Institute of Microbiology, Faculties of a Medicine and Surgery and b Pharmacy, University of Rome La Sapienza, viale Regina Margherita, 255, 00198 Rome, Italy
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Abstract |
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Introduction |
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Materials and methods |
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SPA-S-843 (N-dimethylaminoacetyl-partricin A 2-dimethylaminoethylamide diascorbate), potency 941 mg/g (Società Prodotti Antibiotici, Milan, Italy) and amphotericin B, potency 905 mg/g (Squibb, Rome, Italy) were used throughout. The polyenes were dissolved in dimethylsulphoxide (DMSO, Merck, Darmstadt, Germany) at a concentration of 4 g/L and then serially diluted with water to concentrations ranging from 100 mg/L to 0.002 mg/L. In the stability experiments, the polyene solutions (400 mg/L concentration) were protected from light and stored at 22°C.
Microorganisms
Ten clinical isolates and one stock isolate (ATCC 10261) of C. albicans (from the Microbiology Institute's collection) were tested. The isolates were identified by Microscan panels (Baxter, Milan, Italy) and by conventional methods.5 The cultures were grown in Sabouraud liquid medium overnight at 37°C with shaking, and the growth was estimated with a Thoma Zeiss Camera (Vetro Scientifica S.r.l., Rome, Italy) and controlled by determination of cfu. The final inoculum was 5 x 103 cells/mL in the culture inhibition test, 104 cells/mL in the contact tests and 106 cells/mL in the K+ release tests.
Electrical conductivity
The media conductance was measured in µS/cm with an HI 9032 conductivity meter (Hanna Instruments, S.p.A., Padova, Italy).
Culture inhibition tests
The experiments were performed in Sabouraud dextrose broth (SAB; Becton-Dickinson, Cockeysville, MD, USA) and diluted Sabouraud dextrose broth (1:3) plus 2% glucose. The inocula (5 x 103 cells/mL) were added to culture media containing serial dilutions of the drugs and to a control without drugs. The MIC was determined after 24 h incubation at 37°C.
Contact tests
C. albicans cells were incubated in phosphate buffer, at pH 5.6 and various molarities, in the presence of polyenic drugs for 115 min at 22°C. After contact, the cell suspensions were diluted 103 times, then seeded in SAB agar. The cfu were determined after 48 h incubation at 37°C, and the results were reported as log cells/mL at that time point.
K+ release tests
The tests were performed with a K+ electrode connected to a Microion 2008 (CRISON Instruments S.A., Alella, Barcelona, Spain) with calibration curves adjusted to between 106 mol/L and 5 x 103 mol/L. The polyene concentrations ranged from 200 to 10 mg/L in sodium phosphate buffer 0.1 M and 0.001 M at pH 5.8. K+ ions released were measured after 1, 3, 5, 10 and 15 min and were expressed as percentage of K+ released in the test with respect to the K+ released from control cell suspensions treated at 100°C for 10 min.
Germ-tube inhibition test
The test was carried out in N-acetylglucosamine solution pH 6.6 with an inoculum of 3 x 105 cells/mL.6 After 3 h incubation at 37°C the cultures were examined under a light microscope (Carl Zeiss, x320) and the minimum concentration of the polyenes inhibiting >90% of germ-tube formation was microscopically estimated with respect to controls without drugs, where >95% of germ-tube formation usually occurs after 3 h incubation.
Statistical analysis
K (the velocity constant) was used as a measure of the efficiency of antimicrobial agents. K = (1/t) log(Ni/Nf), where Ni is the initial number of cells, Nf is the final number of cells and t is the time for the viable count to fall from Ni to Nf. Differences between mean MIC values were assessed by the Student's t test. Regression analysis was performed using Excel 95.
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Results and discussion |
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In the study of yeast to mycelium C. albicans cell transition the SPA-S-843 activity was remarkably higher (mean MIC 0.116 mg/L; range 0.0450.31 mg/L) than that of amphotericin B (mean MIC 0.45 mg/L; range 0.250.62 mg/L). The differences between the mean values were statistically significant (P < 0.001).
Because there is evidence that the hyphal form may play a pathogenic role in the initial process of tissue invasion and germ tubes and hyphae adhere better than yeast cells to human buccal and vaginal epithelia,4 the better inhibition of the morphological transition by SPA-S-843 might improve the effectiveness of the drug.
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Notes |
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References |
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2 . Bruzzese, T., Rimaroli, C., Bonabello, A., Ferrari, E. & Signorini, M. (1996). Amide derivatives of partricin A with potent antifungal activity. European Journal of Medical Chemistry 31, 96572.
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Rimaroli, C. & Bruzzese, T. (1998). In vitro antifungal activity of a new polyene, SPA-S-843, against yeast. Antimicrobial Agents and Chemotherapy 42, 301213.
4 . Cutler, J. E. (1991). Putative virulence factors of Candida albicans. Annual Review of Microbiology 45, 187218.[ISI][Medline]
5 . Cooper, B. H. & Silva Hutner, M. (1985). Yeast of medical importance. In Manual of Clinical Microbiology, (Lennette, E. H., Balows, A., Hausler, W. J. & Shadomy, H. J., Eds), pp. 52642. American Society for Microbiology, Washington, DC.
6 . Simonetti, N., Strippoli, V. & Cassone, A. (1974). Yeastmycelial conversion induced by N-acetyl-d-glucosamine in Candida albicans. Nature 250, 3446.[ISI][Medline]
Received 12 August 1998; returned 21 February 1999; revised 19 April 1999; accepted 19 October 1999