Discussion
We propose metagenomic analysis as a quick and accurate diagnostic method for detecting pathogens in bacterial endophthalmitis. We confirmed that the culture-positive results from bacterial cultures matched those from metagenomic analysis in terms of pathogen identity. We also found that causative pathogens were detected by metagenomic analysis even in cases in which bacterial cultures had yielded no results. Patients with a history of uveitis did not exhibit positive results from either of these methods. Confirmation of α and β diversities is both helpful to differentiate between cases involving infection or not. In the cases of endophthalmitis, α diversity was decreased and no bacterial occupancy rate above 25% was observed. The β diversity, which indicates similarity, was different between cases involving infection and those that did not. Metagenomic analysis can detect the pathogen within a few days, allowing for appropriate selection of antibiotic treatment and the subsequent preservation of visual function.
Metagenomic analysis enables the analysis of DNA segments from multiple microorganisms without the need for culture and is carried out using either an amplicon or shotgun-based approach.17 Previously, short-read sequencing of the bacterial 16S rRNA gene has been the standard method for microbiome profiling; however, recently long-read sequencing method has been employed to achieve a more comprehensive microbiome profiling.18 Long-read methods offer higher detection confidence than short-read methods do. Moreover, long-read methods provide strain-level community detection.19 Although long read sequence had not been performed in this study, we analysed the V1–V2 region based on a previous report because of the good performance.14 Both short-read and long-read metagenomic methods have yielded similar results in detecting similar bacterial compositions.14 Considering, the target disease in this study was endophthalmitis, which is an emergency disease,20 the most important factor was the timely detection time for prompt treatment while complete identification of the bacterial strain is desirable.
Detecting bacterial pathogens in endophthalmitis based on bacterial culture poses significant challenges due to specific growth requirements.11–13 In cases of postoperative endophthalmitis, Gram-negative bacteria were identified as the infecting pathogen in almost 50% of the cases, whereas Gram-positive bacteria were present instead in 20% of the cases, and no pathogen was identified in the remaining cases.21 In previous studies, the causative bacterial species varied according to the endophthalmitis type. Notably, bacterial agents could not be identified in numerous cases, highlighting diagnostic challenges.22 Metagenomic analysis makes it possible to detect pathogenic bacteria at the species level.23 24 Previous reports have shown that a 16S rRNA metagenomic analysis encompasses the whole genomic DNA present in the sample, leading to potential issues with contamination.25 As a result, indigenous bacteria, such as Methylobacterium and Corynebacterium, have often been identified in experiments and reported as contamination.26 In this study, negative controls were included, and various types of commensal bacteria were detected. The occupancy rate of these bacteria was very low (<0.05%) and no bacteria suspected to be causative agents were identified. Additionally, if viruses or fungal infection occurred, the number of reads associated with these pathogens could be higher than that of the bacterial species. In addition, due to the presence of specific viruses or fungi in the sample, the predominance of bacterial species may not be accurately detected. Our findings also demonstrate that the pathogenesis for the syndrome can be determined even in cases with negative results from the bacterial culture. In fact, even if endophthalmitis is suspected clinically and the culture yields negative results, treatment for endophthalmitis is often continued until clinical findings improve.11–13 21 If the metagenome yields positive results in culture-negative cases, it can serve as a basis for continuing the use of antibiotics.
It is difficult to clinically determine in which cases infection is present.27–29 This study demonstrates alterations in α and β diversities in endophthalmitis. Considering a 25% occupancy rate as threshold, our results may provide a valuable diagnostic tool for inflammatory ocular conditions such as uveitis. In the patients diagnosed with uveitis, both culture and metagenomic analyses yielded negative results. Interestingly, α diversity was preserved, and no bacterial populations surpassed a 25% occupancy rate. In addition, in the negative controls, all the bacterial occupation rate was below 15% and the average diversity was similar to that of the non-infectious group. These results indicated that the adulteration of genomic remnants from the blood stream of uveitis or contamination may occur during the extraction process. Even if the pathogenic bacteria are present in the vitreous body, their pathogenicity may be low as long as the occupancy rate does not exceed the threshold. While case-specific factors may influence the selected cut-off value, our findings suggest that a decrease in diversity, coupled with an evaluation of bacterial occupancy rates, can serve as potential indicators for the diagnosis of endophthalmitis. Although β-diversity is affected by causative pathogens, it is possible to diagnose endophthalmitis by combining different factors, including compositional differences, occupancy rate of major causative bacteria and decreased α-diversity.
This study had several limitations including its small sample size, which was a direct consequence of the low incidence of endophthalmitis, the small amount of vitreous body available may have resulted in some bacteria being missed during the identification. In the analysis, microbial composition in the vitreous body could have been affected by factors including patient demographics such as age, immunocompromised host and medication. In addition, the occupancy of specific bacteria may differ between the onset and advanced stages of endophthalmitis. Moreover, it was difficult to maintain the same condition across all patients since the reasons for developing infectious endophthalmitis were different for each individual. Regarding technical aspects of metagenomic sequences, taxonomic classification was limited to what was included in reference library, and results could have been affected by contamination and bias. Therefore, negative controls were essential to ensure the integrity and reproducibility of the results. Despite these limitations, the pathogens identified by the metagenomic analysis were consistent with those identified by culture results at the genus and species level.
In summary, metagenomic analysis offers expedited pathogen detection and allows for the discrimination between infectious and non-infectious ocular conditions, particularly in cases with negative results from bacterial culture. This approach facilitates swift pathogen detection, enables precise diagnosis and selection of suitable antibiotic treatment and allows differential diagnosis of uveitis cases.