, 22 strains flucytosine and amphotericin B was not found syner- of Cryptococcus neoformans all of human origin and gistic but additive on the strains of C. albicans used in one strain of Candida parapsilosis, agent of a fungal this study. This combination was found synergistic for endocarditis (Dupont, Drouhet and Lapresle, 1977) a strain of C. parapsilosis and is useful for avoiding were studied. The inoculum for the tests contained resistance to flucytosine. 106 yeast cells/ml cultured by shaking for 18 hr in Sabouraud glucose liquid medium. Introduction The media used were: yeast morphology agar The usual doses of antifungal agents used in (YMA, Difco), a chemically defined medium which human therapy are fungistatic and not fungicidal. permits the testing of flucytosine, amphotericin B Numerous fungi have a high mutation rate in the and imidazole derivatives, and a complex medium presence of flucytosine when this drug is given alone. consisting of casitone Difco 19 g, yeast extract 5 g, http://pmj.bmj.com/ The chromosomal mutation involves development of sodium nitrate 10 g, disodium phosphate one g, char- secondary resistant strains which do not respond to coal purified glucose 10 g, agar 20 g, water 1000 ml. flucytosine. By using 2 antifungal agents, failures in This medium (pH 6.6) is effective for testing ampho- therapy are less likely to occur. Better results are tericin B and econazole but not for flucytosine. produced and the emergence of mutants resistant to The media were poured into Petri dishes (15 ml for one of the drugs is suppressed. However, an associ- a 9-cm diameter dish) and dried for 30 min in the ation of 2 antifungal agents is not always synergistic incubator. Four channels (40 mm long, 2 mm wide) and the result of their action is sometimes only were cut in the agar. The channels were at right on September 26, 2021 by guest. additive or even antagonistic. It is consequently angles to each other but not connected. The con- necessary to test in vitro the effect of the association centrations of the antifungal agents in the channels of 2 antifungal agents on the strain responsible for the were as follows: 10 mg/l for amphotericin B and mycosis (Ansehn, 1977; Dupont and Drouhet, econazole with casitone medium; 5 mg/l for flucy- 1978; Medoff, Comfort and Kobayashi, 1971; tosine; 20 mg/l for amphotericin B; 50 mg/l for Polak, 1974; Shadomy, Wagner and Davis, 1975). econazole on YMA. The Petri dishes were then The usual technique for studying synergy or incubated for 24 hr at 30°C for Candida strains, and antagonism is by making serial dilutions to deter- 48 hr for Cr. neoformans strains. The reading of mine the minimal inhibitory concentration (MIC) inhibition areas states the evaluation of the sensi- for each antifungal agent and then to evaluate the tivity or resistance strain to each antibiotic. In antifungal agents associated by pairs according to particular, it is mainly the growth around the angle every combination of various concentrations. How- of the 2 channels which is important. If the 2 0032-5473/79/0900-0683$02.00 © 1979 The Fellowship of Postgraduate Medicine Postgrad Med J: first published as 10.1136/pgmj.55.647.683 on 1 September 1979. Downloaded from 684 B. Dupont and E. Drouhet Protected by copyright. FIG. 1 (a-d). Channel test - yeast morphology agar (YMA) medium. Ampho. = Amphotericin B; Eco. = Econazole; 5-FC. = Flucytosine. No synergy between flucytosine and econazole: (a). Synergy between flucytosine and econazole: (b, c and d). Antagonism between amphotericin B and http://pmj.bmj.com/ econazole: (a-d). Possible synergy between amphotericin B and flucytosine: (b)' inhibition areas intersect at right angles, the effect is shaken in Sabouraud liquid medium for 18 hr indifferent, if the inhibition areas draw a hyperbole, at 37°C) were inoculated to agar medium by a the combination demonstrates an additive effect. An modified Steers apparatus; this inoculum corre- angle with a notch shows a synergistic effect. A sponds to 100 distinct colonies. The MIC and the on September 26, 2021 by guest. reverse image shows an antagonism which can be concentration of antibiotic which inhibits 50% of reciprocal or predominant for the one or the other the inoculum (IC50) were determined by reading the antifungal agent. growth after incubation for 18 hr at 370C. The results from the channel technique were com- The bacteriological criteria for synergy and pared with results from the conventional technique antagonism adopted were as follows: there was of determination of the MIC, i.e. by serially diluting synergism when growth inhibition was obtained with the antifungal agents alone and in combination in one-quarter the MIC (MIC of one antibiotic the agar media described, and poured into Petri dishes. For this comparison, inocula of 4 strains of combined with the equivalent concentration of the C, albicans (Nos 4, 5, 7, 21, obtained from cultures other. Postgrad Med J: first published as 10.1136/pgmj.55.647.683 on 1 September 1979. Downloaded from Synergy and antagonism ofantifungal agents 685 Results MIC. The sum of the fractional inhibitory concen- Strains of C. albicans responded to drug combi- tration (FIC) for strain no. 4 is > 2 (Table 1). nations as follows: antagonism is always observed Similar results were observed with strain no. 5 with amphotericin B plus econazole; antagonism is (Table 1). observed with both casitone medium and YMA For the other 2 strains (7 and 21) a more accurate medium, but it is more readily observed in the method than the MIC was necessary to observe former. The best time for observation of this is after antagonism: the MIC is sometimes difficult to several days at room temperature. A secondary determine, particularly in the case of econazole, growth can be observed in the inhibition area of when there is a growth of very small colonies, even amphotericin B, which is situated in the diffusion in high concentrations; thus, the determination ofthe zone of econazole. The antagonism is also constant IC50 seems more reliable and reproducible. On the between amphotericin B and miconazole for the 26 other hand, MIC determination by 2-fold serial strains tested (Table 3). dilutions can be misleading. For example, if the MIC is quoted as 0.08, this means that the culture grows TABLE 1. Minimal inhibitory concentration (MIC) and at 0.04 but not at 0.08, the real MIC being between antagonism between amphotericin B (Ampho.) and econ- these 2 values and could, in fact, be nearer to 0.04 azole (Eco.) on 2 strains of Candida albicans than 0O08 although the latter is the quoted MIC. MIC mg/l TABLE 2. Median inhibitory concentration (IC50) and an- Strain No. Ampho. Eco. Ampho. + Eco. FIC* tagonism between amphotericin B (Ampho.) and econazole (Eco.) on 2 strains of Candida albicans 4 0-02 0-04 0-02 + 0.04 No inhibition > 2-0 IC50 5 0-08 0-04 0-08 + 0-02 Ampho. Eco. Ampho. + Eco. FIC* No inhibition > 1-5 Strain No. (mg/l) (mg/l) Protected by copyright. 7 0-04 0-08 0.04+0-08 *FIC, fractional inhibition concentration. No inhibition > 2-0 21 0-04 0-02 0.04+0.04 No inhibition > 3-0 Using the conventional technique of serial dilution of antifungal agents, the authors demonstrated * FIC, fractional inhibition concentration. antagonism in all 4 strains of C. albicans. For strain no. 4, the MIC of amphotericin B is 0.02 mg/l and of Table 2 shows the antagonism between ampho- econazole it is 0-04 mg/l. If the 2 antibiotics had an tericin B and econazole stated in IC50 and the sum of additive effect, the inhibition of growth would be FIC shows clearly an antagonistic effect. obtained with 0-01 mg/l of amphotericin B and The combination of flucytosine and econazole, 0.02 mg/l of econazole. However, there is no inhibi- contrary to the previous one, shows a synergistic tion with 0.02mg/l amphotericin B + 0.04 mg/l effect. This was obtained on 22 of 26 C. albicans econazole (Table 1). strains. For 5 strains (Table 3) the synergism was The antagonistic effect is, nevertheless, limited. particularly intense; this can be controlled by http://pmj.bmj.com/ Inhibition can be seen if the concentration of calculation of MIC and of MIC fractions.
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