Tear film microbiome in Sjogren's and non-Sjogren's aqueous deficiency dry eye.

Purpose: To understand the bacterial microbiome changes associated with Sjogren's syndrome (SS) and non-Sjogren's syndrome (NSS) aqueous-deficient dry eyes compared to healthy eyes. Methods: Bacterial microbiome was generated from the deoxyribonucleic acid of tear film samples in healthy (n = 33), SS (n = 17), and NSS (n = 28) individuals. Sequencing of the V3-V4 region of the 16S rRNA gene was performed on the Illumina HiSeq2500 platform. Quantitative Insights Into Microbial Ecology (QIIME) pipeline was used to assign taxa to sequences. Statistical analysis was performed in R to assess the alpha diversity and beta diversity indices. Significant changes between the healthy, SS, and NSS cohorts were depicted by principal coordinate analysis (PCoA), differential abundance, and network analysis. Results: Tear microbiome was generated in healthy, SS, and NSS samples. Phyla Actinobacteria, Firmicutes, and Bacteroidetes showed significant changes in SS and NSS compared to healthy. Genera Lactobacillus and Bacillus were predominantly present in all samples. PCoA and heat map analysis showed distinct clusters for SS and NSS from the healthy cohort. Genera Prevotella, Coriobacteriaceae UCG-003, Enterococcus, Streptomyces, Rhodobacter, Ezakiella, and Microbacterium significantly increased in abundance in SS and NSS compared to a healthy cohort. Bacteria-bacteria interaction in SS, NSS, and healthy cohorts was predicted by CoNet network analysis. This analysis predicted a major hub of interaction for the pro-inflammatory bacterium Prevotella in the SS and NSS cohorts. Conclusion: The results of the study indicate significant changes in the phyla and genera in SS and NSS compared to healthy. Both discriminative analysis and network analysis indicated a possible association of predominant pro-inflammatory bacteria with SS and NSS.

Characterising the tear bacterial microbiome in young adults.

Conjunctival swabs (CS) are the major source of sampling for ocular microbiome studies, however collecting CS from the diseased eyes is difficult and painful. In this study, as an alternative to CS, a less invasive approach of tear collection was used to establish the bacterial microbiome in healthy eyes. Tear bacterial microbiome was generated from the DNA of tears (n = 24; male = 16 and female = 8) of healthy volunteers aged from 20 to 52 years. Sequencing of V3-V4 region of 16S rRNA gene was performed on the Illumina platform. Reads were processed in QIIME to assign the taxa. Statistical analysis of the tear microbiome was done in R to assess the alphadiversity and betadiversity indices. Tear microbiome was generated in all the 24 tear samples. Eight out of the top 10 predominant bacterial genera remained same in both tear and CS microbiomes, which include genera such as Corynebacterium, Staphylococcus, Streptococcus, Mycobacterium, Escherichia-Shigella, Lactobacillus, Bacillus and Acinetobacter. The similarity network analysis indicates that 144 out of 145 genera of tear cohort matched with conjunctival swabs. However, tear and CS microbiomes differed in the abundance of the predominant bacterial genera. The bacterial microbiome of tears in adults appears to be stable and is comparable with that of CS microbiome.

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.

Intraocular Viral Communities Associated With Post-fever Retinitis.

The virome of ocular fluids is naive. The results of this study highlight the virome in the vitreous fluid of the eye of individuals without any ocular infection and compare it with the virome of the vitreous fluid of individuals with retinitis. A total of 1,016,037 viral reads were generated from 25 vitreous fluid samples comprising control and post-fever retinitis (PFR) samples. The top 10 viral families in the vitreous fluids comprised of Myoviridae, Siphoviridae, Phycodnaviridae, Herpesviridae, Poxviridae, Iridoviridae, Podoviridae, Retroviridae, Baculoviridae, and Flaviviridae. Principal coordinate analysis and heat map analysis clearly discriminated the virome of the vitreous fluid of the controls from that of the PFR virome. The abundance of 10 viral genera increased significantly in the vitreous fluid virome of the post-fever retinitis group compared with the control group. Genus Lymphocryptovirus, comprising the human pathogen Epstein-Barr virus (EBV) that is also implicated in ocular infections was significantly abundant in eight out of the nine vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. Human viruses, such as Hepacivirus, Circovirus, and Kobuvirus, were also significantly increased in abundance in the vitreous fluid viromes of post-fever retinitis group samples compared with the control viromes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analysis and the network analysis depicted an increase in the immune response by the host in the post-fever retinitis group compared with the control group. All together, the results of the study indicate changes in the virome in the vitreous fluid of patients with the post-fever retinitis group compared to the control group.

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.

Monitoring metabolic pathway alterations in Escherichia coli due to applied potentials in microbial electrochemical system.

Redox potential is one of the key regulators in determining the fate of the metabolic pathways of biocatalysts and of their associated product synthesis in microbial electrochemical systems. In the present study, the influence of applied potentials on fermentation products and metabolic flux was investigated using isolated E. coli HP3 as a model organism using pyruvate as a substrate. To provide insights into metabolic shifts, electro-fermentative (EF) systems were constructed and poised at both positive and negative redox potentials of 0.2 V, 0.4 V, 0.6 V and 0.8 V (vs Ag/AgCl) at the anode. The relative expression of genes encoding lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), pyruvate dehydrogenase (aceF), hydrogenase (hycE) and NADH: oxidoreductase (nuoB) enabled assessment of metabolic shifts in addition to cyclic voltammograms and short chain fatty acid profiling. Results showed that poised conditions had a significant effect on product formation and observed up-regulation of key enzymes involved in pyruvate metabolism in comparison to controls. More specifically, EF poised at -0.8 V and -0.2 V enhanced H2 production by 7.9 folds and 5.3 folds respectively, whilst at +0.8 V poised operation enhanced lactate production by 1.9 folds compared to the control. Concomitantly, the key genes involved in the pathway for H2 production viz., plfB, hycE and nuoB were all up-regulated significantly in a reactor poised at -0.8 V compared with other conditions. Similarly, transcripts for gene ldhA were up-regulated significantly in the system poised with +0.8 V. The study elucidated the role of redox potential on the product formation with respect to the relative expression of the genes encoding key enzymes in the metabolic pathway in correlation with bio-electrochemical characteristics.

Gene Targets in Ocular Pathogenic Escherichia coli for Mitigation of Biofilm Formation to Overcome Antibiotic Resistance.

The present work is an attempt to establish the functionality of genes involved in biofilm formation and antibiotic resistance in an ocular strain of Escherichia coli (L-1216/2010) which was isolated and characterized from the Vitreous fluid of a patient with Endophthalmitis. For this purpose, seven separate gene-specific knockout mutants were generated by homologous recombination in ocular E. coli. The genes that were mutated included three transmembrane genes ytfR (ABC transporter ATP-binding protein), mdtO (multidrug efflux system) and tolA (inner membrane protein), ryfA coding for non-coding RNA and three metabolic genes mhpA (3-3-hydroxyphenylpropionate 1,2-dioxygenase), mhpB (2,3-di hydroxyphenylpropionate 1,2-dioxygenase), and bdcR (regulatory gene of bdcA). Mutants were validated by sequencing and Reverse transcription-PCR and monitored for biofilm formation by XTT method and confocal microscopy. The antibiotic susceptibility of the mutants was also ascertained. The results indicated that biofilm formation was inhibited in five mutants (ΔbdcR, ΔmhpA, ΔmhpB, ΔryfA, and ΔtolA) and the thickness of biofilm reduced from 17.2 µm in the wildtype to 1.5 to 4.8 µm in the mutants. Mutants ΔytfR and ΔmdtO retained the potential to form biofilm. Complementation of the mutants with the wild type gene restored biofilm formation potential in all mutants except in ΔmhpB. The 5 mutants which lost their ability to form biofilm (ΔbdcR, ΔmhpA, ΔmhpB, ΔtolA, and ΔryfA) did not exhibit any change in their susceptibility to Ceftazidime, Cefuroxime, Ciprofloxacin, Gentamicin, Cefotaxime, Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin in the planktonic phase compared to wild type ocular E. coli. But ΔmdtO was the only mutant with altered MIC to Sulfamethoxazole, Imipenem, Erythromycin, and Streptomycin both in the planktonic and biofilm phase. This is the first report demonstrating the involvement of the metabolic genes mhpA and mhpB and bdcR (regulatory gene of bdcA) in biofilm formation in ocular E. coli. In addition we provide evidence that tolA and ryfA are required for biofilm formation while ytfR and mdtO are not required. Mitigation of biofilm formation to overcome antibiotic resistance could be achieved by targeting the genes bdcR, mhpA, mhpB, ryfA, and tolA.

Impact of selectively enriched microbial communities on long-term fermentative biohydrogen production.

The effect of selectively enriched inoculum for biohydrogen production and shifts in microbial communities was observed using anaerobic sequencing batch reactor (AnSBR). Significantly, combined pretreatment using acid and iodopropane resulted 3 fold increase in H2 production (8.65mol/kgCODR) over untreated control (2.63mol/kgCODR). Pretreatment showed significant shifts in the microbial communities which are thus accounted for enhanced H2 production. The high-throughput pyrosequence analysis depicted shifts in phylum Firmicutes and Proteobacteria. In the case of Proteobacteria, there was an increase in Betaproteobacterial, decrease in Epsilonproteobacterial and compositional variation in Alphaproteobacterial species. The decreased OTU number after pretreatment indicate, reduction of undesirable microbial populations while favouring conditions for microbiome that are involved in acidogenic, acetogenic and H2 production.

Global gene expression in Escherichia coli, isolated from the diseased ocular surface of the human eye with a potential to form biofilm.

BACKGROUND: Escherichia coli, the gastrointestinal commensal, is also known to cause ocular infections such as conjunctivitis, keratitis and endophthalmitis. These infections are normally resolved by topical application of an appropriate antibiotic. But, at times these E. coli are resistant to the antibiotic and this could be due to formation of a biofilm. In this study ocular E. coli from patients with conjunctivitis, keratitis or endophthalmitis were screened for their antibiotic susceptibility and biofilm formation potential. In addition DNA-microarray analysis was done to identify genes that are involved in biofilm formation and antibiotic resistance. RESULTS: Out of 12 ocular E. coli isolated from patients ten isolates were resistant to one or more of the nine antibiotics tested and majority of the isolates were positive for biofilm formation. In E. coli L-1216/2010, the best biofilm forming isolate, biofilm formation was confirmed by scanning electron microscopy. Confocal laser scanning microscopic studies indicated that the thickness of the biofilm increased up to 72 h of growth. Further, in the biofilm phase, E. coli L-1216/2010 was 100 times more resistant to the eight antibiotics tested compared to planktonic phase. DNA microarray analysis indicated that in biofilm forming E. coli L-1216/2010 genes encoding biofilm formation such as cell adhesion genes, LPS production genes, genes required for biofilm architecture and extracellular matrix remodeling and genes encoding for proteins that are integral to the cell membrane and those that influence antigen presentation are up regulated during biofilm formation. In addition genes that confer antimicrobial resistance such as genes encoding antimicrobial efflux (mdtM and cycA), virulence (insQ, yjgK), toxin production (sat, yjgK, chpS, chpB and ygjN), transport of amino-acids and other metabolites (cbrB, cbrC, hisI and mglB) are also up regulated. These genes could serve as potential targets for developing strategies for hacking biofilms and overcoming antibiotic resistance. CONCLUSIONS: This is the first study on global gene expression in antibiotic resistant ocular E. coli with a potential to form biofilm. Using native ocular isolates for antibiotic susceptibility testing, for biofilm formation and global gene expression is relevant and more acceptable than using type strains or non clinical strains which do not necessarily mimic the native isolate.

Polarized potential and electrode materials implication on electro-fermentative di-hydrogen production: Microbial assemblages and hydrogenase gene copy variation.

This study examined the changes in microbial diversity in response to different electrode materials viz., stainless steel mesh (SS) and graphite plate as anodes in two microbial electrolysis cell (MEC) each poised at 0.2V, 0.4V, 0.6V and 0.8V. Changes in microbiota prior to and after pretreatment along with microbiota enriched in response to various poised potentials with SS and graphite are monitored by 16S rRNA gene based DGGE profiling. Significant shifts in microbial community were noticed at all these experimental conditions. Correspondingly, the level of hydrogenase belonging to genera Bacillus, Pseudomonas, Rhodopseudomonas and Clostridium was studied by quantitative real time PCR (RT-PCR) at various applied potentials. DGGE based 16S rRNA gene profiling revealed enriched members belonging to phylum Firmicutes predominantly present at 0.8V in both MECs contributing to high hydrogen production. This study first time explored the growth behavior of mixed consortia in response to poised potentials and electrode materials.

Pythium insidiosum keratitis: clinical profile and role of DNA sequencing and zoospore formation in diagnosis.

PURPOSE: To report the molecular and microbiological diagnosis and clinical profile of 13 patients with Pythium insidiosum keratitis. METHODS: Phase 1 of the study consisted of DNA sequencing of the ITS region of the rDNA of 162 stocked morphologically unidentified nonconsecutive fungal isolates from corneal scraping of patients with keratitis (2010-2012). Blast and phylogenetic analyses of the sequences showed 9 to be P. insidiosum. A retrospective review of archived photographs of colony and direct microscopy of corneal scrapings and clinical records of the cases were performed. Phase 2 began in 2014, in which a simple method of zoospore formation was used for fungal colonies resembling those of P. insidiosum followed by DNA sequencing. RESULTS: The prevalence of P. insidiosum among unidentified fungal isolates from keratitis was 9/162 (5.5%) in phase 1. In phase 2, 4/102 cases (3.9%) of fungal keratitis were identified as P. insidiosum (January-February, 2014). Phylogenetic analysis of all 13 fungal isolates confirmed the identification of P. insidiosum. Corneal infiltrates exhibited hyphate edges, tentacle-like extensions, and dot-like infiltrates surrounding the main infiltrate. Response to topical 5% natamycin eye drops with or without oral antifungals was poor (penetrating keratoplasty: 9 and evisceration: 2) with a mean follow-up period of 82 days. CONCLUSIONS: P. insidiosum keratitis needs to be considered in the differential diagnosis of severe fungal keratitis. It can be identified using the zoospore formation method and confirmed by ITS DNA sequencing. Lack of response to currently used antifungal drugs calls for evaluation of newer drugs for medical therapy and consideration for early penetrating keratoplasty.

Global gene expression analysis of long-term stationary phase effects in E. coli K12 MG1655.

Global gene expression was monitored in long-term stationary phase (LSP) cells of E. coli K12 MG1655 and compared with stationary phase (SP) cells that were sub-cultured without prolonged delay to get an insight into the survival strategies of LSP cells. The experiments were carried out using both LB medium and LB supplemented with 10% of glycerol. In both the media the LSP cells showed decreased growth rate compared to SP cells. DNA microarray analysis of LSP cells in both the media resulted in the up- and down-regulation of several genes in LSP cells compared to their respective SP cells in the corresponding media. In LSP cells grown in LB 204 genes whereas cells grown in LB plus glycerol 321 genes were differentially regulated compared to the SP cells. Comparison of these differentially regulated genes indicated that irrespective of the medium used for growth in LSP cells expression of 95 genes (22 genes up-regulated and 73 down-regulated) were differentially regulated. These 95 genes could be associated with LSP status of the cells and are likely to influence survival and growth characteristics of LSP cells. This is indeed so since the up- and down-regulated genes include genes that protect E. coli LSP cells from stationary phase stress and genes that would help to recover from stress when transferred into fresh medium. The growth phenotype in LSP cells could be attributed to up-regulation of genes coding for insertion sequences that confer beneficial effects during starvation, genes coding for putative transposases and simultaneous down-regulation of genes coding for ribosomal protein synthesis, transport-related genes, non-coding RNA genes and metabolic genes. As yet we still do not know the role of several unknown genes and genes coding for hypothetical proteins which are either up- or down-regulated in LSP cells compared to SP cells.

Effect of simulated microgravity on E. coli K12 MG1655 growth and gene expression.

This study demonstrates the effects of simulated microgravity on E. coli K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (sufE and ssrA) and simultaneously genes coding for membrane transporters (ompC, exbB, actP, mgtA, cysW and nikB) and carbohydrate catabolic processes (ldcC, ptsA, rhaD and rhaS) are down regulated. The enhanced growth in simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (srmB) and repression of the genes encoding for nucleoside metabolism (dfp, pyrD and spoT). In addition, E. coli cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol.

Candida northwykensis sp. nov., a novel yeast isolated from the gut of the click beetle Melanotus villosus.

Two yeast morphotypes, BET 4(T) and BET 7, were isolated from the gut of click beetle Melanotus villosus. Click beetles were collected from the decaying timber within the woodlands of North Wyke Research, South West England, UK (latitude, 50°46'29″N; longitude, 3°55'23″W). Morphotype BET 7 was identified as Debaryomyces hansenii, and the other morphotype, BET 4(T), was found to differ from Priceomyces castillae and Priceomyces haplophilus, its closest phylogenetic neighbours, by 5.0% with respect to the nucleotide sequence of the D1/D2 domain of the large-subunit (LSU) rRNA gene, and by 8.0% with respect to the ribosomal internal-transcribed spacer (ITS) region. BET 4(T) also differ from P. castillae and P. haplophilus in a number of different phenotypic characteristics. Thus, based on the unique nucleotide sequences of its D1/D2 domain and ITS region, its physiological characteristics and an inability to sporulate, strain BET 4(T) is assigned the status of a new species of Candida, for which the name Candida northwykensis sp. nov., is proposed. The type strain is BET 4(T) (NCYC 3525(T) = CBS 11370(T)).