Enhanced contact activity of fluconazole in association with antioxidants against fluconazole-resistant organisms

G. Simonetti, A. Villa and N. Simonetti*

University of Rome ‘La Sapienza’, Institute of Microbiology, Faculty of Pharmacy, Piazzale Aldo Moro 5, 00185 Rome, Italy

Received 8 May 2001; returned 28 December 2001; revised 12 April 2002; accepted 26 April 2002


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluconazole alone does not demonstrate any contact activity against resistant organisms. Phenolic antioxidants, such as butylated hydroxyanisole, appear to promote fluconazole activity resulting in the killing of 104 cfu/mL of 11 resistant Candida albicans strains within 3–15 min and of 104 cfu/mL of 10 resistant Escherichia coli strains within 6–15 min. Fluconazole activity was increased by the addition of ethyl alcohol (20%). Antioxidants appear to promote fluconazole activity by increasing cell membrane permeability. This combination has potential advantages in the administration of topical fluconazole.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Fluconazole has excellent activity against Candida albicans and the good safety profile of this agent, in common with other triazoles, has led to its extensive use. Concomitant with its widespread use, resistance to fluconazole has been observed.1 Fluconazole is an antifungal agent that acts by inhibiting fungal cytochrome P450, but no contact activity of fluconazole on the microbial membrane has been demonstrated to date.2 Antioxidants such as propylgallate (PG) inhibit cytochrome P450-mediated enzyme activity.3,4 Antioxidants alone have not been shown to affect normal cell growth adversely,4 but an increase in antifungal activity has been observed on their concomitant administration with antifungal agents in vitro. Antioxidants interact with membrane phospholipids5 and may enhance the contact activity of fluconazole by affecting molecular organization.

In this study we tested the contact activity of fluconazole against resistant strains of C. albicans and the Gram-negative bacterium Escherichia coli, which is invariably azole resistant.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Antimicrobial agents

Pure fluconazole (Pfizer Spa, Rome, Italy) was dissolved in buffer [Na2HPO4–citric acid buffer pH 7.2 and 5.6, electrical conductivity (EC) 210 µS/cm] at a concentration of 6000 mg/L. Antioxidants PG (Sigma Chemical Co., St Louis, MO, USA) and butylated hydroxyanisole (BHA) (Sigma) were dissolved in polyethylene glycol 400 (Sigma) at a concentration of 5 g/L.

Test organisms

Isolates were identified by Microscan panels (Baxter, Milan, Italy) and by standard methods, and were selected for their resistance.

One stock strain (ATCC 10231) and 10 isolates of C. albicans, which had fluconazole breakpoints >=128 mg/L after 48 h by the NCCLS macrodilution broth method M27-A, were selected. Ten isolates of fluconazole-resistant E. coli (from the Microbiology Institute’s collection in Rome, Italy), which had fluconazole breakpoints >=128 mg/L after 24 h by the NCCLS macrodilution broth method M7-A2, were selected.

For inoculum preparation, Candida cultures were grown on Sabouraud dextrose agar (Sigma) for 24 h at 35°C and E. coli cultures were grown on Mueller–Hinton agar (Sigma) overnight at 35°C.

The C. albicans cells were determined primarily by direct counting using a Thomas Zeiss Camera (Vetro Scientifica Srl, Rome, Italy), and E. coli cell density was assessed by optical density at 540 nm. The values were confirmed by determination of cfu.

Electrical conductivity (EC)

The EC of the medium was measured in µS/cm with an HI 9032 conductivity meter (Hanna Instruments SpA, Padova, Italy).

Culture inhibition tests

The broth macrodilution reference methods used to assess susceptibility were carried out according to the guidelines of the methods used for yeasts (NCCLS M27-A) and bacteria (NCCLS M7-A2).

Experiments were carried out in media with different ECs to determine the influence of EC on microbial resistance.

Contact tests

C. albicans and E. coli cells (108 cfu/mL) were incubated in phosphate buffer (EC 210 µS/cm, pH 5.6) for 15 min at 22°C. After different times of fluconazole contact (1000 mg/L), cell suspensions were diluted by a further 104 cfu/mL and then seeded in Mueller–Hinton agar (E. coli) or Sabouraud dextrose agar (C. albicans). Cfus were determined after 48 h of incubation at 37°C. The times for complete killing of 104 cfu/mL were indicated by K values. In the experiment with antioxidants, microbial cells were treated with antioxidants for 15 min (PG or BHA 200–500 mg/L) at 37°C before fluconazole contact. In some experiments 20% ethyl alcohol was added. Each experiment was repeated three times.

K+ release tests

The tests were carried out with a K+ electrode connected to a Microion 2008 (Crison Instruments S.A., Alella, Barcelona, Spain).

Statistical analysis

K values represent the time taken to kill 104 cfu/mL and are equal to (1/t)log(Ni/Nf), where Ni is the initial number of cfu/mL, Nf is the final number of cfu/mL and t is the time for the viable count to fall from Ni to Nf. Differences between data values were assessed using the Student’s t-test (significant at P < 0.001). Regression analysis was carried out using Excel 97.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In preliminary experiments C. albicans and E. coli fluconazole-resistant strains were selected by standardized susceptibility tests. The strains were also characterized in media with different ECs;6 in particular the reference strain C. albicans ATCC 10231 in Sabouraud medium (EC 2500 µS/cm) and E. coli 67TR in Mueller–Hinton medium (EC 12 500 µS/cm) had MICs > 1000 mg/L with fluconazole.

The MIC of PG or BHA in Mueller–Hinton for E. coli 67TR was >2000 mg/L, while MICs for C. albicans ATCC 10231 in Sabouraud were >240 mg/L. In contact tests, 200–500 mg/L BHA was used because antioxidants are less active in contact tests than in culture tests.2

When assessing contact activity, low EC (210 µS/cm) and pH 5.6 were used in all experiments.6 In contact experiments, fluconazole (2000 mg/L) did not show any contact activity against resistant C. albicans and E. coli, as assessed by the lack of cfu reduction after 15 min of contact under standard experimental conditions. Pre-treatment of C. albicans with PG (500 mg/L for 15 min) did not influence fluconazole activity. Pre-treatment of C. albicans at 37°C with additional 20% ethyl alcohol resulted in the killing of 104 cfu/mL in 2 min (Figure 1).



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Figure 1. Fluconazole (FLZ) activity against resistant C. albicans (ATCC 10231) after pre-treatment with antioxidants PG and BHA. Key: diamonds, curve 1, FLZ 1000 mg/L; rectangles, curve 2, FLZ 1000 mg/L + PG 500 mg/L pre-treatment; triangles, curve 3, FLZ 1000 mg/L + BHA 500 mg/L pre-treatment; crosses, curve 4, FLZ 1000 mg/L + (BHA 500 mg/L + ethyl alcohol 20%) pre-treatment. Pre-treatment was for 15 min at 37°C. Data of curves 3 and 4 with respect to curve 1, P < 0.001. Results are the means of three experiments. Cfu/mL after pre-treatment for 15 min with PG or BHA at 500 mg/L ± ethyl alcohol was not influenced with respect to the control in the absence of FLZ.

 
Pre-treatment of all 11 strains of C. albicans with BHA (300 mg/L for 15 min) increased fluconazole contact activity and resulted in the killing of 104 cfu/mL in 3–15 min (range of K values 3–15, with K arithmetic mean of 6.3).

Fluconazole alone did not show any activity against fluconazole-resistant E. coli. Pre-treatment of 10 E. coli strains with BHA (250 mg/L for 15 min) increased fluconazole contact activity and resulted in the killing of 104 cfu/mL in 6–15 min (range of K values 6–15, with K arithmetic mean of 10.6). The pre-treatment of E. coli at 37°C with additional 20% ethyl alcohol resulted in enhanced fluconazole activity and the killing of 104 cfu/mL E. coli in 2 min (Figure 2).



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Figure 2. Fluconazole (FLZ) contact activity against resistant E. coli (67 TR) after pre-treatment with antioxidants PG and BHA. Key: diamonds, curve 1, FLZ 1000 mg/L; squares, curve 2, FLZ 1000 mg/L + PG 200 mg/L pre-treatment; triangles, curve 3, FLZ 1000 mg/L + (PG 200 mg/L + ethyl alcohol 20%) pre-treatment; circles, curve 4, FLZ 1000 mg/L + BHA 200 mg/L pre-treatment; crosses, curve 5, FLZ 1000 mg/L + (BHA 200 mg/L + ethyl alcohol 20%) pre-treatment. Pre-treatment was for 15 min at 37°C. Data of curves 4 and 5 with respect to curve 1, P < 0.001. Results are the means of three experiments. Cfu/mL after pre-treatment for 15 min with PG or BHA at 200 mg/L ± ethyl alcohol was not influenced with respect to the control in the absence of FLZ.

 
BHA was more active than PG and the addition of ethyl alcohol further increased fluconazole activity. Microbial pre-treatment with BHA (500 mg/L for 15 min) plus 20% ethyl alcohol caused a 50% reduction in the EC of C. albicans; BHA 200 mg/L plus 20% ethyl alcohol reduced the EC of E. coli by 35%. Moreover, the K+ release test showed a 20% increase of K+ release in cells treated with BHA as compared with controls.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The organisms selected for this study, C. albicans and E. coli, were resistant to fluconazole in standardized susceptibility tests. Fluconazole lacks antimicrobial action in contact tests, even at high concentrations, against resistant C. albicans and E. coli. The presence of antioxidants could affect membrane function,6 which may in turn regulate fluconazole susceptibility.7 In contact experiments, bacteria and yeasts demonstrate resistance to azole action due to outer membrane modifications, not a change in metabolic activity.6 The combination of antioxidant and triazole brought about a marked increase in the inhibitory activity of fluconazole in susceptible isolates in which the MIC was reduced from 1 to 0.125 mg/L (data not shown).

PG and BHA are phenolic antioxidants that are widely used in food and pharmaceutical industries, and are generally regarded as safe substances for human consumption.4,8 The acceptable daily intake (ADI) established for BHA is 0.5 mg/kg body weight. Phenolic antioxidants alone have not been shown to affect normal cell growth adversely.4 In our experiments, they have shown low toxicity against the microorganisms used. However, the interaction with membrane phospholipids could affect molecular organization and promote drug passage into the membrane bilayer5 and could increase the in vitro antimicrobial activity of fluconazole.

Phenolic antioxidants with the addition of 20% ethyl alcohol decreased the EC of the strains tested and increased the efficacy of fluconazole,6,7 consequently increasing fluconazole penetration and increasing the activity of fluconazole in contact tests. Sodium dioctylsulphosuccinate, an anionic surfactant that promotes penetrability of fungal cells, has also been shown to increase fluconazole activity.7 The addition of 20% ethyl alcohol did not have direct antimicrobial activity9 but could increase the antioxidant activity and therefore the activity of fluconazole. Alcohol can cause an alteration in the fatty acid composition, and the lethal effect of BHA could be related to microbial fatty acid composition.10 This compound has been shown to interact with biomembranes and to produce a solubilization of proteins that was significantly higher with the addition of ethanol.

Enhancement of contact activity might constitute a new approach to the use of fluconazole, since contact activity offers the opportunity for a quick clinical response, and may ensure efficacy against resistant organisms.


    Footnotes
 
* Corresponding author. Tel: +39-06-4468622; Fax: +39-06-4468625; E-mail: nicola.simonetti{at}uniroma1.it Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Sandven, P., Bjorneklett, A., Maeland, A. & The Norwegian Yeast Study Group. (1993). Susceptibilities of Norwegian Candida albicans strains to fluconazole: emergence of resistance. Antimicrobial Agents and Chemotherapy 37, 2443–8.[Abstract]

2 . Odds, F. C. (1988). Candida and Candidosis. A Review and Bibliography, 2nd edn. Ballière Tindall, London, UK.

3 . Baer-Dubowska, W., Szaefer, H. & Krajka-Kuzniak, V. (1998). Inhibition of murine hepatic cytochrome P-450 activities by natural and synthetic phenolic compounds. Xenobiotica 28, 735–43.[ISI][Medline]

4 . Andrews, F. A., Beggs, W. H. & Sarosi, G. A. (1977). Influence of antioxidants on the bioactivity of amphotericin B. Antimicrobial Agents and Chemotherapy 11, 615–8.[ISI][Medline]

5 . Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Reviews 56, 395–411.[Abstract]

6 . Simonetti, G., Baffa, S. & Simonetti, N. (2001). Contact imidazole activity against resistant bacteria and fungi. International Journal of Antimicrobial Agents 17, 389–93.[ISI][Medline]

7 . Simonetti, N., D’Auria, F. D. & Strippoli, V. (1991). Increased in vitro sensitivity of Candida albicans to fluconazole. Chemotherapy 37, 32–7.[ISI][Medline]

8 . Shahidi, F., Janitha, P. K. & Wanasundara, P. D. (1992). Phenolic antioxidants. Critical Reviews in Food Science and Nutrition 32, 67–103.[ISI][Medline]

9 . Morton, H. E. (1983). In Alcohols in Disinfection, Sterilization and Preservation, 3rd edn (Block, S. S., Ed.), p. 227. Lea & Febiger, Philadelphia, PA, USA.

10 . Post, L. S. & Davidson, P. M. (1986). Lethal effect of butylated hydroxyanisole as related to bacterial fatty acid composition. Applied and Environmental Microbiology 52, 214–6.[ISI][Medline]





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