I. QUANTITATIVE OBSERVATION OF THE ACTION

Transcription

1 Japan. J. Microb., Vol. 5, No. 4, 1961 UDC: : FUNGICIDAL ACTION OF CAPRYLIC ACID FOR CANDIDA ALBICANS I. QUANTITATIVE OBSERVATION OF THE ACTION TOHRU TSUKAHARA Department Bacteriology, Niigata University School Medicine, Niigata (Received August 14, 1961) The anti-fungal effects certain fatty acids have been demonstrated by several investigators1-6). Above all undecylenic acid 10-hendecenoic acid) has been widely applied in the treatment dermatomycosis. Recently, Neuhauser and Gustus (1954) 6) reported that a new therapeutic agent, consisting saturated complexes an acid-adsorbing resin and certain fatty acids, particularly caprylic acid, appeared to be successful for the treatment severe intestinal candidiasis. The caprylic acid-resin complex exerted an excellent inhibition growth Candida albicans in vitro. The author recently investigated the metabolic behavior Candida albicans. In a study aerobic metabolism fatty acids, straight chain saturated fatty acids from acetic acid to stearic acid (C2 to C18), the endogenous rate the organism was found to be almost completely inhibited by the presence minute amounts (10-5 M) caprylic acid (C8). This phenomenon appears to be attributable to the strong antimycotic effect the acid upon the organism. Hereupon, the author planned to study the anti-fungal action caprylic acid on Candida albicans and its mode action. In the present paper, several aspects the effects caprylic acid on the growth and viability Candida albicans are reported. MATERIALS AND METHODS Candida albicans: Stock culture a strain (406; MGL; 3rd AAL; Duke University) C. albicans was used. The organism was grown on a glucose peptone agar slant for 20 hr at 37 Ž. Cells were harvested and washed twice by resuspending and centrifuging in phosphate buffer (ph 5.91, M/15). The final suspension was diluted to contain approximately 60 thousand viable cells per cc. Culture media employed: A glucose (2 per cent) peptone (1 per cent) agar, adjusted to ph 5.6, was used.

2 384 TSUKAHARA Vol. 5, No. 4 Methods for the determination fungicidal action caprylic acid on C. albicans: The fungicidal activity the acid was tested by the following methods. Colony counts were made by the conventional pour agar plate method. A suspension the test organism, containing about 3,000 viable cells (in yeast phase) per cc, was brought into contact with caprylic acid at 37 Ž for different Fig. 1. Schematic diagram apparatus used for the determination fungicidal activity caprylic acid. AD: the agar disc inoculated with a known number C. albicans was prepared as following procedures. The agar medium contained NaCI, 0.8%; Na2HPO4-12H2O, 0.8%; KH2PO4, 0.25%; and agar, 1.5%. It was melted and then cooled to 48 Ž. Ten cc the medium were mixed with 0.1 cc the yeast cell suspension, poured into a petri dish, and allowed to solidify. Discs with a diameter 7 mm and a thickness 1.5 mm were cut from the agar plate with a corkborer. V : vessels, one was 25 cm above and slightly to the back the other. The vessels contained sterile M/30 phosphate buffer solution which was continuously siphoned from the upper to the lower through strips filter paper. FS : filter paper strip (1 by 30 cm) ran slopewise between the vessels. E : carbon electrodes were immersed in the vessels, and connected to a direct current generator. G : direct current generator was adjusted to supply 0,05 ma per cm strip at 500 v. NS: nutrient solution used was a glucose (1%) peptone (1%) water. C caprylic acid solution in various concentrations. B : phosphate buffer solution ph 5.91.

3 October, 1961 FUNGICIDAL ACTION OF CAPRYLIC ACID 385 lengths time, and, then, it was inoculated into the glucose peptone agar plates. After incubation for 2 days, the number colonies that developed on or in the agar plate was counted. The average number colonies from triplicate tests was calculated to obtain the number viable cells present per cc in the suspension used. The other method used was a modification an electrophoretic technique devised by Miyamura (1958) 7). The apparatus used is shown schematically in Fig. 1. The agar discs, inoculated with C. albicans, were submerged in the solutions caprylic acid for a given time. The discs were then removed and placed. in the center the filter paper strips. Apotential difference 500 V was applied to the system for 30 minutes. The anti-fungal agent, present in the agar discs, was removed by the action ionophoresis and washing. In the case caprylic acid, best results were obtained when the cathode was set in the upper vessel and the anode in the lower vessel. The discs were put into tubes containing 10 cc nutrient solution (1% glucose 1% peptone water) and incubated at 37 Ž for 24 hr. Viable yeast cells in the agar discs developed into colonies during the incubation period. Then, the agar discs were removed and photographed on positive paper to determine the number colonies developed. Caprylic acid and othr homologous saturated fatty acids: All the chemicals used were reagent grade. RESULTS Anti-fungal effects various saturated fatty acids on C. albicans. The anti-fungal powers the saturated fatty acids, from acetic acid with two carbon atoms to stearic acid with 18 carbon atoms, were tested on C. albicans by the serial dilution method. Tubes glucose (1 per cent) peptone (1 per cent) water (ph 5.6), containing various concentrations the fatty acid, were inoculated with fifty thousand cells a twenty hour-old culture, which was propagated on glucose peptone agar and incubated for 24 hr at 37 Ž. The presence or absence growth was determined by macroscopic observation turbidity produced. The results this experiment are summarized in Table 1. It was demonstrated that the minimum concentration fatty acids necessary to inhibit completely the growth the organism varied from 1/100 to 1/1,600 M, depending upon the sort the acid tested, and that caprylic acid with 8 carbon atoms in its chain possessed the greatest fungistatic properties all the fatty acids studied. It completely inhibited the growth C. albicans at a concentration 1/1,600 M. However, acetic, myristic and stearic acids hardly demonstrated any fungistatic properties even in a concentration 1/25 M. Capric acid, having 10 carbon atoms in its chain, exerted a considerable amount inhibitory effect upon the growth the organism.

4 386 TSUKAHARA Vol. 5, No. 4 Table 1. Anti-fungal Effects Straight Chain Saturated Fatty Acids on Candida albicans in Vitro Notes : Anti-fungal activities were examined according to the serial dilution method using a glucose (1%) peptone (1%) water at ph 5.6. Furthermore, additional experiments were performed to determine the fungicidal action the acids. One cc the C. albicans culture, which showed no growth in the previous tests as described above, was subcultured into a fresh medium (glucose peptone water, 10 cc) containing no fatty acids and incubated for 72 hr at 37 Ž. The sterility was determined by the absence growth. The results obtained are also indicated in Table 1. Caprylic acid was found to be the most powerful fungicide all the substances tested. It completely inhibited the growth the organism in a liquid medium at a concentration 1/400 M. Finally, it was established that caprylic acid was most effective against C. albicans in both fungistatic and fungicidal activity among all the homologous saturated fatty acids tested. Anti-fungal effect caprylic acid on several species and strains the genus Candida. From the results obtained above, it was found that caprylic acid exerted a strong anti-fungal effect on C. albicans. For this reason additional investigation was made to determine whether the acid may also act on several other species the genus Candida and several strains C. albicans. Tests were made using 10 strains 7 different species the genus under the same experimental condition as that described above. The results are given in Table 2. It was demonstrated that caprylic acid was effective against all tested species and strains the genus Candida, particularly the 4 strains C. albicans which appeared to be the most sensitive to the anti-fungal agent. The minimum amount acid required to kill the cells those strains C. albicans was equally at a concentration 1/400 M. These antimycolic data strongly suggest that caprylic acid is considered as being fungicidal on the organism.

5 October, 1961 FUNGICIDAL ACTION OF CAPRYLIC ACID 387 Table 2. Anti-fungal Effect Caprylic Acid on Several Species and Strains the Genus Candida in Vitro Notes The experimental methods and conditions were the same as that described in table 1. Fungicidal effect caprylic acid on C. albicans. The fungicidal activity caprylic acid on C. albicans was determined by the ordinary plating method, in respect to the concentration the acid and the time contact with it. The diluted cell suspension the organism, containing 3,000 viable cells per cc in 1/15 M phosphate buffer solution ph 5.91, was kept in contact with the acid for various period time at 37 Ž. Then, the cell suspension (0.1 cc) so treated was inoculated onto the plate medium to test for the viability cells. Coluny counts were made after incubating for 48 hr at 37 Ž. The results obtained are summarized in Table 3. As indicated in this table, the cells were completely killed by treatment with 10-3 M caprylic acid for 10 min. After contact with 10-4 M caprylic acid for 10 min, the number viable cells was reduced to 10.3 per cent, and the cells were completely killed after 1 hr. However, in the presence 10-5 M they still remained alive to a large extent even after 24 hr. No Table 3. Fungicidal Effect Caprylic Acid in. Phosphate Buffer Solution on C. albicans Serial dilutions caprylic acid were distributed in 1/15 M phosphate buffer solution ph Each 1 per cent corresponds to 3.0 colonies.

6 388 TSUKAHARA Vol. 5, No. 4 fungicidal activity the acid was observed when a concentration 10-6 M was used. In these results it was considered that the fungicidal effect caprylic acid on C. albicans was exceedingly powerful and the effect was depended upon the concentration the acid and the time contact. Effect ph on the fungicidal activity caprylic acid for C. albicans. To determine the optimal ph necessary for fungicidal activity caprylic acid, the number viable cells, when treated with the acid at various ph values 1/15 M potassium phosphate buffer solution, was calculated by the same manner as that described above. Table 4 summarizes the results these experiments. The powerful fungicidal property caprylic acid was observed in the acidic ph levels (ph 6.24 to 4.53), but a sharp drop in the fungicidal activity was noted between the ph 6.24 and 6.64 after 10 to 60 min treatment with the acid. At ph values over 6.98 no fungicidal effect the acid was recognizable during the treatment for 1 hr, but the yeast cells were completely suppressed after 24 hr. Thus, for an optimal fungicidal activity, caprylic acid should be mixed in an acid buffer solution, i.e., 1/15 M potassium phosphate buffer solution ph below For this reason further experiments were altogether conducted using a phosphate buffer solution ph Table 4. Fungicidal Activity Caprylic Acid at Varying ph The yeast cells were exposed to 0.5 ~ 10-5 M caprylic acid. Each 1 per cent corresponds to 2.6 colonies. Cell concentration and fungicidal action caprylic acid. The relationship between the cell concentration the test organism and the fungicidal action the acid was examined by using the cells at various concentrations. The cell suspensions, containing 2 ~ 103 to 2 ~ 105 viable cells per cc, were exposed to 10-4 M caprylic acid for 10 min to 24 hr. When a cell suspension, containing 2 ~ 104 viable cells, was exposed to 10-4 M caprylic acid, the cells were completely killed

7 October, 1961 FUNGICIDAL ACTION OF CAPRYLIC ACID 389 after 1 hr as in the case a cell suspension diluted 10 times (2 ~103 viable cells per cc). Similar result was also obtained from the heavier cell suspension 2 ~ 105 viable cells. In other words, no viable cells were observed in any the suspensions, 2 ~ 103 to 2 ~ 105, after treating with 10-4 M caprylic acid for 1 hr. This fact seems to indicate that the fungicidal capacity caprylic acid was hardly suppressed by the cell concentration the organism. Effect washing treated cell suspension on the fungicidal action caprylic acid. In the employment the usual pour plate method for determining the fungicidal effect caprylic acid, the delivery minute amount caprylic acid into the plate medium together with treated cell suspension cannot be avoided. Thus, the presence the minute amount acid may be fungistatic. Therefore, the possible effect the minute amount acid was investigated by using both washd and unwashed cell suspensions after treating them with the appropriate acid. The washed cell suspension was obtained by washing the treated cells in distilled water. In the experiment, 2 concentrations (10-3 and 10-4 M) caprylic acid were used, and the test organisms were exposed to the solutions the acid for 10 to 60 min. Only 10.5% the unwashed cells were viable after treating them with 10-4 M caprylic acid for 10 min, and almost the same number cells (9.3 per cent) was counted in the washed cell suspension. After 60 min no survival was observed both in the unwashed and washed cell suspensions. Similar result was also obtained using a concentration 10-3 M; the cells were completely killed after 10 min, even after washing. From the data, it is considered that once a cell suspension has been exposed to caprylic acid, subsequent washing rarely recovers the viability the cells. Fungicidal property caprylic acid as determined by a modification an electrophoretic method. The fungicidal activity caprylic acid was further studied by a paper electrophoresis procedure. Preliminary experiments were performed in order to determine whether caprylic acid contained in agar discs was completely removed by this method. The potency the acid in the discs was measured microbiologically at 30 min intervals during the period electrophoretic treatment. According to the agar plate diffusion technique, the concentration caprylic acid in the agar discs was assayed by measuring the inhibition zone produced around the agar disc. By the electrophoretic treatment, the content caprylic acid in discs was decreased within 30 min to a level below the minimum amount required to inhibit the growth C. albicans. In another experiment the yeast cells, which were not exposed to anti-fungal agent, were found to be capable growing, even when exposed to the treatment for 2 hr. Hereupon, the fungicidal action caprylic acid in phosphate buffer solution was examined by the procedure described previously. The results obtained are indicated in Table 5 and Fig. 2. By treating with 10-4 M caprylic acid for 10 min the number viable cells was reduced to 29.4 per cent, and they were completely

8 Vol. TSUKAHARA 390 Table 5. Fungicidal by Effect A Modification Caprylic Acid on C. albicans An Electrophoretic 5, No. 4 Determined Method Serial dilutions caprylic acid were distributed in 1/15M phosphate buffer solution ph Fig. 2. Fungicidal activity caprylic acid determined by using an electrophoretic method. Colonies C. albicans in agar discs treated with 10-4M caprylic acid (upper row) and those not treated (lower row). killed after 30 min. 10 min. After all, tained by caprylic method. this With almost the use 10-3 M, no survival was observed after only the same result as that described above was also ob- electrophoretic acid was established method. both The powerful by an agar plating fungicidal method property and an ionophoretic Protective effect various agents upon the fungicidal action caprylic acid. This effect was examined by the electrophoretic method. The agar discs, inoculated with C. albicans, were exposed to the phosphate buffer solution (ph 5.91) containing then As with caprylic examined shown in 0.5 ~10-5 for this M acid and viability. table, caprylic various Table about acid 24 at 6 summarizes per for substances cent 1 hr. The 37 Ž the the results yeasts fungicidal for 1 to was activity 3 hr these viable were experiments. after and the contact acid was

9 October, 1961 FUNGICIDAL ACTION OF CAPRYLIC ACID 391 Table 6. Protective Effect Various Agents upon the Fungicidal Action Caprylic Acid The agar discs inoculated with C. albicans were exposed to 0.5 ~ 10-5M caprylic acid together with the added substances at 37 C for 1 to 3 hr. completely arrested by the presence 10 per cent serum during the 1 hr contact period. Similar arresting effects were observed with carbohydrates, such as glucose, maltose, sucrose, starch; with polyhydric alcohols, such as dulcitol, glycerol and mannitol; or with D-amino acids, such as D-alanine and D-serine. One the other hand, the fungicidal action caprylic acid was not affected by peptone, cholesterol, lecithin, tween-80, biotin, L-amino acids, such as L-alanine, L-aspartic acid, and L-glutamic acid; or by some intermediates the Krebs tricarboxylic acid cycle, such as pyruvate, citrate, and fumarate. However, no arresting effect the agents, serum and carbohydrates, was demonstrated after the treatment for 3 hr and the cells were completely suppressed by caprylic acid. Accordingly, it seems likely that the fungicidal activity caprylic acid was not essentially influenced by the agents.

10 392 TSUKAHARA Vol. 5, No. 4 DISCUSSION This study was performed in order to determine the fungistatic and fungicidal activity the straight chain saturated fatty acids, containing from 2 to 18 carbon atoms, particularly caprylic acid (C8) under various conditions on C. albicans. It has already been indicated that many these fatty acids exhibit a remarkable effect upon the inhibition growth certain molds, such as Aspergillus niger, Aspergillus glaucus, Rhizopus nigricans, Penicillium frequentans, etc. (Kiesel, 1913; Kirby et al., 1937; Hfman et al., 1939; Keeney et al., 1944). FurtherMore, it was concluded by Hfman et al. (1939) 4) that this effectiveness varied according to the chain length, the concentration the acid, and the ph the media employed. Acids containing 8 to 12 carbon atoms were the most effective for the inhibition mold growth. It is evident from the present experiments just cited that caprylic acid has the greatest fungistatic properties all the normal saturated fatty acids (C2 to C18) studied against C. albicans at ph It was fungistatic in a concentration 1/1,600 M, and it was most effective against the 4 strains C. albicans among all the species genus Candida studied. Recently, Neuhauser and Gustus (1954) 6) reported that caprylic acid-resin complex exerted a strong inhibitory effect upon C. albicans in vitro. Also, they stated that the acid-resin complex had been available for the treatment severe intestinal moniliasis, and in none the cases studied were there any undesirable side reactions due to the agent. Therefore, it can be said that caprylic acid is the most powerful fungistat against C. albicans among the normal saturated fatty acids with even numbered carbon atoms. As a characteristic point the anti-fungal properties caprylic acid on C. albicans, the acid acts mainly as a fungicide rather than as a fungistat. Previously, the fungicidal effects the fatty acids from formic acid with one carbon atom to capric acid with 10 carbon atoms were investigated on Trichophyton gypseum by Peck and Rosenfeld (1938) 3). They pointed out that normal acids were more fungicidal than their isomers, and that the acids with even numbered carbon atoms were apparently less fungicidal than those with an odd number in their chain. Also, there was no increase fungicidal properties with the increase in the number carbon atoms. Thus, valeric acid with 5 carbon atoms was the strongest fungicide all the acids. In the present study the fungicidal activity caprylic acid on C. albicans was established by the usual pour plate method any by an electrophoretic method. The acid was found to be the most powerful fungicide all the faty acids examined. It exhibited far stronger fungicidal activity at a ph below 6.24, and it killed the cells completely after it was kept in contact with the organism for 10 min in a concentration 10-3 M. Furthermore, the activity was hardly suppressed by the concentration the cells employed. These results lead to the conclusion that the fungicidal activity caprylic acid is very effective against C. albicans,

11 October, and this activity depends upon its concentration, the period contact and the ph the buffer solution employed. Moreover, the fungicidal action caprylic acid developed after only a few min treatment with the acid, and this action was not reversed by washing with distilled water or by adding such protective agent as serum, carbohydrate, or polyhydric alcohol. Therefore, caprylic acid is believed to be strongly adsorbed to the yeast cells. The fungicidal activity caprylic acid was found to be reduced by the simultaneous addition serum. The protective effect serum seems to be attributable to the decrease in the quantity caprylic acid in the medium by its adsorption to the substance. This effect, however, disappeared after the cells were in contact with 0.5 ~ 10-5 M caprylic acid for 3 hr. Furthermore, the fungicidal power the acid was weakened to some extent by the presence an energy supplying substances such as glucose, maltose, sucrose, starch, dulcitol, glycerol, or mannitol. Also, any protective effect these substances was not observed after 3 hr as in the case serum. After all, once a cell suspension the organism has been exposed to caprylic acid at concentrations over a definite level in an acid buffer solution below ph 6.24, the cells are completely killed after several hr. Even subsequent washing with distilled water or by simultaneout addition uch protective agents as serum, carbohydrate, or polyhydric alcohol does not arrest the fungicidal effect. From these facts, it is believed that caprylic acid must be adsorbed to the yeast cells specifically and strongly. The powerful anti-fungal properties caprylic acid against C. albicans is mainly due to its fungicidal activity and the possible mechanisms the action will be discussed in a following paper. SUMMARY The anti-fungal property caprylic acid on C. albicans, particularly its fungicidal activity, has been studied by colony count using an ordinary plating method and a modification an electrophoretic method. Caprylic acid exhibits the most remarkable fungistatic and fungicidal properties all the normal saturated fatty acids with even numbered carbon atoms studied. The fungicidal activity caprylic acid depends upon the concentration the acid, the period contact and the ph the media. The fungicidal action caprylic acid is considerably reduced by the simultaneous addition several agents such as serum, carbohydrate, and polyhydric alcohol. This protective effect, however, disappears after the contact with the acid for 3 hr. Furthermore, when the cells the organism are once exposed to caprylic acid, its fungicidal activity is not affected by washing them with distilled water. These quantitative and biochemical facts suggest that the adsorption caprylic acid to the yeast cells must be extremely strong and specific.

12 394 TSUKAHARA Vol. 5, No. 4 REFERENCES ( 1 ) Kiesel, A.: Ann. de l'inst. Pasteur, 27, , ( 2 ) Kirby, G. W., Atkin, L. and Frey, C. N.: Cereal Chem., 14, , ( 3 ) Peck, S. M. and Rosenfeld, H.: J. Invest. Dermat., 1, , ( 4 ) Hfman, C., Schweitzer, T. R. and Dalby, G.: Food Res., 4, , ( 5 ) Keeney, E. L., Ajello, L. and Lankford, E.: Bull. Johns Hopkins Hosp., 75, , ( 6 ) Neuhauser, I. and Gustus, E. L.: Arc. Int. Med., 93, 53-60, ( 7 ) Miyamura, S.: J. Bacteriol., 75, , 1958.