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Gut bacterial microbiome dysbiosis in type 2 Diabetes Mellitus (T2DM) has been reported, but such an association with Diabetic Retinopathy (DR) is not known. We explored possible link between gut bacterial microbiome dysbiosis and DR. Using fecal samples of healthy controls (HC) and people with T2DM with/without DR, gut bacterial communities were analysed using 16S rRNA gene sequencing and data analysed using QIIME and R software. Dysbiosis in the gut microbiomes, at phyla and genera level, was observed in people with T2DM and DR compared to HC. People with DR exhibited greater discrimination from HC. Microbiomes of people with T2DM and DR were also significantly different. Both DM and DR microbiomes showed a decrease in anti-inflammatory, probiotic and other bacteria that could be pathogenic, compared to HC, and the observed change was more pronounced in people with DR. This is the first report demonstrating dysbiosis in the gut microbiome (alteration in the diversity and abundance at the phyla and genera level) in people with DR compared to HC. Such studies would help in developing novel and targeted therapies to improve treatment of DR.

Studies have documented dysbiosis in the gut mycobiome in people with Type 2 diabetes mellitus (T2DM). However, it is not known whether dysbiosis in the gut mycobiome of T2DM patients would be reflected in people with diabetic retinopathy (DR) and if so, is the observed mycobiome dysbiosis similar in people with T2DM and DR. Gut mycobiomes were generated from healthy controls (HC), people with T2DM and people with DR through Illumina sequencing of ITS2 region. Data were analysed using QIIME and R software. Dysbiotic changes were observed in people with T2DM and DR compared to HC at the phyla and genera level. Mycobiomes of HC, T2DM and DR could be discriminated by heat map analysis, Beta diversity analysis and LEfSE analysis. Spearman correlation of fungal genera indicated more negative correlation in HC compared to T2DM and DR mycobiomes. This study demonstrates dysbiosis in the gut mycobiomes in people with T2DM and DR compared to HC. These differences were significant both at the phyla and genera level between people with T2DM and DR as well. Such studies on mycobiomes may provide new insights and directions to identification of specific fungi associated with T2DM and DR and help developing novel therapies for Diabetes Mellitus and DR.

The proof-of-concept, study to investigate the presence of microorganisms in presumed infectious endophthalmitis using Next generation sequencing (NGS) was carried out in vitreous biopsies from 34 patients with endophthalmitis, and thirty patients undergoing surgery for non-infectious retinal disorders as controls. Following DNA extraction using the Qiagen mini kit and PCR amplification of the V3–V4 regions of the bacterial 16S rRNA and ITS 2 region of fungus, they samples were sequenced on an Illumina HiSeq 2500 Machine. Paired reads were curated, taxonomically labeled, and filtered. Culture based diagnosis was achieved in 15/34 (44%) patients while NGS diagnosed the presence of microbes in 30/34 (88%) patients (bacteria in 26/30, fungi in 2/30, mixed infections in 2/30 cases). All 30 controls were negative for bacteria or fungus by NGS. There was good agreement between culture and NGS for culture-positive cases. Among culture negative cases, DNA of common culturable bacteria were identified like Streptococcus sp., Staphylococcus sp., Pseudomonas sp., Gemella sp., Haemophilus sp., Acinetobacter sp . The specificity of NGS with culture and clinical diagnosis was found to be 20% and 100% respectively and sensitivity of NGS with culture and clinical diagnosis was found to be 87.5% and 88% respectively. NGS appears to be promising diagnostic platform for the diagnosis of infectious culture negative endophthalmitis.

Dysbiosis in the gut microbiome has been implicated in several diseases including auto-immune diseases, inflammatory diseases, cancers and mental disorders. Keratitis is an inflammatory disease of the eye significantly contributing to corneal blindness in the developing world. It would be worthwhile to investigate the possibility of dysbiosis in the gut microbiome being associated with Keratitis. Here, we have analyzed fungal and bacterial populations in stool samples through high-throughput sequencing of the ITS2 region for fungi and V3-V4 region of 16S rRNA gene for bacteria in healthy controls (HC, n = 31) and patients with fungal keratitis (FK, n = 32). Candida albicans (2 OTUs), Aspergillus (1 OTU) and 3 other denovo-OTUs were enriched in FK samples and an unclassified denovo-OTU was enriched in HC samples. However, the overall abundances of these 'discriminatory' OTUs were very low (< 0.001%) and not indicative of significant dysbiosis in the fungal community inhabiting the gut of FK patients. In contrast, the gut bacterial richness and diversity in FK patients was significantly decreased when compared to HC. 52 OTUs were significantly enriched in HC samples whereas only 5 OTUs in FK. The OTUs prominently enriched in HC were identified as Faecalibacterium prausnitzii, Bifidobacterium adolescentis, Lachnospira, Mitsuokella multacida, Bacteroides plebeius, Megasphaera and Lachnospiraceae. In FK samples, 5 OTUs affiliated to Bacteroides fragilis, Dorea, Treponema, Fusobacteriaceae, and Acidimicrobiales were significantly higher in abundance. The functional implications are that Faecalibacterium prausnitzii, an anti-inflammatory bacterium and Megasphaera, Mitsuokella multacida and Lachnospira are butyrate producers, which were enriched in HC patients, whereas Treponema and Bacteroides fragilis, which are pathogenic were abundant in FK patients, playing a potential pro-inflammatory role. Heatmap, PCoA plots and functional profiles further confirm the distinct patterns of gut bacterial composition in FK and HC samples. Our study demonstrates dysbiosis in the gut bacterial microbiomes of FK patients compared to HC. Further, based on inferred functions, it appears that dysbiosis in the gut of FK subjects is strongly associated with the disease phenotype with decrease in abundance of beneficial bacteria and increase in abundance of pro-inflammatory and pathogenic bacteria.

Fungi have been associated with various diseases of the eye like keratitis, uveitis and endophthalmitis. Despite this fact, fungal microbiome (mycobiome) studies compared to the bacterial microbiome studies have remained neglected. In the present study, using metagenomic sequencing, the mycobiomes of the vitreous of healthy control individuals (VC, n = 15) and individuals with post fever retinitis + non-PFR uveitis (PFR+, n = 9) were analysed and compared. The results indicated that Ascomycota was the most predominant phylum in both VC and PFR+ groups. Further, at the genera level it was observed that the abundance of 17 fungal genera were significantly different in post fever retinitis (PFR, n = 6) group compared to control group. Of these 17 genera, it was observed that 14 genera were relatively more abundant in PFR group and the remaining 3 genera in the VC group. Genus Saccharomyces , a commensal of the gut and skin, was predominantly present in the vitreous of both the cohorts, however it was significantly less abundant in PFR group. Further, significant increase in the genera that have a pathogenic interaction with the host were observed in PFR group. On the whole the mycobiome in both the groups differed significantly and formed two distinct clusters in the heatmap and Principal co-ordinate analysis. These results demonstrate significant changes in the mycobiome from the vitreous of post fever retinitis patients compared to healthy controls thus implying that dysbiotic changes in the fungal vitreous microbiome are associated with PFR.

To evaluate the clinical utility of high-throughput sequencing (HTS) approach-based analysis of the bacterial and fungal genome in vitreous fluids from patients clinically diagnosed as endophthalmitis, we subjected 75 vitreous fluids from clinically presumed infectious endophthalmitis patients to high-throughput sequencing (Illumina HiSeq 2500) after DNA extraction and amplification of the 16S rRNA for the detection of bacteria, and ITS 2 region for detection of fungal pathogens. As controls, we included vitreous biopsies from 70 patients diagnosed with other non-infectious retinal disorders. Following the construction of the curated microbial genome database and filtering steps to reduce ambiguousness/contaminants from the environment, the paired reads were analyzed. Our HTS reads revealed in almost all cases the same organism that was grown in culture (bacterial-14/15, fungal 3/3) by conventional microbiological workup. HTS additionally diagnosed the presence of microbes in 42/57 (73.7%) patients who were conventionally negative (fungal pathogens in 36/57, bacterial pathogens in 11/57, including five cases that showed the presence of both bacterial and fungal organisms). Aspergillus sp., Fusarium sp., Exserohilum sp., and Candida sp. were the most predominant genera in our cohort of culture-negative endophthalmitis cases. Heat map based microbial clustering analysis revealed that these organisms were taxonomically similar to the species identified by conventional culture methods. Interestingly, 4/70 control samples also showed the presence of bacterial reads, although their clinical significance is uncertain. HTS is useful in detecting pathogens in endophthalmitis cases that elude conventional attempts at diagnosis and can provide actionable information relevant to management, especially where there is a high index of suspicion of fungal endophthalmitis, particularly in tropical countries. Outcome analyses and clinical trials addressing the success and cost savings of HTS for the diagnosis of endophthalmitis are recommended.

Keratitis, an inflammatory disease of the eye, when neglected could lead to sight-threatening complications and ultimately blindness. Globally, over a million people are affected by keratitis annually. Keratitis has a microbial etiology and is caused by bacteria, fungi, viruses, etc. The present study compared the ocular surface fungal microbiome of healthy individuals and individuals with fungal keratitis. Fungal microbiomes from the conjunctival swabs of healthy individuals and from conjunctival swabs and corneal scrapings of individuals with fungal keratitis were generated using ITS2 region amplicons. Microbiomes were sequenced using Illumina MiSeq 2 × 250 base pair chemistry with a paired-end protocol. Based on Alpha diversity indices, phylum and genera level diversity, abundance differences, and heat map analysis, the fungal microbiomes of conjunctival swabs and corneal scrapings of individuals with fungal keratitis exhibited dysbiosis (alterations in the diversity and abundance) compared to the ocular surface microbiome of the healthy control individuals. This is the first report indicating dysbiosis in the fungal microbiome of conjunctival swabs and corneal scrapings in individuals with fungal keratitis. A total of 11 genera present in the majority of the eyes constituted the variable core ocular microbiome.

[This corrects the article DOI: 10.1371/journal.pone.0199640.].

To investigate the immunopathogenesis of endophthalmitis, and determine if cytokine profiles could serve as biomarkers of disease severity in infectious endophthalmitis. Vitreous samples of 46 patients clinically diagnosed as endophthalmitis (of which 25 were culture positive) and 20 non-infectious controls from patients with Retinal Detachment (RD) or diabetic retinopathy were included in the study. The cytokine and chemokine expression patterns of 40 immune mediators including 6 antiinflammatory cytokines, 15 proinflammatory cytokines, 9 Growth factors and 10 proinflammatory chemokines in the vitreous were were analyzed by multiplex cytokine immunoassay. In addition, significant immune mediators were correlated with initial and final visual acuity (VA). Our results demonstrated elevated expression of 16 mediators such as GCSF, GRO, IFN-γ, IL-1α, IL-1β, IL-1 RA, IL-6, IL-8, IP-10, MCP-1, MCP-3, MIP-1α, IL-1β, TGF-α, TNF-α in patients with culture positive endophthalmitis. Cytokine profile expression significantly differed between patients with proven endophthalmitis and the non-infectious controls in heat map analysis. PCoA plot indicated five mediators (IL-1RA, IL-6, IL-8, GRO, G-CSF) as biomarkers that could be Independent Predictors of Disease especially in culture negative cases. Correlation of cytokines with VA revealed strong association between the initial VA and intraocular levels of TGF-α, IL-1β and IL-8 but there was no correlation with the severity or visual outcome of infection. In comparison to non-infectious ocular conditions, the pathogenesis of infectious endophthalmitis correlates with increased expression levels of IL-1RA, IL-6, IL-8, GRO, G-CSF. Understanding cytokine profiles in culture negative endophthalmitis patients could aid in therapy in non-responders to empirical antibiotic therapy.

Ocular microbiome research has gained momentum in the recent past and has provided new insights into health and disease conditions. However, studies on sight threatening intraocular inflammatory diseases have remained untouched. In the present study, we attempted to identify the bacterial microbiome associated with post fever retinitis using a metagenomic sequencing approach. For this purpose, bacterial ocular microbiomes were generated from vitreous samples collected from control individuals (VC, n = 19) and individuals with post fever retinitis (PFR, n = 9), and analysed. The results revealed 18 discriminative genera in the microbiomes of the two cohorts out of which 16 genera were enriched in VC and the remaining two in PFR group. These discriminative genera were inferred to have antimicrobial, anti-inflammatory, and probiotic function. Only two pathogenic bacteria were differentially abundant in 20% of the PFR samples. PCoA and heatmap analysis showed that the vitreous microbiomes of VC and PFR formed two distinct clusters indicating dysbiosis in the vitreous bacterial microbiomes. Functional assignments and network analysis also revealed that the vitreous bacterial microbiomes in the control group exhibited more evenness in the bacterial diversity and several bacteria had antimicrobial function compared to the PFR group.