Epidemiology And Azole Resistance Mechanism Of Invasive Tropical Candida Albicans In China For Five Consecutive Years

Background:Candida tropicalis is an important clinical pathogen causing invasive fungal infections with high mortality.In China,the resistance of C.tropicalis to azoles has raised widely awareness.In the present study,we carried out a systematic investigation for molecular epidemiology,trend in in vitro antifungal resistance and azole resistant mechanisms of C.tropicalis in China.Methods:A total of 507 non-duplicated C.tropicalis isolates were collected from unique patients from ten hospitals which continuously participate the CHIF-NET program during 5-year period from August,2009 to July,2014.Based on accurate identification of isolates,minimum inhibitory concentrations(MICs)of nine antifungal agents were determined.Meanwhile,the clinical consumption of fluconazole and voriconazole during the surveillance period were calculated.Genotyping was carried out by using the novel microsatellite molecular typing assay developed in this study for all the 507 C.tropicalis isolates,and phylogenetic analysis was further performed.For resistant mechanism study,the expression of ERG11(encoding the target for azoles),MDR1 and CDR1(encoding the efflux pumps),and CYTb(encoding cytochrome b),were determined by real-time quantitative PCR for fluconazole resistant and susceptible isolates,to explore differences in gene expression between two phenotype groups.Furthermore,full-length sequencing of ERG11 gene was performed for all 507 isolates to screen mutations.Functional verification for missense mutations was conducted by constructing pYES2/CT expression vectors.In addition,we carried out in vitro resistance-inducing experiment by using three azoles on a C.tropical clinical isolate,to observe MIC changes in of isolates induced by different drugs and investigate their resistant mechanisms.Results:Bloodstream infection predominated in all invasive C.tropicalis infections,which accounted for 53.4%of isolates(220/507).In vitro susceptibility testing revealed that 23.1%and 20.7%isolates were non-susceptible to fluconazole and voriconazole respectively.Meanwhile,11.4%isolates were cross-resistant to fluconazole and voriconazole.For echinocandins,above 99%of isolates remained susceptible.The azole non-susceptible rates increased significantly in the five-year surveillance:fluconazole non-susceptible rate increased from 11.2%to 42.7%,voriconazole non-susceptible rate increased from 10.4%to 39.1%,and fluconazole-voriconazole cross-resistant rate rose from 6.6%to 21.7%.However,the increase in azole non-susceptible rate didn't correlate with clinical azole consumption.Compared with pulse field gel electrophoresis,the 'gold-standard' for molecular typing,the new ctml-ctm3-ctm24 tri-locus microsatellite typing assay yielded a discriminatory power of 0.99.Amongst 507 C.tropicalis isolates,296 microsatellite genotypes were found,of which MT178 was the commonest type(4.1%).In general,the genotype of isolates distributed scattered,and no geographic clusters was identified.However,four genotype clusters were observed to be associated with fluconazole non-susceptible phenotype.Particularly,all 24 isolates that belonged to cluster ? were fluconazole and voriconazole cross-resistant isolates.A comprehensive analysis for azole resistant mechanisms of isolates collected in the present study illustrated that,the missense mutations in ERG11 gene were the major cause of azole resistance.Of 65 fluconazole resistant isolates,only three did not revealed any resistant-related mutations.The double-mutation A395T/W&C461T/Y in ERG11 gene had highest frequency of occurrence,accounted for 76.9%(50/65)of resistant isolates.By using vector-construction for functional verification,A395T alone was responsible for significant increase in azole MIC,whilst C461T did not impact isolates' susceptibilities.In addition,mutations T374C,T769C and G1390A in ERG11 gene also significantly elevate azole MIC,but mutation A983G had no correlation with resistance.The real-time PCR results showed that,the expression of ERG11 gene in average in fluconazole-resistant isolates were higher than that in susceptible isolates,but expression of CYTb gene was lower in resistant group.In comparison,no difference in expression of CDR1 and MDR1 genes was observed between the two groups.Conclusions:The C.tropicalis isolates causing invasive fungal infections in China had high azole resistance,and an increasing trend was observed in recent years,particularly in the last two surveillance years.Generally,the genotype background of C.tropicalis clinical isolates was diverse,but clonal clusters related to fluconazole non-susceptible isolates were identified.Missense mutations in ERG11 gene were mainly responsible for azole resistance in C.tropicalis in China,amongst which A395T missense mutation was the commonest.In addition,the overexpression of ERG11 gene and deficiency in mitochondria also contributed to resistance,whereas the role of overexpression of drug efflux pump is not very important.