Genetic diversity and antimicrobial resistance of Candida glabrata clinical isolates from Odisha, India: An ISSR-PCR study

Vulvovaginal candidiasis (VVC) is a common fungal infection in reproductive-age women, mostly caused by Candida albicans . The Non-albicans Candida (NAC) specie species, mainly C. glabrata , have been increasingly becoming opportunistic pathogens due to their growing resistance against antifungal drugs. For monitoring the emergence of infections and designing appropriate strategies against them, it is necessary to understand the genetic variations of C. glabrata . The objective of this study was to evaluate the genetic diversity and relatedness of C. glabrata isolates derived from clinical samples utilizing Inter-Simple Sequence Repeat Polymerase Chain Reaction (ISSR-PCR). A total of 500 vaginal swab samples were collected from suspected patients of IMS and SUM Hospital and Kalinga Hospital in Bhubaneswar, Odisha. Out of these, 120 samples were identified as C. glabrata , and 31 isolates showing unique resistance patterns were selected for further ISSR-PCR and genetic diversity analysis. The samples were cultured on Sabouraud Dextrose Agar (SDA) and then undergoes for Gram staining and Candida Differential Agar (CDA) to find out the species. Disk diffusion method was used to test the antifungal susceptibility of nine antifungal agents. The Phenol, Chloroform and Isoamyl alcohol (PCI) method was used for genomic DNA extraction. ISSR-PCR was conducted using 19 primers but only focusing on the reproducible banding patterns. Genetic diversity indices such as polymorphism, resolving power, primer index, and diversity index were computed. Phylogenetic relationships were determined based on Jaccard similarity coefficient, hierarchical clustering, and Principal Component Analysis (PCA). Among the 500 vaginal swab samples analyzed, 120 C. glabrata isolates were recovered. Antifungal susceptibility testing showed substantial resistance, particularly to fluconazole (70.97%), ketoconazole (67.74%), itraconazole (58.06%), and caspofungin (54.84%), while voriconazole showed comparatively higher activity. Based on distinct antifungal susceptibility profiles and adequate DNA quality, 31 C. glabrata isolates were selected for ISSR-PCR analysis. Among the selected isolates seven ISSR primers generated reproducible amplification profiles, revealing variation in genetic relatedness. Primer-wise diversity index values ranged from 0.674 to 0.998, with ISSR 12 showing the lowest value, while the other primers exhibited higher values, indicating differences in their ability to detect polymorphism among the isolates. Heat map analysis showed distinct separation of the groups under phylogenetic as well as PCA. The findings reveal that there is a large amount of genetic variation between the C. glabrata samples. ISSR-PCR proved to be a reliable and inexpensive technique that could be used for characterizing organisms in molecular biology studies and tracking the emergence of antifungal resistance.