Genetic Diversity And In Vitro Antifungal Susceptibility Of Clinical Scedosporium Isolates From China

BackgroundScedosporium is a group of opportunistic pathogenic fungi which is widely distributed in nature.In taxonomy,Scedosporium belongs to Fungi,Ascomycota,Sordariomycetes,Microcystis Microascales,Microascaceae and Scedosporium spp.which is distributed in high density in human-affected areas such as soil,sewage,fertilizers and rotten vegetation.Since Scedosporium prolificans has been proven to be independent from Scedosporium in taxonomy,it has been re-named Lomentospora prolificans.The Pseudallescheria and Scedosporium are now re-classified as "Scedosporium".Now,the genus Scedosporium that can cause human disease is mainly divided into 7 species,and S.apiospermum,S.boydii and S.aurantiacum are the three major clinically relevant species.In recent years,the incidence of scedosporiosis has been increasing,and it has become the second opportunistic filamentous fungus following Aspergillus fumigatus.Scedosporium is distributed worldwide,but there are obvious regional differences.For example,S.apiospermum and S.boydii are the mainly species in our country,while S.aurantiacum is more common in dry and hot area such as Australia.Scedosporiosis has various types of manifestation,including colonization in cavity,localized and disseminated infections.It can also cause central nervous system infections in drowned people.Early diagnosis of Scedosporiosis is difficult,thus the prognosis is poor.S.apiospermum is the most common species in clinic.In recent years,the incidence of its invasive infections has been increasing,and the treatment of scedosporiosis is difficult.Moreover,different species of Scedosporium have different sensitivities to commonly used antifungal drugss.However,the current clinical microbiology laboratory’s identification methods for Scedosporium are limited,leading to the consequences that the isolates cannot be identified to the level of strains early,and only empirical antifungal treatment can be performed.Amphotericin B is a broad-spectrum antifungal drug which is often used in empirical antifungal treatment,but most strains of Scedosporium are naturally resistant to it.At present,the genetic diversity and antifungal drug susceptibility monitoring of Scedosporium strains isolated from China are still insufficient,and the clinical and epidemiological breakpoints of their antifungal susceptibility to common antifungal drugs have not yet been identified.Its genotype distribution characteristics and geographic distribution are not clear either.In this study we sought to perform new molecular markers such as multilocus sequence typing(MLST)and whole-genome sequencing analysis on the clinical isolates of Scedosporium from Fujian,Shandong,Beijing,Hunan and other provinces that have been collected in the past 20 years.This study aimed to provide scientific basis and research foundation for accurate antifungal treatment of Scedosporiosis.ObjectiveFirstly,use MLST to identify Scedosporium strains that were collected from China at the genotype level and to analyze its phylogeny,explore genetic diversity such as the distribution of genotypes and the evolutionary relationship.Secondly,conduct an in vitro antifungal drug susceptibility test on Scedosporium to reveal the susceptibility characteristics of Scedosporium to different antifungal drugs in China,and provide a reference for the study of clinically accurate antifungal treatment plans.Thirdly,use nextgeneration genome sequencing(NGS)technology to conduct a genome study on the unique genotype or phenotype of Scedosporium strains,and analyze the origin and molecular mechanism of antifungal drug resistance of it.This study eventually aimed to provide a scientific basis for the research on the diagnosis and treatment of Scedosporiosis in China.MethodsPart one: The clinical and epidemiological information of Scedosporium strains collected from Fujian,Shandong,Beijing,Hunan,Anhui and Guangxi were summarized and sorted.And we amplify the internal transcribed spacer region(ITS)and β-tubulin(BT2)sequence and the sequences were used to identify Scedosporium species by BLAST in NCBI database,and use MLST which was recommended by the International Society for Human and Animal Mycology(ISHAM)to identify it’s sequence type(ST).We use ITS,and ITS combined with BT2 to construct phylogenetic tree to explore the evolution relationship of Scedosporium.Part two: Using M-38 method recommended by CLSI to test the antifungal susceptibility of Scedosporium strains and find the antifungal drug-resistant strain.Part three: The whole genome of Scedosporium was sequenced using Pac Bio platform as a high-quality reference genome.The genome was annotated combining ab initio methods and de novo RNA-seq.Genomes of other clinical isolates were also sequenced using the second-generation sequencing platform.With these data,the phylogenetic relationship of the Cyp51 gene were analyzed.Also,the single nucleotide polymorphism(SNP)was investigated across all genomes of Scedosporium.ResultsPart one: The six Scedosporium species collected in this study included S.apiospermum,S.boydii,S.aurantiacum,S.aurantiacum,S.dehoogii,S.ellipsoidea and S.angusta.S.apiospermum(37%,10/27)and S.boydii(37%,10/27)were the most common Scedosporium species.The strains were mainly isolated from lung-related tissue specimens(37.0%,10/27).S.apiospermum(40.0%,4/10)was the dominant pathogenic strain for lung infections,but S.boydii(47.1%,8/17)was more than other species in other sites.In this study,4 strains of S.anrautiacum(14.8%,4/27)were collected,among which 3 strains(75%,3/4)were isolated from pulmonary infection,and 1 strain(25%,1/4)was isolated from central nervous system infection.S.aurantiacum was firstly reported here in China.The predominant ST types of S.apiospermum,S.boydii and S.anrantiacum in China are ST17,ST3 and ST92 respectively,and in this study we discovered 3 new ST types in S.apiospermum(ST40,ST41,ST42).Part two: Among the 8 antifungal drugs,Voriconzole(VOR)(MIC50=0.25ug/ml;MIC90=0.5ug/ml)and Micafungin(MCF)(MIC50=0.25ug)/ml;MIC90=0.5ug/ml)had the highest antifungal activity;Amphotericin B(AMB)has the lowest antibacterial activity(MIC50=8ug/ml;MIC90 >16ug/ml),which is worthy of further research and next vigilant monitoring.Part three: In this study,we obtained the reference genome with the best quality compared with the Scedosporium genome in the public database.Using a combination of bioinformatics and RNA-seq,10,423 non-redundant protein-coding genomes were annotated with COG,KOG,KEGG and GO terms providing a comprehensive bioinformatic analysis.Secondly,a phylogenetic tree was constructed on the Cyp51 gene of the isolated clinical samples,and it was found that the antifungal drug resistance of Scedosporium has a certain correlation with the evolution of the Cyp51 gene.This study found that the classification based on whole-genome SNPs is significantly different from the classification of MLST based on multiple housekeeping gene loci.By comparing with the population of European Scedosporium strains,we found that the genetic diversity of clinical isolates of Scedosporium in China is relatively low,which is obviously different from the distribution of Scedosporium strains from Europe.At the same time,the S.apiospermum isolated from China is more genetically diverse than S.boydii.ConclusionIn this study,the clinical isolates of Scedosporium collected from different provinces in China included S.apiospermum,S.boydii,S.aurantiacum,S.dehoogii,S.ellipsoidea,S.angusta.Among them,S.apiospermum and S.boydii were the most common species of Scedosporium in China.Among S.apiospermum,3 new STs(ST40,ST41,ST42)were firstly reported in this sutdy.The results of the in vitro antifungal drug susceptibility test of Scedosporium suggested that voriconazole and micafungin had the highest antifungal activity for Scedosporium in this study,and Scedosporium has the lowest sensitivity to amphotericin B.In the future,systematic research and monitoring of the antifungal drug resistance trend and genetic diversity of clinical and environmental isolates of Scedosporium in China is necessary for the clinical diagnosis and treatment of Scedosporiosis.