Rational strategy for studying microbiome of the ocular surface of people using hard contact lenses by method of 16S rRNA gene metabarcoding

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. MATERIAL AND METHODS 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 16S rRNA gene sequencing using 454 GS Junior (Ion Torrent) and Illumina MiSeq platforms. RESULTS Results obtained by the Illumina platform (analysis of the V3-V4 variable region of the 16S rRNA gene) showed better convergence with the data of culture tests reported in the literature. The major microorganism groups found were: Acinetobacter (39%), Gluconacetobacter (10.8%), Propionibacterium (9.3%), Corynebacterium (9.3%), Staphylococcus (7.2%), Streptococcus (7%), Pseudomonas (4.1%), Micrococcus (3.3%), Yersinia (3%), Chondromyces (2.4%), Serratia (2.3%), and Bacillus (2.1%). Analysis of the samples obtained directly from the mucosa revealed dominance of typical skin-associated microorganisms. CONCLUSION 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.