Investigation On Phylogenetic Characters And Of Important Candida Species Causing Invasive Diseases And Association With Antifungal Resistance In China

[Objective]Invasive fungal diseases have been considered as a major clinical challenge and cause of morbidity and mortality that linked to advances in medical technology.Meanwhile,emergence and spread of antifungal resistance further poses great threats to management of invasive fungal diseases.A better understanding for population genetic characters of medically important fungi pathogens could help illustrate evolution and adaptation of species,and explore if certain phylogenetic clades with antifungal resistance or other adaptive advantage phenotypes undergo population expansions.In this study,we carried out systematic population genetic study for selected major Candida species pathogens responsible for invasive fungal diseases in China.[Methods]This study relied on resources accumulated in China Hospital Invasive Fungal Surveillance Net(CHIF-NET)study.Part Ⅰ.The study updated recent surveillance results of candidemia from CHIF-NET program.A total of 77 hospitals across China over 1 August,2014 to 31 July,2017 was enrolled.Species identification was carried out by MALDI-TOF MS supplemented with DNA sequencing.Antifungal susceptibility was determined using YeastOne kits.Epidemiology and antifungal resistance of different Candida species was analyzed.Part Ⅱ.Population genetics investigation of Candida krusei isolates in China.We developed a novel set of microsatellite markers for molecular typing of C.krusei isolates.The assay was further used for typing 48 C.krusei clinical stains that from 15 hospitals distributed in 10 cities.Correlation between genetic diversity,clinical characters and antifungal susceptibility among the studied strains were evaluated.Part Ⅲ.Population genomics study of Candida albicans isolates in China.From 33 cities,370 C.albicans isolates causing invasive fungal diseases were collected,and 181 global C.albicans genomes were involved as comparators.Association between clonal structures of isolates and antifungal non-susceptible phenotypes was illustrated,and molecular mechanisms responsible for resistance was predicted.Part Ⅳ.Population genomics investigation of Candida tropicalis isolates in China.The study comprised 398 C.tropicalis isolates from 27 cities,and 78 C.tropicalis genomes from previous international research.Population structure,polyploidy and aneuploidy characters were evaluated.Further,association between phylogenetic groups and antifungal resistance,and isolates’ resistant mechanisms was further analyzed[Results]Part Ⅰ.Amongst 4,010 isolates collected,although C.albicans remained the most common species,non-albicans Candida species accounted for over two-thirds of isolates.Most C.albicans and C.parapsilosis complex isolates were susceptible to all antifungal agents(resistance rate<5%).However,there was a consistent decrease in voriconazole susceptibility to C.glabrata sensu stricto over the three years(the non-wild-type phenotype rose from<20%to～50%).In addition,the fluconazole resistance rate in C.tropicalis tripled from<10%in 2009-2010 to>30%in 2016-2017.Moreover,amongst less common Candida species,over one-third of C.pelliculosa isolates were co-resistant to fluconazole and 5-flucytocine,and>56%of C.haemulonii isolates were multidrug resistance.Part Ⅱ.We developed a set of microsatellite markers for this organism from genomic analysis,which contained 33 selected loci.The new system had a cumulative discriminatory power of 1.000.Population structure analyses by this assay revealed that C.krusei could be separated into two clusters,namely Cluster A(n=17)and Cluster B(n=31),which was also supported by PCoA.Patients infected by Clusters A strains were mainly children<10 years old or aged people>50 years old.While the hosts of Cluster B covered all age groups.Cluster B strains showed higher genetic diversity,which exhibited wider MIC ranges for six antifungal agents.In addition,the highest MIC values of the six antifungal agents were discovered in Cluster B.Furthermore,Part Ⅲ.It revealed that 66.6%of azole-non-susceptible strains belonged to C.albicans Clade 1.Of note,two sub-clades in Clade 1,namely Clade 1-R(n=44)and Clade 1-R-α(a unique sub-group of Clade 1-R,n=9)were identified,and phylogenetic analysis and loss-of-heterozygosity events indicated two recent population expansions.Clade 1-R isolates exhibited high azole-non-susceptible rate(75.0%),while all Clade 1-R-α isolates were pan-azole-non-susceptible,which carried unique A114S and Y257H double substitutions in Erg11p.In addition,Clade 1-R-α isolates had substitution A736V in transcript factor Tac1 that regulation expression of Cdr drug efflux pumps.Worldwide,Clade 1-R-α isolates were exclusively identified in China,but from a large span of geographic origins.Part Ⅳ.Fourteen phylogenetic groups were identified from 476 C.tropicalis genomes,with geographic variation in proportion of local and international isolates.Over 95%of isolates were diploid,while some haploid,triploid and octoploid isolates(n=13)were also identified.In addition,aneuploidies were observed,and loss-of-heterozygous events were common.Of 105 fluconazole-resistant C.tropicalis isolates in China,75%belonged to a conserved CNR clade.All CNR clade isolates were collected in China,with>98%exhibiting azole non-susceptible phenotypes and carrying Erg11p Y132F and S154F double amino acid substitutions.Moreover,certain CNR sub-branch isolates also had Tac1 substitutions A446E and E507D.Overall,CNR isolates were widely distributed in different geographic regions of China.[Conclusions]Amongst Candida species causing invasive fungal diseases in China,decreasing in antifungal susceptibility caused by intrinsic and acquired resistance has become a notable challenge.By population genetic analysis,we found there were certain phylogenetic populations in representative pathogenic Candida species,including C.albicans,C.tropicalis and C.krusei,exhibited strong associations with antifungal non-susceptible phenotype.Therefore,along with previous epidemiology and antifungal susceptibility surveillance,there’s substantial needs to develop a close monitoring system for spreading of antifungal resistant Candida clonal populations,and carry out further research to illustrate the mechanism behind population expansion.