Challenges and insights in the exploration of the low abundance human ocular surface microbiome.

PURPOSE The low microbial abundance on the ocular surface results in challenges in the characterization of its microbiome. The purpose of this study was to reveal factors introducing bias in the pipeline from sample collection to data analysis of low-abundant microbiomes. METHODS Lower conjunctiva and lower lid swabs were collected from six participants using either standard cotton or flocked nylon swabs. Microbial DNA was isolated with two different kits (with or without prior host DNA depletion and mechanical lysis), followed by whole-metagenome shotgun sequencing with a high sequencing depth set at 60 million reads per sample. The relative microbial compositions were generated using the two different tools MetaPhlan3 and Kraken2. RESULTS The total amount of extracted DNA was increased by using nylon flocked swabs on the lower conjunctiva. In total, 269 microbial species were detected. The most abundant bacterial phyla were Actinobacteria, Firmicutes and Proteobacteria. Depending on the DNA extraction kit and tool used for profiling, the microbial composition and the relative abundance of viruses varied. CONCLUSION The microbial composition on the ocular surface is not dependent on the swab type, but on the DNA extraction method and profiling tool. These factors have to be considered in further studies about the ocular surface microbiome and other sparsely colonized microbiomes in order to improve data reproducibility. Understanding challenges and biases in the characterization of the ocular surface microbiome may set the basis for microbiome-altering interventions for treatment of ocular surface associated diseases

The role of the gut microbiome in eye diseases.

The gut microbiome is a complex ecosystem of microorganisms and their genetic entities colonizing the gastrointestinal tract. When in balanced composition, the gut microbiome is in symbiotic interaction with its host and maintains intestinal homeostasis. It is involved in essential functions such as nutrient metabolism, inhibition of pathogens and regulation of immune function. Through translocation of microbes and their metabolites along the epithelial barrier, microbial dysbiosis induces systemic inflammation that may lead to tissue destruction and promote the onset of various diseases. Using whole-metagenome shotgun sequencing, several studies have shown that the composition and associated functional capacities of the gut microbiome are associated with age-related macular degeneration, retinal artery occlusion, central serous chorioretinopathy and uveitis. In this review, we provide an overview of the current knowledge about the gut microbiome in eye diseases, with a focus on interactions between the microbiome, specific microbial-derived metabolites and the immune system. We explain how these interactions may be involved in the pathogenesis of age-related macular degeneration, retinal artery occlusion, central serous chorioretinopathy and uveitis and guide the development of new therapeutic approaches by microbiome-altering interventions for these diseases

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri

Background The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. Results Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. Conclusions In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.publishedVersio

Investigating the Ocular Surface Microbiome: What Can It Tell Us?

While pathogens of the eye have been studied for a very long time, the existence of resident microbes on the surface of healthy eyes has gained interest only recently. It appears that commensal microbes are a normal feature of the healthy eye, whose role and properties are currently the subject of extensive research. This review provides an overview of studies that have used 16s rRNA gene sequencing and whole metagenome shotgun sequencing to characterize microbial communities associated with the healthy ocular surface from kingdom to genus level. Bacteria are the primary colonizers of the healthy ocular surface, with three predominant phyla: Proteobacteria, Actinobacteria, and Firmicutes, regardless of the host, environment, and method used. Refining the microbial classification to the genus level reveals a highly variable distribution from one individual and study to another. Factors accounting for this variability are intriguing - it is currently unknown to what extent this is attributable to the individuals and their environment and how much is artifactual. Clearly, it is technically challenging to accurately describe the microorganisms of the ocular surface because their abundance is relatively low, thus, permitting substantial contaminations. More research is needed, including better experimental standards to prevent biases, and the exploration of the ocular surface microbiome's role in a spectrum of healthy to pathological states. Outcomes from such research include the opportunity for therapeutic interventions targeting the microbiome

The importance of age in compositional and functional profiling of the human intestinal microbiome.

The intestinal microbiome plays a central role in human health and disease. While its composition is relatively stable throughout adulthood, the microbial balance starts to decrease in later life stages. Thus, in order to maintain a good quality of life, including the prevention of age-associated diseases in the elderly, it is important to understand the dynamics of the intestinal microbiome. In this study, stool samples of 278 participants were sequenced by whole metagenome shotgun sequencing and their taxonomic and functional profiles characterized. The two age groups, below65 and above65, could be separated based on taxonomic and associated functional features using Multivariate Association of Linear Models. In a second approach, through machine learning, biomarkers connecting the intestinal microbiome with age were identified. These results reflect the importance to select age-matched study groups for unbiased metagenomic data analysis and the possibility to generate robust data by applying independent algorithms for data analysis. Furthermore, since the intestinal microbiome can be modulated by antibiotics and probiotics, the data of this study may have implications on preventive strategies of age-associated degradation processes and diseases by microbiome-altering interventions

Associations of the intestinal microbiome with the complement system in neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of severe vision loss in the aged population. The etiology of AMD is multifactorial including nutritional factors, genetic variants mainly in the complement pathway, environmental risk factors and alterations in the intestinal microbiome. However, it remains unexplored whether there is an interdependency of these factors leading to the development of AMD. To investigate this issue, a shotgun metagenomics analysis of 57 neovascular AMD and 58 healthy controls as well as of 16 complement C3-deficient mice and 16 wildtypes was performed. Whereas the class Negativicutes was more abundant in patients, the genus Oscillibacter and species Bacteroides had a significantly higher prevalence in persons without AMD. Similar taxonomic features were identified that distinguished wildtype mice from C3-deficient mice. Moreover, several purine signaling pathways were associated with both, neovascular AMD and C3 deficiency. While SNPs within the complement factor B gene were more abundant in controls, SNPs within the high temperature requirement A serine peptidase 1 and complement factor H (CFH) genes were associated with neovascular AMD. Using a classification model, Negativicutes was identified as a potential biomarker for AMD and furthermore, it positively correlated with CFH. This study suggests an association between the intestinal microbiome and the complement system in neovascular AMD

The gut microbiome and HLA-B27-associated anterior uveitis: a case-control study.

BACKGROUND The human gut microbiome (GM) is involved in inflammation and immune response regulation. Dysbiosis, an imbalance in this ecosystem, facilitates pathogenic invasion, disrupts immune equilibrium, and potentially triggers diseases including various human leucocyte antigen (HLA)-B27-associated autoinflammatory and autoimmune diseases such as inflammatory bowel disease (IBD) and spondyloarthropathy (SpA). This study assesses compositional and functional alterations of the GM in patients with HLA-B27-associated non-infectious anterior uveitis (AU) compared to healthy controls. METHODS The gut metagenomes of 20 patients with HLA-B27-associated non-infectious AU, 21 age- and sex-matched HLA-B27-negative controls, and 6 HLA-B27-positive healthy controls without a history of AU were sequenced using the Illumina NovaSeq 6000 platform for whole metagenome shotgun sequencing. To identify taxonomic and functional features with significantly different relative abundances between groups and to identify associations with clinical metadata, the multivariate association by linear models (MaAsLin) R package was applied. RESULTS Significantly higher levels of the Eubacterium ramulus species were found in HLA-B27-negative controls (p = 0.0085, Mann-Whitney U-test). No significant differences in microbial composition were observed at all other taxonomic levels. Functionally, the lipid IVA biosynthesis pathway was upregulated in patients (p < 0.0001, Mann-Whitney U-test). A subgroup analysis comparing patients with an active non-infectious AU to their age- and sex-matched HLA-B27-negative controls, showed an increase of the species Phocaeicola vulgatus in active AU (p = 0.0530, Mann-Whitney U-test). An additional analysis comparing AU patients to age- and sex-matched HLA-B27-positive controls, showed an increase of the species Bacteroides caccae in controls (p = 0.0022, Mann-Whitney U-test). CONCLUSION In our cohort, non-infectious AU development is associated with compositional and functional alterations of the GM. Further research is needed to assess the causality of these associations, offering potentially novel therapeutic strategies

Microbiome and retinal vascular diseases.

The gut microbiome consists of more than thousand different microbes and their associated genes and microbial metabolites. It influences various host metabolic pathways and is therefore important for homeostasis. In recent years, its influence on health and disease was extensively researched. In case of a microbiome disequilibrium called dysbiosis, the gut microbiome is associated with several diseases. Consequent chronic inflammation may lead to or promote inflammatory bowel disease, obesity, diabetes mellitus, atherosclerosis, alcoholic and non-alcoholic liver disease, cirrhosis, hepatocellular carcinoma and other diseases. The pathogenesis of the three most common retinal vascular diseases, diabetic retinopathy, retinal vein and artery occlusion, may also be influenced by an altered microbiome and associated risk factors such as diabetes mellitus, atherosclerosis, hypertension and obesity. Direct cause-effect relationships remain less well understood. A potential prevention or treatment modality for these diseases could be targeting and modulating the individual's gut microbiome

The importance of age in compositional and functional profiling of the human intestinal microbiome.

The intestinal microbiome plays a central role in human health and disease. While its composition is relatively stable throughout adulthood, the microbial balance starts to decrease in later life stages. Thus, in order to maintain a good quality of life, including the prevention of age-associated diseases in the elderly, it is important to understand the dynamics of the intestinal microbiome. In this study, stool samples of 278 participants were sequenced by whole metagenome shotgun sequencing and their taxonomic and functional profiles characterized. The two age groups, below65 and above65, could be separated based on taxonomic and associated functional features using Multivariate Association of Linear Models. In a second approach, through machine learning, biomarkers connecting the intestinal microbiome with age were identified. These results reflect the importance to select age-matched study groups for unbiased metagenomic data analysis and the possibility to generate robust data by applying independent algorithms for data analysis. Furthermore, since the intestinal microbiome can be modulated by antibiotics and probiotics, the data of this study may have implications on preventive strategies of age-associated degradation processes and diseases by microbiome-altering interventions

Retinal artery occlusion is associated with compositional and functional shifts in the gut microbiome and altered trimethylamine-N-oxide levels.

Retinal artery occlusion (RAO) is a sight threatening complication of cardiovascular disease and commonly occurs due to underlying atherosclerosis. As cardiovascular disease and atherosclerosis in particular has been associated with compositional alterations in the gut microbiome, we investigated this association in patients with clinically confirmed non-arteritic RAO compared to age- and sex-matched controls. On the phylum level, the relative abundance of Bacteroidetes was decreased in patients with RAO compared to controls, whereas the opposite applied for the phylum of Proteobacteria. Several genera and species such as Actinobacter, Bifidobacterium spp., Bacteroides stercoris, Faecalibacterium prausnitzii were relatively enriched in patients with RAO, whereas others such as Odoribacter, Parasutterella or Lachnospiraceae were significantly lower. Patient's gut microbiomes were enriched in genes of the cholesterol metabolism pathway. The gut derived, pro-atherogenic metabolite trimethylamine-N-oxide (TMAO) was significantly higher in patients with RAO compared to controls (p = 0.023) and a negative correlation between relative abundances of genera Parasutterella and Lachnospiraceae and TMAO levels and a positive correlation between relative abundance of genus Akkermansia and TMAO levels was found in study subjects. Our findings proposes that RAO is associated with alterations in the gut microbiome and with elevated TMAO levels, suggesting that RAO could be targeted by microbiome-altering interventions