| Objective:To understand the clinical epidemiological characteristics of Candida haemulonii complex causing invasive infections.To comprehensively evaluate the identification ability of clinical laboratories for C.haemulonii complex.To elucidate the phylogenetic relationship between Chinese C.haemulonii complex and international strains using a population genomics approach.To investigate the potential mechanisms underlying the unique multidrug-resistant phenotype of C.haemulonii complex through genomic information analysis.Methods:A total of 72 invasive C.haemulonii complex were collected from twentythree hospitals that participate in the China Hospital Invasive Fungal Surveillance Net(CHIF-NET)Programs between 2009 and 2017.The study compared and analyzed the identification ability of clinical isolated Candida duobushaemulonii and Candida haemulonii var.vulnera by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)and VITEK 2.Additionally,antifungal susceptibility testing was performed on all strains of C.haemulonii complex.A genomics-based approach was adopted to perform phylogenetic analysis and predict the population structure of 72 C.haemulonii complex collected through CHIF-NET,as well as 97 international strains available in the National Center for Biotechnology Information(NCBI)Sequence Read Archive(SRA).The genomic differences among strains were analyzed using pairwise single nucleotide polymorphism(SNP)and combined with clinical information to perform genomic epidemiological analysis.The potential underlying antifungal drug resistance mechanisms were explored by correlating the drug resistance phenotype with genomic information.Results:In our study,the C.haemulonii complex was comprised of four main species,which included 58 strains of C.haemulonii,11 strains of C.duobushaemulonii,two strains of C.haemulonii var.vulnera,and one strain of Candida pseduobushaemulonii.The identification accuracy rate of VITEK MS for C.duobushaemulonii was 100%(11/11).However,the VITEK MS was unable to identify the C.haemulonii var.vulnera.The VITEK 2 correctly identified 72.73%(8/11)of isolates from C.duobushaemulonii,but it exhibits a lack of accuracy in identifying two C.haemulonii var.vulnera.One strain was misidentified as C.haemulonii and the other with low discrimination identification result.Internal transcribed spacer(ITS)sequencing enables the precise identification of C.haemulonii var.vulnera.The C.haemulonii complex have various degrees of susceptibility to antifungal drugs.55.17%(32/58)and 34.48%(20/58)of C.haemulonii were non-wild-type for fluconazole and voriconazole,respectively.All 58 strains showed wild-type for posaconazole,caspofungin,and anidulafungin,and exhibited higher susceptibility to itraconazole and micafungin(MIC[minimum inhibitory concentration]≤2 μg/mL for itraconazole and MIC<0.12 μg/mL for micafungin).However,37.93%(22/58)and 53.45%(31/58)strains of C.haemulonii exhibited resistance to amphotericin B and 5-fluorocytosine,respectively.All 11 strains of C.duobushaemulonii were non-wild type for azoles and echinocandins,and 72.73%(8/11)of the strains exhibited high MIC(≥8 μg/mL)for amphotericin B.One C.duobushaemulonii exhibited high-level resistance to 5-fluorocytosine(MIC>64μg/mL).Among the two strains of C.haemulonii var.vulnera,one with elevated MIC to fluconazole(>256 μg/mL)and the other exhibited high MIC to 5-fluorocytosine(>64 μg/mL).The MIC to amphotericin B was 4 μg/mL for two C.haemulonii var.vulnera.One strain of C.pseduobushaemulonii had low MICs to azoles,echinocandins and amphotericin B,but this strain exhibited resistance to 5-fluorocytosine(MIC>64μg/mL).Phylogenetic and population structure analysis revealed low levels of genome variations between 58 isolates collected from 23 hospitals in China and 36 previously published international strains.The maximum single nucleotide polymorphisms(SNPs)pairwise between global isolates were less than 653,accounting for<0.005%of the 13.31 Mb genome.Phylogenetic analysis revealed that C.haemulonii can be divided into four clades(clade 1-4).Clade 1 comprised strains from China and other global strains;clades 2-4 contained only isolates from China,were more recently evolved,and showed higher antifungal resistance.Four epidemic clusters(A,B,C,and D)were identified in China,each comprising ≥5 cases(largest intracluster pairwise SNP differences<50 bp).Among these potential epidemic clusters,Cluster A was identified in 2 hospitals located in the same city,suggesting potential intracity transmissions.Cluster D was resistant to 3 classes of antifungals(fluconazole,amphotericin B,and 5fluorocytosine).There is no extensive genomic variation existed in the C.duobushaemulonii,but its variability is higher than that of C.haemulonii.The phylogenetic analysis of 11 C.duobushaemulonii isolates and 55 international strains revealed two major distinct clades,with Chinese strains distributed in both clade without any clustering tendency.The pairwise SNP differences between Chinese C.haemulonii var.vulnera and Brazilian strains is only 665 SNPs.The phylogenetic analysis of C.pseduobushaemulonii revealed that Chinese strains are clustered in the same branch as strains isolated from Panama and Colombia,while strain from Venezuela form a separate branch.In terms of potential drug resistance mechanisms,all fluconazole-resistant C.haemulonii strains harboring a Y132F substitution in Erg11p.Additionally,we found 56.25%(18/32)of fluconazole-resistant strains in China harbored>1 of the substitutions V125A,F126L,and G307A.For 5-fluorocytosine-resistant strains,the A143T mutation in FUR1 gene was detected in all cluster C strains.No previously described amphotericin B-resistance mutations were identified in C.haemulonii.We identified a start codon mutation within the FUR1 gene in C.duobushaemulonii strain that were resistant to 5-fluorocytosine.Y132F and G307A amino acid substitutions in Erg11p were found in the fluconazole-resistant C.haemulonii var.vulnera.Conclusion:In China,invasive C.haemulonii complex infections are mainly caused by C.haemulonii,and the two cases of bloodstream infections caused by C.haemulonii var.vulnera infection were first reported in China.Compared to other C.haemulonii complexes,accurate identification of C.haemulonii var.vulnera requires the use of molecular sequencing technology.The phylogenetic analysis incorporated a total of 94 C.haemulonii genomes,and observed four phylogenetic clades,three of which were identified exclusively in China and exhibited higher antifungal resistance.The four regional cluster cases were caused by specific clones,among which were a potential interfacility transmission within the same city and the spread of multidrug-resistant clones.The genomes of C.duobushaemulonii and C.haemulonii var.vulnera are conserved,while the genome of C.pseudohaemulonii exhibits a high degree of genetic variability.All fluconazole-resistant strains carried the Y132F substitution in Erg11p.In addition,Chinese fluconazole non-wild type strains had polymorphisms in the ERG11.The resistance of C.haemulonii var.vulnera to fluconazole is also associated with mutations in the ERG11 gene.Resistance to 5-fluorocytosine in the C.haemulonii complex is associated with mutations in the FUR1 gene.Currently,the isolation rate of C.haemulonii complex is still low,and there are problems with misidentification,making the true isolation rate unknown.Similar to C.auris,C.haemulonii had been reported to exhibit antifungal resistance and clonal dissemination.C.haemulonii should be recognized as a potential threat to global health,and further monitoring and stewardship steps to limit excessive antifungal usage are warranted. |
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