Combined effect of furanone fluconazole and amphotericin B against biofilms formed from Cryptococcus neoformans

It is of interest to document the combined effect of furanone fluconazole and amphotericin B against the biofilm formed by Cryptococcus neoformans. The MIC values of amphotericine B and Fluconazole were observed as 20µg/ml and 60µg/ml, respectively. The MIC for the Combination (Amphotericin B/ Fluconazole) was found to be at (15/20) µg/ml drug concentration. Thus, data shows the combined effect of furanone fluconazole and amphotericine B derivative against C. neoformans.


Background:
Cryptococcus neoformans are the encapsulated fungus groups that cause meningoencephalitis in immuno-compromised individuals [1,3]. Cryptococcal spores and/or dried yeast cells are very small in size and can deposit deep in the respiratory tract following inhalation [2]. Amphotericin B is an antifungal drug given intravenously for serious fungal infections. Amphotericin B is effective with side effects to lungs and kidneys [4][5][6][7][8][9][10]. Polysaccharide capsule of C. neoformans enlarge the size of capsule during infection and the mechanism of growth was unknown [11][12][13]. Therefore, it is of interest to document the combined effect of furanone fluconazole and amphotericin B against the biofilm formed by Cryptococcus neoformans.
©Biomedical Informatics (2021) 537 Preparation of drug stock solutions: 20mg of amphotericin B and 20mg of fluconazole was weighted and dissolved in 1ml of Dimethyl sulfoxide (DMSO) and stored at 2ºC in vials. Drug stock solution was diluted according to the culture and the DMSO concentration was equally maintained in all experiments (<1%) as shown in Table 1.

Individual drug Treatment:
The MIC of antifungal drugs against C. neoformans biofilm is inoculated in a 96-well plate along with positive and negative control. This was incubated at 37ºC for 7days. The 96 well plates are washed twice with Phosphate Buffer Saline (PBS) to remove freeliving cells from the plate after the biofilm formation. Drugs were then added into the wells and incubated at 37ºC for 48 hours. This was washed twice with PBS and the absorbance was measured using ELISA plate reader at 620nm.

Combined drug treatment:
Two fold dilution method of the drug both above and below the MIC value were prepared. The individual MIC value of amphotericin B and Fluconazole is shown in Table 2. The antifungal drugs were serially diluted. Equal volume of inoculums was added using the Checkerboard method. The culture were added equally to the all wells and incubated at 37ºC for 48 hours.

Concentration of amphotericin B (µg/ml):
Amphotericin B was added in the concentration range of 0-5µg/ml. Fluconazole was added in the range of 0-25µg/ml. The plates are then incubated at 37ºC for 48 hours. The incubation plates are washed twice with phosphate buffer saline (PBS) and the absorbance was measured using a ELISA plate reader at 620nm (Automatic ELISA Reader -Sunrise)

Fluorescent Microscopy:
The cell were washed twice with PBS, the cells were stained with FITC and propidium Iodine that were prepared at the concentration of 30µg/ml respectively. The staining solutions were added and let to standing for 15min in dark. The staining solution was removed and washed with PBS. The samples were then analyzed using a Nikon Trinocular microscope (Nikon Eclipse Ni-U Japan) [14].

Results & Discussion:
The concentration range of Amphotericin B and Fluconazole used for ATCC strain was 16µg/ml to 240µg/ml and 60µg/ml to 100µg/ml. The MIC value was found to be 25/15 µg/ml (Amphotericin B/Fluconazole) (Figure 1).

Conclusion:
The data shows the combined effect of furanone fluconazole and amphotericine B derivative against C. neoformans by disrupting the formation of biofilms.