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

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

Long-term exposure to low 17α-ethinylestradiol (EE2) concentrations disrupts both the reproductive and the immune system of juvenile rainbow trout, Oncorhynchus mykiss

Estrogenic endocrine disrupting compounds (EEDCs), such as ethinylestradiol (EE2), are well studied for their impact on the reproductive system of fish. EEDCs may also impact the immune system and, as a consequence, the disease susceptibility of fish. It is currently not yet known whether the low concentrations of EEDCs that are able to disrupt the reproductive system of trout are effective in disrupting the immune system and the fish host resistance towards pathogens, too, or whether such immunodisruptive effects would occur only at higher EEDC concentrations. Therefore, in the present study we compare the effect thresholds of low 17α-ethinylestradiol concentrations (1.5 and 5.5 EE2 ng/L) on the reproductive system, the immune system, the energy expenditures and the resistance of juvenile rainbow trout (Oncorhynchus mykiss) against the parasite Tetracapsuloides bryosalmonae – the etiological agent of proliferative kidney disease (PKD) of salmonids. The parasite infection was conducted without injection and under low pathogen exposure concentrations. The disease development was followed over 130 days post infection – in the presence or absence of EE2 exposure. The results show that the long-term EE2 exposure affected, at both concentrations, reproductive parameters like the mRNA levels of hepatic vitellogenin and estrogen receptors. At the same concentrations, EE2 exposure modulated the immune parameters: mRNA levels of several immune genes were altered and the parasite intensity as well as the disease severity (histopathology) were significantly reduced in EE2-exposed fish compared to infected control fish. The combination of EE2 exposure and parasite infection was energetically costly, as indicated by the decreased values of the swim tunnel respirometry. Although further substantiation is needed, our findings suggest that EE2 exerts endocrine disruptive and immunomodulating activities at comparable effect thresholds, since reproductive and immune parameters were affected by the same, low EE2 concentrations

Estrogens as immunotoxicants: 17α-ethinylestradiol exposure retards thymus development in zebrafish (Danio rerio)

Estrogenic endocrine disrupting compounds (EEDCs) can cause alterations in sexual development and reproductive function of fish. Growing evidence suggests that EEDCs can also interfere with development and function of innate immunity of fish. The present study examined a potential disruptive effect of EEDCs at field-relevant concentrations on the development of adaptive immunity, more specifically the thymus. Zebrafish (Danio rerio) were exposed from fertilization until 64 days post-fertilization (dpf) to environmentally relevant (3 and 10 ng/L) concentrations of the synthetic estrogen 17α-ethinylestradiol (EE2). The exposure duration covered the period of initial thymus differentiation to maximum growth. Thymus development was assessed by histological and morphometric (thymus area) analysis, thymocyte number, and transcript levels of thymocyte marker genes. Additionally, transcript levels of the estrogen receptors (esr1 and esr2a) were determined. The EE2 exposure altered sexual development (gonad differentiation, transcript levels of hepatic vitellogenin and estrogen receptors) of zebrafish, as expected. At the same time, the EE2 treatment reduced the thymus growth (thymus area, thymocyte number) and transcript levels of thymus marker genes. The expression of the thymic estrogen receptors responded to the EE2 exposure but in a different pattern than the hepatic estrogen receptors. After the 64-day-exposure period, the juvenile fish were transferred into clean water for another 95 days to assess the reversibility of EE2-induced effects. The thymic alterations were found to be reversible in female zebrafish but persisted in males. The present study provides the first evidence that the development of the fish adaptive immune system is sensitive to EEDCs, and that this takes place at concentrations similar to those that disrupt sexual development

Trade-Offs Underwater: Physiological Plasticity of Rainbow Trout (<i>Onchorhynchus mykiss</i>) Confronted by Multiple Stressors

Organisms have evolved mechanisms to partition the available resources between fitness-relevant physiological functions. Organisms possess phenotypic plasticity to acclimate to changing environmental conditions. However, this comes at a cost that can cause negative correlations or &#8220;trade-offs&#8222;, whereby increasing investments in one function lead to decreased investments in another function. The aim of the present study was to investigate the prioritization of resource allocation between growth, pathogen defense, and contaminant response in juvenile rainbow trout (Oncorhynchus mykiss) exposed to changes of resource income or expenditure. We performed a multifactorial experiment with three resource-impacting stressors&#8212;limited food availability, a parasitic infection, exposure to a vitellogenesis-inducing contaminant&#8212;and combinations thereof. Treatment with the individual stressors evoked the expected responses in the respective physiological target systems&#8212;body growth, immune system, and hepatic vitellogenin transcription&#8212;but we found little evidence for significant negative relations (trade-offs) between the three systems. This also applied to fish exposed to combinations of the stressors. This high phenotypic flexibility of trout in their resource allocation suggests that linear resource allocations as mechanisms of phenotypic plasticity may be too simplistic, but it also may point to a greater capacity of ectothermic than endothermic vertebrates to maintain key physiological processes under competing resource needs due to lower maintenance costs