Date of Award
12-2025
Document Type
Thesis
Degree Name
Master of Science (MS)
Department
Biochemistry and Molecular Biology
Committee Chair/Advisor
Dr. Lukasz Kozubowski
Committee Member
Dr. Cheryl Ingram Smith
Committee Member
Dr. Stephen Kevin Dolan
Abstract
Cryptococcus neoformans, an opportunistic fungal pathogen is responsible for cryptococcosis, leading to approximately 181,000 deaths annually, predominantly in regions such as Sub-Saharan Africa. Fluconazole, a widely used fungistatic azole, is often administered due to its relative affordability and lower toxicity. However, resistance to fluconazole in C. neoformans can arise through aneuploidy, complicating treatment efforts. This study aims to deepen our understanding of resistance mechanisms to azole drugs by comparing fluconazole with other azoles, including both clinical and agricultural azole antifungals. Disk diffusion assays were performed to evaluate the ability of C. neoformans to develop resistance to these compounds, focusing on the zone of inhibition (ZOI) and emergence of resistant colonies. Notably, significant differences were observed in the formation of resistant/tolerant colonies in response to equalized inhibitory concentrations of the tested azoles. To rule out the possibility that these colonies arose due to drug instability; a stability test was conducted for each compound. To address the potential for azoles to exert a killing effect, each drugs killing/inhibitory effect was assessed by incubating C. neoformans at minimum inhibitory concentrations, followed by growth evaluation on drug-free media. Our findings indicate that while some azoles exhibit lower stability, this alone does not fully account for the emergence of resistant/tolerant colonies. Strikingly, isavuconazole stood out among other azoles as it didn’t form any tolerance or resistance during the initial treatment, and a similar effect was seen when tested with various clinical isolates. Flow cytometry was employed to assess the effects of the azoles on plasma membrane integrity, morphological changes, and viability. Notably, while the azoles were largely inhibitory, compromised plasma membrane integrity was detected, based on increased fluorescence of the propidium iodide (PI)dye. The latter was consistent with drug-induced changes in morphology, most notably in isavuconazole-treated cells, in which increased branching and clumping was accompanied by high fluorescence PI signals. Following repeated exposure, we were able to establish 5 independent isavuconazole-resistant strains which displayed a much-reduced zone of inhibition in the disk diffusion assays and increased MIC in Etest assays when compared to exposures of the original strain. The whole-genome sequences of these strains are complete, and these data will be utilized to identify the underlying genetic mutations. This will enable comparative assessment of isavuconazole resistance mechanisms in parallel with those previously established for fluconazole and potentially identify same or any different adaptation pathways involved in development of resistance. Taken together, our data indicate that isavuconazole may be a superior drug against cryptococcosis compared to commonly utilized fluconazole.
Recommended Citation
Sindhe, Gnanambika, "Investigating the Impacts of Azole Antifungals on Growth of Cryptococcus Neoformans" (2025). All Theses. 4638.
https://open.clemson.edu/all_theses/4638