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Abstract 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.

Abstract Microbes residing in biofilms confer several fold higher antimicrobial resistances than their planktonic counterparts. Compared to monomicrobial biofilms, polymicrobial biofilms involving multiple bacteria, multiple fungi or both are more dominant in nature. Paradoxically, polymicrobial biofilms are less studied. In this study, ocular isolates of Staphylococcus aureus, S. epidermidis and Candida albicans, the etiological agents of several ocular infections, were used to demonstrate their potential to form mono- and polymicrobial biofilms both in vitro and on human cadaveric corneas. Quantitative (crystal violet and XTT methods) and qualitative (confocal and scanning electron microscopy) methods demonstrated that they form polymicrobial biofilms. The extent of biofilm formation was dependent on whether bacteria and fungi were incubated simultaneously or added to a preformed biofilm. Additionally, the polymicrobial biofilms exhibited increased resistance to different antimicrobials compared to planktonic cells. When the MBECs of different antibacterial and antifungal agents were monitored it was observed that the MBECs in the polymicrobial biofilms was either identical or decreased compared to the monomicrobial biofilms. The results are relevant in planning treatment strategies for the eye. This study demonstrates that ocular bacteria and fungi form polymicrobial biofilms and exhibit increase in antimicrobial resistance compared to the planktonic cells.

Fungal endophthalmitis is a potentially blinding condition. It is more often reported from Asia, including India. The incidence is lower than bacterial endophthalmitis. But it is relatively more challenging to treat than bacterial endophthalmitis. Many eyes may need therapeutic keratoplasty and/or evisceration. The current mainstays of treatment are vitrectomy irrespective of the presenting vision, intravitreal antifungal agents, and systemic therapy; additionally, the patients could require prolonged treatment with repeat vitreous surgeries and intravitreal injections. Difficulty in clinical diagnosis, delay in microbiological culture, and limited options of antifungal drugs make the treatment more difficult and less rewarding. Three common fungi causing endophthalmitis are Aspergillus, Fusarium, and Candida. The former two are molds, often identified in exogenous endophthalmitis, postoperative and traumatic; the latter is yeast and is more often identified in endogenous endophthalmitis. A faster diagnosis with newer molecular microbiological technologies might help institute treatment earlier than it is currently possible. A target trial using big data from different regions of the world might emulate a randomized clinical trial to design a definite treatment strategy. Given fewer antifungal drugs, one must be mindful of antifungal stewardship to prevent resistance to the existing drugs.

Abstract 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.

The primary focus of this review was to establish the possible association of dysbiotic changes in the gut bacterial microbiomes with both intestinal and extra-intestinal diseases with emphasis on ocular diseases such as bacterial keratitis, fungal keratitis, uveitis, age-related macular degeneration, and ocular mucosal diseases. For this particular purpose, a systematic search was conducted using PubMed and Google Scholar for publications related to gut microbiome and human health (using the keywords: gut microbiome, ocular disease, dysbiosis, keratitis, uveitis, and AMD). The predictions are that microbiome studies would help to unravel dysbiotic changes in the gut bacterial microbiome at the taxonomic and functional level and thus form the basis to mitigate inflammatory diseases of the eye by using nutritional supplements or fecal microbiota transplantation.

The current study compared the gut mycobiomes of diabetic rats generated by a streptozotocin chemical challenge, diabetic rats with retinal changes and normal control rats over a period of 4 months. Sustained increase in blood sugar levels (>150 mg/dL) confirmed the induction of diabetes. Histology and immunohistochemistry were used to identify changes in the retinal tissues in the diabetic rats indicative of the animals progressing into diabetic retinopathy. Gut mycobiomes generated using faecal DNA, indicated dysbiosis at the genus level in both diabetic (DM) and diabetic rats with retinal changes (DRC) when compared with the control rats. In Tables 3-6 the specific genera that were significantly increased/decreased in DM1 and DM2 and in DRC1 and DRC2 respectively compared to the respective controls CT1-CT4 rats are listed. Further, the mycobiomes of the DM and DRC rats separated into distinct clusters following heat-map analysis of the discriminating genera. In addition, β-diversity analysis separated the mycobiomes of DM and DRC rats from that of the control rats, but the mycobiomes of diabetic rats and diabetic rats with retinal changes showed an overlap. Based on the inferred functions of the discriminating genera in the mycobiomes, we speculated that increase in pathogenic fungi might contribute to the inflammatory status both in diabetic rats and rats showing retinal changes.

Biofilms confer several advantages to the organisms associated with them, such as increased resistances to antibacterial and antifungal compounds compared to free living cells. Compared to monomicrobial biofilms involving a single microorganism, biofilms composed of microorganisms affiliated to bacterial and fungal kingdoms are predominant in nature. Despite the predominance of polymicrobial biofilms, and more so mixed polymicrobial biofilms, they are rarely studied. The objective of the current study is to evaluate the potential of ocular bacteria and a filamentous fungus to form monomicrobial and mixed polymicrobial biofilms on synthetic and natural substrates and to monitor their response to antibiotics. In this sense, we demonstrated that the ocular pathogens Staphylococcus aureus, S. epidermidis, and Fusarium solani form monomicrobial and mixed polymicrobial biofilms both on tissue culture polystyrene plates and on ex vivo human corneas from cadavers using confocal microscopy and scanning electron microscopy. Additionally, the mixed polymicrobial biofilms involving the above ocular bacteria and a filamentous fungus were less susceptible to different antibacterials and antifungals in relation to the corresponding control planktonic cells. Further, the MICs to the screened antibacterials and antifungals in polymicrobial biofilms involving a bacterium or a fungus was either increased, decreased, or unchanged compared to the corresponding individual bacterial or fungal biofilm. The results would be useful to the ophthalmologist to plan effective treatment regimens for the eye since these are common pathogens of the eye causing keratitis, endophthalmitis, conjunctivitis, etc.

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.

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.

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.

Abstract 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.

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.

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

The biofilm-forming potential of Staphylococcus aureus and Staphylococcus epidermidis, isolated from patients with Endophthalmitis, was monitored using glass cover slips and cadaveric corneas as substrata. Both the ocular fluid isolates exhibited biofilm-forming potential by the Congo red agar, Crystal violet and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-(phenylamino) carbonyl-2H-tetra-zolium hydroxide (XTT) methods. Confocal microscopy demonstrated that the thickness of the biofilm increased from 4–120 h of biofilm formation. Scanning electron microscopic studies indicated that the biofilms grown on cover slips and ex vivo corneas of both the isolates go through an adhesion phase at 4 h followed by multilayer clumping of cells with intercellular connections and copious amounts of extracellular polymeric substance. Clumps subsequently formed columns and eventually single cells were visible indicative of dispersal phase. Biofilm formation was more rapid when the cornea was used as a substratum. In the biofilms grown on corneas, clumping of cells, formation of 3D structures and final appearance of single cells indicative of dispersal phase occurred by 48 h compared to 96–120 h when biofilms were grown on cover slips. In the biofilm phase, both were several-fold more resistant to antibiotics compared to planktonic cells. This is the first study on biofilm forming potential of ocular fluid S. aureus and S. epidermidis on cadaveric cornea, from attachment to dispersal phase of biofilm formation.

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.

Escherichia coli is a predominant bacterium in the intestinal tracts of animals. Phylogenetically, strains have been classified into seven phylogroups, A, B1, B2, C, D, E, and F. Pathogenic strains have been categorized into several pathotypes such as Enteropathogenic (EPEC), Enterotoxigenic (ETEC), Enteroinvasive (EIEC), Enteroaggregative (EAEC), Diffusely adherent (DAEC), Uropathogenic (UPEC), Shiga-toxin producing (STEC) or Enterohemorrhagic (EHEC) and Extra-intestinal pathogenic E. coli (ExPEC). E. coli also survives as a commensal on the ocular surface. However, under conditions of trauma and immune-compromised states, E. coli causes conjunctivitis, keratitis, endopthalmitis, dacyrocystitis, etc. The phylogenetic affiliation and the pathotype status of these ocular E. coli strains is not known. For this purpose, the whole-genome sequencing of the 10 ocular E. coli strains was accomplished. Based on whole-genome SNP variation, the ocular E. coli strains were assigned to phylogenetic groups A (two isolates), B2 (seven isolates), and C (one isolate). Furthermore, results indicated that ocular E. coli originated either from feces (enteropathogenic and enterotoxigenic), urine (uropathogenic), or from extra-intestinal sources (extra-intestinal pathogenic). A high concordance was observed between the presence of AMR (Antimicrobial Resistance) genes and antibiotic resistance in the ocular E. coli strains. Furthermore, several virulent genes (fimB to fimI, papB to papX, etc.) and prophages (Enterobacteria phage HK97, Enterobacteria phage P1, Escherichia phage D108 etc.) were unique to ocular E. coli. This is the first report on a whole-genome analysis of ocular E. coli strains.

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.

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.

The bacterial diversity of the water sample, collected from Ny-Ålesund, Arctic Archipelago Svalbard was analyzed by a phenotypic as well as a genotypic approach. Pure colonies of the culturable bacteria were established and grown at a range of temperatures: 4ºC, 15ºC, 22ºC and 37ºC. Optimum growth was found at 15ºC, and around 28 colonies were obtained. The library was dominated by 16S rDNAs of Gram-negative bacteria ( γ -Proteobacteria). Twenty two isolates exhibited sequences were similar to that of known bacterial isolates (>97% sequence similarity), represented by the species of the genera Psychrobacter , Pseudomonas , and Acinetobacter . Six isolates exhibited sequences showed less affi liation with known taxa (

We report the 4.86-Mb draft genome sequence of Bacillus okhensis strain Kh10-101T, a halo-alkali tolerant rod shaped bacterium isolated from a salt pan near port of Okha, India. This bacterium is a potential model to study the molecular response of bacteria to salt as well as alkaline stress, as it thrives under both high salt and high pH conditions. The draft genome consist of 4,865,284 bp with 38.2% G + C, 4952 predicted CDS, 157 tRNAs and 8 rRNAs. Sequence was deposited at DDBJ/EMBL/GenBank under the project accession JRJU00000000.

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.

BACKGROUND/AIMS: The importance of sperm capacitation for mammalian fertilization has been confirmed in the present study via sperm metabolism. Involvement of the metabolic enzymes pyruvate dehydrogenase complex (PDHc) and its E3 subunit, dihydrolipoamide dehydrogenase (DLD) in hamster in vitro fertilization (IVF) via in vitro sperm capacitation is being proposed through regulation of sperm intracellular lactate, pH and calcium. METHODOLOGY AND PRINCIPAL FINDINGS: Capacitated hamster spermatozoa were allowed to fertilize hamster oocytes in vitro which were then assessed for fertilization, microscopically. PDHc/DLD was inhibited by the use of the specific DLD-inhibitor, MICA (5-methoxyindole-2-carboxylic acid). Oocytes fertilized with MICA-treated (MT) [and thus PDHc/DLD-inhibited] spermatozoa showed defective fertilization where 2nd polar body release and pronuclei formation were not observed. Defective fertilization was attributable to capacitation failure owing to high lactate and low intracellular pH and calcium in MT-spermatozoa during capacitation. Moreover, this defect could be overcome by alkalinizing spermatozoa, before fertilization. Increasing intracellular calcium in spermatozoa pre-IVF and in defectively-fertilized oocytes, post-fertilization rescued the arrest seen, suggesting the role of intracellular calcium from either of the gametes in fertilization. Parallel experiments carried out with control spermatozoa capacitated in medium with low extracellular pH or high lactate substantiated the necessity of optimal sperm intracellular lactate levels, intracellular pH and calcium during sperm capacitation, for proper fertilization. CONCLUSIONS: This study confirms the importance of pyruvate/lactate metabolism in capacitating spermatozoa for successful fertilization, besides revealing for the first time the importance of sperm PDHc/ DLD in fertilization, via the modulation of sperm intracellular lactate, pH and calcium during capacitation. In addition, the observations made in the IVF studies in hamsters suggest that capacitation failures could be a plausible cause of unsuccessful fertilization encountered during human assisted reproductive technologies, like IVF and ICSI. Our studies indicate a role of sperm capacitation in the post-penetration events during fertilization.

BACKGROUND: Understanding changes in the host-parasite relationship due to habitat fragmentation is necessary for better management and conservation of endangered species in fragmented landscapes. Pathogens and parasites can pose severe threat to species in restricted environments such as forest fragments where there is increased contact of wildlife with human and livestock populations. Environmental stress and reduced nutritional level in forest fragments can influence parasite infection and intensity on the native species. In this study, we examine the impact of habitat fragmentation on the prevalence of gastrointestinal parasites in lion-tailed macaques in a fragmented rainforest in Western Ghats. METHODS: The prevalence of different gastrointestinal parasites was estimated from 91 fecal samples collected from 9 lion-tailed macaque groups in nine forest fragments. The parasites were identified up to genus level on the basis of the morphology and coloration of the egg, larva and cyst. The covariates included forest fragment area, group size and the presence/absence of human settlements and livestock in proximity. We used a linear regression model to identify the covariates that significantly influenced the prevalence of different parasite taxa. RESULTS: Nine gastrointestinal parasite taxa were detected in lion-tailed macaque groups. The groups near human settlements had greater prevalence and number of taxa, and these variables also had significant positive correlations with group size. We found that these parameters were also greater in groups near human settlements after controlling for group size. Livestock were present in all five fragments that had human settlements in proximity. CONCLUSION: The present study suggests that high prevalence and species richness of gastrointestinal parasites in lion-tailed macaque groups are directly related to habitat fragmentation, high anthropogenic activities and high host density. The parasite load partially explains the reason for the decline in immature survival and birth rate in small and isolated rainforest fragments in Anamalai Hills.

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.

Tiger populations are dwindling rapidly making it increasingly difficult to study their dispersal and mating behaviour in the wild, more so tiger being a secretive and solitary carnivore.We used non-invasively obtained genetic data to establish the presence of 28 tigers, 22 females and 6 males, within the core area of Pench tiger reserve, Madhya Pradesh. This data was evaluated along with spatial autocorrelation and relatedness analyses to understand patterns of dispersal and philopatry in tigers within this well-managed and healthy tiger habitat in India.We established male-biased dispersal and female philopatry in tigers and reiterated this finding with multiple analyses. Females show positive correlation up to 7 kms (which corresponds to an area of approximately 160 km(2)) however this correlation is significantly positive only upto 4 kms, or 50 km(2) (r = 0.129, p

BackgroundNon-invasively collected samples allow a variety of genetic studies on endangered and elusive species. However due to low amplification success and high genotyping error rates fewer samples can be identified up to the individual level. Number of PCRs needed to obtain reliable genotypes also noticeably increase.MethodsWe developed a quantitative PCR assay to measure and grade amplifiable nuclear DNA in feline faecal extracts. We determined DNA degradation in experimentally aged faecal samples and tested a suite of pre-PCR protocols to considerably improve DNA retrieval.ResultsAverage DNA concentrations of Grade I, II and III extracts were 982pg/µl, 9.5pg/µl and 0.4pg/µl respectively. Nearly 10% of extracts had no amplifiable DNA. Microsatellite PCR success and allelic dropout rates were 92% and 1.5% in Grade I, 79% and 5% in Grade II, and 54% and 16% in Grade III respectively. Our results on experimentally aged faecal samples showed that ageing has a significant effect on quantity and quality of amplifiable DNA (p0.05). DNA concentrations of fresh tiger and leopard faecal extracts without addition of carrier RNA were 816.5pg/µl (±115.5) and 690.1pg/µl (±207.1), while concentrations with addition of carrier RNA were 49414.5pg/µl (±9370.6) and 20982.7pg/µl (±6835.8) respectively.ConclusionsOur results indicate that carnivore faecal samples should be collected as freshly as possible, are better preserved by two-step method and should be extracted with addition of carrier RNA. We recommend quantification of template DNA as this facilitates several downstream protocols.

Majority of the tiger habitat in Indian subcontinent lies within high human density landscapes and is highly sensitive to surrounding pressures. These forests are unable to sustain healthy tiger populations within a tiger-hostile matrix, despite considerable conservation efforts. Ranthambore Tiger Reserve (RTR) in Northwest India is one such isolated forest which is rapidly losing its links with other tiger territories in the Central Indian landscape. Non-invasive genetic sampling for individual identification is a potent technique to understand the relationships between threatened tiger populations in degraded habitats. This study is an attempt to establish tiger movement across a fragmented landscape between RTR and its neighboring forests, Kuno-Palpur Wildlife Sanctuary (KPWLS) and Madhav National Park (MNP) based on non-invasively obtained genetic data.Data from twelve microsatellite loci was used to define population structure and also to identify first generation migrants and admixed individuals in the above forests.Population structure was consistent with the Central Indian landscape and we could determine significant gene flow between RTR and MNP. We could identify individuals of admixed ancestry in both these forests, as well as first generation migrants from RTR to KPWLS and MNP.Our results indicate reproductive mixing between animals of RTR and MNP in the recent past and migration of animals even today, despite fragmentation and poaching risk, from RTR towards MNP. Substantial conservation efforts should be made to maintain connectivity between these two subpopulations and also higher protection status should be conferred on Madhav National Park.

Endometriosis is an estrogen-dependent disease causing pelvic pain and infertility in 10% of reproductive-aged women. Despite a long history of the disease the pathogenesis of endometriosis is poorly understood. It is known that the expression of several proteins is either up or down regulated during endometriosis, but their precise role remains to be determined. DJ-1 is one such protein that is upregulated in eutopic endometrium of women having endometriosis suggesting that DJ-1 may be involved in the pathogenesis of endometriosis.The role of DJ-1 in the pathogenesis of endometriosis was investigated. For this purpose the influence of DJ-1 on endometrial cell survival, attachment, proliferation, migration, and invasion either by overexpressing DJ-1 in normal endometrial cells or by knocking down DJ-1 expression in endometriotic cells using siRNA was investigated. The results indicated that DJ-1 protects endometrial cells from oxidative stress mediated apoptosis. Overexpression of DJ-1 in normal endometrial epithelial cells increases the adhesion on collagen type IV. However, no significant difference was observed incase of stromal cells. It was further demonstrated that DJ-1 regulates cell proliferation, migration, and invasion in normal endometrial and endometriotic epithelial cells whereas in the case of normal endometrial and endometriotic stromal cells, it regulates cell proliferation and invasion but not migration. Furthermore, the present study also indicated that DJ-1 regulates these cellular processes by modulating PI3K/Akt pathway by interacting and negatively regulating PTEN.Abnormally high levels of DJ-1 expression may be involved in endometriosis, possibly by stimulating endometrial cell survival, proliferation, migration, and invasion.