Epidemiology,Fluconazole Resistance And Pathogenic Mechanisms Of Sporidiobolaceae Causing Invasive Infection In China:A Multicenter Study

Objective:To investigate the clinical characteristics of invasive infections caused by Sporidiobolaceae.To comprehensively evaluate the performance of laboratory identification and 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 mechanismsMethods: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 ERG 11 sequences encoding azoles target in Rhodosporidiobolus species and R.mucilaginosa.Transcriptome analysis and heterologous expression were used to evaluate ERG 11 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≥64μg/mL.In terms of drug resistance mechanism,there are 2 and 4 sequences of ERG 11 on the genomes ofR.fluvialis andR.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 crtYBΔstrain 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 ERG 11 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.