Intraocular Microbiome in Diabetes and Diabetic Retinopathy: A Pilot Study.

INTRODUCTION: The objective of this study was to compare the microbiome in the aqueous humour and gut of people with diabetes mellitus (DM) with and without diabetic retinopathy (DR). METHODS: This was a prospective controlled study. The study included 17 people undergoing intraocular surgery in their naïve eyes. Stool samples were obtained in the perioperative period; aqueous humour samples of sufficient quantity were obtained in 12 people during intraocular surgery. Dietary information was obtained using a previously validated questionnaire. The gut and aqueous humour samples were assessed for microbiome using 16S rRNA gene sequencing coupled with QIIME and R software. RESULTS: Aqueous humour was analysed in 12 people: 4 each healthy controls, people with DM, and people with DR. There were minor differences at the phyla levels, but the aqueous humour microbiomes of healthy controls, DM, and DR formed three distinct clusters on heat map analysis with discriminatory genera. This genera-level clustering was more apparent for the intraocular than the gut microbiome. In people with DM and DR, we identified genera unique to the eye or the gut. There was a consistent reduction in the abundance of anti-inflammatory bacteria in people with DR than DM. CONCLUSIONS: There is a difference in intraocular and gut microbiome regardless of disease or health. Our preliminary findings indicate distinctive features of the intraocular microbiome in people with DR compared with those without it. While this distinctiveness appears more evident in aqueous humour than in the gut, it needs further confirmation with larger studies.

Unconventional avenues to decelerate diabetic retinopathy.

Diabetic retinopathy (DR) is an important microvascular complication of diabetes mellitus (DM), causing significant visual impairment worldwide. Current gold standards for retarding the progress of DR include blood sugar control and regular fundus screening. Despite these measures, the incidence and prevalence of DR and vision-threatening DR remain high. Given its slowly progressive course and long latent period, opportunities to contain or slow DR before it threatens vision must be explored. This narrative review assesses the recently described unconventional strategies to retard DR progression. These include gut-ocular flow, gene therapy, mitochondrial dysfunction-oxidative stress, stem cell therapeutics, neurodegeneration, anti-inflammatory treatments, lifestyle modification, and usage of phytochemicals. These therapies impact DR directly, while some of them also influence DM control. Most of these strategies are currently in the preclinical stage, and clinical evidence remains low. Nevertheless, our review suggests that these approaches have the potential for human use to prevent the progression of DR.

Elucidating the clinical, microbiological and molecular diagnostic aspects of Macrophomina phaseolina keratitis.

This study reports the clinico-microbiological features of Macrophomina phaseolina keratitis. Clinically diagnosed as microbial keratitis, six patients underwent microbiological evaluation. Fungal culture isolates from cornea were subjected to DNA sequencing of the ITS region, phylogenetic analysis and reconfirmation by polymerase chain reaction (PCR). Minimum inhibitory concentrations (MICs) of six antifungal drugs were determined by microbroth dilution method against the six isolates. All patients were treated with antifungals. Failed medical therapy necessitated therapeutic penetrating keratoplasty (TPK). Corneal buttons were processed for histopathology. In all patients, the corneal scraping showed septate hyaline fungal filaments. The BLAST analysis for ITS sequences of all six fungal isolates suggested M. phaseolina, however, when limited to sequences from type material, they matched M. pseudophaseolina. Phylogenetic analysis could not differentiate between these two species and clustered in a single clade. PCR assay of specific gene sequence [MpCal (calmodulin)] reconfirmed all isolates as M. phaseolina. The MICs of voriconazole and posaconazole were lowest (0.03 to 2 and 0.1 to 2µg/mL respectively) and all isolates were susceptible to natamycin. Except for case 1, which healed with a scar on treatment, all other cases worsened, despite medical treatment, necessitating TPK. Histopathology of 3 out of 4 buttons showed the presence of fungal filaments. While direct microscopic examination of corneal scrapings is helpful in diagnosis, identification of M. phaseolina in culture is challenging. Although MICs of commonly used antifungals are low response to medical therapy is not encouraging; patients may require TPK for resolution of infection in M. phaseolina keratitis.

DNA sequencing, phylogenetic analysis and specific PCR confirmed Macrophomina phaseolina keratitis in six patients. Although antifungal susceptibility showed the organisms to be susceptible to natamycin five patients did not respond to treatment and needed keratoplasty.

Fungi of the human eye: Culture to mycobiome.

The focus of the current review is multi-fold and compares the diversity and abundance of fungi on the ocular surface by the conventional culture-based method with the more sensitive, high throughput, culture-independent NGS method. The aim is to highlight the existence of a core ocular mycobiome and explore the transition of the ocular fungal microbiota from the normal eye to the diseased eye. PubMed, Google Scholar and Medline were used to search for publications and reviews related to cultivable fungi and the mycobiome of the normal and diseased eye. The conventional cultivable approach and the NGS approach confirm that the eye has its own mycobiome and several confounding factors (age, gender, ethnicity etc.) influence the mycobiome. Further, dysbiosis in the mycobiome appears to be associated with ocular diseases and thus impacts the health of the human eye. Considering that the mycobiome of the eye is influenced by several confounding factors and also varies with respect to the disease status of the eye there is a need to extensively explore the mycobiome under different physiological conditions, different ethnicities, geographical regions etc. Such studies would unravel the diversity and abundance of the mycobiomes and contribute to our understanding of ocular health. Research focused on ocular mycobiomes may eventually help to build a targeted and individualized treatment.

Alterations in the gut bacterial microbiome in people with type 2 diabetes mellitus and diabetic retinopathy.

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.

Alterations in the conjunctival surface bacterial microbiome in bacterial keratitis patients.

Microbial keratitis is an infectious disease of the eye, in which the cornea is inflamed. Under severe conditions, keratitis can lead to significant loss of vision and enucleation of the eye. Ocular trauma is the major risk factor causing keratitis and microorganisms viz., bacteria, fungi, viruses are the causative agents. The current study characterized the conjunctival bacterial microbiomes of healthy individuals and individuals with bacterial keratitis (BK) and assessed whether ocular microbiome dysbiosis is prevalent in BK patients. Ocular bacterial microbiomes were generated from the conjunctival swabs of healthy controls (HC-SW) and conjunctival swabs (BK-SW) and corneal scrapings (BK-CR) of BK patients using V3-V4 amplicon sequencing and data analysed using QIIME and R software. The Alpha diversity indices, diversity and abundance of different phyla and genera, heat map analysis, NMDS plots and inferred functional pathway analysis clearly discriminated the bacterial microbiomes of conjunctival swabs of healthy controls from that of BK patients. Preponderance of negative interactions in the hub genera were observed in BK-CR and BK-SW compared to HC-SW. In addition, a consistent increase in the abundance of pathogenic bacteria, as inferred from published literature, was observed in the conjunctiva of BK patients compared to HC and this may be related to causing or exacerbating ocular surface inflammation. This is the first study demonstrating dysbiosis in the ocular bacterial microbiome of conjunctiva of bacterial keratitis patients compared to the eye of healthy controls. The bacterial microbiome associated with the corneal scrapings of keratitis individuals is also described for the first time.

Gut mycobiomes are altered in people with type 2 Diabetes Mellitus and Diabetic Retinopathy.

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.

Mycobiome changes in the vitreous of post fever retinitis patients.

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.

Comparison of the Vitreous Fluid Bacterial Microbiomes between Individuals with Post Fever Retinitis and Healthy Controls.

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.

Targeted High-Throughput Sequencing Identifies Predominantly Fungal Pathogens in Patients with Clinically Infectious, Culture-Negative Endophthalmitis in South India.

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.

Alterations in the Ocular Surface Fungal Microbiome in Fungal Keratitis Patients.

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.

Implicating Dysbiosis of the Gut Fungal Microbiome in Uveitis, an Inflammatory Disease of the Eye.

Purpose: In this study, the gut fungal microbiome of uveitis (UVT) patients was generated and compared with healthy controls (HC) to identify dysbiosis in UVT patients and ascertain the role of gut fungal microbiome in disease pathology. Methods: In the present study, gut fungal microbiomes were analyzed in the fecal samples of HC (n = 24) and UVT patients (n = 14) using high-throughput Illumina sequencing of ITS2 region of the fungal ribosomal RNA. QIIME and R software were used for data analysis. Results: The gut fungal richness and diversity were significantly decreased in UVT patients compared to HC. Our analyses showed enrichment of several pathogenic fungi including Malassezia restricta, Candida albicans, Candida glabrata, and Aspergillus gracilis in UVT patients. Heatmap and discriminatory OTUs further confirmed the disparities between UVT and HC microbiomes. Conclusions: This is the first study demonstrating dysbiosis in the gut fungal communities of UVT patients indicating the importance of fungal microbiome in the disease pathology. These initial findings might warrant further investigation into the fungal microbiome, especially interactions between fungal and bacterial that then might give further insight into how probiotics or fecal transplants might benefit.

The Human Ocular Surface Fungal Microbiome.

Purpose: To enumerate the ocular surface fungal microbiome of healthy human eyes by using next-generation sequencing (NGS). Methods: Tarsal and fornix conjunctiva from the lower and upper lids of both eyes of healthy individuals were swabbed in duplicate separately. A total of 34 samples were collected from both the eyes of 17 individuals, which were used for the generation of ocular surface fungal microbiomes by NGS. Twenty-four swabs were used for the detection of culturable fungi by the conventional cultivable method. Microbiome generation involved DNA extraction, internal transcribed spacer 2 (ITS2) amplification, library preparation, amplicon sequencing, taxonomic assignment of sequences, diversity analyses, and identification of genera. Results: The cultivable method detected fungi in 3 out of 24 (12.5 %) ocular surface swabs, whereas NGS identified fungi in 25 of the 34 (73.5 %) swabs. In the cultivable method Aspergillus was the only genus detected, whereas NGS detected 65 distinct genera with 12 to 24 genera per microbiome. Genera Aspergillus, Setosphaeria, Malassezia, and Haematonectria were present in the 25 eyes in which fungi were detected. Alpha diversity in the two eyes was similar and sex had no effect, but Chao1 and Simpson indices were altered by age. Conclusions: This study explored the ocular surface fungal microbiome of healthy individuals using NGS and identified a greater degree of diversity of fungi than with the conventional cultivable method. It was observed that several fungal genera were associated with the healthy conjunctiva.

New insights into culture negative endophthalmitis by unbiased next generation sequencing.

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.

Correction: Alterations in the gut bacterial microbiome in fungal Keratitis patients.

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

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis, an inflammatory disease of the human eye.

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Elevated cytokine levels in vitreous as biomarkers of disease severity in infectious endophthalmitis.

PURPOSE: To investigate the immunopathogenesis of endophthalmitis, and determine if cytokine profiles could serve as biomarkers of disease severity in infectious endophthalmitis. MATERIALS AND METHODS: 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). RESULTS: Our results demonstrated elevated expression of 16 mediators such as GCSF, GRO, IFN-γ, IL-1α, IL-1ß, IL-1 RA, IL-6, IL-8, IP-10, MCP-1, MCP-3, MIP-1α, IL-1ß, 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-1ß and IL-8 but there was no correlation with the severity or visual outcome of infection. CONCLUSION: 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.

Dysbiosis in the Gut Bacterial Microbiome of Patients with Uveitis, an Inflammatory Disease of the Eye.

Uveitis (UVT), an inflammatory disease of the eye significantly contributes to vision impairment and blindness. Uveitis is associated with systemic infectious and autoimmune diseases, but in most cases, the aetiology remains unidentified. Dysbiosis in the gut microbiome has been implicated in autoimmune diseases, inflammatory diseases, cancers and mental disorders. In a mice model of autoimmune UVT, it was observed that manipulating the gut microbiome reduces the inflammation and disease severity. Further, alterations in the bacterial gut microbiome and their metabolites were reported in UVT patients from a Chinese cohort. Hence, it is worth comparing the bacterial gut microbiome of UVT patients with that of healthy controls (HC) to ascertain whether dysbiosis of the gut microbiome has implications in UVT. Our analyses showed reduced diversity of several anti-inflammatory organisms including Faecalibacterium, Bacteroides, Lachnospira, Ruminococcus and members of Lachnospiraceae and Ruminococcaceae families, and enrichment of Prevotella (proinflammatory) and Streptococcus (pathogenic) OTUs in UVT microbiomes compared to HC. In addition, decrease in probiotic and antibacterial organisms was observed in UVT compared to HC microbiomes. Heatmap and PCoA plots also indicated significant variations in the microbiomes of UVT versus HC. This is the first study demonstrating dysbiosis in the gut bacterial communities of UVT patients in an Indian cohort and suggests a role of the gut microbiome in the pathophysiology of UVT.

Alterations in the gut bacterial microbiome in fungal Keratitis patients.

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.

Polybacterial community analysis in human conjunctiva through 16S rRNA gene libraries.

The conjunctival sac of healthy human harbours a variety of microorganisms. When the eye is compromised, an occasional inadvertent spread happens to the adjacent tissue, resulting in bacterial ocular infections. Microbiological investigation of the conjunctival swab is one of the broadly used modality to study the aetiological agent of conjunctiva. However, most of the time such methods yield unsatisfactory results. Hence, the present study intends to identify the bacterial community in human conjunctiva of pre-operative subjects through 16S rRNA gene libraries. Out of 45 samples collected from preoperative patients undergoing cataract surgery, 36 libraries were constructed with bacterial nested-PCR-positive samples. The representative clones with unique restriction pattern were generated through Amplified Ribosomal DNA Restriction Analysis (ARDRA) which were sequenced for phylogenetic affiliation. A total of 211 representative clones were obtained which were distributed in phyla Actinobacteria, Firmicutes, α-Proteobacteria, ß-Proteobacteria, γ-Proteobacteria, Bacteroidetes, and Deinococcus-Thermus. Findings revealed the presence of polybacterial community, especially in some cases even though no bacterium or a single bacterium alone was identified through cultivable method. Remarkably, we identified 17 species which have never been reported in any ocular infections. The sequencing data reported 6 unidentified bacteria suggesting the possibility of novel organisms in the sample. Since, polybacterial community has been identified consisting of both gram positive and gram negative bacteria, a broad spectrum antibiotic therapy is advisable to the patients who are undergoing cataract surgery. Consolidated effort would significantly improve a clear understanding of the nature of microbial community in the human conjunctiva which will promote administration of appropriate antibiotic regimen and also help in the development of oligonucleotide probes to screen the predominant pathogens for early predisposition.