A Genomic Approach to Investigating Ocular Surface Microorganisms: Monitoring Core Microbiota on Eyelid Margin with a Dot hybridization Assay

Abstract

A sound ocular surface microbiota has been recognized as a part of ocular surface health following a growing body of evidence from next-generation sequencing technique and metagenomic analysis. However, even from the perspective of contemporary precision medicine, it is difficult to directly apply these new technologies to clinical practice. Therefore, we proposed a model based on dot hybridization assay (DHA) to bridge conventional culture with a metagenomic approach in investigating and monitoring ocular surface microbiota. Endophthalmitis, mostly caused by bacterial infection, is the most severe complication of many intraocular surgeries, such as cataract surgery. Hazardous microorganisms hiding and proliferating in the ocular surface microbiota not only increase the risk of endophthalmitis but also jeopardize the effectiveness of the preoperative aseptic procedure and postoperative topical antibiotics. The DHA model enables the simultaneous assessment of bacterial bioburden, detection of target pathogens and microorganisms, and surveillance of methicillin/oxacillin resistance gene mecA in the ocular surface microbiota. This assay revealed heavier bacterial bioburden in men, compatible with a higher risk of endophthalmitis in male patients who underwent cataract surgery. No occurrence of endophthalmitis for these patients was compatible with non-hazardous microorganisms identified by specific dots for target pathogens. Moreover, the mecA dot detected oxacillin-resistant strains, of which culture failed to isolate. Therefore, the DHA model could provide an alternative genomic approach to investigate and monitor ocular surface microorganisms in clinical practice nowadays.

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

“…Of course, 16S rDNA amplicon sequencing is not a perfect technology that always provides accurate and satisfactory results: it can usually identify bacteria in the genus level instead of species through the V3 to V4 sequences of 16S rDNA; several procedures of the process including DNA extraction, PCR amplification, matching in the databases for identification would introduce bias to influence the results. 18 – 21 Those defaults could be improved gradually through detecting more ocular samples. Conversely, our findings proved that the culture method is not always reliable to diagnose pathogenic bacteria even when samples show positive culture results: sometimes, bacteria from the environment may provide false-positive results; sometimes, the pathogenic bacteria that cannot grow easily under classical culture conditions may show false-negative findings; sometimes, the commensal bacteria that adapted well to the culture conditions may grow extremely vigorously and provide a false-positive result.…”

“…Moreover, new and improved approaches based on NGS will be explored with the accumulation of increasing discoveries from more parts of the world. 21 …”

Characterization and Comparison of Ocular Surface Microbiome in Newborns

“…Respect with to newborn OSM, the microbiota of adults was extensively investigated [ 27 , 42 ]. Several evidences report the Proteobacteria as the main phylum in the ocular microbiota of adults.…”

Application Progress of High-Throughput Sequencing in Ocular Diseases

“…Although this study has some differences from the findings of Dong et al [ 40 ], it shows the relevance of HTS in identifying normal conjunctival microbiota ( Table 1 ). Recently, Kuo et al [ 41 ] constructed a model based on a dot hybridization assay (DHA) that combines traditional culture with emerging HTS technologies to study ocular surface microbiota. DHA revealed a higher bacterial bioburden in men than in women, enabled the detection of target pathogens and microbiota, and can monitor ocular surface microbiota for antibiotic resistance [ 41 ].…”

“…Recently, Kuo et al [ 41 ] constructed a model based on a dot hybridization assay (DHA) that combines traditional culture with emerging HTS technologies to study ocular surface microbiota. DHA revealed a higher bacterial bioburden in men than in women, enabled the detection of target pathogens and microbiota, and can monitor ocular surface microbiota for antibiotic resistance [ 41 ]. Shivaji et al [ 42 ] were the first to use the NGS technology to explore fungal microbiota on the ocular surface of healthy humans.…”