Antifungal resistance: why are we losing this battle?

The emergence of fungal pathogens and changes in the epidemiological landscape are prevalent issues in clinical mycology. Reports of resistance to antifungals have been reported. This review aims to evaluate molecular and nonmolecular mechanisms related to antifungal resistance. Mutations in the ERG genes and overexpression of the efflux pump (MDR1, CDR1 and CDR2 genes) were the most reported molecular mechanisms of resistance in clinical isolates, mainly related to Azoles. For echinocandins, a molecular mechanism described was mutation in the FSK genes. Furthermore, nonmolecular virulence factors contributed to therapeutic failure, such as biofilm formation and selective pressure due to previous exposure to antifungals. Thus, there are many public health challenges in treating fungal infections.
The emergence of fungal pathogens and changes in scenery are a prevalent subject in clinical mycology.
Species of non-albicans Candida spp. have been reported to cause invasive infection, surpassing the rates of Candida albicans, worldwide.
Furthermore, there is also a constant report of resistance during the treatment of fungal infections.
Presently, the clinical arsenal for systemic antifungals counts with different mechanisms of action and many classes.
This review aims to group the molecular and nonmolecular mechanisms correlated with the resistance of nosocomial infections like Candida spp., Aspergillus spp. and Cryptococcus spp., to antifungals currently used in health services.
The articles passed through three processes: identification, screening and eligibility. The identification was carried out in the following databases: EMBASE; MEDLINE; and COCHRANE. It included 74 articles.
ERG's genes are responsible for regulating the synthesis of ergosterol, which is the major component of the fungal cell membrane and contributing with the fluidity and integrity of the membrane and the proper function of membrane-bound enzymes.
The azole drugs block the synthesis by inhibiting the activity of the genes ERG, binding and inhibiting the lanosterol demethylase enzyme, causing damage to the cell membrane.
In literature, we have a lot of findings with overexpression and mutation of different ERG genes, but the most frequent is ERG11.
Mutations and super expression in the ERG genes, that are responsible for the ergosterol biosynthesis pathway, promote a resistance mechanism against azoles or Amphotericin b.
The efflux pump is a mechanism where pathogens can export azole drugs from the inside of the cell.
The expression is regulated by the genes CDR2, CDR1 and MDR1, which coordinate the synthesis of ABC transporter proteins that act on the cell membrane exporting the azole substances.
In cases of overexpression the synthesis of ABC transporters is superior causing bigger action of efflux pump, it contributes to a bigger exporting activite of azoles substances outside the cell conferring resistance.
The fungal cell wall contains β-1,3-Glucan, which is synthesized by a membrane-integrated synthase encoded by FKS genes.
For echinocandins, a molecular mechanism described was mutation in the FSK genes, these kinds of mutations have been shown to influence the sensibility of the GS enzyme complex to inhibition by the individual echinocandins.
Furthermore, nonmolecular virulence factors contributed to therapeutic failure, such as biofilm formation, fungal melanins and selective pressure due to previous exposure to antifungals, and other factors that involved virulence factors and the host immunity system. Thus, there are many public health challenges in treating fungal infections.
This study warns of the emergence of fungi resistant to the main antifungals of medical interest.
Multiple molecular and nonmolecular mechanisms of antifungal resistance have been identified in several fungal pathogens, emphasizing the need for new strategies to contain fungal resistance.
The use of tools for accurate laboratory diagnosis of fungal infections is necessary.

临床真菌学领域中，真菌病原体的出现以及流行病学格局的变化是普遍存在的核心问题。抗真菌药物耐药性的相关报道也屡见不鲜。本综述旨在评估与抗真菌药物耐药性相关的分子及非分子耐药机制。ERG基因（ERG genes）的突变以及外排泵（由MDR1、CDR1与CDR2基因编码）的过表达，是临床分离株中报道最多的分子耐药机制，且该类耐药主要与唑类抗真菌药（Azoles）相关。针对棘白菌素类（echinocandins），已报道的分子耐药机制为FSK基因的突变。此外，非分子毒力因子也会导致治疗失败，例如生物膜形成以及既往接触抗真菌药物所引发的选择压力。因此，真菌感染的治疗面临诸多公共卫生层面的挑战。

真菌病原体的出现以及研究场景的变化是临床真菌学领域的热门议题。全球范围内，非白假丝酵母菌（non-albicans Candida spp.）引发侵袭性感染的病例率已超越白假丝酵母菌（Candida albicans）。此外，真菌感染治疗过程中出现耐药性的相关报道也持续不断。目前，临床用于全身治疗的抗真菌药物拥有多样的作用机制与众多类别。本综述旨在梳理与假丝酵母菌属（Candida spp.）、曲霉菌属（Aspergillus spp.）以及隐球菌属（Cryptococcus spp.）等医院感染相关病原体对当前医疗服务中常用抗真菌药物产生耐药性的分子及非分子机制。

所纳入的文献均经过三步筛选流程：文献识别、筛查与资格评估。文献识别工作在EMBASE、MEDLINE以及COCHRANE三个数据库中开展，最终纳入74篇文献。

ERG基因（ERG genes）负责调控麦角固醇的合成，麦角固醇是真菌细胞膜的主要组成成分，对维持细胞膜的流动性、完整性以及膜结合酶的正常功能至关重要。唑类抗真菌药通过抑制ERG基因的表达活性，结合并抑制羊毛甾醇去甲基化酶的活性，从而阻断麦角固醇的合成，最终造成细胞膜损伤。现有文献中已有诸多关于不同ERG基因过表达与突变的研究发现，其中最为常见的是ERG11基因。参与麦角固醇生物合成通路的ERG基因发生突变或过表达，会引发针对唑类抗真菌药或两性霉素B（Amphotericin b）的耐药机制。

外排泵是病原体可将细胞内的唑类抗真菌药物排出体外的耐药机制。外排泵的表达由CDR2、CDR1与MDR1基因调控，这些基因可协同合成ABC转运蛋白，该蛋白定位于细胞膜，负责将唑类物质排出细胞外。当ABC转运蛋白的合成出现过表达时，外排泵的活性会增强，从而提升唑类物质向细胞外的排出效率，最终赋予病原体耐药性。

真菌细胞壁含有β-1,3-葡聚糖，该物质由FKS基因编码的膜整合合酶合成。针对棘白菌素类，已报道的分子耐药机制为FSK基因的突变，此类突变已被证实会影响β-1,3-葡聚糖合酶（GS enzyme complex）复合物对各棘白菌素类药物抑制作用的敏感性。此外，非分子毒力因子也会导致治疗失败，例如生物膜形成、真菌黑色素生成、既往接触抗真菌药物所引发的选择压力，以及其他与毒力因子和宿主免疫系统相关的因素。因此，真菌感染的治疗仍面临诸多公共卫生层面的挑战。

本研究警示了对临床主流抗真菌药物产生耐药性的真菌菌株的出现。目前已在多种真菌病原体中发现了抗真菌药物耐药性的多种分子与非分子机制，这凸显了研发新策略以遏制真菌耐药性的必要性。精准的实验室诊断工具对于真菌感染的诊疗至关重要。