Department of Biological Sciences, University of North Texas, Denton, Texas, USA.
Invest Ophthalmol Vis Sci. 2011 Jul 20;52(8):5408-13. doi: 10.1167/iovs.10-6939.
Ocular surface (OS) microbiota contributes to infectious and autoimmune diseases of the eye. Comprehensive analysis of microbial diversity at the OS has been impossible because of the limitations of conventional cultivation techniques. This pilot study aimed to explore true diversity of human OS microbiota using DNA sequencing-based detection and identification of bacteria.
Composition of the bacterial community was characterized using deep sequencing of the 16S rRNA gene amplicon libraries generated from total conjunctival swab DNA. The DNA sequences were classified and the diversity parameters measured using bioinformatics software ESPRIT and MOTHUR and tools available through the Ribosomal Database Project-II (RDP-II).
Deep sequencing of conjunctival rDNA from four subjects yielded a total of 115,003 quality DNA reads, corresponding to 221 species-level phylotypes per subject. The combined bacterial community classified into 5 phyla and 59 distinct genera. However, 31% of all DNA reads belonged to unclassified or novel bacteria. The intersubject variability of individual OS microbiomes was very significant. Regardless, 12 genera-Pseudomonas, Propionibacterium, Bradyrhizobium, Corynebacterium, Acinetobacter, Brevundimonas, Staphylococci, Aquabacterium, Sphingomonas, Streptococcus, Streptophyta, and Methylobacterium-were ubiquitous among the analyzed cohort and represented the putative "core" of conjunctival microbiota. The other 47 genera accounted for <4% of the classified portion of this microbiome. Unexpectedly, healthy conjunctiva contained many genera that are commonly identified as ocular surface pathogens.
The first DNA sequencing-based survey of bacterial population at the conjunctiva have revealed an unexpectedly diverse microbial community. All analyzed samples contained ubiquitous (core) genera that included commensal, environmental, and opportunistic pathogenic bacteria.
眼表（OS）微生物群有助于眼部的感染和自身免疫性疾病。由于传统培养技术的限制，对 OS 微生物多样性的综合分析一直无法实现。本初步研究旨在使用基于 DNA 测序的细菌检测和鉴定方法来探索人类 OS 微生物组的真实多样性。
使用从总结膜拭子 DNA 生成的 16S rRNA 基因扩增子文库的深度测序来描述细菌群落的组成。使用生物信息学软件 ESPRIT 和 MOTHUR 以及通过核糖体数据库项目-II（RDP-II）提供的工具对 DNA 序列进行分类并测量多样性参数。
对四名受试者的结膜 rDNA 进行深度测序共产生了 115,003 个质量 DNA 读段，每个受试者对应 221 个种级别的分类群。组合细菌群落分为 5 个门和 59 个不同属。然而，所有 DNA 读段的 31％属于未分类或新型细菌。个体 OS 微生物组的个体间可变性非常显著。尽管如此，12 个属 - 假单胞菌、丙酸杆菌、慢生根瘤菌、棒状杆菌、不动杆菌、短小杆菌、葡萄球菌、Aquabacterium、鞘氨醇单胞菌、链球菌、链形植物和甲基杆菌 - 在分析队列中普遍存在，代表了结膜微生物组的“核心”。其余 47 个属仅占该微生物组分类部分的<4％。出乎意料的是，健康的结膜包含许多通常被认为是眼表病原体的属。
基于 DNA 测序的首次对结膜细菌种群的调查揭示了一个出人意料的多样化微生物群落。所有分析的样本均含有普遍存在的（核心）属，包括共生菌、环境菌和机会致病菌。
