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Shotgun metagenomic sequencing in culture negative microbial keratitis
Davide Borroni1, Chiara Bonzano2, José-María Sánchez-González3
1Department of Ophthalmology, Riga Stradins University, Riga, Latvia.
Purpose:
To evaluate the microbiota of culture negative Corneal Impression Membrane (CIM) microbial keratitis samples with the use of shotgun metagenomics analysis.
Methods:
DNA of microbial keratitis samples were collected with CIM and extracted using the MasterPure™ Complete DNA and RNA Purification Kit (Epicentre). DNA was fragmented by sonication into fragments of 300 to 400 base pairs (bp) using Bioruptor® (Diagenode, Belgium) and then used as a template for library preparation. DNA libraries were sequenced on Illumina® HiSeq2500. The resulting reads were quality controlled, trimmed and mapped against the human reference genome. The unmapped reads were taxonomically classified using the Kraken software.
Results:
18 microbial keratitis samples were included in the study. Brevundimonas diminuta was found in 5 samples while 6 samples showed the presence of viral infections. Cutibacterium acnes, Staphylococcus aureus, Moraxella lacunata and Pseudomonas alcaligenes were also identified as the presumed putative cause of the infection in 7 samples.
Conclusions:
Shotgun sequencing can be used as a diagnostic tool in microbial keratitis samples. This diagnostic method expands the available tests to diagnose eye infections and could be clinically significant in culture negative samples.
