Protein expression and antifungal effect of fluconazole-resistant Candida species following effective in vitro treatment with K21, a novel antifungal agent

Abstract

Background: Oropharyngeal candidiasis, caused by the fungus Candida, is the most common opportunistic infection affecting the quality of life of immunocompromised patients. Fluconazole is widely used as the first line of treatment for fungal infections. However, the inappropriate and misguided use of the drug has led to the evolvement of fluconazole-resistant Candida organisms. This arising resistance resulted in the urgent need for the development of new antimicrobial drugs. The aim of the present study was to investigate the antifungal action of K21, a novel antimicrobial quarternary ammonium compound, on fluconazole-resistant Candida species. Materials and Methods: An in vitro study was conducted using a total of 143 Candida isolates obtained from HIV-positive patients. The ethical aspects of the study complied with the declaration of Helsinki (2013). The time-kill assay was used to evaluate the rate of action of K21 over time on Candida while the fungicidal effect of K21 against C. albicans (ATCC 90028) and C. glabrata (ATCC 26512) was observed at 2 hours. The minimum inhibitory concentration (MIC) of K21 was compared with the MICs of fluconazole. Synergy between K21 and fluconazole was evaluated by both the checkerboard microdilution method and time-kill assay. The modes of action of K21 and drug delivery were determined by performing postembedding immunogold labelling and for the sight-specific target and protein expression of Sap 1-3 and Sap 4-6 within the Candida cell. Results: Of the 143 isolates, 108 were fluconazole-resistant, 15 were fluconazole-intermediate and 20 were fluconazole-susceptible using the broth microdilution assay and breakpoint values recommended by the Clinical and Laboratory Standards Institute (CLSI). The MIC of K21 was 31.24 µg/mL for C. albicans when determined by the broth microdilution assay. About 103 Candida species were resistant and 13 were categorised as intermediate to FCZ with a MIC range between 64 µg/mL - 256 µg/mL and 16 µg/mL - 32 µg/mL respectively. However, the majority of the Candida species (n = 86) showed intermediate susceptibility to K21 with a MIC range between 62.48 µg/mL - 124.95 µg/mL and only 9 of the Candida species were resistant to K21 with a MIC value of ≥249.89 µg/mL. A statistically significant (p value = 0.000) was observed when the MIC values of K21 and FCZ were compared. No antagonism was observed in the study among the Candida strains. The time-kill and synergism assays showed significant differences over time with synergy between K21 and FCZ demonstrated for C. albicans (ATCC 90028 and NCPF 3281) C. dubliniensis (NCPF 3949a), C. tropicalis (ATCC 950) and C. lusitaniae (ATCC 34449). Scanning electron microscopy displayed major alterations in the morphology of Candida species between 2 hours and 24 hours, exhibiting cell lysis and cell death. Transmission micrographs of C. albicans (ATCC 90028) treated with K21 showed shrunken nuclei with disruption of cell walls and cell membranes. Immunogold labelling of C. albicans (ATCC 90028) and C. dubliniensis (NCPF 3949a) with Sap 1-3 antibodies exhibited the presence of gold particles confined to the cell wall and cell membrane, but, when exposed to Sap 4-6 antibodies there were a few non-specific gold particles found in C. albicans (ATCC 90028) and an absence of gold particles in C. dubliniensis (NCPF 3949a). C. tropicalis (ATCC 950) showed few gold particles along the cell membrane when treated with Sap 1-3 antibodies and no gold particels were found when treated with Sap 4-6 antibodies. Conclusion: The present study suggests that K21 acts as a potent antifungal agent and can be considered for development as an alternative treatment for fluconazole-resistant Candida species especially in immunocompromised patients.