Avetisov S E, Abramova N D, Gogoleva N E, Gusev O A, Mitichkina T S, Novikov I A, Subbot A M, Shagimardanova E I
Research Institute of Eye Diseases, Moscow, Russia.
I.M. Sechenov First Moscow State Medical University (Sechenov University), Moscow, Russia.
Vestn Oftalmol. 2020;136(3):3-9. doi: 10.17116/oftalma20201360313.
The study is based on the hypothesis that high taxonomic diversity of bacteria detectable on the eye surface by molecular genetic methods is attributed to the high level of its contamination by skin microflora. Such contamination would make it problematic to identify the fractions of real ocular surface microbiome, which remains behind the one-percent cut-off threshold adopted in the metagenomic analysis. Hard contact lenses for long-wearing act as a physical filter preventing DNA contamination from random microorganisms, and at the same time providing adhesion to the living cells of bacteria and fungi. To confirm this assumption, a detailed analysis of references was carried out, supplemented by original laboratory research.
The analysis included 16 hard contact lenses obtained from 11 patients with impaired refraction (myopia). Additionally, conjunctival mucosa scrapings were collected from 42 patients. Samples were cross-analyzed by rRNA gene sequencing using 454 GS Junior (Ion Torrent) and Illumina MiSeq platforms.
Results obtained by the Illumina platform (analysis of the V3-V4 variable region of the rRNA gene) showed better convergence with the data of culture tests reported in the literature. The major microorganism groups found were: (39%), (10.8%), (9.3%), (9.3%), (7.2%), (7%), (4.1%), (3.3%), (3%), (2.4%), (2.3%), and (2.1%). Analysis of the samples obtained directly from the mucosa revealed dominance of typical skin-associated microorganisms.
The present study proposes a contamination-reduction algorithm for microbiological testing of the ocular surface using hard contact lenses for prolonged wearing as a carrier for microbial DNA.
本研究基于这样一种假设，即通过分子遗传学方法在眼表检测到的细菌高分类多样性归因于其被皮肤微生物群高度污染。这种污染会使识别真正的眼表微生物组部分变得有问题，而这部分微生物组在宏基因组分析中低于采用的百分之一截止阈值。长时间佩戴的硬性隐形眼镜起到物理过滤器的作用，可防止随机微生物的DNA污染，同时为细菌和真菌的活细胞提供附着。为证实这一假设，进行了详细的参考文献分析，并辅以原始实验室研究。
分析包括从11名屈光不正（近视）患者处获得的16副硬性隐形眼镜。此外，还从42名患者处收集了结膜黏膜刮片。使用454 GS Junior（Ion Torrent）和Illumina MiSeq平台通过rRNA基因测序对样本进行交叉分析。
通过Illumina平台获得的结果（rRNA基因V3 - V4可变区分析）与文献报道的培养试验数据显示出更好的一致性。发现的主要微生物类群有：（39%）、（10.8%）、（9.3%）、（9.3%）、（7.2%）、（7%）、（4.1%）、（3.3%）、（3%）、（2.4%）、（2.3%）和（2.1%）。对直接从黏膜获得的样本分析显示典型的皮肤相关微生物占主导。
本研究提出一种用于眼表微生物检测的污染减少算法，使用长时间佩戴的硬性隐形眼镜作为微生物DNA的载体。
