Antifungal resistance remains a significant public health threat, yet poorly investigated. This study for the first time investigated the antifungal profile of moulds isolated from groundwaters in a rural community of Osun State, Southwest Nigeria. Groundwater samples were collected during dry and rainy seasons, and processed for mould isolation using standard methods. The isolates were presumptively identified and screened for in vitro susceptibility using 4 antifungals conventionally employed in clinical cases. Multiple antifungal resistance phenotypes and indices were equally evaluated. Representative of each putatively identified species were confirmed using molecular techniques. A total of 29 and 27 moulds were obtained during the dry and rainy seasons, respectively. Genus Aspergillus was the most prevalent (30.4%). Of the 56 isolates subjected to antifungal susceptibility testing, 55(98%) were resistant to fluconazole, followed by flucytosine 49(88%), ketoconazole 45(80%) and amphotericin B 31(55%). Exactly 46 isolates were multidrug-resistant, 23(50%) each, to 3 and 4 drugs, respectively. The multiple antifungal resistance index (MARI) evaluated was 1 in all the samples A-Q, except C (0.75), transcending the threshold limit of 0.2, indicating the isolates to be of high antifungal usage origin. The molecularly identified species had 99–100% similarity with reference strains from GenBank and evolutionary relationship of taxa revealed 10 distinct clades. The outcome of this study reveals a rise in the frequency of antifungal resistance in moulds towards standard drugs, thus, advocating for adequate investigations targeted at monitoring antifungal resistance in groundwater milieus.
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