目的：了解锁掷酵母科真菌侵袭性感染的临床特征；综合评价实验室鉴定性能、体外药物敏感性并探究其唑类耐药机制；建立胶红酵母高分辨率分子分型方法并结合全基因组序列进行分子流行病学调查；系统性评价红冬孢锁掷孢酵母的毒力表型及探索相关机制。

方法：本研究纳入中国医院侵袭性真菌病监测网（CHIF-NET）2010年至2019年21家监测中心收集的侵袭性感染非重复锁掷酵母科真菌77株，并将可获得的16个环境菌株和3个动物分离株用于与临床菌株比较分析。比较基质辅助激光解吸-电离飞行时间质谱（MALDI-TOF MS）对锁掷酵母临床分离株的鉴定能力，并检测菌株对抗真菌药物的敏感性。耐药机制方面，通过高质量基因组组装及注释，获取红冬孢锁掷孢酵母和胶红酵母唑类药物靶基因ERG11序列，利用转录组分析和异源表达评价ERG11表达水平和获得性突变对唑类药物敏感性的影响，并使用AlphaFold2预测Erg11蛋白结构。胶红酵母分子流行病学调查中，建立微卫星分型方法，并结合rDNA序列分型（ITS+D1/D2）和全基因组单核苷酸多态性（SNP）对84株3种来源的胶红酵母进行亲缘关系分析。毒力评价方面，综合检测体外毒力因子和适应力表型，构建免疫低下小鼠血流感染模型，检测红冬孢锁掷孢酵母感染小鼠的器官真菌负荷及生存曲线，并结合组织病理学改变判断菌株侵袭力；通过crtYB和Ace2基因敲除分别构建色素缺失株和假菌丝形态菌株并评价其对毒力和耐药性的影响。

结果：77株锁掷酵母中，胶红酵母（Rhodotorula mucilaginosa）占比94.8%（73/77），另有小红酵母（R. minuta）、R. diobovata 、河道红冬孢锁掷孢酵母（Rhodosporidiobolus fluvialis）和天津红冬孢锁掷孢酵母（R. tianjinensis）各1株。MALDI-TOF MS可以100%正确鉴定胶红酵母和小红酵母，但罕见的红酵母及红冬孢锁掷孢酵母需依赖分子生物学进行鉴定。胶红酵母除棘白菌素天然耐药外，分别有6.8%（5/73）和98.6%（72/73）的临床菌株存在唑类全耐药和交叉耐药，并有12.3%（9/73）分离株新出现了5-氟胞嘧啶（5-FC）非敏感表型；4个非胶红-锁掷酵母临床株氟康唑（FZ）的最小抑菌浓度（MIC）均≥64 µg/mL。

耐药机制方面，河道红冬孢锁掷孢酵母、天津红冬孢锁掷孢酵母临床株基因组上分别存在2个和4个ERG11序列，并且总体表达水平显著高于唑类敏感菌株新型隐球菌；锁掷酵母单一ERG11序列异源表达后氟康唑MIC均显著升高至耐药水平（64 - >256 µg/mL）；河道红冬孢锁掷孢酵母crtYBΔ菌株唑类MIC比野生型（WT）菌株下降1 - 2个浓度梯度。

分子流行病学调查发现，68.5%（50/73）的临床分离株集中在rDNA序列2型；15个分型位点组合的微卫星（MT）分型以0.979的DP值将73个临床分离株鉴定至50个MT基因型；MT最小生成树和全基因组SNP系统发育树均发现一个聚集了48个临床株、2个动物株和1个环境株的流行簇。该流行簇菌株来源于国内不同地区的多家医院，并且分离年从2012年连续到2019年；其中，天津一家医院单一MT流行基因型（MT03 - MT06）分离株达3 - 6个，甚至一个分支上的MT05和MT06型共计9个分离株都对5-FC不敏感。此外，2个动物株、1个环境株与2个临床株同属MT02型，并在SNP进化树上聚集在一起。

毒力方面，红冬孢锁掷孢酵母存在荚膜并分泌脲酶，其对CO₂的耐受性与感染小鼠的毒力表型一致；河道红冬孢锁掷孢酵母Ace2Δ菌株致病力强于WT菌株；小鼠感染天津红冬孢锁掷孢酵母5 d内死亡，组织病理显示肺泡上皮基本脱落，肺泡间隔及支气管周围可见大量淋巴细胞及出血；早期伏立康唑治疗虽然可将脑组织中的真菌负荷降低50%，但肺组织菌载量无显著改变。

结论：我国侵袭性锁掷酵母科真菌感染以胶红酵母血流感染为主，国际上首次出现红冬孢锁掷孢酵母人类血流感染病例2例，并命名了其中一个新种——天津红冬孢锁掷孢酵母。ERG11复制、点突变和过表达联合导致了锁掷酵母对唑类特别是氟康唑高度耐药，类胡萝卜素产生也是其多药耐药的基础。首次建立胶红酵母微卫星分型方法并发现了中国地区广泛存在的胶红酵母流行簇，以及一个新出现的棘白菌素-唑类-5-FC耐药的克隆株。红冬孢锁掷孢酵母是条件致病菌，可引发小鼠侵袭性感染及死亡；体外敏感的伏立康唑在感染早期对肺部菌体清除无益；菌丝形态是河道红冬孢锁掷孢酵母的高毒力形态。

Objective: To investigate the clinical characteristics of invasive infections caused by Sporidiobolaceae. To comprehensively evaluate the performance of laboratory identification and in vitro antifungal susceptibility profiles, and to explore its azole resistance mechanism. To establish a high-resolution molecular genotyping method of Rhodotorula mucilaginosa and compare it with the whole genome sequence for investigating molecular epidemiology. To systematically evaluate the virulence phenotypes of Rhodosporidiobolus species and explore their related mechanisms

Methods: The study included non-duplicated 77 strains of invasive Sporidiobolaceae collected from 2020 to 2019, as a part of National China Hospital Invasive Fungal Surveillance Net (CHIF-NET) Programs. And a total of 16 environmental strains and 3 animal strains were also used for comparative analysis with clinical strains. The identification of clinical Sporidiobolaceae strains with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and antifungal susceptibility testing were performed. In terms of azole resistance mechanism, high-quality genome assembly and annotation were used to obtain the ERG11 sequences encoding azoles target in Rhodosporidiobolus species and R. mucilaginosa. Transcriptome analysis and heterologous expression were used to evaluate ERG11 expression levels and the effect of its acquired mutations on azole susceptibility, respectively. AlphaFold2 was also used to predict Erg11 protein structure. In the molecular epidemiological investigation of R. mucilaginosa, a newly established microsatellite genotyping was combined with ribosome DNA (rDNA) genotyping (internal transcribed spacers [ITS]+D1/D2) and whole-genome sequence (WGS) single nucleotide polymorphism (SNP) for phylogenetic analysis of 84 R. mucilaginosa strains collected from three different kinds of sources. In terms of virulence evaluation, we comprehensive detected in vitro virulence factors and fitness phenotypes, and established a bloodstream infection model in immunocompromised mice. We determined the invasiveness of Rhodosporidiobolus species based on the fungal burden of mice organs, survival curves of the mice post challenge and their histopathological change. A pigment-deficient strain and a pseudohyphal morphological strain were constructed by knocking out crtYB and Ace2, respectively, and their effects on virulence and drug resistance were evaluated.

Results: Among the 77 strains of Sporidiobolaceae, R. mucilaginosa accounted for 94.8% (73/77). There is one strain each of R. minuta, R. diobovata, R. fluvialis and R. tianjinensis. All R. mucilaginosa and R. minuta strains were correctly identified by MALDI-TOF MS, but the identification of rare species of Sporidiobolaceae must rely on molecular sequences. In addition to the inherent resistance to echinocandins, 6.8% (5/73) and 98.6% (72/73) of the R. mucilaginosa clinical strains were pan-resistant and cross-resistant to azoles, respectively. And 12.3% (9/73) of clinical strains had a newly acquired 5-fluorocytosine (5-FC) non-susceptible profile. The minimum inhibitory concentration (MIC) of fluconazole (FZ) against four non-R. mucilaginosa Sporidiobolaceae clinical strains were all ≥64 µg /mL.

In terms of drug resistance mechanism, there are 2 and 4 sequences of ERG11 on the genomes of R. fluvialis and R. tianjinensis clinical strains, respectively. The their overall expression levels are significantly higher than Cryptococcus neoformans susceptible to azoles. Heterologous expression of a single sequence of ERG11 in C. neoformans, the MIC of fluconazole was significantly increased to the resistance level (64 - >256 µg/mL). The MIC of azoles in R. fluvialis crtYBΔ strain decreased by 1 to 2 concentration gradients compared with its wild type (WT) strain.

Molecular epidemiological investigation found that 68.5% (50/73) of R. mucilaginosa clinical isolates were identified as rDNA type 2. The microsatellite (MT) genotyping with 15 tandem repeat loci identified 50 different MT genotypes in all 73 clinical strains with 0.97 discriminatory power. The minimum spanning tree of the microsatellite and phylogenetic tree of genomic SNP found that an epidemic cluster included 48 clinical strains, 2 animal strains and 1 environmental strain. The strains in epidemic cluster were collected from various hospitals in different regions of China from 2012 to 2019. Among them, there were 3 to 6 strains identified as the same MT epidemic genotype (MT03 - MT06) collected from a hospital in Tianjin, and even a branch including 9 strains of MT05 and MT06 genotypes were all resistant to 5-FC. In addition, 2 animal strains, 1 environmental strain and 2 clinical strains of MT02 genotype were clustered together in the SNP tree.

In terms of virulence, Rhodosporidiobolus spp. had capsule and can secrete urease. Their phenotype of CO₂ tolerance is consistent with the virulence phenotype in infected mice. R. fluvialis Ace2Δ strain had a higher virulence than the WT strain. Mice infected with R. tianjinensis died within 5 days, and their histopathology showed that the alveolar epithelium was basically sloughed off, and a large number of lymphocytes and erythrocytes were seen in the alveolar septa and around the bronchi. The fungal burden in the brain was reduced to 50% fater treatment with voriconazole, but the fungal burden in the lung was not significantly changed.

Conclusions: The invasive fungal infection of Sporidiobolaceae in China is dominated by bloodstream infection of R. mucilaginosa. There were 2 cases of human bloodstream infection of Rhodosporidiobolus spp. for the first international discovery, including a newly named species —R. tianjinensis. The combination of ERG11 duplication, point mutation and overexpression in Sporidiobolaceae resulted in high resistance to azoles, especially fluconazole. Carotenoid production was also the basis of its multidrug resistance. The microsatellite typing of R. mucilaginosa established in this study discovered an epidemic cluster of R. mucilaginosa in China, and an emergence of an echinocandin-azole-5-FC-non-susceptible clone. Rhodosporidiobolus spp. were opportunistic pathogens and caused invasive infections and even death in mice. Voriconazole, which is susceptible in vitro, is not beneficial to the clearance of pulmonary fungi in the early stage of infection. Pseudohypha is the hypervirulent morphotype in Rhodosporidiobolus species.