ANTIMICROBIiAL AGzNTs AND CIzmOrHzRAPY, Aug. 1977, p. 206-212 Copyright C 1977 American Society for Microbiology

Vol. 12, No. 2

Printed in U.S.A.

Anti-Candida Activity of Clotrimazole in Combination with Dioctyl Sodium Sulfosuccinate and Other Surfactants KAZUO IWATA AND HIDEYO YAMAGUCHI* Department of Microbiology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113,

Japan Received for publication 4 February 1977

Six anionic and five nonionic surfactants were tested for their effect on the fungistatic action of clotrimazole against Candida albicans. All of the anionic agents that did not contain an ethylene oxide group were capable of potentiating the anti-Candida activity of clotrimazole, whereas all five members of the polyoxyethylene surfactant group, including four nonionic agents and one anionic agent, acted in an antagonistic fashion. The combination of clotrimazole and the anionic surfactant dioctyl sodium sulfosuccinate was the most potent in synergy and, thus, more precise studies were made with this combination. Although appropriate combinations of the two drugs showed a potent fungicidal activity against proliferating cultures, none of these combinations tested was lethal when cell growth was restricted by nutritional deficiency. The lethal effect of the combined drugs was partly reversed when growing cultures were treated in the presence of an osmotic stabilizer. Whether cells were treated with moderate and higher concentrations of clotrimazole and dioctyl sodium sulfosuccinate, alone or in combination, there was little change in cell wall content of total protein, carbohydrate, or lipid from that in untreated control cells. However, there was a significant decrease in the cell wall content of phospholipid when moderate concentrations of the two drugs were combined. An anionic surfactant, dioctyl sodium sulfosuccinate (DSS), is mainly used as a wetting agent in industrial, pharmaceutical, cosmetic, and food applications and as an ingredient in some laxatives. This surfactant has also been known to have bactericidal action against various bacteria, in particular, gram-positive bacteria, and has been used as a topical antiseptic. We have previously reported that DSS exhibits a limited antifungal activity against Candida albicans and several other yeasts, whereas in combination with the imidazole antimycotic clotrimazole, it shows effective, synergistic fungicidal action (8, 19). In the present study, we first attempted to examine the effect of various other surfactants, both anionic and nonionic, used in combination with clotrimazole on the viability of C. albicans cultures. Although several anionic surfactants were also found to be synergistic, none of them was as potent as DSS. Since many in vitro studies on the evaluation of the antifungal activity of clotrimazole revealed that this drug is basically fungistatic rather than fungicidal at clinically attainable concentrations in tissues (7, 13, 16, 17), appropriate combinations of clotrimazole with DSS may provide a formulation for more effective preparations of this antimycotic.

The potential usefulness of the combined drugs for therapy of human mycoses prompted us to investigate further the mechanism of their synergistic action. In this study, we have characterized several factors that are involved in the in vitro antifungal activity of DSS plus clotrimazole against C. albicans. In addition, experiments were also carried out to examine changes in the chemical composition of cell walls. MATERIALS AND METHODS Organisms. C. albicans MTU 12021 was originally

isolated from a patient with systemic candidiasis and has been subcultured in this laboratory. Growth studies. Unless otherwise noted, experiments were carried out at 37rC, and cultures were grown in GPY broth consisting of (on a weight basis: 4% glucose, 1% peptone (Difco), and 0.5% yeast extract (Difco). In some experiments, a chemically defined medium (7) was substituted for GPY broth. Sterile Monod tubes were inoculated with an overnight culture that had been diluted with the same medium to give the indicated cell concentrations in a range between 105 and 107 cells/ml and were then incubated on a wrist-action shaker. Clotrimazole was dissolved in dimethyl sulfoxide, and all the surfactants were in distilled water. The final concentration of this organic solvent was 1% (vol/ vol). The two drugs were added simultaneously to cultures at zero time, unless otherwise specified.

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COMBINED EFFECT OF CLOTRIMAZOLE AND SURFACTANT

Samples were taken at indicated times during a 24-h experimental period for assay of viability. Fungal viability was estimated by plating dilutions (in saline) of cultures on candida GS agar (Eiken Co., Tokyo) plates as described previously (7). In studies of osmotic effects on synergy, growing cultures were exposed to clotrimazole and DSS in combination in broth containing 0.6 M mannitol as an osmotic stabilizer. Control cultures without stabilizer were also studied. After 8 h of incubation, samples were taken and diluted in broth plus stabilizer, and viable counts were determined on candida GS agar plates containing stabilizer. For an investigation of the effect of the growth phase of cells on synergy, the cells grown on GPY broth were depleted of endogenous nitrogen as well as reserve carbohydrates by subsequent incubation at 37°C in the complete synthetic medium (7) minus (NH4)2SO4 and glucose until no further change in turbidity was produced. At this time, (NH4)2SO4 and glucose were added to final concentrations of 0.8 and 1%, respectively, to control cultures that had been incubated at 37°C. Clotrimazole (1 ,ug/ml) and DSS (0.025%, wt/vol) were added together to two other portions of the cell suspension, which were then incubated at 37°C, one with, and the other without, supplementation of nitrogen and carbon sources. The numbers of viable units in the untreated control and in the two treated cultures were determined after the indicated times of incubation. Fractionation and chemical analysis of cell walls. Growing cells were incubated with or without indicated concentrations of clotrimazole and DSS, either singly or in combination, at 37°C for 6 h in a GPY broth. Cells were harvested by centrifugation and washed thoroughly with distilled water; cell walls were then isolated and purified according to the method described in our previous paper (18). Methods for determination of proteins, ribonucleic acid, and deoxyribonucleic acid were those reported by Brown and Rose (2). Total carbohydrate was measured by the anthrone method (5) with 1-glucose as a standard. Total lipid was determined gravimetrically after extraction of purified cell wall materials by the method of Longley et al. (11). Lipid phosphorus was estimated by the modified Bartlett procedure described by Marinetti (12). Chemicals. Clotrimazole was generously provided by Bayer Yakuhin Ltd., Osaka, Japan. RESULTS

Anti-Candida action of clotrimazole and various surfactants. Under the present assay conditions, in which a relatively large inoculum (10w cells/ml) was employed, clotrimazole concentrations of 10 to 20 ,g/ml were required to completely arrest the increase in viable cell counts during a 24-h incubation, but no killing effect was exerted even at 80 ,ug of the drug per ml (Fig. 1). Of the six anionic and five nonionic surfactants under study (Table 1), two anionic agents, i.e., sodium lauryl polyoxyethylene sulfate

I

207

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0.0

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100

10

0.i

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100

Clotrimaole .,g/ml

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FIG. 1. Viable counts ofC. albicans cultures after 24-h exposure to graded concentrations of clotrimazole.

TABLE 1. List of surfactants tested Ionic type Anionic

Compound (Trademark) Sodium laurylbenzene sulfonatea Sodium lauryl sulfatea Sodium lauryl polyoxyethylene sulfate (EO, 3)b (Sannol NES)c Dioctyl sodium sulfosuccinate (Aerosol OT)a Sodium tetradecanoyl sulfonate (Lipolan 1400)C Sodium deoxycholatea

Nonionic

Polyoxyethylene stearyl ether (EO, 20) (Liponox OCC)Y Polyoxyethylene caprylphenyl ether (EO, 9) (Triton X-100)a Polyoxyethylene sorbitan monooleate (EO, 20) (Tween 80)a Sorbitan monooleate (Span 80)a Polyoxyethylene monooleate (EO, 10) (Ethofat 0/20)C a Purchased from Wako Chemical Industries, Ltd., Osaka, Japan. b EO, Mean number of ethylene oxide groups per molecule. c Generous gifts from Lion Armour Co., Ltd., Tokyo, Japan.

(Sannol NES) and sodium deoxycholate (DOC), and all of the nonionic agents had almost no effect on the growth ofC. albicans at concentrations ranging from 0.001 to 0.4% (wt/vol). DSS and the other three anionic agents, viz., sodium laurylbenzene sulfonate (LBS), sodium lauryl sulfate (SLS), and sodium tetradecanoyl sulfonate (Lipolan 1400), were not inhibitory at concentrations of 0.02% or less. However, they showed varying extents of fungistatic and/or fungicidal activities at higher concentrations (Fig. 2). Effect of various combinations of clotrimazole and surfactant on viability. The combination study was performed to examine whether or not a given surfactant would potentiate (or

208

ANTIMICROB. AGENTS CHEMOTHER.

IWATA AND YAMAGUCHI No Swfoi

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LBS 0.01%

SLS 0.01%

Uplm 1400 DSS 0.01%

DOC

0.025% 0.05%

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,I 10

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FIG. 2. Viable counts ofC. albicans cultures after 24-h exposure to graded concentrations of various types of anionic and nonionic surfactants.

counteract) the anti-Candida action of clotrimazole. Based on the results from the foregoing experiments (Fig. 1 and 2), clotrimazole and any surfactant combined were tested, for purposes of convenience, at concentrations too low to affect the growth when used alone. The results are summarized in Fig. 3. It shows that, with the only exception of Sannol NES, all the anionic surfactants were more or less capable of potentiating the anti-Candida action of clotrimazole; when combined with 0.13 or 0.5 ug of the antimycotic per ml, substantial synergy was attained by 0.01% LBS, 0.01% SLS, 0.025% Lipolan 1400, 0.05% DOC, and 0.025% DSS. In general, combinations of clotrimazole with LBS or SLS exhibited fungistatic synergy, whereas those with Lipolan 1400, DOC, or DSS were effective in fungicidal synergy. A similar effect was observed with a half-lower concentration of each surfactant in combination with 0.5 ,ug of clotrimazole per ml (data not presented). Of all the combinations tested, clotrimazole plus DSS showed the most potent fungicidal activity. In contrast, a polyoxyethylene surfactant of anionic type, Sannol NES, counteracted clotrimazole. This antagonistic phenomenon was particularly prominent when relatively high concentrations of the surfactant (0.1%) and of clotrimazole (10 ,ug/ml) were combined. A similar degree of antagonism was also observed with four nonionic polyoxyethylene surfactants; viable counts of cultures receiving combinations of 0.1% of either of these surfactants and 10 ,tg of clotrimazole per ml were about 10fold higher than those of cultures receiving the same concentration of clotrimazole alone. All of these surfactants were still effective in antago-

FIG. 3. Viable counts of C. albicans cultures after 24-h exposure to concentrations of various surfactants and clotrimazole, alone and in combination. Clotrimazole concentrations (micrograms per milliliter) were: 0 (0); 0.13 (a); 0.5 (ED); and 10 (WI).

nism, although to a lesser extent, in those combinations that contained lower clotrimazole concentrations (0.13 or 0.5 ,ug/ml). However, none of the nonionic agents showed synergistic action with clotrimazole, irrespective of the concentration of both drugs used in combination. Fungitoxic activity of clotrimazole and DSS used singly and in various combinations. Varying combinations of clotrimazole and DSS at concentrations ofeach drug that were too low to affect growth when used alone exhibited significant fungistatic or even a synergistic fungicidal action, the extent of which increased with increasing concentrations of either clotrimazole or DSS or both in combination. Although 0.05 ,ug of clotrimitzole per ml was without antifungal effect, whatever concentration of DSS was present, clotrimazole at concentrations of 0.1 ,ug or more per ml exerted a definite fungistatic action when combined with 0.013% of DSS (Fig. 4). To produce a significant killing effect, 0.025% or higher concentrations of DSS were required, irrespective of the level of clotrimazole. Time-dependent changes in viable cell counts after exposure to clotrimazole and DSS, alone and in combination. Under the present growth conditions, in which drug-free control cultures showed a logarithmic increase in the viable cell count until h 8 of incubation after the first 2-h lag period, addition of either clotrimazole (0.5 ,ug/ml) or DSS (0.025%) scarcely affected the growth rate (Fig. 5). However, when the two drugs were added together, a drastic reduction occurred in the viable population within 3 h after the onset of incubation; this reduction eventually resulted in a loss of more than 99.99% of viability after 16 h of incu-

COMBINED EFFECT OF CLOTRIMAZOLE AND SURFACTANT

VOL. 12, 1977

bation. Figure 5 also shows that a higher concentration (0.1%) of DSS alone caused a transient loss of about 90% of the viable population during the course ofthe first 10 h. This decrease was followed by a gradual restoration of viable number, which reached a level as high as that in the untreated control cultures within 24 h. Effect of an osmotic stabilizer on synergistic fungitoxicity of combination of clotrimazole and DSS. Plate counts of both of the untreated control cultures grown in the presence or absence of an osmotic stabilizer increased approximately 5- and 10-fold, respectively, after 8 h of incubation (Table 2). Upon treatment with 1 ,ug of clotrimazole per ml and 0.025% DSS in combination, those cultures that

209

incubated in the stabilizer-depleted medium showed a 5,000-fold decrease in the viable count, whereas only a 200-fold decrease was produced in the osmotically stabilized cultures. Effect of combining clotrimazole and DSS on nongrowing cells. Susceptibility to the fungicidal combination of clotrimazole and DSS in growing cells incubated in complete medium was compared with susceptibility in nongrowing cells placed in medium depleted of nitrogen and carbon sources. The results showed that nongrowing cells in deficient medium withstood almost completely the fungicidal action of the combined drugs, in contrast with growing cells in complete medium, which were highly susceptible to the combination (Table 3). Effect of clotrimazole and DSS, alone and in combination, on cell wall composition. were

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FIG. 4. Fungicidal synergy between clotrimazole and DSS as a function of the concentrations ofeither or both ofthe two drugs. Numbers ofviable cells were measured after 24-h exposure of C. albicans cultures in nutrient broth to various combinations of clotrimazole and DSS. Clotrimazole was used at concentrations (micrograms per milliliter) of: (x) 0, (O) 0.05, (0) 0.1, (O) 0.2, (A) 1, and (A) 5.

Time ( Hor)

FIG. 5. Time-dependent changes of the viability of C. albicans cultures in nutrient broth as influenced by clotrimazole and DSS, alone and in combination. To C. albicans cultures were added at zero-time: (O) no addition, (0) clotrimazole (0.5 L/ml), (O) DSS (0.025%), (A) DSS (01 %), and (A) clotrimazole (0.5 pg/ml) plus DSS (0.025%).

TABLE 2. Effect ofosmotic stabilizer on antifungal action of clotrimazole and DSS, alone and in combination, against C. albicansa Viable cell count/ml

Treated with:

With stabilizer Oh

Without stabilizer 8h

Oh

8h

None 4.8 x 106 2.3 x 107 5.5 x 106 5.2 x 107 1.1 x 107 4.8 x 106 Clotrimazole (1 ,ug/ml) 5.5 x 106 2.1 x 107 4.8 x 106 2.0 x 107 DSS (0.025%) 5.5 x 106 4.0 x 107 4.8 x 106 2.5 x 104 Combination 5.5 x 10ff 1.0 x 103 a Cultures were treated as indicated for 8 h in nutrient broth with and without osmotic stabilizer.

210 IWATA AND YAMAGUCHI Since the foregoing experimental data suggested that impairment of cell wall structure may be involved in the synergistic fungicidal action of clotrimazole and DSS, experiments were conducted to characterize the chemical composition of cell walls. No significant change occurred in the wall content, express6i on a dry weight basis, of protein, carbohydrate; or lipid after 6 h of exposure of growing cells to clotrimazole (1 and 40 ,ug/ml), DSS (0.025 and 0.1%), 'and a combination of clotrimazole (1 Ag/ml) and DSS (0.025%) (Table 4). However, measurement of the quantity of phospholipid revealed that its content was markedly, diminished when cells were treated with clotrimazole plus DSS. A similar change, although to a less extent, was also produced by 0.1% DSS alone, whereas clotrimizole alone had no such effect.

ANTIMIcROB. AGENTS CHEMOTHER.

agent: on one hand, they cause some potentiation of the drug activity, possibly through their surface effect which modifies the permeability of the cell membrane so as to enhance the entry of the drug molecule; but, on the other hand, inactivation of the drug occurs after it becomes bound to micelles, since the drug is kept from contact with the surface of the cells. It may be, therefore, that in synergistic combinations of surfactant and antimicrobial agent, the first effect may predominate, whereas in antagonistic combinations, the second effect may predominate. The eleven surfactants of anionic or nonionic type tested here can be classified into three groups on the basis of their effect in combination with clotrimazole: (i) synergistic, (ii) antagonistic, and (iii) indifferent. The five agents belonging to first group share two characterisDISCUSSION tics: they are all anionic in nature and none conan ethylene oxide group. The five members There is a general notion that surfactants tains of the second group were all polyoxyethylene a have dual effect on an antimicrobial usually surfactants of the anionic or nonionic type. The only agent indifferent to clotrimazole action is TABLE 3. Failure of clotrimazole plus DSS of the nonionic type and not a derivative of combination to affect viability on nongrowing polyoxyethylene. These data make it possible to cultures of C. albicans correlate the antagonistic action of a given surfactant toward clotrimazole with the presence Viable cell count/nml of an ethylene oxide group in its molecular Treated with clotrimazole structure. Partially consistent results were reTreatment Untreated: (1 JLg/ml) plus ported by Kurisu and Kawahara (10), who DSS (h) (0.025%) complete worked on varying combinations of 7 surfacmedium Complete Deficient tants alid 15 germicides. There are also several medium mediuma papers in which physico-chemical as well as 0 5.6 x 106 5.6 x 106 biological aspects ofthe interaction of Tween 80 5.6 x 106 1 5.6 x 106 5.2 x 106 4.9 x 106 or other polyoxyethylene surfactants with some 2 1.5x 106 1.Ox 107 4.7x 10O cationic antimicrobial agents are investigated 4 1.4 x 107 3.5 x 105 4.5 x 10O (1, 3, 6). These investigators showed that inac6 3.1x107 1.2x103 4.1x106 tivation of the germicides is augmented when 24 9.6 x 107