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
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Abstract |
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Introduction |
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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.
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Materials and methods |
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Pure fluconazole (Pfizer Spa, Rome, Italy) was dissolved in buffer [Na2HPO4citric 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 Institutes 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 MuellerHinton 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 MuellerHinton 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 200500 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 Students t-test (significant at P < 0.001). Regression analysis was carried out using Excel 97.
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Results |
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The MIC of PG or BHA in MuellerHinton for E. coli 67TR was >2000 mg/L, while MICs for C. albicans ATCC 10231 in Sabouraud were >240 mg/L. In contact tests, 200500 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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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 615 min (range of K values 615, 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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Discussion |
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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.
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Footnotes |
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References |
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2 . Odds, F. C. (1988). Candida and Candidosis. A Review and Bibliography, 2nd edn. Ballière Tindall, London, UK.
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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, 2146.[ISI][Medline]