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Review

Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance

by
Yanyu Su
1,2,3,
Yi Li
1,2,
Qiaolian Yi
1,2,
Yingchun Xu
1,2,
Tianshu Sun
2,4,5,* and
Yingxing Li
2,5,6,*
1
Department of Laboratory Medicine, State Key Laboratory of Complex Severe and Rare Diseases, Peking Union Medical College Hospital, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China
2
Beijing Key Laboratory for Mechanisms Research and Precision Diagnosis of Invasive Fungal Diseases (BZ0447), Beijing 100730, China
3
Graduate School, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China
4
Clinical Biobank, Center for Biomedical Technology, National Science and Technology Key Infrastructure on Translational Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
5
State Key Laboratory of Complex, Severe, and Rare Diseases, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100730, China
6
Biomedical Engineering Facility of National Infrastructures for Translational Medicine, Institute of Clinical Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100730, China
*
Authors to whom correspondence should be addressed.
J. Fungi 2025, 11(2), 143; https://doi.org/10.3390/jof11020143
Submission received: 4 January 2025 / Revised: 11 February 2025 / Accepted: 11 February 2025 / Published: 13 February 2025
(This article belongs to the Section Fungal Pathogenesis and Disease Control)
Browse Figures
Figure 1
<p>Antifungal mode of action of azoles, AMB, 5-FC and echinocandins. Azoles inhibit ergosterol biosynthesis by targeting sterol 14α-demethylase, producing toxic 14-methyl sterols, altering fungal cell membrane permeability and metabolic state [<a href="#B74-jof-11-00143" class="html-bibr">74</a>,<a href="#B75-jof-11-00143" class="html-bibr">75</a>]. Echinocandins target β-1,3-glucan synthase, impairing fungal cell wall integrity and stress resistance [<a href="#B76-jof-11-00143" class="html-bibr">76</a>]. Amphotericin B binds to membrane ergosterol, forming pores that disrupt permeability and cause reactive oxygen species (ROS) accumulation, leading to cell death [<a href="#B77-jof-11-00143" class="html-bibr">77</a>,<a href="#B78-jof-11-00143" class="html-bibr">78</a>,<a href="#B79-jof-11-00143" class="html-bibr">79</a>]. 5-FC is converted intracellularly to 5-FU, inhibiting DNA and RNA synthesis [<a href="#B80-jof-11-00143" class="html-bibr">80</a>,<a href="#B81-jof-11-00143" class="html-bibr">81</a>].</p> "> Figure 2
<p>(By Figdraw 2.0). Explanation of resistance, susceptible, tolerance, persistence and heteroresistance from a cell population perspective. The state of cell proliferation is represented by the number of cells in the figure. Individual colors indicate different genotypes and phenotypes, which are red (genetically stable resistant), blue (susceptible), yellow (genetically unstable or phenotypically resistant), green (phenotypically tolerant).</p> ">
Review Reports Versions Notes

Abstract

:
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.

1. Introduction

Fungal infections pose a significant and growing threat to public health worldwide, particularly in immunocompromised patients [1,2]. These infections are associated with high mortality rates, especially in vulnerable populations, such as neonates, those with HIV/AIDS, cancer patients undergoing chemotherapy, and transplant recipients [3,4,5,6,7]. For instance, invasive fungal infections have mortality rates ranging from 40% to 70% in candidiasis, 40% to 80% in cryptococcosis and can reach over 90% in aspergillosis, depending on populations and infection types [8,9,10].The economic cost of fungal infections is substantial, exceeding billions of dollars annually due to prolonged hospital stays, the expense of antifungal therapies, and complex management strategies [11,12,13]. Moreover, the increasing prevalence of antifungal-resistant infections complicates treatment, making fungal infection a major global health challenge [14]. Traditionally, antifungal resistance has been considered as being due to inherent and stable genetic mutations, with elevated MIC (Minimum Inhibitory Concentration) values, that diminish drug efficacy. However, recent research has uncovered a more complex and dynamic form of resistance: heteroresistance. Fungal heteroresistance, in which a small subpopulation of a fungal strain exhibits resistance to antifungal drugs while the majority remain susceptible, presents a significant global threat. This phenomenon complicates treatment, as it can lead to treatment failure despite the apparent efficacy of the drug against most of the fungal cells, further exacerbating the public health crisis caused by fungal pathogens.
Heteroresistance was first discovered in 1947 in Haemophilus influenzae [15]. In bacteriology, it refers to the presence of a heterogeneous population composed of one or several subpopulations with increased levels of antibiotic resistance compared with the major population. This can be due to clonal heterogeneity during co-infection by colonies with different levels of resistance, or microevolution of a single colony [16,17]. The heteroresistance phenotype can either be sufficiently stable or not in rapidly reverting to a susceptible phenotype, or show instability that may affect laboratory test results [18,19]. Under persistent selective pressure, it may even evolve heritable properties, causing higher rate of treatment failure, and create an antibiotic resistance reservoir [20]. As for mechanisms, they vary among different pathogens in response to various kinds of drugs. For example, Pseudomonas aeruginosa shows heteroresistance to imipenem due to biofilm formation and OprD gene mutation [21], while heteroresistance to levofloxacin is linked to elevated expression of genes involved in DNA replication and repair [22]. Research on H. influenzae showed that imipenem heteroresistance was linked to alteration of PBP3 (penicillin-binding protein 3), drug influx and efflux changes [23]. It can also be induced by antibiotic exposure, through upregulation of stress-related pathways, etc. [24,25].
While extensively studied in bacteria, research on fungal heteroresistance remains comparatively limited. From a clinical perspective, heteroresistance has been implicated in treatment failure in murine models [26], but clinically important mechanisms have not been studied in sufficient depth. However, the clinical impact of heteroresistance leading to drug resistance during treatment should not be underestimated, as it may lead to prophylaxis failure, recurrent infection or relapse [3,27,28,29,30]. In this review, we summarize current findings to outline the characteristics, mechanisms, and clinical prevalence of heteroresistance in fungal pathogens, with the goal of providing a foundation for future investigations.

2. Methodology

This narrative review was undertaken to provide a comprehensive overview of antifungal heteroresistance, focusing on its molecular mechanisms and clinical implications. Relevant literature was identified through searches in PubMed and Web of Science using the keywords “antifungal heteroresistance”, “antifungal resistance”, “antifungal tolerance”, and “antifungal persistence”. Articles published from 1995 to 2025 were considered, with a focus on studies investigating genetic, epigenetic, and phenotypic mechanisms underlying heteroresistance, as well as its clinical outcomes and treatment methods. Priority was given to peer-reviewed research articles, reviews, and case reports relevant to clinical microbiology and antifungal therapy. Non-English articles, conference abstracts, and studies lacking direct relevance to antifungal tolerance/persistence/heteroresistance, or expressing the definition ambiguously, were excluded.

3. Antifungal Resistance Mechanisms

Fungi are significant eukaryotic pathogens in humans capable of causing infections ranging from superficial to life-threatening systemic diseases [8,31,32,33,34,35,36,37,38,39,40]. Their pathogenicity stems from immune evasion [41,42,43,44,45,46,47,48,49,50,51], adaptation to changing pH conditions and nutrient availability [45,47,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67], tissue invasions [40,42,43,44,68,69], etc. These characteristics make fungal infections particularly challenging to treat, especially in immunocompromised individuals [4,7,70,71,72,73]. Currently, there are four main classes of drugs for treatment of fungal infections: azoles, echinocandins, polyenes (Amphotericin B, AmB), and 5-fluorocytosine (5-FC). Azoles act on fungal 14α-demethylases and inhibit ergosterol biosynthesis, producing toxic methyl sterols and leading to alterations in the permeability and metabolic state of fungal cells, which may result in growth inhibition or cell death [74,75]. Echinocandins target and inhibit β-1,3-glucan synthase, thereby inhibiting β-1,3-glucan production and impairing fungal cells in maintaining their shape and resisting external stresses [76]. AmB targets membrane ergosterol, leading to pore formation, altered permeability, and reactive oxygen species (ROS) accumulation, all of which led to eventual fungal cell death [77,78,79]. 5-FC is a prodrug that is imported into cells by cytosine permease, which is encoded by the FCY2 gene. Once inside the cell, 5-FC is converted to 5-fluorouracil (5-FU) through the action of cytosine deaminase and uracil phosphoribosyl transferase, both encoded by the FCY1 gene, inhibiting DNA and RNA biosynthesis [80,81]. Specific modes of action of these antifungal drugs are illustrated Figure 1.
In recent years, a concerning surge in the isolation of antifungal resistant strains has raised a critical alarm for public health. Mechanisms associated with azole resistance mainly include mutations in azole targets, efflux pumps and biofilm formation, etc. [82,83,84]. In Candida spp., upregulation of the efflux transporter genes CDR1, MDR1 and mutations in transcriptional factor TAC1B and in the genes ERG11 and ERG3 [82,84,85,86,87], which result in for example, primary sequence changes Y132F and R398I in C. parapsilosis lanosterol 14α-demethylase [85], have been associated with fluconazole resistance. A point mutation in C. neoformans sterol 14α-demethylase 484S gives drug resistance to fluconazole only [88]. In A. fumigatus, mutations in the cyp51A gene encoding sterol 14α-demethylase and mutations in the ABC transporter gene atrF encode common azole resistance mechanisms [84,89].
Currently, resistance to echinocandins is mainly associated with mutations in the FKS1/FKS2 genes [82,90,91]. Other molecular mechanisms associated with echinocandin resistance have also been reported. Yu et al. found that the ADA2 gene of Nakaseomyces glabrata (formerly Candida glabrata) is associated with resistance to three classes of antifungals, as evidenced by Δada2 knockout strains having significantly reduced MIC values [51]. Singh et al. found that deletion of seven genes, including MOH1, GPH1, CDC6, TCB1/2, DOT6, MRPL11 and SUI2, increased levels of resistance to echinocandins, but considering their small effect on caspofungin resistance or the concurrent occurrence with FKS2 mutations, it is more likely that they create a genetic background in which the FKS2 mutations are adaptive and less harmful, rather than a direct cause of resistance [92].
5-FC resistance is highly correlated with its pharmacological mechanism, i.e., mutations in any one or more of the key enzyme genes of the pyrimidine salvage pathway, which may involve mutations in the genes encoding the cytosine permease FCY2, purine FCY1 encoding the cytosine deaminase, FUR1 encoding the uracil phosphoribosyl transferase, and ADE17 [82,93]. However, other mechanisms have also been reported: Kannan et al. found that V668G substitution of a putative transcriptional activator (MRR1) led to the upregulation of MFS7, a multidrug transporter protein that mediated azole-5-FC cross-resistance in Clavispora lusitaniae (formerly Candida lusitaniae) [94] Billmyre et al. found that deletion of the mismatch repair gene, MSH2, in C. deuterogattii also led to an elevated rate of 5-FC resistance [95].
Fungal resistance to AmB is less common but has been reported in species such as Candidazymus auris (formerly Candida auris) and the Candidazymus haemulonii (formerly Candida haemulonii) complex [82,96]. Resistance to AmB in both species involves a variety of mechanisms, including alterations in cell membrane composition, cellular metabolic state, iron homeostasis, and ROS metabolism [58,77,78,96]. The molecular mechanisms of resistance to AmB in C. haemulonii were systematically discussed in the author’s previous review [40].

4. Heteroresistance, Tolerance and Persistence of Antifungal Drugs

At the cell population level, not all survival with fungicidal concentrations of antifungals can be attributed to elevated MICs. This is when heteroresistance, tolerance and persistence come into play. Here, we describe the differences between the three from the perspective of the performance of cell populations under drug stress, to minimize confusion about these concepts (Figure 2).
Heteroresistance, a concept first introduced for bacteria, is a form in which a minority subpopulation of resistant phenotype cells has a higher MIC, sometimes involving as few as one in one million cells, that coexists with a majority population of susceptible cells with a lower MIC [15]. Clinically, it refers to persistent infection, treatment failure, or relapse, despite in vitro susceptibility results indicating sensitivity [97,98]. It may be polyclonal or monoclonal. Polyclonal heteroresistance involves heterogeneous consortia with genetically distinct subpopulations or to the appearance of rare resistant mutants whose frequency increases with antibiotic exposure. Monoclonal heteroresistance is pure clones [18]. As a variant, it is more of continuous than binary (on/off). A study of heteroresistance properties showed that the incremental effects of multiple binary genetic switches, including on the efflux pumps CDR1, PDH1, PDR1 and SNQ1, etc. produced a spectrum of heteroresistance rather than binary states, in N. glabrata [99]. From the perspective of population evolution, it can be seen as a strategy that helps the fungal population survive diverse environments [100]. In vitro, heteroresistance can be obtained through antifungal induction or non-antifungal agents and artificial selection, the last of which may produce the longest-lasting phenotype [57,101]. Repeated culture in stress-free environments can abolish heteroresistance [48]. Interestingly, this majority subpopulation can never be purified, meaning that a minority remains phenotypically resistant [102].
Tolerance is defined as an extension of the killing time, characterized by an increase in the MDK99 (Minimum Duration of Killing 99%) time [103]. It refers to the whole population that can survive a transient exposure to antifungals at otherwise lethal concentrations, without an increase in the antifungal MIC [104]. Furthermore, some 5–90% of the tolerant cells can slowly proliferate [105]. This definition applies to fungicides such as echinocandins and polyenes. However, for fungistatic drugs (e.g., azoles), tolerance has a different definition, i.e., the ability of a tolerant strain to overcome growth inhibition faster than a sensitive strain at drug concentrations above the MIC [106]. This phenomenon can be measured using broth microdilution or disk diffusion assays [98]. The reversibility of this property has been confirmed for several fungi species [107,108].
Persistence and persister cells have been extensively studied in bacteria [109]. For fungi, persistence refers to a subpopulation of genetically susceptible fungal cells that survives fungicidal concentrations of antifungals and may lead to the emergence of genetically resistant isolates [110,111,112]. This phenomenon has been observed in intra-macrophage N. glabrata cells, in which a subpopulation survives and gradually evolves heritable resistance mutations in FKS [110]. Sometimes “persistence” is used interchangeably with “tolerance”, as the MIC values of the fungal cell population do not change in both phenotypes. Importantly, persistence is characterized by a biphasic killing curve, whereas tolerance is not [109]. In other words, “tolerance” means that all survive and some may proliferate, whereas “persistence” means that some survive but none proliferates. Comparison of these concepts can be seen in Table 1.

5. Mechanisms of Antifungal Tolerance

Fungal tolerance to antifungals arises from a variety of molecular mechanisms. For example, azole tolerance in Candida populations is often characterized by the ability to survive and proliferate slowly at concentrations above the minimum inhibitory concentration (MIC). This tolerance is typically temperature-independent and frequently associated with aneuploidy, which may be lost upon temperature changes or ploidy recovery [114,115]. Similarly, in C. neoformans, brain glucose has been shown to induce AmB tolerance without affecting the MIC. This process is mediated by the zinc-finger transcription factor Mig1 and involves more complex mechanisms, such as the inhibition of ergosterol synthesis and the promotion of competing compounds that contribute to antifungal tolerance [116]. In C. neoformans, altered mitochondrial metabolism caused by cell ageing drives increased ergosterol synthesis and ABC transporter upregulation and leads to azole tolerance [117].
Alterations in cellular structure and metabolic levels also play a key role in antifungal tolerance. Cell wall remodeling contributes to echinocandin tolerance, with changes in β-glucan synthesis or increased production of compensatory components, like chitin, strengthening the cell wall and reducing drug efficacy [118,119]. In addition, activation of cellular stress response pathways, such as the heat shock protein (Hsp) and calcineurin pathways, has been shown to contribute to echinocandin tolerance in C. albicans [120,121]. Moreover, single-cell transcriptomics have shown that the ribosome assembly stress response supports survival under fluconazole exposure above the MIC, highlighting the importance of stress responses in antifungal tolerance [122]. Together, these mechanisms illustrate how fungi adapt to antifungal treatments through a combination of genetic, transcriptional, and physiological changes, making effective treatment more challenging.

6. Mechanisms of Antifungal Persistence

Persistence is always linked to cell dormancy, a state in which the cellular metabolism temporarily ceases [111,123,124]. As the fungal pathogen enters the host organism, it is often phagocytosed by immune cells, such as macrophages. Studies on pathogen–phagocyte interactions have revealed the inducing effect of the phagocytic intracellular environment on the formation of persisters [110]. Comparing the metabolism of planktonic cells and persisters affected by ROS or pH-related environmental stress suggested a possible role for cellular stress response in persistence [110]. Ke et al. constructed a mouse model of pulmonary infection and demonstrated that AmB-tolerant persisters are enriched in C. neoformans cells with high levels of the stationary-phase protein Sps1 and the metabolic marker ergothioneine [112]. This provides insight into the critical role of the EGT gene, responsible for the synthesis of ergothioneine, in regulating AmB susceptibility [112].
There are also several key factors that enable persisters. The formation of persister cells does not require biofilm formation, but biofilm-containing persister populations are more tolerant of oxidative stress and better resist the fungicidal effects of AmB [125]. Inhibiting biofilms helps eradicate C. tropicalis persister cells [126]. As antifungals can increase levels of ROS, persister cells might obtain stronger antioxidant capacity by upregulating enzymes, like superoxide dismutases (SODs) or alkyl hydroperoxide reductase 1, etc. [127]. Such mechanisms have been confirmed via SOD inhibition which reduces persistence levels [128]. Formation of persister cells should be considered important, as they may cause chronic or recurrent infections that complicate treatment.

7. Mechanisms of Antifungal Heteroresistance

The molecular mechanisms underlying fungal heteroresistance are diverse and involve a complex interplay of genetic, transcriptional, and physiological factors without always involving heritable change. Detailed mechanisms can be seen in Table 2. In many cases, heteroresistance arises via transient change that allows the fungus to survive antifungal stress without permanent genetic change. This adaptability complicates treatment strategies, as the resistant phenotype may not be stable and may fluctuate in response to environmental conditions.

7.1. Aneuploidy and Copy Number Variations (CNVs)

Aneuploidy is a common heritable mechanism responsible for the most widely studied form of antifungal heteroresistance [148,149,150]. The presence of an abnormal number of chromosomes plays a crucial role in fungal survival and evolution by providing rapid adaptability in response to environmental stress, such as antifungal drug exposure [151,152]. Yang et al. found that exposure to fluconazole at subinhibitory concentrations for a short period of time (48 h) may cause C. neoformans to acquire different aneuploid chromosomes that confer heteroresistance to fluconazole and cross-resistance to 5-FC [153]. Thus, exposure to one class of antifungal can promote adaptation to similar or different antifungal agents, highlighting the plasticity of the fungal genome and raising serious public health concerns. By increasing the copy number of chromosomes containing drug resistance genes, such as those encoding transcription factors, efflux pumps or involved in ergosterol biosynthesis, aneuploidy leads to resistance against multiple antifungals. including azoles and echinocandins [99,129,154]. Non-antifungal agents can also induce heteroresistance. Zhang et al. found that the endoplasmic reticulum stress chemo inducer Brefeldin A led to aneuploidy in C. neoformans: disomy in chromosome 1 led to cross-resistance to two classes of antifungal drugs, fluconazole and 5-FC, plus hypersensitivity to AmB [139]. By altering gene dosage or expression levels, aneuploidy allows fungi to quickly adjust to adverse conditions without permanent genetic mutations, promoting survival in fluctuating environments [99,129,145,152,154]. Importantly, its reversibility offers a dynamic mechanism for fungi to balance adaptation and stability, in the population and for individuals [151,155]. As a result, aneuploidy plays a pivotal role in both fungal adaptability and the evolution of drug resistance, making it a key factor in heteroresistance and its clinical effects.

7.1.1. C. albicans

Mechanisms associated with azole heteroresistance in C. albicans are diverse. Under fluconazole exposure, C. albicans rapidly evolves CNVs and aneuploidy in vitro [135]. Associated resistance is mainly related to chr5, which has the ERG11 and TAC1 gene [129,130]. TAC1 encodes a transcription regulator of ABC transporter genes located on chr3 [156]. Loss of chr5 can result in enhanced susceptibility to azoles [130], and can also lead to enhanced susceptibility to AmB and increased resistance to 5-FC. The latter effect is due to the location of the negative regulator (s) of anti 5-fluorocytosin on chr5, while the former needs clarification [130]. Harrison et al. reported the appearance of “trimeras”, connected cells composed of a mother, a daughter, and a granddaughter bud, after exposure to fluconazole [131]. The same morphology is not found in genetically resistant strains, suggesting the potential role of trimeras in heteroresistance. The trimeras produce progeny with different chromosome numbers, increasing the chances of developing heteroresistance [131].
C. albicans’ heteroresistance to echinocandins is related to chr2, as chr2 trisomy can be induced after exposure to caspofungin [132]. It is also related to chr5, as chr5 aneuploidy after caspofungin exposure gave cross-resistance to caspofungin, micafungin and anidulafungin [133]. Yang et al. found three negative regulators of echinocandin susceptibility on chr5. These are CHT2, encoding a glycosylphosphatidylinositol (GPI)-dependent chitinase, a covalently bound cell wall protein, PGA4, encoding a GPI-anchored cell surface 1,3-β-d-glucanosyltransferase, and CSU51 encoding another putative GPI-anchored protein [132]. They also found positive regulators, i.e., CNB1 encoding a regulatory subunit of calcineurin B, and MID1 encoding a putative stretch-activated Ca2+ channel of the high-affinity calcium uptake system [118]. However, the genes involved and whether they act together in echinocandin heteroresistance is not clear [118].

7.1.2. N. glabrata

N. glabrata was considered a haploid and an asexual organism for decades, but recent studies have reported the instability of clinical isolate genomes, mainly due to the frequent change in ploidy [157]. Ploidy variation might promote the rapid adaptation of N. glabrata to the changing environment and support the evolution of new traits, like heteroresistance. Ksiezopolska et al. found chrE aneuploidy contributes to heteroresistance after exposing clinical isolates to anidulafungin [134]. Heteroresistance due to environmental stress may remain even after the stress [134]. N. glabrata can also form “trimeras” on exposure to azoles. This might be related to heteroresistance, and its mechanistic basis needs further investigation [131].

7.1.3. C. parapsilosis

In C. parapsilosis, multi-center studies have shown that heteroresistance facilitates breakthrough infections in immunocompromised patients and may cause prophylaxis failure [3]. However, unlike C. neoformans, exposure of C. parapsilosis to tunicamycin did not reveal any relationship between aneuploidy and heteroresistance [3,153]. Evidence of aneuploidy in the heteroresistance was obtained by Harrison et al., and showed that C. parapsilosis can form “trimeras” when exposed to azoles [131].

7.1.4. C. auris

For highly resistant pathogen C. auris, Zhai et al. reported heteroresistance towards echinocandins, the first-line treatment for C. auris infection [3]. In vitro evolution during fluconazole exposure is relatively slow, compared to C. albicans, and mainly composed of a single nucleotide polymorphism (SNP), with aneuploidy a minority effect [135,136]. Due to its haploid genome, some SNP changes in C. auris may have immediate phenotypic effects [137].

7.1.5. C. neoformans and C. gattii

For Cryptococcus spp., heteroresistance is a critical factor in the treatment failure of cryptococcosis and involves a subpopulation that appears under antifungal exposure and retains the potential to grow under continuous drug stress [98]. Because of the broad definition, many studies have reported “tolerance” as cryptococcal azole heteroresistance and vice versa [158]. For C. neoformans and C. gattii, the heteroresistance to fluconazole is intrinsic and increased resistance can be induced [19,102]. A comparative study showed that C. gattii showed a higher level of heteroresistance to fluconazole than C. neoformans [159]. The main mechanisms that could be related to C. gattii heteroresistance are copy number increase in chr10, chr9 and chr11 for the VGI genotype and copy number increase in chr3 for the VGII genotype [138]. For C. neoformans, heteroresistance can be reduced or inhibited by several environmental factors, including temperature, media type, growth phase, and cell age [147]. One of its mechanisms is associated with the multiple types of aneuploid daughter cells produced by titan cells [56,140,141]. A genomic analysis of clinical C. neoformans isolates found a high rate of aneuploidy in heteroresistant colonies and recurrent isolates, with a predominance of chr1 disomy [142]. Strains with chr1 disomy can also be isolated from mouse brain during treatment with fluconazole [160]. Ngamskulrungroj et al. demonstrated a high incidence of chr4 disomy, which may be related to SEY1 (GTPase with a role in Endoplasmic Reticulum morphology), GCS2 (ADP-ribosylation factor GTPase activating proteins) or GLO3 (ADP-ribosylation factor GTPase activating proteins) genes [143]. If any of the ERG11, SEY1, GCS2 or GLO3 genes are relocated to chr3, then the frequency of chr3 disomy could increase [143,144]. This is due to the presence on chr1 of ERG11, which is the drug target of fluconazole, and AFR1, which encodes the drug efflux pump. However, the AFR1 gene does not directly lead to heteroresistance [102]. Other research on C. neoformans’ ploidy found that exposure to inhibitory concentrations of fluconazole diminished budding and growth while permitting nuclear events. This resulted in populations with increased DNA, whose content grow better in the presence of fluconazole, increasing aneuploidy and survival [161]. The mechanisms of C. neoformans heteroresistance need to be defined to identify clinical therapies that will prevent and eliminate drug-resistant Cryptococcus subpopulations.

7.2. Alterations in Gene Expression

Efflux pumps, such as those belonging to the ABC transporter and major facilitator superfamily (MFS), play a crucial role in antifungal resistance by actively expelling drugs from the fungal cell. In heteroresistant subpopulations, the overexpression of efflux pumps can confer a temporary survival advantage. A genomic analysis of clinical C. neoformans strains found a high rate of aneuploidy in heteroresistant colonies and recurrent isolates, with a predominance of chr1 disomy and up-regulated activity of efflux pumps [142]. Sykes et al. obtained serial isolates from the same patient and found that overexpression of the efflux pump PDR11 contributed greatly to the induced fluconazole heteroresistance [146]. Marr et al. demonstrated that the induction effect of fluconazole on heteroresistance involved elevation of mRNA levels for ABC superfamily CDR genes [145].

7.3. Environmental Stress Induction

In addition to specific antifungals inducing aneuploidy, non-pharmacological stressors can also impact antifungal heteroresistance and even cross-heteroresistance. Although the resistance phenotype is not stable due to the intrinsic instability of aneuploidy, it has been suggested that external inducing factors and genomic instability have vital roles in drug resistance in C. neoformans. For example it was found that C. neoformans heteroresistance was detected at 35 °C but not at 30 °C, indicating the importance of environmental temperature [19]. “Titanization” in C. neoformans also plays a role in the formation of aneuploidy. Gerstein et al. found that, during cryptococcosis treatment, newly emerged polyploid titan cells produced daughter cells that were more resistant to fluconazole and thus adapted to the host environment. Furthermore, a single titan mother cell produced multiple types of aneuploid daughter cells, which might contribute to the survivability of progeny under different environmental stresses [56]. This process was associated with intracellular ROS accumulation and mitochondrial responses [141]. For C. neoformans, heteroresistance can be reduced or inhibited by several environmental factors, including temperature, media type, growth phase, and the age of cells [147]. We speculate that stress response pathways contribute to the generation of antifungal heteroresistance. Bosch et al. demonstrated that environmental stress, such as the nitrogen limitation commonly encountered in its natural habitats, increases the resistance of C. neoformans to AmB and fluconazole, and increases the frequency of heterogeneous resistance to fluconazole [57]. For A. fumigatus, clinically, long-term itraconazole treatment may cause decreased susceptibility. Progressive itraconazole exposure in vitro can reproduce such phenomena, independent of the mutation in cyp51A gene [162]. This is called secondary resistance, suggesting the existence of heteroresistance. However, there are also studies showing the existence of intrinsic heteroresistance [48,102], and mechanisms of stress induction need to be further explored.

8. Clinical Relevance of Antifungal Heteroresistance

8.1. Outcomes of Antifungal Heteroresistance

Several studies have confirmed the increased emergence of heteroresistance in clinical, compared with environmental, isolates, supporting the induction effect of antifungal exposure [101,158]. Infections caused by heteroresistant strains increase risk to patients and make clinical treatment more difficult. One study demonstrated that Trimethoprim-Sulfamethoxazole heteroresistant Staphylococcus aureus strains gave near identical mortality as the resistant strains, indicating clinical consequences [163]. For fungi, a mouse model showed that heteroresistant C. cryptococcus isolates suppress the host immune response to enhance evasion, achieve higher fungal burden in different organs, and increase virulence [48,102]. As for high-level heteroresistant strains in brain-infected mice, if untreated their 20-day mortality rate can reach 100% [160]. Heteroresistant N. glabrata isolates can lead to higher fungal burden in mice kidneys, making it harder to thoroughly clear the pathogen and resulting in persistent infection [99].
In bacteria, heteroresistance may progress during antibiotic therapy, leading to changes in clinical tests and treatment failures and even creating an antimicrobial resistance reservoir [20,164]. Similarly, the transient nature of antifungal heteroresistance allows pathogenic fungi to survive conventional antifungal treatment, even when most of the population is inhibited. Prior to the onset of infection, this can facilitate breakthrough infection or result in prophylaxis failure [3]. For diagnosed fungal infections, it may lead to persistent infections that require longer or more aggressive treatment regimens [27,28,29,30]. Despite initial susceptibility to the drug, heteroresistant subpopulations can expand during therapy, partially due to antifungal exposure, characterized as the emergence of resistance-related genetic mutations, and clinically as relapse or progression of the infection [110,151,152,153]. To make things worse, antifungal heteroresistance can also contribute to cross-resistance between different classes of antifungal drugs. For example, C. albicans chr5 aneuploidy caused by caspofungin exposure can result in cross-resistance to caspofungin, micafungin and anidulafungin [133] and, in C. neoformans, chr1 disomy may confer cross-resistance to both azoles and 5-FC [139]. This makes treatment options even more restrictive.

8.2. Diagnosis of Antifungal Heteroresistance

Conventional antifungal susceptibility testing methods, such as broth microdilution and disk diffusion assays, may not detect heteroresistant subpopulations [165,166]. An in vivo experiment with K. pneumoniae strain SWMUF35-infected mice showed contradictory results compared to in vitro experiments, which classified the strain as amikacin susceptible [28]. These tests typically measure the MIC for the bulk population, potentially overlooking small subpopulations that can survive higher drug concentrations. This limitation arises because heteroresistant subpopulations may exist at low frequencies or exhibit resistance only under specific conditions, such as prolonged exposure to antifungal agents or within certain host environments [153,165,167,168]. As a result, infections caused by heteroresistant strains may be mistakenly classified as susceptible based on conventional testing. This misclassification can lead to the selection of antifungal therapies that are ineffective against resistant subpopulations, resulting in treatment failure, persistent infections, or even the emergence of fully resistant strains [99,165]. In clinical practice, this underscores the need for more sensitive diagnostic approaches to accurately detect and address heteroresistance, ensuring appropriate therapeutic strategies and improved patient outcomes.
For heteroresistance, the population analysis profile (PAP) assay is recommended as the gold standard [169]. PAP has been proved to efficiently test the heteroresistance to azoles and echinocandins in C. albicans, C. haemulonii, C. tropicalis, N. glabrata, C. parapsilosis, C. neoformans and S. cerevisiae, etc. [30]. Recently the novel Kirby–Bauer disk diffusion method demonstrated superior diagnostic performance for heteroresistance detection, though the PAP test remains the current standard method [97]. Single-cell assays can also be used in heteroresistance detection. These include techniques such as flow cytometry, single-cell RNA sequencing (scRNA-seq), and microfluidics-based assays, allowing for the analysis of gene expression, cell viability, and drug tolerance in individual fungal cells [170,171]. Though the protocols are lengthier, they may reveal a wide spectrum of adaptation mechanisms [30]. Time-kill assays, conventionally used to monitor the survival of fungal cells over time in the presence of an antifungal drug, can also be used to detect heteroresistance. By measuring the rate of cell death at various time points, this method can identify delayed growth or survival of heteroresistant subpopulations undetected by static MIC assays, and even evaluate the kinetics of heteroresistance and assess whether heteroresistant cells eventually adapt to drug pressure, which makes for wide clinical applicability [172,173,174]. In C. neoformans, time kill assays have been used to study azole and AmB heteroresistance, revealing that subpopulations of cells can survive for prolonged periods despite the presence of high drug concentrations [57]. These make time-kill assays a valuable tool in understanding heteroresistance dynamics.

8.3. Treatments of Antifungal Heteroresistance

There are currently no approved first-line therapies for heteroresistance. For bacteria, a combination of several antibiotics serves as the preferred strategy [28,174,175,176]. For example, combination polymyxin B and tigecycline therapy was shown to kill both the polymyxin B- and tigecycline-heteroresistant Klebsiella pneumoniae [174]. Similarly in Acinetobacter baumannii, combined tigecycline and colistin therapy was bactericidal on TGC and colistin multi-heteroresistance [175]. For fungi, combination of drugs or dosage escalation may aid in alleviating treatment failure. A study of C. neoformans showed that 5-FC effectively suppress fluconazole-induced heteroresistance, suggesting the importance of combined therapy [142]. Other research suggested the feasibility of fluconazole dose escalation and combination therapy in the treatment of cryptococcal meningitis but also revealed limitations in combination options, for which only 5-FC was widely available [165]. However, comparative studies and clinical trials remain limited. Expanding the scope to all failed antifungal treatments, such as adding 5-FC or amphotericin B to fluconazole monotherapy, alleviates mortality rate and achieves up to 90% efficacy [177,178,179]. Considering the evolution of resistance or cross-resistance, even suitable combination therapies or escalation dose therapies can increase the risk of toxicity and adverse effects. Inspired by well-studied combination antibiotic therapy and results from the limited use of combination antifungal therapy, addition of different classes of antifungals has the potential to clear heteroresistance. In-depth study of the mechanisms of heteroresistance may help develop novel drugs and providing low-toxicity combination therapies.

9. Conclusions

Heteroresistance is a reversible form of antifungal resistance that fluctuates in response to varying conditions, primarily driven by genome aneuploidy and changes in gene expression. Unlike bacterial resistance, fungal heteroresistance remains largely underexplored, with most research focusing on azole resistance. A unique characteristic of heteroresistance is its inducibility, with resistant phenotypes emerging in response to antifungal exposure or other factors, leading to reduced drug efficacy over time.
Molecularly, aneuploidy and CNVs play a crucial role, influencing mechanisms such as gene loss, gene amplification, modifications of antifungal-binding sites, and upregulation of efflux pumps. These genetic changes enhance fungal adaptability to antifungal pressure. One point worth exploring is that CNVs can be stably inherited and may occur in both resistance and heteroresistance, which means that they may contribute greatly to the evolution of antifungal resistance. Understanding the evolutionary and molecular mechanisms of heteroresistance is crucial for predicting and controlling clinical resistance.
Antifungal heteroresistance poses a significant clinical challenge, as traditional diagnostic techniques may mistakenly report it as susceptibility and lead to misuse of antifungals. To make things worse, it can develop during treatment and even evolve into stable and heritable resistance, leading to therapeutic failure and recurrent infections. While escalating drug dosages or using combination therapies may sometimes be effective, comparative clinical studies remain limited to date. Moreover, these approaches come with limited options and increased toxicity risks. Therefore, the development of novel antifungal agents is critical, and is expected to provide more therapeutic choices and combination possibilities. In the final analysis, gaining deeper insights into the specific mechanisms of heteroresistance could help to gain deeper and clearer insights into phenotype, thus aiding the development of innovative treatment strategies targeting multiple pathways, ultimately improving patient outcomes and reducing healthcare costs.

Author Contributions

Y.S., Y.L. (Yi Li) and Q.Y. devised the project and wrote the draft of the manuscript; T.S., Y.L. (Yingxing Li) and Y.X. edited the draft of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National High Level Hospital Clinical Research Funding [Zhao Ying, 2022-PUMCH-C-052].

Data Availability Statement

This is a review article, and no new data were created or analyzed. All data discussed in this study are publicly available in the references cited.

Acknowledgments

We apologize to all colleagues whose work could not be cited in this Review owing to space constraints.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
MICMinimum Inhibitory Concentration
CNVsCopy Number Variations
AmBAmphotericin B
5-FC5-fluorocytosine
5-FU5-fluorouracil
ROSReactive Oxygen Species
MDK99Minimum Duration of Killing 99%
SODSuperoxide Dismutases
chrChromosome
GPIGlycosylphosphatidylinositol
PAPPopulation Analysis Profile
scRNA-seqSingle-Cell RNA Sequencing
SNPSingle Nucleotide Polyphorism
ABCATP-Binding Cassette
PAPPopulation Analysis Profile

References

  1. Spallone, A.; Schwartz, I.S. Emerging Fungal Infections. Infect. Dis. Clin. N. Am. 2021, 35, 261–277. [Google Scholar] [CrossRef] [PubMed]
  2. Lockhart, S.R.; Guarner, J. Emerging and reemerging fungal infections. Semin. Diagn. Pathol. 2019, 36, 177–181. [Google Scholar] [CrossRef] [PubMed]
  3. Zhai, B.; Liao, C.; Jaggavarapu, S.; Tang, Y.; Rolling, T.; Ning, Y.; Sun, T.; Bergin, S.A.; Gjonbalaj, M.; Miranda, E.; et al. Antifungal heteroresistance causes prophylaxis failure and facilitates breakthrough Candida parapsilosis infections. Nat. Med. 2024, 30, 3163–3172. [Google Scholar] [CrossRef] [PubMed]
  4. Godoy, M.C.B.; Ferreira Dalla Pria, H.R.; Truong, M.T.; Shroff, G.S.; Marom, E.M. Invasive Fungal Pneumonia in Immunocompromised Patients. Radiol. Clin. N. Am. 2022, 60, 497–506. [Google Scholar] [CrossRef]
  5. Huang, S.F.; Ying-Jung Wu, A.; Shin-Jung Lee, S.; Huang, Y.S.; Lee, C.Y.; Yang, T.L.; Wang, H.W.; Chen, H.J.; Chen, Y.C.; Ho, T.S.; et al. COVID-19 associated mold infections: Review of COVID-19 associated pulmonary aspergillosis and mucormycosis. J. Microbiol. Immunol. Infect. 2023, 56, 442–454. [Google Scholar] [CrossRef]
  6. Piano, S.; Bunchorntavakul, C.; Marciano, S.; Rajender Reddy, K. Infections in cirrhosis. Lancet Gastroenterol. Hepatol. 2024, 9, 745–757. [Google Scholar] [CrossRef]
  7. Weimer, K.E.D.; Smith, P.B.; Puia-Dumitrescu, M.; Aleem, S. Invasive fungal infections in neonates: A review. Pediatr. Res. 2022, 91, 404–412. [Google Scholar] [CrossRef]
  8. Denning, D.W. Global incidence and mortality of severe fungal disease. Lancet Infect. Dis. 2024, 24, e428–e438. [Google Scholar] [CrossRef]
  9. Krzych, Ł.J.; Putowski, Z.; Gruca, K.; Pluta, M.P. Mortality in critically ill COVID-19 patients with fungal infections: A comprehensive systematic review and meta-analysis. Pol. Arch. Intern. Med. 2022, 132, 16221. [Google Scholar] [CrossRef]
  10. Garcia-Vidal, C.; Sanjuan, G.; Moreno-García, E.; Puerta-Alcalde, P.; Garcia-Pouton, N.; Chumbita, M.; Fernandez-Pittol, M.; Pitart, C.; Inciarte, A.; Bodro, M.; et al. Incidence of co-infections and superinfections in hospitalized patients with COVID-19: A retrospective cohort study. Clin. Microbiol. Infect. 2021, 27, 83–88. [Google Scholar] [CrossRef]
  11. Kruithoff, C.; Gamal, A.; McCormick, T.S.; Ghannoum, M.A. Dermatophyte Infections Worldwide: Increase in Incidence and Associated Antifungal Resistance. Life 2023, 14, 1. [Google Scholar] [CrossRef] [PubMed]
  12. Rayens, E.; Norris, K.A. Prevalence and Healthcare Burden of Fungal Infections in the United States, 2018. Open Forum. Infect. Dis 2022, 9, ofab593. [Google Scholar] [CrossRef] [PubMed]
  13. Liu, J.Y.; Dickter, J.K. Nosocomial Infections: A History of Hospital-Acquired Infections. Gastrointest. Endosc. Clin. N. Am. 2020, 30, 637–652. [Google Scholar] [CrossRef] [PubMed]
  14. Fisher, M.C.; Hawkins, N.J.; Sanglard, D.; Gurr, S.J. Worldwide emergence of resistance to antifungal drugs challenges human health and food security. Science 2018, 360, 739–742. [Google Scholar] [CrossRef]
  15. Alexander, H.E.; Leidy, G. Mode of action of streptomycin on type b Hemophilus influenzae: II. Nature of resistant variants. J. Exp. Med. 1947, 85, 607. [Google Scholar] [CrossRef]
  16. Zheng, C.; Li, S.; Luo, Z.; Pi, R.; Sun, H.; He, Q.; Tang, K.; Luo, M.; Li, Y.; Couvin, D.; et al. Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates. J. Clin. Microbiol. 2015, 53, 2138–2147. [Google Scholar] [CrossRef]
  17. Liu, Q.; Via, L.E.; Luo, T.; Liang, L.; Liu, X.; Wu, S.; Shen, Q.; Wei, W.; Ruan, X.; Yuan, X.; et al. Within patient microevolution of Mycobacterium tuberculosis correlates with heterogeneous responses to treatment. Sci. Rep. 2015, 5, 17507. [Google Scholar] [CrossRef]
  18. Andersson, D.I.; Nicoloff, H.; Hjort, K. Mechanisms and clinical relevance of bacterial heteroresistance. Nat. Rev. Microbiol. 2019, 17, 479–496. [Google Scholar] [CrossRef]
  19. Yamazumi, T.; Pfaller, M.A.; Messer, S.A.; Houston, A.K.; Boyken, L.; Hollis, R.J.; Furuta, I.; Jones, R.N. Characterization of heteroresistance to fluconazole among clinical isolates of Cryptococcus neoformans. J. Clin. Microbiol. 2003, 41, 267–272. [Google Scholar] [CrossRef]
  20. Kon, H.; Hameir, A.; Nutman, A.; Temkin, E.; Keren Paz, A.; Lellouche, J.; Schwartz, D.; Weiss, D.S.; Kaye, K.S.; Daikos, G.L.; et al. Prevalence and Clinical Consequences of Colistin Heteroresistance and Evolution into Full Resistance in Carbapenem-Resistant Acinetobacter baumannii. Microbiol. Spectr. 2023, 11, e0509322. [Google Scholar] [CrossRef]
  21. Xu, Y.; Zheng, X.; Zeng, W.; Chen, T.; Liao, W.; Qian, J.; Lin, J.; Zhou, C.; Tian, X.; Cao, J.; et al. Mechanisms of Heteroresistance and Resistance to Imipenem in Pseudomonas aeruginosa. Infect. Drug Resist. 2020, 13, 1419–1428. [Google Scholar] [CrossRef] [PubMed]
  22. Li, W.R.; Zhang, Z.Q.; Liao, K.; Wang, B.B.; Liu, H.Z.; Shi, Q.S.; Huang, X.B.; Xie, X.B. Pseudomonas aeruginosa heteroresistance to levofloxacin caused by upregulated expression of essential genes for DNA replication and repair. Front. Microbiol. 2022, 13, 1105921. [Google Scholar] [CrossRef] [PubMed]
  23. Cherkaoui, A.; Diene, S.M.; Renzoni, A.; Emonet, S.; Renzi, G.; François, P.; Schrenzel, J. Imipenem heteroresistance in nontypeable Haemophilus influenzae is linked to a combination of altered PBP3, slow drug influx and direct efflux regulation. Clin. Microbiol. Infect. 2017, 23, 118.e9–118.e19. [Google Scholar] [CrossRef] [PubMed]
  24. Adams-Sapper, S.; Gayoso, A.; Riley, L.W. Stress-Adaptive Responses Associated with High-Level Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae. J. Pathog. 2018, 2018, 3028290. [Google Scholar] [CrossRef]
  25. Fukuzawa, S.; Sato, T.; Aoki, K.; Yamamoto, S.; Ogasawara, N.; Nakajima, C.; Suzuki, Y.; Horiuchi, M.; Takahashi, S.; Yokota, S.I. High prevalence of colistin heteroresistance in specific species and lineages of Enterobacter cloacae complex derived from human clinical specimens. Ann. Clin. Microbiol. Antimicrob. 2023, 22, 60. [Google Scholar] [CrossRef]
  26. Xiong, Y.; Han, Y.; Zhao, Z.; Gao, W.; Ma, Y.; Jiang, S.; Wang, M.; Zhang, Q.; Zhou, Y.; Chen, Y. Impact of Carbapenem Heteroresistance Among Multidrug-Resistant ESBL/AmpC-Producing Klebsiella pneumoniae Clinical Isolates on Antibiotic Treatment in Experimentally Infected Mice. Infect. Drug Resist. 2021, 14, 5639–5650. [Google Scholar] [CrossRef]
  27. Kotilea, K.; Iliadis, E.; Nguyen, J.; Salame, A.; Mahler, T.; Miendje Deyi, V.Y.; Bontems, P. Antibiotic resistance, heteroresistance, and eradication success of Helicobacter pylori infection in children. Helicobacter 2023, 28, e13006. [Google Scholar] [CrossRef]
  28. Zhang, F.; Ding, M.; Yan, X.; Bai, J.; Li, Q.; Zhang, B.; Liang, Q.; Liang, S.; Wang, G.; Zhou, Y. Carbapenem-resistant K. pneumoniae exhibiting clinically undetected amikacin and meropenem heteroresistance leads to treatment failure in a murine model of infection. Microb. Pathog. 2021, 160, 105162. [Google Scholar] [CrossRef]
  29. Kargarpour Kamakoli, M.; Sadegh, H.R.; Farmanfarmaei, G.; Masoumi, M.; Fateh, A.; Javadi, G.; Rahimi Jamnani, F.; Vaziri, F.; Siadat, S.D. Evaluation of the impact of polyclonal infection and heteroresistance on treatment of tuberculosis patients. Sci. Rep. 2017, 7, 41410. [Google Scholar] [CrossRef]
  30. Gautier, C.; Maciel, E.I.; Ene, I.V. Approaches for identifying and measuring heteroresistance in azole-susceptible Candida isolates. Microbiol. Spectr. 2024, 12, e0404123. [Google Scholar] [CrossRef]
  31. Kessel, J.; Rossaert, A.C.; Lingscheid, T.; Grothe, J.; Harrer, T.; Wyen, C.; Tominski, D.; Bollinger, T.; Kehr, A.K.; Kalbitz, S.; et al. Survival after cryptococcosis in Germany: A retrospective multicenter cohort study of patients diagnosed between 2004 and 2021. Int. J. Med. Microbiol. 2024, 314, 151614. [Google Scholar] [CrossRef] [PubMed]
  32. Omura, M.; Komori, A.; Tamura, T.; Han, H.S.; Kano, R.; Makimura, K. Molecular epidemiological investigation of Cryptococcus spp. isolated from cats in Japan using multi-locus sequence typing. Med. Mycol. 2024, 62, myae085. [Google Scholar] [CrossRef] [PubMed]
  33. Sababoglu Baytaroglu, E.; Ipek, V.; Gokce, H.I.; Ayozger, L.E.O.; Olguner, B. Natural disasters and the rise of zoonotic diseases: A case of post-earthquake disseminated cryptococcosis in a dog. J. Mycol. Med. 2024, 34, 101501. [Google Scholar] [CrossRef] [PubMed]
  34. Lee, Y.M.; Liu, Y.M.; Chen, T.C. Disseminated Cryptococcus neoformans infection involving multiple bones and lung in an immunocompetent patient: A case report. BMC Infect. Dis. 2024, 24, 397. [Google Scholar] [CrossRef]
  35. Min, J.; Zhao, Y.; Du, J.; Ning, Y.; Wu, Z. Arthritis caused by Cryptococcus neoformans infection: A case report. Biomed. Rep. 2024, 20, 6. [Google Scholar] [CrossRef]
  36. Morgan, J.; Vardanega, J.; Yue, M.; Gassiep, I. Haematologic and Urologic Manifestations of Cryptococcus neoformans. Case Rep. Infect. Dis. 2024, 2024, 2245391. [Google Scholar] [CrossRef]
  37. Qin, Y.; Zou, X.; Jin, Y.; Li, J.; Cai, Q. Cryptococcus neoformans Osteomyelitis of the Right Ankle Diagnosed by Metagenomic Next-Generation Sequencing in a HIV-Negative Patient with Tuberculous Lymphadenitis and Pulmonary Tuberculosis: A Case Report and Recent Literature Review. Infect. Drug Resist. 2024, 17, 3805–3812. [Google Scholar] [CrossRef]
  38. Francis, V.I.; Liddle, C.; Camacho, E.; Kulkarni, M.; Junior, S.R.S.; Harvey, J.A.; Ballou, E.R.; Thomson, D.D.; Brown, G.D.; Hardwick, J.M.; et al. Cryptococcus neoformans rapidly invades the murine brain by sequential breaching of airway and endothelial tissues barriers, followed by engulfment by microglia. mBio 2024, 15, e0307823. [Google Scholar] [CrossRef]
  39. Bing, J.; Du, H.; Guo, P.; Hu, T.; Xiao, M.; Lu, S.; Nobile, C.J.; Chu, H.; Huang, G. Candida auris-associated hospitalizations and outbreaks, China, 2018–2023. Emerg. Microbes Infect. 2024, 13, 2302843. [Google Scholar] [CrossRef]
  40. Huang, Y.; Su, Y.; Chen, X.; Xiao, M.; Xu, Y. Insight into Virulence and Mechanisms of Amphotericin B Resistance in the Candida haemulonii Complex. J. Fungi 2024, 10, 615. [Google Scholar] [CrossRef]
  41. Cherniak, R.; Sundstrom, J.B. Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect. Immun. 1994, 62, 1507–1512. [Google Scholar] [CrossRef] [PubMed]
  42. Cox, G.M.; Mukherjee, J.; Cole, G.T.; Casadevall, A.; Perfect, J.R. Urease as a virulence factor in experimental cryptococcosis. Infect. Immun. 2000, 68, 443–448. [Google Scholar] [CrossRef]
  43. Cox, G.M.; McDade, H.C.; Chen, S.C.; Tucker, S.C.; Gottfredsson, M.; Wright, L.C.; Sorrell, T.C.; Leidich, S.D.; Casadevall, A.; Ghannoum, M.A.; et al. Extracellular phospholipase activity is a virulence factor for Cryptococcus neoformans. Mol. Microbiol. 2001, 39, 166–175. [Google Scholar] [CrossRef] [PubMed]
  44. Olszewski, M.A.; Noverr, M.C.; Chen, G.H.; Toews, G.B.; Cox, G.M.; Perfect, J.R.; Huffnagle, G.B. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion. Am. J. Pathol. 2004, 164, 1761–1771. [Google Scholar] [CrossRef] [PubMed]
  45. Santangelo, R.; Zoellner, H.; Sorrell, T.; Wilson, C.; Donald, C.; Djordjevic, J.; Shounan, Y.; Wright, L. Role of extracellular phospholipases and mononuclear phagocytes in dissemination of cryptococcosis in a murine model. Infect. Immun. 2004, 72, 2229–2239. [Google Scholar] [CrossRef]
  46. Villena, S.N.; Pinheiro, R.O.; Pinheiro, C.S.; Nunes, M.P.; Takiya, C.M.; DosReis, G.A.; Previato, J.O.; Mendonça-Previato, L.; Freire-de-Lima, C.G. Capsular polysaccharides galactoxylomannan and glucuronoxylomannan from Cryptococcus neoformans induce macrophage apoptosis mediated by Fas ligand. Cell Microbiol. 2008, 10, 1274–1285. [Google Scholar] [CrossRef]
  47. Chrisman, C.J.; Albuquerque, P.; Guimaraes, A.J.; Nieves, E.; Casadevall, A. Phospholipids trigger Cryptococcus neoformans capsular enlargement during interactions with amoebae and macrophages. PLoS Pathog. 2011, 7, e1002047. [Google Scholar] [CrossRef]
  48. Ferreira, G.F.; Santos, J.R.; Costa, M.C.; Holanda, R.A.; Denadai, Â.M.; Freitas, G.J.; Santos, Á.R.; Tavares, P.B.; Paixão, T.A.; Santos, D.A. Heteroresistance to Itraconazole Alters the Morphology and Increases the Virulence of Cryptococcus gattii. Antimicrob. Agents Chemother. 2015, 59, 4600–4609. [Google Scholar] [CrossRef]
  49. Dambuza, I.M.; Drake, T.; Chapuis, A.; Zhou, X.; Correia, J.; Taylor-Smith, L.; LeGrave, N.; Rasmussen, T.; Fisher, M.C.; Bicanic, T.; et al. The Cryptococcus neoformans Titan cell is an inducible and regulated morphotype underlying pathogenesis. PLoS Pathog. 2018, 14, e1006978. [Google Scholar] [CrossRef]
  50. Vij, R.; Cordero, R.J.B.; Casadevall, A. The Buoyancy of Cryptococcus neoformans Is Affected by Capsule Size. mSphere 2018, 3, e00534-18. [Google Scholar] [CrossRef]
  51. Yu, S.J.; Chang, Y.L.; Chen, Y.L. Deletion of ADA2 Increases Antifungal Drug Susceptibility and Virulence in Candida glabrata. Antimicrob. Agents Chemother. 2018, 62, e01924-17. [Google Scholar] [CrossRef] [PubMed]
  52. Giles, S.S.; Batinic-Haberle, I.; Perfect, J.R.; Cox, G.M. Cryptococcus neoformans mitochondrial superoxide dismutase: An essential link between antioxidant function and high-temperature growth. Eukaryot. Cell 2005, 4, 46–54. [Google Scholar] [CrossRef]
  53. Giles, S.S.; Perfect, J.R.; Cox, G.M. Cytochrome c peroxidase contributes to the antioxidant defense of Cryptococcus neoformans. Fungal Genet. Biol. 2005, 42, 20–29. [Google Scholar] [CrossRef] [PubMed]
  54. Sun, T.S.; Ju, X.; Gao, H.L.; Wang, T.; Thiele, D.J.; Li, J.Y.; Wang, Z.Y.; Ding, C. Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis. Nat. Commun. 2014, 5, 5550. [Google Scholar] [CrossRef]
  55. Evans, R.J.; Li, Z.; Hughes, W.S.; Djordjevic, J.T.; Nielsen, K.; May, R.C. Cryptococcal phospholipase B1 is required for intracellular proliferation and control of titan cell morphology during macrophage infection. Infect. Immun. 2015, 83, 1296–1304. [Google Scholar] [CrossRef]
  56. Gerstein, A.C.; Fu, M.S.; Mukaremera, L.; Li, Z.; Ormerod, K.L.; Fraser, J.A.; Berman, J.; Nielsen, K. Polyploid titan cells produce haploid and aneuploid progeny to promote stress adaptation. mBio 2015, 6, e01340-15. [Google Scholar] [CrossRef]
  57. Bosch, C.; Toplis, B.; Vreulink, J.M.; Volschenk, H.; Botha, A. Nitrogen concentration affects amphotericin B and fluconazole tolerance of pathogenic cryptococci. FEMS Yeast Res. 2020, 20, foaa010. [Google Scholar] [CrossRef]
  58. Schatzman, S.S.; Peterson, R.L.; Teka, M.; He, B.; Cabelli, D.E.; Cormack, B.P.; Culotta, V.C. Copper-only superoxide dismutase enzymes and iron starvation stress in Candida fungal pathogens. J. Biol. Chem. 2020, 295, 570–583. [Google Scholar] [CrossRef]
  59. Horianopoulos, L.C.; Lee, C.W.J.; Hu, G.; Caza, M.; Kronstad, J.W. Dnj1 Promotes Virulence in Cryptococcus neoformans by Maintaining Robust Endoplasmic Reticulum Homeostasis Under Temperature Stress. Front. Microbiol. 2021, 12, 727039. [Google Scholar] [CrossRef]
  60. Sun, T.; Li, Y.; Li, Y.; Li, H.; Gong, Y.; Wu, J.; Ning, Y.; Ding, C.; Xu, Y. Proteomic Analysis of Copper Toxicity in Human Fungal Pathogen Cryptococcus neoformans. Front. Cell Infect. Microbiol. 2021, 11, 662404. [Google Scholar] [CrossRef]
  61. Bosch, C.; Volschenk, H.; Botha, A. The copper transporter, Ctr4, and the microtubule-associated protein, Cgp1, are important for Cryptococcus neoformans adaptation to nitrogen availability. FEMS Microbiol. Lett. 2021, 368, fnab134. [Google Scholar] [CrossRef] [PubMed]
  62. Hamed, M.F.; Araújo, G.R.S.; Munzen, M.E.; Reguera-Gomez, M.; Epstein, C.; Lee, H.H.; Frases, S.; Martinez, L.R. Phospholipase B Is Critical for Cryptococcus neoformans Survival in the Central Nervous System. mBio 2023, 14, e0264022. [Google Scholar] [CrossRef] [PubMed]
  63. Xue, P.; Sánchez-León, E.; Hu, G.; Lee, C.W.J.; Black, B.; Brisland, A.; Li, H.; Jung, W.H.; Kronstad, J.W. The interplay between electron transport chain function and iron regulatory factors influences melanin formation in Cryptococcus neoformans. mSphere 2024, 9, e0025024. [Google Scholar] [CrossRef] [PubMed]
  64. Chadwick, B.J.; Ristow, L.C.; Xie, X.; Krysan, D.J.; Lin, X. Discovery of CO2 tolerance genes associated with virulence in the fungal pathogen Cryptococcus neoformans. Nat. Microbiol. 2024, 9, 2684–2695. [Google Scholar] [CrossRef] [PubMed]
  65. Folorunso, O.S.; Sebolai, O.M. A Limited Number of Amino Acid Permeases Are Crucial for Cryptococcus neoformans Survival and Virulence. Int. J. Microbiol. 2024, 2024, 5566438. [Google Scholar] [CrossRef]
  66. Lee, C.W.J.; Brisland, A.; Qu, X.; Horianopoulos, L.C.; Hu, G.; Mayer, F.L.; Kronstad, J.W. Loss of Opi3 causes a lipid imbalance that influences the virulence traits of Cryptococcus neoformans but not cryptococcosis. Front. Cell Infect. Microbiol. 2024, 14, 1448229. [Google Scholar] [CrossRef]
  67. Peterson, P.P.; Choi, J.T.; Fu, C.; Cowen, L.E.; Sun, S.; Bahn, Y.S.; Heitman, J. The Cryptococcus neoformans STRIPAK complex controls genome stability, sexual development, and virulence. PLoS Pathog. 2024, 20, e1012735. [Google Scholar] [CrossRef]
  68. Ni, Y.; Qiao, Y.; Tian, X.; Li, H.; Meng, Y.; Li, C.; Du, W.; Sun, T.; Zhu, K.; Huang, W.; et al. Unraveling the mechanism of thermotolerance by Set302 in Cryptococcus neoformans. Microbiol. Spectr. 2024, 12, e0420223. [Google Scholar] [CrossRef]
  69. Hoffman, H.J.; McClelland, E.E. Measuring Urease and Phospholipase Secretion in Cryptococcus neoformans. Methods Mol. Biol. 2024, 2775, 269–275. [Google Scholar] [CrossRef]
  70. Baomo, L.; Guofen, Z.; Jie, D.; Liu, X.; Shuru, C.; Jing, L. Disseminated cryptococcosis in a patient with idiopathic CD4 + T lymphocytopenia presenting as prostate and adrenal nodules: Diagnosis from pathology and mNGS, a case report. BMC Infect. Dis. 2024, 24, 26. [Google Scholar] [CrossRef]
  71. Horton Embrey, P.; Long, A.; Alfattal, R.; Qiu, S.; Hornak, J.P. Isolated cryptococcal osteomyelitis in the setting of immune reconstitution inflammatory syndrome. Ther. Adv. Infect. Dis. 2024, 11, 20499361241230149. [Google Scholar] [CrossRef] [PubMed]
  72. Saito, Z.; Ito, T.; Imakita, T.; Oi, I.; Kanai, O.; Fujita, K. Organizing pneumonia secondary to pulmonary cryptococcosis in immunocompromised patient. Respir. Med. Case Rep. 2024, 47, 101975. [Google Scholar] [CrossRef]
  73. Mbangiwa, T.; Sturny-Leclère, A.; Lechiile, K.; Kajanga, C.; Boyer-Chammard, T.; Hoving, J.C.; Leeme, T.; Moyo, M.; Youssouf, N.; Lawrence, D.S.; et al. Development and validation of quantitative PCR assays for HIV-associated cryptococcal meningitis in sub-Saharan Africa: A diagnostic accuracy study. Lancet Microbe 2024, 5, e261–e271. [Google Scholar] [CrossRef]
  74. Odds, F.C.; Cheesman, S.L.; Abbott, A.B. Suppression of ATP in Candida albicans by imidazole and derivative antifungal agents. Sabouraudia 1985, 23, 415–424. [Google Scholar] [CrossRef]
  75. Saag, M.S.; Dismukes, W.E. Azole antifungal agents: Emphasis on new triazoles. Antimicrob. Agents Chemother. 1988, 32, 1–8. [Google Scholar] [CrossRef]
  76. Patil, A.; Majumdar, S. Echinocandins in antifungal pharmacotherapy. J. Pharm. Pharmacol. 2017, 69, 1635–1660. [Google Scholar] [CrossRef]
  77. Mesa-Arango, A.C.; Scorzoni, L.; Zaragoza, O. It only takes one to do many jobs: Amphotericin B as antifungal and immunomodulatory drug. Front. Microbiol. 2012, 3, 286. [Google Scholar] [CrossRef]
  78. Shivarathri, R.; Jenull, S.; Chauhan, M.; Singh, A.; Mazumdar, R.; Chowdhary, A.; Kuchler, K.; Chauhan, N. Comparative Transcriptomics Reveal Possible Mechanisms of Amphotericin B Resistance in Candida auris. Antimicrob. Agents Chemother. 2022, 66, e0227621. [Google Scholar] [CrossRef]
  79. Perlin, D.S.; Rautemaa-Richardson, R.; Alastruey-Izquierdo, A. The global problem of antifungal resistance: Prevalence, mechanisms, and management. Lancet Infect. Dis. 2017, 17, e383–e392. [Google Scholar] [CrossRef]
  80. Polak, A.; Scholer, H.J. Mode of action of 5-fluorocytosine and mechanisms of resistance. Chemotherapy 1975, 21, 113–130. [Google Scholar] [CrossRef]
  81. Gleason, M.K.; Fraenkel-Conrat, H. Biological consequences of incorporation of 5-fluorocytidine in the RNA of 5-fluorouracil-treated eukaryotic cells. Proc. Natl. Acad. Sci. USA 1976, 73, 1528–1531. [Google Scholar] [CrossRef] [PubMed]
  82. Jacobs, S.E.; Jacobs, J.L.; Dennis, E.K.; Taimur, S.; Rana, M.; Patel, D.; Gitman, M.; Patel, G.; Schaefer, S.; Iyer, K.; et al. Candida auris Pan-Drug-Resistant to Four Classes of Antifungal Agents. Antimicrob. Agents Chemother. 2022, 66, e0005322. [Google Scholar] [CrossRef] [PubMed]
  83. Oliveira, N.K.; Bhattacharya, S.; Gambhir, R.; Joshi, M.; Fries, B.C. Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans. J Fungi 2022, 8, 677. [Google Scholar] [CrossRef] [PubMed]
  84. Rogers, T.R.; Verweij, P.E.; Castanheira, M.; Dannaoui, E.; White, P.L.; Arendrup, M.C. Molecular mechanisms of acquired antifungal drug resistance in principal fungal pathogens and EUCAST guidance for their laboratory detection and clinical implications. J. Antimicrob. Chemother. 2022, 77, 2053–2073. [Google Scholar] [CrossRef]
  85. Štefánek, M.; Garaiová, M.; Valček, A.; Jordao, L.; Bujdáková, H. Comparative Analysis of Two Candida parapsilosis Isolates Originating from the Same Patient Harbouring the Y132F and R398I Mutations in the ERG11 Gene. Cells 2023, 12, 1579. [Google Scholar] [CrossRef]
  86. Pristov, K.E.; Ghannoum, M.A. Resistance of Candida to azoles and echinocandins worldwide. Clin. Microbiol. Infect. 2019, 25, 792–798. [Google Scholar] [CrossRef]
  87. Fattouh, N.; Hdayed, D.; Geukgeuzian, G.; Tokajian, S.; Khalaf, R.A. Molecular mechanism of fluconazole resistance and pathogenicity attributes of Lebanese Candida albicans hospital isolates. Fungal Genet. Biol. 2021, 153, 103575. [Google Scholar] [CrossRef]
  88. Bosco-Borgeat, M.E.; Mazza, M.; Taverna, C.G.; Córdoba, S.; Murisengo, O.A.; Vivot, W.; Davel, G. Amino acid substitution in Cryptococcus neoformans lanosterol 14-α-demethylase involved in fluconazole resistance in clinical isolates. Rev. Argent. Microbiol. 2016, 48, 137–142. [Google Scholar] [CrossRef]
  89. Diaz-Guerra, T.M.; Mellado, E.; Cuenca-Estrella, M.; Rodriguez-Tudela, J.L. A point mutation in the 14alpha-sterol demethylase gene cyp51A contributes to itraconazole resistance in Aspergillus fumigatus. Antimicrob. Agents Chemother. 2003, 47, 1120–1124. [Google Scholar] [CrossRef]
  90. Garcia-Effron, G.; Katiyar, S.K.; Park, S.; Edlind, T.D.; Perlin, D.S. A naturally occurring proline-to-alanine amino acid change in Fks1p in Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis accounts for reduced echinocandin susceptibility. Antimicrob. Agents Chemother. 2008, 52, 2305–2312. [Google Scholar] [CrossRef]
  91. Healey, K.R.; Perlin, D.S. Fungal Resistance to Echinocandins and the MDR Phenomenon in Candida glabrata. J. Fungi 2018, 4, 105. [Google Scholar] [CrossRef] [PubMed]
  92. Singh-Babak, S.D.; Babak, T.; Diezmann, S.; Hill, J.A.; Xie, J.L.; Chen, Y.L.; Poutanen, S.M.; Rennie, R.P.; Heitman, J.; Cowen, L.E. Global analysis of the evolution and mechanism of echinocandin resistance in Candida glabrata. PLoS Pathog. 2012, 8, e1002718. [Google Scholar] [CrossRef]
  93. Delma, F.Z.; Al-Hatmi, A.M.S.; Brüggemann, R.J.M.; Melchers, W.J.G.; de Hoog, S.; Verweij, P.E.; Buil, J.B. Molecular Mechanisms of 5-Fluorocytosine Resistance in Yeasts and Filamentous Fungi. J. Fungi 2021, 7, 909. [Google Scholar] [CrossRef] [PubMed]
  94. Kannan, A.; Asner, S.A.; Trachsel, E.; Kelly, S.; Parker, J.; Sanglard, D. Comparative Genomics for the Elucidation of Multidrug Resistance in Candida lusitaniae. mBio 2019, 10, e02512-19. [Google Scholar] [CrossRef] [PubMed]
  95. Billmyre, R.B.; Applen Clancey, S.; Li, L.X.; Doering, T.L.; Heitman, J. 5-fluorocytosine resistance is associated with hypermutation and alterations in capsule biosynthesis in Cryptococcus. Nat. Commun. 2020, 11, 127. [Google Scholar] [CrossRef]
  96. Silva, L.N.; Oliveira, S.S.C.; Magalhães, L.B.; Andrade Neto, V.V.; Torres-Santos, E.C.; Carvalho, M.D.C.; Pereira, M.D.; Branquinha, M.H.; Santos, A.L.S. Unmasking the Amphotericin B Resistance Mechanisms in Candida haemulonii Species Complex. ACS Infect. Dis. 2020, 6, 1273–1282. [Google Scholar] [CrossRef]
  97. Huang, Y.; Zhang, Y.; Yang, S.; Lu, H.; Yu, H.; Wang, X.; Jia, X.; Tang, D.; Wu, L.; Huang, S.; et al. Epidemiology of cryptococcal meningitis and fluconazole heteroresistance in Cryptococcus neoformans isolates from a teaching hospital in southwestern China. Microbiol. Spectr. 2024, 12, e0072524. [Google Scholar] [CrossRef]
  98. Lyons, N.; Berman, J. Protocols for Measuring Tolerant and Heteroresistant Drug Responses of Pathogenic Yeasts. Methods Mol. Biol. 2023, 2658, 67–79. [Google Scholar] [CrossRef]
  99. Ben-Ami, R.; Zimmerman, O.; Finn, T.; Amit, S.; Novikov, A.; Wertheimer, N.; Lurie-Weinberger, M.; Berman, J. Heteroresistance to Fluconazole Is a Continuously Distributed Phenotype among Candida glabrata Clinical Strains Associated with In Vivo Persistence. mBio 2016, 7, e00655-16. [Google Scholar] [CrossRef]
  100. Levy, S.F.; Ziv, N.; Siegal, M.L. Bet hedging in yeast by heterogeneous, age-correlated expression of a stress protectant. PLoS Biol. 2012, 10, e1001325. [Google Scholar] [CrossRef]
  101. Claudino, A.L.; Peixoto Junior, R.F.; Melhem, M.S.; Szeszs, M.W.; Lyon, J.P.; Chavasco, J.K.; Franco, M.C. Mutants with heteroresistance to amphotericin B and fluconazole in Candida. Braz. J. Microbiol. 2009, 40, 943–951. [Google Scholar] [CrossRef] [PubMed]
  102. Sionov, E.; Chang, Y.C.; Garraffo, H.M.; Kwon-Chung, K.J. Heteroresistance to fluconazole in Cryptococcus neoformans is intrinsic and associated with virulence. Antimicrob. Agents Chemother. 2009, 53, 2804–2815. [Google Scholar] [CrossRef] [PubMed]
  103. Balaban, N.Q.; Helaine, S.; Lewis, K.; Ackermann, M.; Aldridge, B.; Andersson, D.I.; Brynildsen, M.P.; Bumann, D.; Camilli, A.; Collins, J.J.; et al. Publisher Correction: Definitions and guidelines for research on antibiotic persistence. Nat. Rev. Microbiol. 2019, 17, 460. [Google Scholar] [CrossRef] [PubMed]
  104. Kester, J.C.; Fortune, S.M. Persisters and beyond: Mechanisms of phenotypic drug resistance and drug tolerance in bacteria. Crit. Rev. Biochem. Mol. Biol. 2014, 49, 91–101. [Google Scholar] [CrossRef]
  105. Berman, J.; Krysan, D.J. Drug resistance and tolerance in fungi. Nat. Rev. Microbiol. 2020, 18, 319–331. [Google Scholar] [CrossRef]
  106. Chen, L.; Zhang, L.; Xie, Y.; Wang, Y.; Tian, X.; Fang, W.; Xue, X.; Wang, L. Confronting antifungal resistance, tolerance, and persistence: Advances in drug target discovery and delivery systems. Adv. Drug Deliv. Rev. 2023, 200, 115007. [Google Scholar] [CrossRef]
  107. Rasouli Koohi, S.; Shankarnarayan, S.A.; Galon, C.M.; Charlebois, D.A. Identification and Elimination of Antifungal Tolerance in Candida auris. Biomedicines 2023, 11, 898. [Google Scholar] [CrossRef]
  108. Rosenberg, A.; Ene, I.V.; Bibi, M.; Zakin, S.; Segal, E.S.; Ziv, N.; Dahan, A.M.; Colombo, A.L.; Bennett, R.J.; Berman, J. Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemia. Nat. Commun. 2018, 9, 2470. [Google Scholar] [CrossRef]
  109. Moldoveanu, A.L.; Rycroft, J.A.; Helaine, S. Impact of bacterial persisters on their host. Curr. Opin. Microbiol. 2021, 59, 65–71. [Google Scholar] [CrossRef]
  110. Arastehfar, A.; Daneshnia, F.; Cabrera, N.; Penalva-Lopez, S.; Sarathy, J.; Zimmerman, M.; Shor, E.; Perlin, D.S. Macrophage internalization creates a multidrug-tolerant fungal persister reservoir and facilitates the emergence of drug resistance. Nat. Commun. 2023, 14, 1183. [Google Scholar] [CrossRef]
  111. Lewis, K. Persister cells. Annu. Rev. Microbiol. 2010, 64, 357–372. [Google Scholar] [CrossRef] [PubMed]
  112. Ke, W.; Xie, Y.; Chen, Y.; Ding, H.; Ye, L.; Qiu, H.; Li, H.; Zhang, L.; Chen, L.; Tian, X.; et al. Fungicide-tolerant persister formation during cryptococcal pulmonary infection. Cell Host Microbe 2024, 32, 276–289.e7. [Google Scholar] [CrossRef]
  113. Levinson, T.; Dahan, A.; Novikov, A.; Paran, Y.; Berman, J.; Ben-Ami, R. Impact of tolerance to fluconazole on treatment response in Candida albicans bloodstream infection. Mycoses 2021, 64, 78–85. [Google Scholar] [CrossRef] [PubMed]
  114. Yang, F.; Scopel, E.F.C.; Li, H.; Sun, L.L.; Kawar, N.; Cao, Y.B.; Jiang, Y.Y.; Berman, J. Antifungal Tolerance and Resistance Emerge at Distinct Drug Concentrations and Rely upon Different Aneuploid Chromosomes. mBio 2023, 14, e0022723. [Google Scholar] [CrossRef] [PubMed]
  115. Yang, F.; Gritsenko, V.; Slor Futterman, Y.; Gao, L.; Zhen, C.; Lu, H.; Jiang, Y.Y.; Berman, J. Tunicamycin Potentiates Antifungal Drug Tolerance via Aneuploidy in Candida albicans. mBio 2021, 12, e0227221. [Google Scholar] [CrossRef]
  116. Chen, L.; Tian, X.; Zhang, L.; Wang, W.; Hu, P.; Ma, Z.; Li, Y.; Li, S.; Shen, Z.; Fan, X.; et al. Brain glucose induces tolerance of Cryptococcus neoformans to amphotericin B during meningitis. Nat. Microbiol. 2024, 9, 346–358. [Google Scholar] [CrossRef]
  117. Yoo, K.; Bhattacharya, S.; Oliveira, N.K.; Pereira de Sa, N.; Matos, G.S.; Del Poeta, M.; Fries, B.C. With age comes resilience: How mitochondrial modulation drives age-associated fluconazole tolerance in Cryptococcus neoformans. mBio 2024, 15, e0184724. [Google Scholar] [CrossRef]
  118. Yang, F.; Zhang, L.; Wakabayashi, H.; Myers, J.; Jiang, Y.; Cao, Y.; Jimenez-Ortigosa, C.; Perlin, D.S.; Rustchenko, E. Tolerance to Caspofungin in Candida albicans Is Associated with at Least Three Distinctive Mechanisms That Govern Expression of FKS Genes and Cell Wall Remodeling. Antimicrob. Agents Chemother. 2017, 61, e00071-17. [Google Scholar] [CrossRef]
  119. Prasetyoputri, A.; Jarrad, A.M.; Cooper, M.A.; Blaskovich, M.A.T. The Eagle Effect and Antibiotic-Induced Persistence: Two Sides of the Same Coin? Trends Microbiol. 2019, 27, 339–354. [Google Scholar] [CrossRef]
  120. Singh, S.D.; Robbins, N.; Zaas, A.K.; Schell, W.A.; Perfect, J.R.; Cowen, L.E. Hsp90 governs echinocandin resistance in the pathogenic yeast Candida albicans via calcineurin. PLoS Pathog. 2009, 5, e1000532. [Google Scholar] [CrossRef]
  121. Iyer, K.R.; Robbins, N.; Cowen, L.E. The role of Candida albicans stress response pathways in antifungal tolerance and resistance. iScience 2022, 25, 103953. [Google Scholar] [CrossRef] [PubMed]
  122. Dumeaux, V.; Massahi, S.; Bettauer, V.; Mottola, A.; Dukovny, A.; Khurdia, S.S.; Costa, A.; Omran, R.P.; Simpson, S.; Xie, J.L.; et al. Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to antifungal compounds. eLife 2023, 12, e81406. [Google Scholar] [CrossRef] [PubMed]
  123. Defraine, V.; Fauvart, M.; Michiels, J. Fighting bacterial persistence: Current and emerging anti-persister strategies and therapeutics. Drug Resist. Updat. 2018, 38, 12–26. [Google Scholar] [CrossRef]
  124. Lewis, K. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 2007, 5, 48–56. [Google Scholar] [CrossRef]
  125. da Silva, M.A.; Baronetti, J.L.; Páez, P.L.; Paraje, M.G. Oxidative Imbalance in Candida tropicalis Biofilms and Its Relation with Persister Cells. Front. Microbiol. 2020, 11, 598834. [Google Scholar] [CrossRef]
  126. Dasilva, M.A.; Andrada, K.F.C.; Torales, M.M.; Hughes, I.M.; Pez, P.; García-Martínez, J.C.; Paraje, M.G. Synergistic activity of gold nanoparticles with amphotericin B on persister cells of Candida tropicalis biofilms. J. Nanobiotechnol. 2024, 22, 254. [Google Scholar] [CrossRef]
  127. El Meouche, I.; Jain, P.; Jolly, M.K.; Capp, J.P. Drug tolerance and persistence in bacteria, fungi and cancer cells: Role of non-genetic heterogeneity. Transl. Oncol. 2024, 49, 102069. [Google Scholar] [CrossRef]
  128. Zou, P.; Liu, J.; Li, P.; Luan, Q. Antifungal Activity, Synergism with Fluconazole or Amphotericin B and Potential Mechanism of Direct Current against Candida albicans Biofilms and Persisters. Antibiotics 2024, 13, 521. [Google Scholar] [CrossRef]
  129. Selmecki, A.; Forche, A.; Berman, J. Aneuploidy and isochromosome formation in drug-resistant Candida albicans. Science 2006, 313, 367–370. [Google Scholar] [CrossRef]
  130. Sah, S.K.; Hayes, J.J.; Rustchenko, E. The role of aneuploidy in the emergence of echinocandin resistance in human fungal pathogen Candida albicans. PLoS Pathog. 2021, 17, e1009564. [Google Scholar] [CrossRef]
  131. Harrison, B.D.; Hashemi, J.; Bibi, M.; Pulver, R.; Bavli, D.; Nahmias, Y.; Wellington, M.; Sapiro, G.; Berman, J. A tetraploid intermediate precedes aneuploid formation in yeasts exposed to fluconazole. PLoS Biol. 2014, 12, e1001815. [Google Scholar] [CrossRef] [PubMed]
  132. Yang, F.; Teoh, F.; Tan, A.S.M.; Cao, Y.; Pavelka, N.; Berman, J. Aneuploidy Enables Cross-Adaptation to Unrelated Drugs. Mol. Biol. Evol. 2019, 36, 1768–1782. [Google Scholar] [CrossRef] [PubMed]
  133. Husain, F.; Yadav, A.; Sah, S.K.; Hayes, J.J.; Rustchenko, E. Candida albicans Strains Adapted to Caspofungin Due to Aneuploidy Become Highly Tolerant under Continued Drug Pressure. Microorganisms 2022, 11, 23. [Google Scholar] [CrossRef] [PubMed]
  134. Ksiezopolska, E.; Schikora-Tamarit, M.; Carlos Nunez-Rodriguez, J.; Gabaldón, T. Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata). Front. Cell Infect. Microbiol. 2024, 14, 1416509. [Google Scholar] [CrossRef]
  135. Todd, R.T.; Selmecki, A. Expandable and reversible copy number amplification drives rapid adaptation to antifungal drugs. Elife 2020, 9, e58349. [Google Scholar] [CrossRef]
  136. Burrack, L.S.; Todd, R.T.; Soisangwan, N.; Wiederhold, N.P.; Selmecki, A. Genomic Diversity across Candida auris Clinical Isolates Shapes Rapid Development of Antifungal Resistance In Vitro and In Vivo. mBio 2022, 13, e0084222. [Google Scholar] [CrossRef]
  137. Anderson, J.B.; Sirjusingh, C.; Ricker, N. Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae. Genetics 2004, 168, 1915–1923. [Google Scholar] [CrossRef]
  138. D’Souza, C.A.; Kronstad, J.W.; Taylor, G.; Warren, R.; Yuen, M.; Hu, G.; Jung, W.H.; Sham, A.; Kidd, S.E.; Tangen, K.; et al. Genome variation in Cryptococcus gattii, an emerging pathogen of immunocompetent hosts. mBio 2011, 2, e00342-10. [Google Scholar] [CrossRef]
  139. Zhang, Z.H.; Sun, L.L.; Fu, B.Q.; Deng, J.; Jia, C.L.; Miao, M.X.; Yang, F.; Cao, Y.B.; Yan, T.H. Aneuploidy underlies brefeldin A-induced antifungal drug resistance in Cryptococcus neoformans. Front. Cell Infect. Microbiol. 2024, 14, 1397724. [Google Scholar] [CrossRef]
  140. Cao, C.; Wang, K.; Wang, Y.; Liu, T.B.; Rivera, A.; Xue, C. Ubiquitin proteolysis of a CDK-related kinase regulates titan cell formation and virulence in the fungal pathogen Cryptococcus neoformans. Nat. Commun. 2022, 13, 6397. [Google Scholar] [CrossRef]
  141. García-Barbazán, I.; Torres-Cano, A.; García-Rodas, R.; Sachse, M.; Luque, D.; Megías, D.; Zaragoza, O. Accumulation of endogenous free radicals is required to induce titan-like cell formation in Cryptococcus neoformans. mBio 2024, 15, e0254923. [Google Scholar] [CrossRef] [PubMed]
  142. Stone, N.R.; Rhodes, J.; Fisher, M.C.; Mfinanga, S.; Kivuyo, S.; Rugemalila, J.; Segal, E.S.; Needleman, L.; Molloy, S.F.; Kwon-Chung, J.; et al. Dynamic ploidy changes drive fluconazole resistance in human cryptococcal meningitis. J. Clin. Investig. 2019, 129, 999–1014. [Google Scholar] [CrossRef]
  143. Ngamskulrungroj, P.; Chang, Y.; Hansen, B.; Bugge, C.; Fischer, E.; Kwon-Chung, K.J. Characterization of the chromosome 4 genes that affect fluconazole-induced disomy formation in Cryptococcus neoformans. PLoS ONE 2012, 7, e33022. [Google Scholar] [CrossRef] [PubMed]
  144. Sionov, E.; Lee, H.; Chang, Y.C.; Kwon-Chung, K.J. Cryptococcus neoformans overcomes stress of azole drugs by formation of disomy in specific multiple chromosomes. PLoS Pathog. 2010, 6, e1000848. [Google Scholar] [CrossRef] [PubMed]
  145. Marr, K.A.; Lyons, C.N.; Rustad, T.R.; Bowden, R.A.; White, T.C. Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR. Antimicrob. Agents Chemother. 1998, 42, 2584–2589. [Google Scholar] [CrossRef]
  146. Sykes, J.E.; Hodge, G.; Singapuri, A.; Yang, M.L.; Gelli, A.; Thompson, G.R., 3rd. In vivo development of fluconazole resistance in serial Cryptococcus gattii isolates from a cat. Med. Mycol. 2017, 55, 396–401. [Google Scholar] [CrossRef]
  147. Altamirano, S.; Simmons, C.; Kozubowski, L. Colony and Single Cell Level Analysis of the Heterogeneous Response of Cryptococcus neoformans to Fluconazole. Front. Cell Infect. Microbiol. 2018, 8, 203. [Google Scholar] [CrossRef]
  148. Ning, Y.; Dai, R.; Zhang, L.; Xu, Y.; Xiao, M. Copy number variants of ERG11: Mechanism of azole resistance in Candida parapsilosis. Lancet Microbe 2024, 5, e10. [Google Scholar] [CrossRef]
  149. Fan, X.; Dai, R.C.; Zhang, S.; Geng, Y.Y.; Kang, M.; Guo, D.W.; Mei, Y.N.; Pan, Y.H.; Sun, Z.Y.; Xu, Y.C.; et al. Tandem gene duplications contributed to high-level azole resistance in a rapidly expanding Candida tropicalis population. Nat. Commun. 2023, 14, 8369. [Google Scholar] [CrossRef]
  150. Yang, F.; Berman, J. Beyond resistance: Antifungal heteroresistance and antifungal tolerance in fungal pathogens. Curr. Opin. Microbiol. 2024, 78, 102439. [Google Scholar] [CrossRef]
  151. Tsai, H.J.; Nelliat, A. A Double-Edged Sword: Aneuploidy is a Prevalent Strategy in Fungal Adaptation. Genes 2019, 10, 787. [Google Scholar] [CrossRef] [PubMed]
  152. Vande Zande, P.; Zhou, X.; Selmecki, A. The Dynamic Fungal Genome: Polyploidy, Aneuploidy and Copy Number Variation in Response to Stress. Annu. Rev. Microbiol. 2023, 77, 341–361. [Google Scholar] [CrossRef] [PubMed]
  153. Yang, F.; Gritsenko, V.; Lu, H.; Zhen, C.; Gao, L.; Berman, J.; Jiang, Y.Y. Adaptation to Fluconazole via Aneuploidy Enables Cross-Adaptation to Amphotericin B and Flucytosine in Cryptococcus neoformans. Microbiol. Spectr. 2021, 9, e0072321. [Google Scholar] [CrossRef] [PubMed]
  154. Gohar, A.A.; Badali, H.; Shokohi, T.; Nabili, M.; Amirrajab, N.; Moazeni, M. Expression Patterns of ABC Transporter Genes in Fluconazole-Resistant Candida glabrata. Mycopathologia 2017, 182, 273–284. [Google Scholar] [CrossRef]
  155. Beach, R.R.; Ricci-Tam, C.; Brennan, C.M.; Moomau, C.A.; Hsu, P.H.; Hua, B.; Silberman, R.E.; Springer, M.; Amon, A. Aneuploidy Causes Non-genetic Individuality. Cell 2017, 169, 229–242.e21. [Google Scholar] [CrossRef]
  156. Coste, A.T.; Karababa, M.; Ischer, F.; Bille, J.; Sanglard, D. TAC1, transcriptional activator of CDR genes, is a new transcription factor involved in the regulation of Candida albicans ABC transporters CDR1 and CDR2. Eukaryot. Cell 2004, 3, 1639–1652. [Google Scholar] [CrossRef]
  157. Zheng, Q.; Liu, J.; Qin, J.; Wang, B.; Bing, J.; Du, H.; Li, M.; Yu, F.; Huang, G. Ploidy Variation and Spontaneous Haploid-Diploid Switching of Candida glabrata Clinical Isolates. mSphere 2022, 7, e0026022. [Google Scholar] [CrossRef]
  158. Moreira, I.M.B.; Cortez, A.C.A.; de Souza, É.S.; Pinheiro, S.B.; de Souza Oliveira, J.G.; Sadahiro, A.; Cruz, K.S.; Matsuura, A.B.J.; Melhem, M.S.C.; Frickmann, H.; et al. Investigation of fluconazole heteroresistance in clinical and environmental isolates of Cryptococcus neoformans complex and Cryptococcus gattii complex in the state of Amazonas, Brazil. Med. Mycol. 2022, 60, myac005. [Google Scholar] [CrossRef]
  159. Varma, A.; Kwon-Chung, K.J. Heteroresistance of Cryptococcus gattii to fluconazole. Antimicrob. Agents Chemother. 2010, 54, 2303–2311. [Google Scholar] [CrossRef]
  160. Sionov, E.; Chang, Y.C.; Kwon-Chung, K.J. Azole heteroresistance in Cryptococcus neoformans: Emergence of resistant clones with chromosomal disomy in the mouse brain during fluconazole treatment. Antimicrob. Agents Chemother. 2013, 57, 5127–5130. [Google Scholar] [CrossRef]
  161. Altamirano, S.; Fang, D.; Simmons, C.; Sridhar, S.; Wu, P.; Sanyal, K.; Kozubowski, L. Fluconazole-Induced Ploidy Change in Cryptococcus neoformans Results from the Uncoupling of Cell Growth and Nuclear Division. mSphere 2017, 2, e00205-17. [Google Scholar] [CrossRef] [PubMed]
  162. Escribano, P.; Recio, S.; Peláez, T.; González-Rivera, M.; Bouza, E.; Guinea, J. In vitro acquisition of secondary azole resistance in Aspergillus fumigatus isolates after prolonged exposure to itraconazole: Presence of heteroresistant populations. Antimicrob. Agents Chemother. 2012, 56, 174–178. [Google Scholar] [CrossRef] [PubMed]
  163. Nurjadi, D.; Chanthalangsy, Q.; Zizmann, E.; Stuermer, V.; Moll, M.; Klein, S.; Boutin, S.; Heeg, K.; Zanger, P. Phenotypic Detection of Hemin-Inducible Trimethoprim-Sulfamethoxazole Heteroresistance in Staphylococcus aureus. Microbiol. Spectr. 2021, 9, e0151021. [Google Scholar] [CrossRef]
  164. Chen, Y.; Ji, L.; Liu, Q.; Li, J.; Hong, C.; Jiang, Q.; Gan, M.; Takiff, H.E.; Yu, W.; Tan, W.; et al. Lesion Heterogeneity and Long-Term Heteroresistance in Multidrug-Resistant Tuberculosis. J. Infect. Dis. 2021, 224, 889–893. [Google Scholar] [CrossRef]
  165. Hope, W.; Stone, N.R.H.; Johnson, A.; McEntee, L.; Farrington, N.; Santoro-Castelazo, A.; Liu, X.; Lucaci, A.; Hughes, M.; Oliver, J.D.; et al. Fluconazole Monotherapy Is a Suboptimal Option for Initial Treatment of Cryptococcal Meningitis Because of Emergence of Resistance. mBio 2019, 10, e02575-19. [Google Scholar] [CrossRef]
  166. Banerjee, T.; Adwityama, A.; Sharma, S.; Mishra, K.; Prusti, P.; Maitra, U. Comparative evaluation of colistin broth disc elution (CBDE) and broth microdilution (BMD) in clinical isolates of Pseudomonas aeruginosa with special reference to heteroresistance. Indian. J. Med. Microbiol. 2024, 47, 100494. [Google Scholar] [CrossRef]
  167. Chang, Y.C.; Khanal Lamichhane, A.; Kwon-Chung, K.J. Cryptococcus neoformans, Unlike Candida albicans, Forms Aneuploid Clones Directly from Uninucleated Cells under Fluconazole Stress. mBio 2018, 9, e01290-18. [Google Scholar] [CrossRef]
  168. Winski, C.J.; Qian, Y.; Mobashery, S.; Santiago-Tirado, F.H. An Atypical ABC Transporter Is Involved in Antifungal Resistance and Host Interactions in the Pathogenic Fungus Cryptococcus neoformans. mBio 2022, 13, e0153922. [Google Scholar] [CrossRef]
  169. El-Halfawy, O.M.; Valvano, M.A. Antimicrobial heteroresistance: An emerging field in need of clarity. Clin. Microbiol. Rev. 2015, 28, 191–207. [Google Scholar] [CrossRef]
  170. Zhu, Y.; Li, J.; Lin, X.; Huang, X.; Hoffmann, M.R. Single-Cell Phenotypic Analysis and Digital Molecular Detection Linkable by a Hydrogel Bead-Based Platform. ACS Appl. Bio Mater. 2021, 4, 2664–2674. [Google Scholar] [CrossRef]
  171. Dai, Y.; Li, C.; Yi, J.; Qin, Q.; Liu, B.; Qiao, L. Plasmonic Colloidosome-Coupled MALDI-TOF MS for Bacterial Heteroresistance Study at Single-Cell Level. Anal. Chem. 2020, 92, 8051–8057. [Google Scholar] [CrossRef] [PubMed]
  172. Mezcord, V.; Escalante, J.; Nishimura, B.; Traglia, G.M.; Sharma, R.; Vallé, Q.; Tuttobene, M.R.; Subils, T.; Marin, I.; Pasteran, F.; et al. Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility. Int. J. Mol. Sci. 2023, 24, 11752. [Google Scholar] [CrossRef] [PubMed]
  173. Wang, Y.; Ma, X.; Zhao, L.; He, Y.; Yu, W.; Fu, S.; Ni, W.; Gao, Z. Heteroresistance Is Associated With in vitro Regrowth During Colistin Treatment in Carbapenem-Resistant Klebsiella pneumoniae. Front. Microbiol. 2022, 13, 868991. [Google Scholar] [CrossRef] [PubMed]
  174. Tian, Y.; Zhang, Q.; Wen, L.; Chen, J. Combined effect of Polymyxin B and Tigecycline to overcome Heteroresistance in Carbapenem-Resistant Klebsiella pneumoniae. Microbiol. Spectr. 2021, 9, e0015221. [Google Scholar] [CrossRef]
  175. Jo, J.; Kwon, K.T.; Ko, K.S. Multiple heteroresistance to tigecycline and colistin in Acinetobacter baumannii isolates and its implications for combined antibiotic treatment. J. Biomed. Sci. 2023, 30, 37. [Google Scholar] [CrossRef]
  176. Rajakani, S.G.; Xavier, B.B.; Sey, A.; Mariem, E.B.; Lammens, C.; Goossens, H.; Glupczynski, Y.; Malhotra-Kumar, S. Insight into Antibiotic Synergy Combinations for Eliminating Colistin Heteroresistant Klebsiella pneumoniae. Genes 2023, 14, 1426. [Google Scholar] [CrossRef]
  177. Yamamoto, Y.; Maesaki, S.; Kakeya, H.; Yanagihara, K.; Ohno, H.; Ogawa, K.; Hirakata, Y.; Tomono, K.; Tashiro, T.; Kohno, S. Combination therapy with fluconazole and flucytosine for pulmonary cryptococcosis. Chemotherapy 1997, 43, 436–441. [Google Scholar] [CrossRef]
  178. Jackson, A.T.; Nussbaum, J.C.; Phulusa, J.; Namarika, D.; Chikasema, M.; Kanyemba, C.; Jarvis, J.N.; Jaffar, S.; Hosseinipour, M.C.; van der Horst, C.; et al. A phase II randomized controlled trial adding oral flucytosine to high-dose fluconazole, with short-course amphotericin B, for cryptococcal meningitis. Aids 2012, 26, 1363–1370. [Google Scholar] [CrossRef]
  179. Liu, J.; Liu, J.; Su, X.; Yang, L.; Wang, Y.; Wang, A.; Xu, X.; Li, M.; Jiang, Y.; Peng, F. Amphotericin B plus fluorocytosine combined with voriconazole for the treatment of non-HIV and non-transplant-associated cryptococcal meningitis: A retrospective study. BMC Neurol. 2022, 22, 274. [Google Scholar] [CrossRef]
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Figure 1. Antifungal mode of action of azoles, AMB, 5-FC and echinocandins. Azoles inhibit ergosterol biosynthesis by targeting sterol 14α-demethylase, producing toxic 14-methyl sterols, altering fungal cell membrane permeability and metabolic state [74,75]. Echinocandins target β-1,3-glucan synthase, impairing fungal cell wall integrity and stress resistance [76]. Amphotericin B binds to membrane ergosterol, forming pores that disrupt permeability and cause reactive oxygen species (ROS) accumulation, leading to cell death [77,78,79]. 5-FC is converted intracellularly to 5-FU, inhibiting DNA and RNA synthesis [80,81].
Figure 1. Antifungal mode of action of azoles, AMB, 5-FC and echinocandins. Azoles inhibit ergosterol biosynthesis by targeting sterol 14α-demethylase, producing toxic 14-methyl sterols, altering fungal cell membrane permeability and metabolic state [74,75]. Echinocandins target β-1,3-glucan synthase, impairing fungal cell wall integrity and stress resistance [76]. Amphotericin B binds to membrane ergosterol, forming pores that disrupt permeability and cause reactive oxygen species (ROS) accumulation, leading to cell death [77,78,79]. 5-FC is converted intracellularly to 5-FU, inhibiting DNA and RNA synthesis [80,81].
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Figure 2. (By Figdraw 2.0). Explanation of resistance, susceptible, tolerance, persistence and heteroresistance from a cell population perspective. The state of cell proliferation is represented by the number of cells in the figure. Individual colors indicate different genotypes and phenotypes, which are red (genetically stable resistant), blue (susceptible), yellow (genetically unstable or phenotypically resistant), green (phenotypically tolerant).
Figure 2. (By Figdraw 2.0). Explanation of resistance, susceptible, tolerance, persistence and heteroresistance from a cell population perspective. The state of cell proliferation is represented by the number of cells in the figure. Individual colors indicate different genotypes and phenotypes, which are red (genetically stable resistant), blue (susceptible), yellow (genetically unstable or phenotypically resistant), green (phenotypically tolerant).
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Table 1. Differences between resistance, tolerance, persistence and heteroresistance.
Table 1. Differences between resistance, tolerance, persistence and heteroresistance.
ConceptDescriptionMechanismsClinical Features Referrence
ResistanceThe ability to withstand the effects of an antifungal drug whose concentration is typically effective.Heritable mutations are associated with drug targets or efflux pumps expression, etc.Treatment failure and increased risk of infection spread.[82,83,84]
ToleranceThe whole population survives antifungal exposure at concentrations that would otherwise be lethal.Altered cell growth or metabolism, etc.High mortality rates, persistent infection and treatment failure.[98,103,104,105,106,107,108,113]
PersistenceA subpopulation of genetically susceptible fungal cells survives fungicidal concentrations of drugs.Altered cell growth and metabolism, etc.Treatment failure and recurrence.[109,110,111,112]
HeteroresistanceA minority of phenotypically resistant cells with elevated MICs coexist with the most susceptible cells.Altered expression levels, aneuploidy and copy number variation, etc.High mortality rates, treatment failure, potential to evolve into full resistance.[15,18,48,57,99,100,101,102]
Table 2. Heteroresistance Mechanisms in Common Fungal Pathogens.
Table 2. Heteroresistance Mechanisms in Common Fungal Pathogens.
TypesSpeciesAntifungalsMechanismsRelated ComponentsReferences
Aneuploidy and CNVsC. albicansFluconazoleChr5 disomyERG11 and TAC1[129,130]
C. albicans5-FlucytosineLoss of chr5, due to the location of negative regulator (s) of anti 5-FC resistanceUnknown.[130]
C. albicansFluconazole“Trimeras”, three connected cells composed of a mother, daughter, and granddaughter budUnknown.[131]
C. albicansEchinocandinsChr2 trisomyRNR1, RNR21[132]
C. albicansEchinocandins (caspofungin, micafungin and anidulafungin)Chr5 aneuploidy after caspofungin exposure can produce cross-resistanceThree negative regulators CHT2, PGA4 and CSU51, and two positive regulators, CNB1 and MID1.[118,133]
N. glabrataEchinocandins (anidulafungin)ChrE aneuploidy contributes to heteroresistance after exposing clinical isolates to anidulafungin.Unknown.[134]
N. glabrataAzolesIncremental effects of multiple binary genetic switchesCDR1, PDH1, PDR1 and SNQ1[99]
N. glabrataAzolesFormation of “trimeras”Unknown.[131]
C. parapsilosisAzolesFormation of “trimeras”Unknown.[131]
C. aurisAzoles (fluconazole)Genome changes mainly
involving SNP, with aneuploidy a minority. But due to its haploid genome, SNPs may have immediate phenotypic impact.		SNPs[135,136,137]
C. gattiiAzoles (fluconazole)Copy number increase in chr10, chr9 and chr11 in VGI genotype and copy number increase in chr3 in VGII genotypeUnknown.[138]
C. neoformansCross-resistance to 5-FC and FluconazoleChr1 disomyERG11, AFR1[139]
C. neoformansFluconazoleOverexpression of AFR1 on chr1 and GEA2 on chr3AFR1, GEA2[139]
C. neoformansAzoles (fluconazole)Titan cells that produce multiple types of aneuploid daughter cellsUnknown.[56,140,141]
C. neoformansAzoles (fluconazole)Chr1 disomyERG11, AFR1[142]
C. neoformansAzoles (fluconazole)Chr4 disomySEY1, GCS2, GLO3[143]
C. neoformansAzoles (fluconazole)Chr3 disomy caused by gene relocationERG11, SEY1, GCS2, GLO3[143,144]
Alterations in Gene ExpressionC. albicansAzolesElevation of mRNAATP Binding Cassette superfamily CDR genes[145]
C. gattiiAzoles (fluconazole)Up-regulated activity of efflux pumpsPDR11[146]
C. neoformansAzoles (fluconazole)Up-regulated activity of efflux pumpsAFR1[142]
Environmental StressC. neoformansPolyene (AMB) and Azoles (fluconazole)Nitrogen limitationUnknown.[57]
C. neoformansAzoles (fluconazole)Environmental temperatureUnknown.[19]
C. neoformansAzolesTemperature, media type, growth phase, and the age of cellsUnknown.[147]
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Su, Y.; Li, Y.; Yi, Q.; Xu, Y.; Sun, T.; Li, Y. Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance. J. Fungi 2025, 11, 143. https://doi.org/10.3390/jof11020143

AMA Style

Su Y, Li Y, Yi Q, Xu Y, Sun T, Li Y. Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance. Journal of Fungi. 2025; 11(2):143. https://doi.org/10.3390/jof11020143

Chicago/Turabian Style

Su, Yanyu, Yi Li, Qiaolian Yi, Yingchun Xu, Tianshu Sun, and Yingxing Li. 2025. "Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance" Journal of Fungi 11, no. 2: 143. https://doi.org/10.3390/jof11020143

APA Style

Su, Y., Li, Y., Yi, Q., Xu, Y., Sun, T., & Li, Y. (2025). Insight into the Mechanisms and Clinical Relevance of Antifungal Heteroresistance. Journal of Fungi, 11(2), 143. https://doi.org/10.3390/jof11020143

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