الفهرس يوجد فقط 14 صفحة متاحة للعرض العام
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المستخلص
This study was done in Microbiology and Immunology Department, Faculty of Medicine, Zagazig University in the period from May 2004 to April 2005. The aim of this work is to study Candida albicans biofilm from its whole aspects; formation, architecture, and antifungal susceptibility. Also, testing antifungal susceptibility of planktonic counterparts of biofilm forming isolates to fluconazole and amphotericin B by broth microdilution technique of NCCLS, spectrophotometric, and colorimetric methods. All samples were transmitted within 1-2 h to the laboratory where each was examined microscopically by Gram’s stain. Samples with yeast cells were inoculated on the surface of plates of Sabouraud dextrose agar and incubated for at least 48 h at 30° C. Then Candida albicans isolates were identified according to colonial morphology, microscopic examination, germ tube test, chlamydospore production, and sucrose assimilation. All Candida albicans isolates were subjected to biofilm formation using microtitre plate method. The already formed biofilms were quantified using XTT reduction assay and crystal violet (CV) assay . All Candida albicans biofilms (51) were subjected to susceptibility testing to fluconazole and amphotericin B using XTT reduction assay (colorimetric modified microdilution method). The data of susceptibility were reported as MIC ranges and the MICs at which 50 and 90% of the isolates are inhibited (MIC50s and MIC90s, respectively). Out of the identified Candida albicans isolates (the planktonic forms of biofilm forming strains), 51 isolates were picked up and subjected to susceptibility testing to fluconazole and amphotericin B using broth microdilution, spectrophotometric broth microdilution, and XTT reduction assay. The percentage of germ tube positive samples was 56.7% while sucrose assimilation and chlamydospore production positive samples had a very closed percentages that were 60.7% and 61.3%, respectively. Also, the sucrose assimilation had a higher sensitivity (98.9%) than germ tube (95.7%) on comparing them with the definitively diagnostic chlamydospore formation. The percentage of Candida albicans biofilm positive isolates was 55.4%. There was no statistically significant differences between biofilm forming abilities by Candida albicans from different sources. XTT reduction assay method revealed a relatively narrow range of variation among different isolates. On the other hand, crystal violet staining method revealed a greater range of variation of biofilm production among different isolates. However, there was a significant correlation between XTT reduction assay method from one side to CV assay method from the other side. Biofilms from all Candida albicans strains tested were intrinsically resistant to fluconazole. The resistance to amphotericin B was less pronounced and more variable between the isolates tested. This polyene antifungal still demonstrates some activity against Candida albicans biofilms when measured using XTT reduction 50%. However, most of Candida albicans biofilm fell within the resistant range for this antifungal agent (>1 µg/ml) when measured using XTT reduction 80%. The three methods of fluconazole MICs determination demonstrated a broad range of susceptibility. MIC50 value of fluconazole using broth microdilution method at 48 h was higher than that of broth microdilution method determined visually at 24 h , spectrophotometric method and XTT reduction assay. While MIC50 of broth microdilution method determined visually at 24 h was equal to that determined by Spectro 50% and XTT reduction 80% . MIC50 determined by XTT reduction 50% and XTT reduction 80% was one fold lower than that determined by Spectro 50% and Spectro 80% respectively. The MIC90 value of fluconazole determined visually by broth microdilution method at 48 h was higher than that determined by all other methods. MIC90 of fluconazole determined visually by broth microdilution method at 24 h was equal to that determined by Spectro 50% and XTT reduction 50%. While MIC90 determined by XTT reduction 80% was two folds lower than that determined by Spectro 80%. The MIC50 value of amphotericin B determined visually at 48 h using broth microdilution method was equal to that determined visually at 24 h, Spectro 80% and XTT reduction 80% and two folds higher than that determined by Spectro 50% and XTT reduction 50% at 48 h. The MIC90 value of amphotericin B determined visually at 48 h using the microdilution method was equal only to that determined visually at 24 h and higher than that determined by all other methods. While The MIC90 determined by Spectro 50% and Spectro 80% was equal to that determined by XTT reduction 50% and XTT reduction 80% respectively. The present study showed that Candida albicans isolates were more susceptible for amphotericin B than fluconazole. In conclusion, our study indicated that there was high percentage of Candida albicans had the ability to grow within biofilms and statistically, these biofilms did not be different among Candida albicans isolated from different sources. This biofilm mode of growth was highly resistant to the classic antifungals. In comparison, the susceptibility of planktonic counterparts of Candida albicans isolates could be easily determined and they were more susceptible to both fluconazole and amphotericin B than Candida albicans biofilms.