| Objective:Most researches on fungal keratitis,a sight-threating infectious corneal disease,have focused on the pathogenesis and treatment of the disease.However,the composition and diversity of fungal microbiome on normal ocular surface is still unclear.Our objective is to investigate and characterize the fungal microbiota on normal ocular surface of humans with high-throughput sequencing method and the culture-based method.Methods:(1)A total of 45 adult subjects were recruited from an urban community.Conjunctival swabs acquired from both eyes of each subject were randomly allocated to either ITS sequencing or fungal cultivation(2)The samples in sequencing group were sent to undertake DNA extraction and examination,PCR amplification and purification,library preparation,and those passed library test were sequenced on Miseq platform.After the completion of sequencing,the double-ended raw data were spliced by overlap,and high quality and effective data were obtained by quality control and chimera filtering,then the sequences were clustered with the 97% similarity,and the species annotation and diversity analysis are carried out according to the final OTU abundance and representative sequence.(3)Another set of samples were inoculated on Sabouraud glucose agar,and incubated at 27 ? for 4 weeks and observed daily.The fungi were identified according to the morphological characteristics of colony and the characteristics of mycelium and spores under microscope.Results:(1)In the sequencing group,18 samples were positive for PCR amplication and sent for the subsequent sequencing.A total of 568,285 raw data were generated,and 518,703 valid data were obtained after quality control and chimera filtration,then 467 different OTU were clustered according to the similarity of 97%.According to the abundance of different OTU in each sample and OUTs in all tested samples,the composition and diversity of fungal community at the level of phylum,class,order,family,genus,and species were obtained by annotation respectively.(2)At the level of phyla,4 fungal phyla were identified.The predominant phylum was Basidiomycota(78.67%,29.45%-99.39%),followed by Ascomycota(19.54%,0.39%-69.74%),Zygomycota(0.13%,0-1.26%),and Neocallimastigomycota(0.01%,0-0.05%).(3)At the class level,nineteen fungal class were identified,of which,8 fungal class had the average abundance over the 0.5%.In descending order: Ustilaginomycotina Incertae sedis(74.65%,25.79%-97.43%),Eurotiomycetes(6.22%,0.04%-56.6%),Dothideomycetes(5.98%,0.03%-23.37%),Saccharomycetes(5.48%,0.03%-23.37%),Microbotryomycetes(2.03%,0.04%-15%),Sordariomycetes(1.01%,0-3.76%),Tremellomycetes(0.79%,0-5.29%),and Wallemiomycetes(0.78%,0-4.27%).Nine fungal class were identified in more than 50% samples,including Ustilaginomycotina incertae sedis,Eurotiomycetes,Dothideomycetes,Microbotryomycetes were detected in all tested samples,and followed by Saccharomycetes(94.44%,17/18),Tremellomycetes(88.89%,16/18),Sordariomycetes(77.78%,14/18),Wallemiomycetes(66.67%,12/18).Among them,Ustilaginomycotina incertae sedis,Eurotiomycetes,Dothideomycetes,Microbotrymycetes,Saccharomycetes and Tremellomycetes,which accounted for 95.15% of all detected fungi and were identified in more than 80% of the tested subjects,may constitute the core fungal class on normal ocular surface.(4)At the order level,thirty-eight fungal order were found,and eight fungal order: Malasseziales(74.65%,25.79%-97.43%),Eurotiales(5.96%,0.04%-56.60%),Saccharomycetales(5.48%,0-68.57%),Capnodiales(2.96%,0.01%-15.43%),Pleosporales(2.24%,0-8.46%),Sporidiobolales(2.03%,0.04%-15.00%),Wallemiales(0.78%,0-4.27%),and Dothideales(0.77%,0-6.52%)had the average abundance >0.5%?A total of nine fungal order were detected in more than 50% samples,of which,Malasseziales,Eurotiales,Capnodiales and Sporidiobolales appeared in all subjects,Saccharomycetales was detected in 94.44%(17/18)of the subjects,Pleosporales was detected in 88.89%(16/18)samples,Tremellales was detected in 77.78%(14 /18)samples,Wallemiales and Hypocreales were detected in 66.67%(12/18)samples.Among them,Malasseziales,Eurotiales,Capnodiales,Sporidiobolales,Saccharomycetales and Pleosporales accounted for 93.32% of all the fungal order,and were detected in more than 80% of the tested subjects,possibly consisting the core fungal order on the normal ocular surface.(5)At the level of family,sixty-four fungal family were identified,among which,ten family with an average abundance over 0.5%.In desending order: Malasseziales Incertae sedis(74.65%,25.79%-97.43),Trichocomaceae(5.96%,0.04%-56.6%),Saccharomycetaceae(4.14%,0-15.02%),Sporidiobolales Incertae sedis(1.95%,0-14.27%),Pleosporaceae(0.89%,0-4.77%),Wallemiaceae(0.78%,0-4.27%),Dothioraceae(0.77%,0-6.52%),Debaryomycetaceae(0.66%,0-6.93%),and Saccharomycetales Incertae sedis(0.60%,0-5.34%)(6)At the genus level,94 fungal genera were identified,of which,12 genera had average abundance>0.5% of all genera.The most abundant genera was Malassezia(74.65%,25.79%-97.43%),followed by Petromyces(4.32%,0-56.31%),Kazachstania(3.80%,0-68.29%),Rhodotorula(1.93%,0.03%-14.00%),Davidiella(1.89,0-7.77%),Kabatiella(0.77%,0-6.52%),Cladosporium(0.73%,0-6.03%),Meyerozyma(0.66%,0-6.93%),Alternaria(0.61%,0-3.31%)and Candida(0.59%,0-5.34%).(7)At the level of species,113 species were identified,and 9 fungal species had average abundance over 0.5%,among which,Malassezia restrica(55.39%,2.21%-90.81%)were most abundant species,followed by Malassezia globose(5.29%,0.22%-20.78%),Aspergillus flavus(4.27%,0-55.76%),Kazachstania telluris(3.80%,0-68.29%),Rhodotorula mucilaginasa(1.67%,0.03%-13.94%),Cladosporium haloterans(0.73%,0-6.03%),Alternaria alternate(0.61%,0-3.31%),Wallemia mellicola(0.61%,0-4.27%)and Aspergillus vitricola(0.52%,0-5.92%).(8)Four in 45 conjunctival swabs were positive for fungal cultivation: Penicillium citrinum,Aspergillus niger,Phialophora,and Trichoderma.Conclusion : 1.The fungal microbiome on normal ocular surface were expored preliminarily,which strengthened the recognization of microbiota on ocular surface,and provided a basis for further investigation of the relationship between microbiota and ocular health and diseases.2.Compared to fungal cultivation,the identification of fungal microbiome on ocular surface with high-throughput sequencing was more diverse,moreover,it can provide a more comprehensive overview of structure of fungal microbiome on ocular surface. |
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