Accurate prediction of the host phenotypes from a microbial sample and identification of the associated microbial markers are important in understanding the impact of the microbiome on the ...pathogenesis and progression of various diseases within the host. A deep learning tool, PopPhy-CNN, has been developed for the task of predicting host phenotypes using a convolutional neural network (CNN). By representing samples as annotated taxonomic trees and further representing these trees as matrices, PopPhy-CNN utilizes the CNN's innate ability to explore locally similar microbes on the taxonomic tree. Furthermore, PopPhy-CNN can be used to evaluate the importance of each taxon in the prediction of host status. Here, we describe the underlying methodology, architecture, and core utility of PopPhy-CNN. We also demonstrate the use of PopPhy-CNN on a microbial dataset.
The objective of this study was to evaluate the most effective method of DNA extraction of oral mouthwash samples for use in microbiome studies that utilize next generation sequencing (NGS). Eight ...enzymatic and mechanical DNA extraction methods were tested. Extracted DNA was amplified using barcoded primers targeting the V6 variable region of the bacterial 16S rRNA gene and the ITS1 region of the fungal ribosomal gene cluster and sequenced using the Illumina NGS platform. Sequenced reads were analyzed using QIIME and R. The eight methods yielded significantly different quantities of DNA (p < 0.001), with the phenol-chloroform extraction method producing the highest total yield. There were no significant differences in observed bacterial or fungal Shannon diversity (p = 0.64, p = 0.93 respectively) by extraction method. Bray-Curtis beta-diversity did not demonstrate statistically significant differences between the eight extraction methods based on bacterial (R
= 0.086, p = 1.00) and fungal (R
= 0.039, p = 1.00) assays. No differences were seen between methods with or without bead-beating. These data indicate that choice of DNA extraction method affect total DNA recovery without significantly affecting the observed microbiome.
Germ-Free (GF) research has required highly technical pressurized HEPA-ventilation anchored systems for decades. Herein, we validated a GF system that can be easily implemented and portable using ...Nested Isolation (NesTiso). GF-standards can be achieved housing mice in non-HEPA-static cages, which only need to be nested 'one-cage-inside-another' resembling 'Russian dolls'. After 2 years of monitoring ~100,000 GF-mouse-days, NesTiso showed mice can be maintained GF for life (>1.3 years), with low animal daily-contamination-probability risk (1 every 867 days), allowing the expansion of GF research with unprecedented freedom and mobility. At the cage level, with 23,360 GF cage-days, the probability of having a cage contamination in NesTiso cages opened in biosafety hoods was statistically identical to that of opening cages inside (the 'gold standard') multi-cage pressurized GF isolators. When validating the benefits of using NesTiso in mouse microbiome research, our experiments unexpectedly revealed that the mouse fecal microbiota composition within the 'bedding material' of conventional SPF-cages suffers cyclical selection bias as moist/feces/diet/organic content ('soiledness') increases over time (e.g., favoring microbiome abundances of Bacillales, Burkholderiales, Pseudomonadales; and cultivable Enterococcus faecalis over Lactobacillus murinus and Escherichia coli), which in turn cyclically influences the gut microbiome dynamics of caged mice. Culture 'co-streaking' assays showed that cohoused mice exhibiting different fecal microbiota/hemolytic profiles in clean bedding (high-within-cage individual diversity) 'cyclically and transiently appear identical' (less diverse) as bedding soiledness increases, and recurs. Strategies are proposed to minimize this novel functional form of cyclical bedding-dependent microbiome selection bias.
The mammalian gut microbiome can potentially impact host health and disease state. It is known that the mouse-genome, eating-behavior, and exercise-status promotes higher taxonomic rank-level ...alterations (e.g. family to phyla-level) of the gut microbiota. Here, host genotype or activity status was investigated to determine if selection of individual bacterial species or strains could be discerned within the murine digestive system. For this study, the fecal bacterial community of adenylyl cyclase 5 knock-out (AC5KO, n = 7) mice or their wild-type (WT, n = 10) littermates under exercise or sedentary conditions were profiled by sequencing rRNA operons. AC5KO mice were chosen since this genotype displays enhanced longevity/exercise capacity and protects against cardiovascular/metabolic disease. Profiling of rRNA operons using the Oxford MinION yielded 65,706 2-D sequences (after size selection of 3.7-5.7 kb) which were screened against an NCBI 16S rRNA gene database. These sequences were binned into 1,566 different best BLAST hits (BBHs) and counted for each mouse sample. Non-metric multidimensional scaling (NMDS) of the gut microbial community demonstrated clustering by physical activity (p = 0.001) but not by host genotype. Additionally, sequence similarity and phylogenetic analysis demonstrated that different bacterial species (closely related to Muribaculum intestinale and Parasutterella excrementihominis) inhabit AC5KO or WT mice depending on activity status. Other bacterial species of the gut microbiota did not follow such patterning (e.g. Turicibacter sanguinis and Turicimonas muris). Our results support the need of improved taxonomic resolution for better characterization of bacterial communities to deepen our understanding of the role of the gut microbiome on host health.
In the last few years, a significant increase of childhood obesity incidence unequally distributed within countries and population groups has been observed, thus representing an important public ...health problem associated with several health and social consequences. Obese children have more than a 50% probability of becoming obese adults, and to develop pathologies typical of obese adults, that include type 2-diabetes, dyslipidemia and hypertension. Also environmental factors, such as reduced physical activity and increased sedentary activities, may also result in increased caloric intake and/or decreased caloric expenditure. In the present review, we aimed to identify and describe a specific panel of parameters in order to evaluate and characterize the childhood obesity status useful in setting up a preventive diagnostic approach directed at improving health-related behaviors and identifying predisposing risk factors. An early identification of risk factors for childhood obesity could definitely help in setting up adequate and specific clinical treatments.
In addition to Helicobacter pylori infection, nitrosating/nitrate-reducing bacteria and type IV secretion system (T4SS) protein gene-contributing bacteria have been proposed as potential causes of ...gastric cancer development. However, bacterial modules related with gastric carcinogenesis have not been clarified. In this study, we analyzed gastric microbiome using the gastric mucosal samples obtained from the Hanyang University Gastric Microbiome Cohort by 16S rRNA gene sequencing. Weighted correlation network analysis was performed to construct a microbiome network and to identify microbial modules associated with gastric carcinogenesis. At the family level, 420 bacterial taxa were identified in the gastric microbiome of 83 participants. Through network analysis, 18 microbial modules were organized. Among them, two modules-pink and brown-were positively correlated with a higher-risk of gastric cancer development such as intestinal metaplasia with no current H. pylori infection (correlation coefficient γ: pink module, 0.31 P = 0.004, brown module, 0.26 P = 0.02). At the family level, twenty-two and thirty-two bacterial taxa belonged to the pink and brown modules, respectively. They included nitrosating/nitrate-reducing bacteria, T4SS protein gene-contributing bacteria, and various other bacteria, including Gordoniaceae, Tsukamurellaceae, Prevotellaceae, Cellulomonadaceae, Methylococcaceae, and Procabacteriaceae. The blue module, which included H. pylori, was correlated negatively with intestinal metaplasia (γ = -0.49 P < 0.001). In conclusion, intragastric bacterial taxa associated with gastric carcinogenesis can be classified by network analysis. Microbial modules may provide an integrative view of the microbial ecology relevant to precancerous lesions in the stomach.
Gastrointestinal helminth-microbiota associations are shaped by various ecological processes. The effect of the ecological context of the host on the bacterial microbiome and gastrointestinal ...helminth parasites has been tested in a number of ecosystems and experimentally. This study takes the important step to look at these two groups at the same time and to start to examine how these communities interact in a changing host environment. Fresh faecal samples (N = 335) from eight wild Eulemur populations were collected over 2 years across Madagascar. We used 16S ribosomal RNA gene sequencing to characterise the bacterial microbiota composition, and faecal flotation to isolate and morphologically identify nematode eggs. Infections with nematodes of the genera Callistoura and Lemuricola occurred in all lemur populations. Seasonality significantly contributed to the observed variation in microbiota composition, especially in the dry deciduous forest. Microbial richness and Lemuricola spp. infection prevalence were highest in a previously intensely logged site, whereas Callistoura spp. showed no such pattern. In addition, we observed significant correlations between gastrointestinal parasites and bacterial microbiota composition in these lemurs, with 0.4-0.7% of the variation in faecal bacterial microbiota composition being explained by helminth infections. With this study, we show effects of environmental conditions on gastrointestinal nematodes and bacterial interactions in wild lemurs and believe it is essential to consider the potential role of microbiome-parasite associations on the hosts' GI stability, health, and survival.
Currently, increasing attention cancer treatment has focused on molecularly targeted therapies and more recently on immunotherapies targeting immune checkpoints. However, even such advanced treatment ...may be ineffective. The reasons for this are sought, inter alia, in the human microbiome. In our intestines, there are bacteria that are beneficial to us, but pathogenic microorganisms may also be present. Microbial imbalance (dysbiosis) is now perceived as one of the gateways to cancer. However, it is feasible to use bacteria and their metabolites to restore the natural, beneficial microbiome during oncological treatment.
Akkermansia mucinifila
,
Enterococcus hirae
, or
Faecalibacterium prausnitzii
are bacteria that exhibit this beneficial potential. Greater benefits of therapy can be observed in cancer patients enriched in these bacterial species and treated with anti-PD-1, anti-PD-L1, or anti-CTLA-4 monoclonal antibodies. In this review, we present issues related to the role of bacteria in carcinogenesis and their therapeutic potential “supporting” modern anti-cancer therapies.
Key Points
• Bacteria can be directly or indirectly a cancer trigger.
• Bacterial metabolites regulate the pathways associated with carcinogenesis.
• Intestinal bacteria activate the immune system to fight cancer.
Owing to the increased cost-effectiveness of high-throughput technologies, the number of studies focusing on the human microbiome and its connections to human health and disease has recently surged. ...However, best practices in microbiology and clinical research have yet to be clearly established. Here, we present an overview of the challenges and opportunities involved in conducting a metagenomic study, with a particular focus on data processing and analytical methods.
Collembola are a key component of the soil biota globally, playing an important role in community and ecosystem dynamics. Equally significant are their associated microbiomes, that can contribute to ...key metabolic functions. In the present study, we investigated the bacterial community composition of four Antarctic springtail species to assess if and how the extreme Antarctic environment has shaped the collembolans' microbiomes. Springtails were collected from two biogeographical regions, the maritime and the continental Antarctic. From each region, two endemic species, belonging to the genera Cryptopygus (Isotomidae, Entomobryomorpha) and Friesea (Neanuridae, Poduromorpha), were included. This experimental design allowed us to quantify the relative importance of ecological factors (different regions of occurrence) and/or phylogenetic divergence in the host (different Orders) in shaping the Collembola microbiome. The diversity and richness of springtail microbiomes was lower in the Antarctic taxa compared to published information from species from temperate regions. The microbiome composition was predominantly species-specific, with a limited core microbiome shared across the four species examined. While both geographic origin and host species influenced the associated microbiomes, the former was the prevalent driver, with closer similarity between springtails from the same bioregion than between those belonging to the same genus.
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