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

Abstract

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.

Achieving diagnostic excellence for infectious keratitis: A future roadmap

“…In addition, NGS can result in non-selective amplifications of the RNA / DNA of the causative organisms and normal ocular surface commensals, which will require special bioinformatic consideration and analysis (hence the need for additional resources, facilities and expertise). Another issue that has been highlighted is that NGS may produce inconsistent results to the conventional culture methods, which may complicate the clinical findings and decision making in IK (An et al, 2022 ).…”

Microbiological Culture Versus 16S/18S Ribosomal RNA PCR-Sanger Sequencing for Infectious Keratitis: A Three-Arm, Diagnostic Cross-Sectional Study

“…In the future, multiplex PCR or metagenomic next generation sequencing may also further improve the diagnosis of IK, though its routine use in clinic is currently limited by the cost and the availability of technical expertise and resources. [44][45][46][47][48] However, it has also been reported that by adopting an intensive antibiotic treatment regime for sight threatening IK, the clinical outcome of culture positive and culture negative cases was not significantly different. 4,49 Our study observed a moderate diagnostic yield in both culture and PCR methods (ranged 40-50%), which might be related to the inclusion of less severe infection cases (~60% of the cases have an infiltrate size of <3mm).…”

Bacillus velezensis Strain GUMT319 Reshapes Soil Microbiome Biodiversity and Increases Grape Yields

“…The 16S rDNA sequencing followed previously described protocols [ 36 , 37 ]. The V3-V4 region of 16S rDNA was cloned and prepared for high-throughput sequencing on an Illumina HiSeq 2500 platform in Novogene Co., Ltd. (Novogene, Beijing, China).…”