Whole genome analysis of the common pathogens associated with microbial keratitis

Abstract

Background Microbial Keratitis is a serious infection of the cornea and a major cause of corneal opacity and vision loss worldwide. Whole-genome sequencing (WGS) of keratitis bacterial isolates has the ability to provide a wealth of clinically-valuable information. The potential of metagenomics has also been evaluated as current techniques for pathogen identification are laborious, time-consuming and associated with poor culture rates, making metagenomics an exciting future prospect. Methods/Results Samples from keratitis patients were collected and isolates grown in agar before DNA was extracted from pure-cultures and WGS data was obtained from a total of 30 isolates, including common causative agents Pseudomonas aeruginosa and Staphylococcus aureus. Libraries were prepared using a miniaturised Nextera XT protocol and sequencing was carried out on Illumina’s NextSeq platform. Genomic analysis was then based around MLST-typing, comparative genomics, and the identification of key genes inferring antibiotic resistance and virulence. Our pathogens exhibited diverse evolutionary lineages and a range of virulence-associated genes, providing insight into pathogenicity. The ability to obtain sufficient DNA quantities for metagenomics sequencing was also assessed using various host depletion and microbiome enrichment techniques. Discussion/Conclusion The clinical role of next-generation sequencing is currently limited due to associated costs and required computational resources, however in the future this method could replace current techniques for pathogen identification. WGS provides important insights into the genomes of the common keratitis infectious agents, improving understanding of the aetiology of corneal infections and metagenomics shows future potential to be the centre of culture-independent pathogen identification.