Emergence of Triazole-Resistant <i>Cryptococcus neoformans</i> after Exposure to Environmentally Relevant Concentrations of Difenoconazole in Liquid Medium and Soil

The rapid global emergence and spread of resistance to clinical triazoles in <i>Cryptococcus neoformans</i> have been associated with the use of agricultural triazole fungicides. However, there is no direct evidence currently linking the emergence of triazole-resistant <i>C. neoformans</i> (TRCN) strains to the application of triazole fungicides in soil. This study investigated whether triazole resistance in <i>C. neoformans</i> could be induced by difenoconazole, an agricultural triazole fungicide, in liquid medium and soil. Our findings reveal that environmentally relevant concentrations of difenoconazole can drive cross-resistance to clinical triazoles in <i>C. neoformans</i> through the upregulation of <i>ERG11</i> and efflux pump genes (<i>AFR1</i>, <i>AFR3</i>, and <i>MDR1</i>). Notably, the prevalence of TRCN strains in soil correlates with residual difenoconazole levels, with significantly more TRCN isolates observed at two- and five-fold the recommended dose than at the standard dose. These results provide direct evidence linking agricultural triazole use to the emergence of TRCN and highlight the importance of applying difenoconazole at or below the recommended dosage to mitigate resistance development in soil environments. This study addresses a critical gap in the understanding of the environmental drivers of triazole resistance and underscores the need for responsible fungicide use to prevent the spread of resistant pathogens.