Antifungal resistance poses a critical global health threat, particularly in immuno-compromised patients. Beyond the traditional resistance mechanisms rooted in heritable and stable mutations, a distinct phenomenon known as heteroresistance has been identified, wherein a minority of resistant fungal cells coexist within a predominantly susceptible population. Heteroresistance may be induced by pharmacological factors or non-pharmacological agents. The reversible nature of it presents significant clinical challenges, as it can lead to undetected resistance during standard susceptibility testing. As heteroresistance allows fungal pathogens to survive antifungal treatment, this adaptive strategy often leads to treatment failure and recurring infection. Though extensively studied in bacteria, limited research has explored its occurrence in fungi. This review summarizes the current findings on antifungal heteroresistance mechanisms, highlighting the clinical implications of fungal heteroresistance and the pressing need for deeper mechanism insights. We aim to bring together the latest research advances in the field of antifungal heteroresistance, summarizing in detail its known characteristics, inducing factors, molecular mechanisms, and clinical significance, and describing the similarities and differences between heteroresistance, tolerance and persistence. Further research is needed to understand this phenomenon and develop more effective antifungal therapies to combat fungal infections.

Antifungal heteroresistance has emerged as a clinical challenge across diverse species. In Candida glabrata , however, it has rarely been reported. Here, we characterized heteroresistance in a multicenter collection of 156 C. glabrata isolates, revealing a 25% prevalence with caspofungin specificity and 37°C dependent phenotypes. Transcriptomic profiling of the heteroresistant subpopulation under escalating drug pressure revealed an adaptive program centered on cell cycle and cell wall integrity, including several members of the calcineurin pathway. Mechanically, we identified that the phenotype was abolished by pharmacological inhibition or Δ cnb1 , Δ crz1 genetic deletion, confirming the regulatory role of calcineurin. Crucially, heteroresistance functioned as a reservoir for resistance, with in vitro descendant strains spanning a spectrum of MIC alterations. Mechanistically, we found the heteroresistance phenotype mostly independent of aneuploidy. To decipher the genetic basis, we applied an unbiased machine‐learning framework to genomic data, which not only identified the canonical FKS2_F659del mutation but also prioritized novel candidate PIR2_G149_I167del , demonstrating its power to uncover drivers of resistance from complex datasets. In summary, our study established a stepwise model of heteroresistance in C. glabrata , wherein calcineurin serves as a master regulator that promotes a resistance reservoir, revealing a potential vulnerability that could be exploited to prevent treatment failure.

Antifungal resistance poses a critical global health threat, particularly in immuno-compromised patients. Beyond the traditional resistance mechanisms rooted in heritable and stable mutations, a distinct phenomenon known as heteroresistance has been concerned, wherein a minority of resistant fungal cells coexist within a predominantly susceptible population. It may be induced by pharmacological factors or non-pharmacological agents. The reversible nature of it presents significant clinical challenges, as it can lead to undetected resistance during standard susceptibility testing. As heteroresistance allows fungal pathogens to survive antifungal treatment, this adaptive strategy often leads to treatment failure and infection recurrence. Though extensively studied in bacteria, limited research has explored its occurrence in fungi. This review synthesizes the current findings on antifungal heteroresistance mechanisms, highlighting the clinical implications of fungal heteroresistance and the pressing need for deeper mechanism insights. We aim to combine the latest research advances in the field of antifungal heteroresistance, summarizing in detail the characteristics, inducing factors, molecular mechanisms, and clinical significance of it, and describing the similarities and differences between heteroresistance, tolerance and persistence. Further research is essential to elucidate this phenomenon and develop more effective antifungal therapies to combat fungal infections.