| Objective The aim of the present study is thus to interrogate the mechanism underpinning the synergistic effect of fluconazole and calcineurin inhibitors.Methods 24 FLC sensitive C. albicans clinical strains were isolated from bloodstream samples and collected from the department of laboratory medicine of the General Hospital of Ningxia Medical university between January 2014 and December 2014, which were identified by harnessing the VITEK-2 COMPACT fully automated microbiological system. A biofilm model was generated using a clinical biofilm-producing isolate of C. albicans isolated from bloodstream infections. Growth dynamics are analyzed, in order to specify the logarithmic phase of growth.The inhibition of biofilm formation of C. albicans was determined by a fluorescent microscopy assay, and viability of bacteria against agents was ascertained by an XTT method. The expression of drugresistant and adhesive genes ALS3, HWP1, CDR1, MDR, ERG11 was evaluated by a q RT-PCR assay. C. albicans cellular surface hydrophobicity(CSH) was assessed using a water hydrocarbon two-phase assay as described previously. The calcium levels were determined by flow cytometry in a FACS can flow cytometer using a parameter of the excitation/emission wave lengths(485nm/530nm) with reading sensitivity level.Results 24 FLC sensitive C. albicans clinical strains were isolated from bloodstream samples and collected from the department of laboratory medicine of the General Hospital of Ningxia Medical university between January 2014 and December 2014. A distinct capacity of biofilm formation was observed among the 24 clinical C. albicans isolates, including 4 HBF(high biofilm formation capacity) C. albicans isolate?6 IBF(Intermediate biofilm formation capacity) C. albicans isolate? 14 LBF(low biofilm formation capacity) C. albicans isolate. The biofilms were then stained with crystal violet, and visualized and imaged under a light microscope. Most of the cell adhered to the bottom of the culture plate with yeast,representative images of biofilm produced by a LBF(low biofilm formation capacity) C. albicans isolate. Hypha growth of mutual crisscross can be seen in most of the cells, basal yeast cells vaguely can be seen, representative images of biofilm produced by a HBF(high biofilm formation capacity) C. albicans isolate. A high biofilm-producing C. albicans strain was isolated from bloodstream infections, which was designated as CA NX05; Calcineurin inhibitors was found to be able to enhance the susceptibility of biofilm-producing C. albicans to FLC. In comparison with the treatment of calcineurin inhibitors or FLC alone, the expression of ALS3, HWP1, CDR1, MDR, ERG11 genes were dramatically down-regulated in the treatment of a combination of calcineurin inhibitors and FLC for the clinical biofilm-producing C. albicans isolate, which was statistically significant(P<0.05). Clinical C. albicans isolates were cultured in the presence of 32?g/m L FLC or 75?g/m L of Cs A alone, or a combination of Cs A and FLC for 24 h prior to be used for CSH measurement. The combination of Cs A and FLC caused significantly decrease of CSH of C. albicans cells, although Cs A or FLC along also had a moderate effect on the reduction of CSH. Clinical C. albicans isolates were exposed to 32 ?g/m L FLC or 75 ?g/m L of Cs A alone, or a combination of Cs A and FLC for 24 h prior to be used for determining intracellular calcium concentration by a flow cytometric assay(FACS). The result showed that both Cs A and FLC alone could reduce intracellular calcium, but a combination of them acaused a time-dependently evoked intracellular calcium concentration.Conclusions 1.We can build the model of Candida albicans biofilm in vitro on the cell culture plate and can be detected by fluorescence microscopy.2 Calcineurin inhibitor cyclosporine A synergistically enhances the susceptibility Candida albicans to fluconazole.3 The results presented in this report demonstrated that the synergism of Cs A and FLC against C. albicans biofilms is in part through a mechanism involved in suppressing the expression of biofilm related and drug-resistant genes, and reducing cellular surface hydrophobicity, as well as evoking high intracellular calcium concentration. |
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