Comparison of 16S RDNA Amplicon Sequencing With the Culture Method for Diagnosing Causative Pathogens in Bacterial Corneal Infections

Purpose: The purpose of this study was to explore if 16S rDNA amplicon sequencing can improve the conventional diagnosis of causative pathogens for bacterial corneal infection.

Methods: Corneal scraping and conjunctiva and eyelid margin swab samples from infected eyes of patients diagnosed with "bacterial corneal infection" and conjunctiva and eyelid margin swab samples from a random eye of healthy participants were collected. Each swab was used for both aerobic and anaerobic cultures and 16S rDNA amplicon sequencing. The V3 to V4 region of the 16S rDNA was amplified using polymerase chain reaction (PCR) and sequenced on the Illumina HiSeq 2500 Sequencing Platform.

Results: The overall culture positivity rate for all 72 samples was 69% (72% in the bacterial keratitis group and 67% in the healthy control group), whereas 1719 operational taxonomic units in total were generated using 16S rDNA amplicon sequencing with each sample showing 123 to 337 different genera. Staphylococcus, Corynebacterium, Propionibacterium, and Micrococcus most frequently appeared in culture, whereas Streptococcus, Acinetobacter, and Lactobacillus were the most common genera, with large ratios in 16S rDNA amplicon sequencing. The causative pathogens detected by the two methods were inconsistent for most samples, except for several corneal samples.

Conclusions: We suggest that a combination of different techniques, such as clinical observation, microscopic analysis, culture, and next-generation sequencing techniques including 16S rDNA amplicon sequencing, should be used to comprehensively analyze pathogens in corneal and external ocular infections.

Translational Relevance: This paper uses a basic research methodology for studying the microbiome in ocular samples to help improve the diagnostic accuracy of corneal and external ocular infections.