Decreased echinocandin susceptibility in <i>Candida parapsilosis</i> causing candidemia and emergence of a pan-echinocandin resistant case in China

Candida parapsilosis is becoming a predominant non-albicans cause of invasive candidiasis (IC). Echinocandins are the preferred choice for IC treatment and prophylaxis. Resistance to echinocandins in C. parapsilosis has emerged in several countries, but little is known about the susceptibility profile in China or about mechanisms of resistance. Here, we investigated the echinocandin susceptibilities of 2523 C. parapsilosis isolates collected from China and further explored the resistance mechanism among echinocandin-resistant isolates. Anidulafungin exhibited the highest MICs (MIC50/90, 1 and 2 µg/mL; GM, 0.948 µg/mL), while caspofungin showed better activity (0.5 and 1 µg/mL; 0.498 µg/mL). Significantly higher echinocandin MICs were observed among blood-derived isolates compared to others, especially for caspofungin (GM, 1.348 µg/mL vs 0.478 µg/mL). Isolates from ICU and surgical wards also showed higher MICs. Twenty isolates showed intermediate phenotypes for at least one echinocandin. One was resistant to all three echinocandins, fluconazole and voriconazole, which caused breakthrough IC during long-term exposure to micafungin. WGS revealed this isolate carried a mutation S656P in hotspot1 region of Fks1. Bioinformatics analyses suggested that this mutation might lead to an altered protein conformation. CRISPR Cas9-mediated introduction of this mutation into a susceptible reference C. parapsilosis strain increased MICs of all echinocandins 64-fold, with similar results found in the subspecies, C. orthopsilosis and C. metapsilosis. This is the first report of a multi-azole resistant and pan-echinocandin resistant C. parapsilosis isolate, and the identification of a FKS1S656P conferring pan-echinocandin resistance. Our study underscores the necessity of rigorous management of antifungal use and of monitoring for antifungal susceptibility.近平滑念珠菌（Candida parapsilosis）正逐渐成为引发侵袭性念珠菌病（invasive candidiasis, IC）的主要非白念珠菌致病菌。棘白菌素类（Echinocandins）是侵袭性念珠菌病治疗与预防的首选药物。目前，多个国家已报道近平滑念珠菌对棘白菌素类的耐药性，但国内关于该菌的药敏谱及耐药机制的研究仍较为有限。本研究针对我国临床分离的2523株近平滑念珠菌，开展了棘白菌素类药敏性检测，并进一步探究了棘白菌素类耐药分离株的耐药机制。药敏结果显示，阿尼芬净（Anidulafungin）的最低抑菌浓度（Minimum Inhibitory Concentration, MIC）水平最高，其MIC50/90分别为1 μg/mL与2 μg/mL，几何平均浓度（geometric mean, GM）为0.948 μg/mL；而卡泊芬净（caspofungin）的抗菌活性更优，MIC50/90为0.5 μg/mL与1 μg/mL，GM为0.498 μg/mL。与非血液来源分离株相比，血液来源分离株的棘白菌素类MIC值显著升高，尤以卡泊芬净最为显著（GM分别为1.348 μg/mL与0.478 μg/mL）。来自重症监护病房（Intensive Care Unit, ICU）及外科病房的分离株同样表现出较高的MIC值。共计20株分离株对至少一种棘白菌素类药物呈现中介表型。其中1株对3种棘白菌素类、氟康唑（fluconazole）及伏立康唑（voriconazole）均表现为耐药，该菌株在长期暴露于米卡芬净（micafungin）期间引发了突破性侵袭性念珠菌病。全基因组测序（Whole Genome Sequencing, WGS）结果显示，该耐药株的Fks1基因热点1区域存在S656P突变。生物信息学分析提示，该突变可能导致蛋白质构象发生改变。通过CRISPR-Cas9介导将此突变引入敏感参考近平滑念珠菌菌株后，所有棘白菌素类药物的MIC值均升高了64倍，该结果在奥氏假丝酵母（Candida orthopsilosis）及中间假丝酵母（Candida metapsilosis）两个亚种中均得到验证。本研究首次报道了一株多唑类耐药且泛棘白菌素类耐药的近平滑念珠菌分离株，并鉴定出FKS1基因S656P突变可赋予泛棘白菌素类耐药性。本研究强调了严格规范抗真菌药物使用以及开展抗真菌药敏性监测的必要性。