ORIGINAL RESEARCH ARTICLE

Drug Invest. 3 (3): 201-204, 1991 0114-2402/91/0003-0210/$02.00/0 © Adis International Limited. All rights reserved.

DRI1 0258

Use of Alkylaminoethylglycine for Testing Fluconazole Activity in Sabouraud Dextrose

F.D. D'Auria, N. Simonetti, V. Strippoli and A. Villa Institute of Microbiology, Department of Pharmacy, 'La Sapienza' University of Rome, Rome, Italy

Summary Alkylaminoethylglycine (AAEG) overcomes the resistance of Candida albicans to triazole anti-fungal agents in Sabouraud dextrose medium. When supplemented with AAEG at pH 5.5, Sabouraud dextrose was found to be a reliable, precise medium for testing the sensitivity of C. albicans towards fluconazole.

Fluconazole is a triazole antifungal agent whose potential is based on its intrinsic characteristics that allow oral administration (Brammer & Tarbit 1987), solubility in water, a wide spectrum of ac­tion (Troke 1987) and low toxicity (Dupont & Drouhet 1987). In vitro, however, demonstrating activity against Candida albicans strains presents difficulties because of the inhibiting agents found in usual culture media (Marriott & Richardson 1987). It has been shown that it is possible to over­come this inhibitory action of complex substances in Sabouraud dextrose with sodium dioctylsul­phosuccinate (Simonetti et at. 1990). The purpose of the present study was to demonstrate an anal­ogous activity using alkylaminoethylglycine (AAEG) [Villa et at. 1989] to remove the inhibitory action of Sabouraud dextrose.

Materials and Methods Antimicrobial Agents

Fluconazole (Pfizer) was dissolved in water at a concentration of I gjL and brought to the desired concentrations (0.1 to 100 mgjL) in Sabouraud dextrose (Phillips & Nash 1985). AAEG (Uniderm)

was added to the culture medium containing flu­conazole, at concentrations of 5 and 20 mgjL, i.e. below the minimum inhibitory concentration (MIC).

Microorganisms

The tests were conducted on 50 strains of C. albicans and 20 strains of other Candida species (4 of C. krusei, 4 of C. glabrata and 12 of C. trop­icalis) freshly isolated from biological material and recovered on Sabouraud dextrose agar slants.

The inoculum suspensions (3 X 103 cells/ml for the broth tests, 105 cells/ml for the agar tests) in saline (0.9% NaCl) were obtained from cultures grown for 18 hours at 37°C in Sabouraud dextrose broth (Jefcoate 1978) and measured with a spec­trophotometer (Carl Zeiss) at 540nm. In the tests used to measure the influence of inoculum size in broth, the suspension ranged from 3 X 103 to 3 X

106 cells/mt.

Antimicrobial Activity Tests

The tests were conducted in Sabouraud dex­trose broth and in Sabouraud dextrose agar both at pH 5.5. In some tests, the pH ranged between

202 Drug Invest. 3 (3) 1991

Table I. Effects of alkylamlnoethylglyclne (AAEG) 20 mg/L on the activity of fluconazole in Sabouraud dextrose broth (SOB) and in Sabouraud dextrose agar (SOA) against 32 strains of Candida albicans at pH 5.5 after incubation for 48h at 37°C

Medium Xg Xa ± SO MICso MICeo Range R(%)

SOA 23.1 89.9 ± 91 25 >100 0.2->100 48 SOA + AAEG 1.6 2.1 ± 1.5 1.6 3.1 0.1-6.25 0 SOB 87 148.8 ± 83.2 >100 >100 3.1->100 70 SOB + AAEG 1.5 1.7 ± 0.9 1.6 3.1 0.2-3.1 0

Abbreviations: Xa = mean of MIC values; Xg = geometric mean MIC; R = resistant strains (MIC ;!> 100).

4.5 and 7.5. In the broth tests, 0.2ml of the culture medium containing serial dilutions of fluconazole between 0.1 and 100 mg!L either alone or associ­ated with AAEG were dispensed into microdilu­tion trays. The inoculum was 33#£1 (3 X 103 cells/ ml). The minimum inhibitory concentration (MIC) was determined after 48 hours at 37°C.

In the Sabouraud dextrose agar tests, Candida suspensions (lOs cells/ml) were seeded with a multi­pointer onto plates of Sabouraud agar containing serial dilutions of fluconazole from 0.1 to 100 mg! L. The MIC levels were determined after incubat­ing for 48 hours at 37°C.

Results

In Sabouraud broth, the C. albicans strains were generally resistant to the action of fluconazole (table

Table II. Effect of inoculum size on AAEG activity In fluconazole in Sabouraud dextrose broth (conditions as in table I)

Inoculum Xg MICso MICeo Range R

(cells/ml) (%)

SOB 3 x 1()3 35.6 100 >100 1.6->100 67 3 x 1()4 39.8 100 >100 1.6->100 67 3 x 10S >100 >100 >100 >100 100 3 x 108 >100 >100 >100 >100 100

SOB + AAEG 3 x 1()3 1.9 1.6 3.12 0.8-3.12 0 3 x 1()4 2.5 3.12 3.12 1.6-3.12 0 3 x 10S 2.5 3.12 3.12 1.6-3.12 0 3 x 10S 11.2 3.12 >100 1.6->100 34

Abbreviations: as for table I.

I). This resistance was overcome when subinhibi­tory concentrations of AAEG (20 mg/L) were added to the medium. All experimental values (MICso, MIC90 and the geometric mean MIC) showed that the strains used were sensitive to fluconazole in the presence of AAEG. AAEG seemed to enhance the activity of fluconazole more in Sabouraud dextrose broth than in agar (table II), although the validity of its use in testing the sensitivity to fluconazole was essentially unchanged. The inoculum size greater than 3 x lOS cells/ml could be a limiting factor in the action of AAEG (table II). Saline added to Sabouraud dextrose reduced the effect of AAEG on fluconazole (table III).

The effect of fluconazole in Sabouraud dextrose broth at different pH values showed that the ac­tivity of AAEG against Candida may be dependent on the pH of the medium. Table IV indicates that C. albicans strains behaved similarly at pH levels between 5.5 and 7.5. As shown in table V, adding AAEG increased the activity of fluconazole on other Candida species equally well, although some strains were inhibited by AAEG at a concentration of 20 mg!L.

Discussion

Despite a different chemical structure, flucon­azole does not differ widely from other azole com­pounds in its mechanism of antifungal action. The drug has an affinity for the haem moiety of cyto­chrome P450, thus preventing the fixation and ac­tivation of oxygen (Jefcoate 1978), for the fungal enzymes dependent on cytochrome P450 and for the C-14 dimethylase that determines the dime-

Alkylaminoethylglycine and fluconazole Activity 203

Table III. Effect of saline (NaCI) on fluconazole + AAEG (20 mg/l) activity against 10 C. alb/cans strains (inoculum size 3 x lOS cells/ml)

Medium Xg MIC50

SOB 183.4 100 SOB + AAEG 1.88 1.6 SOB + NaCI 200 100 SOB + NaCI + AAEG 2.87 3.12

Abbreviations: as for table I.

thylation of lanosterol into ergosterol (Shaw et al. 1987). The resistance of C. albieans in a medium containing complex substances, like Sabouraud dextrose, could be analogous to the phenotypic re­sistance towards the polyene antifungal agents un­der certain conditions, because in both cases the cell wall prevents the antibiotic molecule from reaching the target for its full inhibitory action. In the case of the polyene antifungal agents, however, structural cell wall changes, even if transitory, can be observed (Gale 1986). These changes might al­ter the hydrophobic interaction between the super­ficial structures at the molecular level. On the other hand, with fluconazole the characteristics of the medium do not appear to be able to alter mycotic cell wall structures much less the target of the anti­fungal activity. Thus, excluding structural vari­ations, one must consider functional changes that would block the interaction between the mycotic cell and the drug.

The complex substances present in Sabouraud

Table IV. Effect of AAEG in Sabouraud dextrose broth at vary­ing pH on the activity of fluconazole against 10 strains of C. alb/cans (inoculum size 3 x lOS cells/ml)

pH Xg MICso MICgo Range

SOB + MEG 20 mg/l 4.5 13.6 12.5 25 6.25-25 5.0 10.5 12.5 12.5 6.25-12.5

5.5 2.0 3.12 3.12 0.4-3.12

SOB + MEG 5 mg/l 6.0 2.4 3.12 3.12 1.8-3.12 6.5 2.2 1.6 3.12 1.6-3.12 7.0 2.5 3.12 3.12 1.6-3.12 7.5 2 1.6 3.12 1.6-3.12

MICgo Range R(%)

100 100 3.12 0.8-3.12 0 100 100 3.12 1.6-3.12 0

medium may interfere with the ionic concentration at the microbial surface and thus with the possible interaction of fluconazole with the cellular target. The presence of AAEG, an amphoteric surfactant, at noninhibitory concentrations, could cause this ionic interference to be overcome at the microbial membrane level. As shown in the present study an

increase in ionic concentration with sodium chlor­ide can, in fact, lessen the activity of fluconazole.

At higher concentrations, AAEG working at the membrane level can exert an antifungal effect. In our study, subinhibitory doses of AAEG were used to avoid additive inhibitory effects with flucona­zole. An antimicrobial effect of AAEG at the con­centration used can be excluded because in TC me­dium AAEG reduces the activity of fluconazole (unpublished data). In addition, AAEG does not work as well in environments between pH 5.5 and 7.5 where there is no strict relation of its activity to the antimicrobial action of the amphoteric sur­factant. AAEG activity in Sabouraud medium, therefore, appears antagonistic with complex mo­lecules that negatively influence the activity of flu­conazole, probably by physicochemical means rather than antimicrobial in nature. The activity of AAEG towards fluconazole was slightly greater in the liquid medium than in the solid form, an effect that can also be observed with polyene antifungal agents (Odds et al. 1986).

In conclusion, AAEG added to Sabouraud dex­trose allows the testing of the activity of flucona­zole on C. albieans in this medium. The use of Sabouraud plus AAEG, furthermore, presents the advantages of better reproducibility of the sensi­tivity tests.

204 Drug Invest. 3 (3) 1991

Table V. Effect of AAEG on fluconazole activity in Sabouraud dextrose agar (SOA) and Sabouraud dextrose broth (SOB) against 20 strains of Candida spp.: 4 C. g/abrata, 4 C. krusei and 12 C. troplcalis. Inoculum size 105 cells/ml in SOA and 3 x 1()3 cells/ml

in SOB

Medium Xg Xa ± SO

SOA 82.17 132.7 ± 76.46 SOA + AAEG 8.04 19.3 ± 21.6 SOB 189.57 176.2 ± 43.6 SOB + AAEG 4.7 42.8 ± 71.8

Abbreviations: as for table I.

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Correspondence and reprints: Professor Nicola Simonetti, Via

Nazionale, 18, 00147 Rome RM, Italy.