BackgroundAlcohol abuse has deleterious effects on human health by disrupting the functions of many organs and systems. Gut microbiota has been implicated in the pathogenesis of alcohol-related liver diseases, with its composition manifesting expressed dysbiosis in patients suffering from alcoholic dependence. Due to its inherent plasticity, gut microbiota is an important target for prevention and treatment of these diseases. Identification of the impact of alcohol abuse with associated psychiatric symptoms on the gut community structure is confounded by the liver dysfunction. In order to differentiate the effects of these two factors, we conducted a comparative “shotgun” metagenomic survey of 99 patients with the alcohol dependence syndrome represented by two cohorts—with and without liver cirrhosis. The taxonomic and functional composition of the gut microbiota was subjected to a multifactor analysis including comparison with the external control group.ResultsAlcoholic dependence and liver cirrhosis were associated with profound shifts in gut community structures and metabolic potential across the patients. The specific effects on species-level community composition were remarkably different between cohorts with and without liver cirrhosis. In both cases, the commensal microbiota was found to be depleted. Alcoholic dependence was inversely associated with the levels of butyrate-producing species from the Clostridiales order, while the cirrhosis—with multiple members of the Bacteroidales order. The opportunist pathogens linked to alcoholic dependence included pro-inflammatory Enterobacteriaceae, while the hallmarks of cirrhosis included an increase of oral microbes in the gut and more frequent occurrence of abnormal community structures. Interestingly, each of the two factors was associated with the expressed enrichment in many Bifidobacterium and Lactobacillus—but the exact set of the species was different between alcoholic dependence and liver cirrhosis. At the level of functional potential, the patients showed different patterns of increase in functions related to alcohol metabolism and virulence factors, as well as pathways related to inflammation.ConclusionsMultiple shifts in the community structure and metabolic potential suggest strong negative influence of alcohol dependence and associated liver dysfunction on gut microbiota. The identified differences in patterns of impact between these two factors are important for planning of personalized treatment and prevention of these pathologies via microbiota modulation. Particularly, the expansion of Bifidobacterium and Lactobacillus suggests that probiotic interventions for patients with alcohol-related disorders using representatives of the same taxa should be considered with caution. Taxonomic and functional analysis shows an increased propensity of the gut microbiota to synthesis of the toxic acetaldehyde, suggesting higher risk of colorectal cancer and other pathologies in alcoholics.Electronic supplementary materialThe online version of this article (10.1...

Microbiomes are vast communities of microbes and viruses that populate all natural ecosystems. Viruses have been considered the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared to other environments. Here we investigate the origin, evolution, and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboratory, we obtained DNA sequences of crAssphage from over one-third of the world's countries, and showed that its phylogeography is locally clustered within countries, cities, and individuals. We also found colinear crAssphage-like genomes in both Old-World and New-World primates, challenging genomic mosaicism and suggesting that the association of crAssphage with primates may be millions of years old. We conclude that crAssphage is a benign globetrotter virus that may have co-evolved with the human lineage and an integral part of the normal human gut virome.

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-tocell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slowgrowing oral microbiota species that suppress the growth of a common pathogen, Staphylococcus aureus, and predicted which genera were associated with inhibitory activity.ultrahigh-throughput screening | microfluidic encapsulation | butyrylcholinesterase | Staphylococcus aureus | cell-cell interactions T he ultrahigh-throughput (1, 2) technique of screening (uHTS) in a double emulsion was applied in directed enzyme evolution (3, 4) to investigate the idea that a universal genotypephenotype linkage was provided by compartmentalization. Artificial compartments of double emulsions were produced with high polydispersity by shear stress (5, 6), which significantly decreased the portion of uniform droplets and thereby reduced the sensitivity and the maximal sorting rate. By contrast, sophisticated custom sorters demonstrated the screening of precise monodisperse droplets of water-in-oil emulsions generated by microfluidic technology (1, 7). However, it is not always convenient to use custom devices, and the use of oil as a continuous phase limits the sorting rate. Alternatively, compartmentalization in microfluidic double emulsion (MDE) enables uHTS of >10,000 events/s using commercially available cell sorters (8, 9). Furthermore, biocompatible oil and water phases provide viability and proliferation of Escherichia coli cells (10) inside the microenvironment of a double emulsion.Here, we propose an MDE-FACS platform that combines the benefits of previously reported systems based on compartmentalization in MDE and FACS selection together with modern omics (Fig. 1). We succeeded in assembling this platform using commercially available parts, which included straightforward microfluidics (Fig. S1) for MDE generation, multiparametric FACS for uHTS, next-generation sequencing (NGS) for bioinformatic predictions, and mass spectrometry for proteome and secretome analysis. We demonstrated this idea with several single-cell methods (Fig. S2), including the selection of di...

Due to its rapid spread and association with the numerous outbreaks, the global spread of East Asian lineage of Mycobacterium tuberculosis strains presents a global concern. Although there were many attempts to describe its population structure, no consensus has been reached yet. To define unbiased classification that will facilitate future studies of this lineage, we analyzed the performance and congruence of eight different genotyping schemes based on phylogenetic analysis of 1,398 strains from 32 countries using whole-genome sequencing (WGS) data. We confirm that East Asian lineage comprises two major clades, designated proto-Beijing, which harbors unusual 43-signal spoligoprofile, and Beijing, with well-known spoligoprofile (deleted signals from 1 to 34). We show that different genotyping methods give high consistency results in description of ancient Beijing strains while the classification of modern Beijing strains is significantly divergent due to star-shaped phylogeny. Using WGS data we intersect different studies and for the first time provide balanced classification with well-defined major groups and their genetic markers. Our reconstructed phylogenetic tree can also be used for further analysis of epidemiologically important clusters and their ancestors as well as white spots of unclassified strains, which are prospective areas of research.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a rapid, cost-effective alternative for bacterial species identification. Identifying 60 blind-coded nonfermenting bacteria samples, this international study (using eight laboratories) achieved 98.75% interlaboratory reproducibility. Only 6 of the 480 samples were misidentified due to interchanges (4 samples) or contamination (1 sample) or not identified because of insufficient signal intensity (1 sample).Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has emerged as a fast and costeffective alternative for bacterial species identification in microbiology. On the basis of mass analysis of the protein composition of a bacterial cell, which is assumed to be characteristic for each bacterial species, it is possible to determine the species within few minutes, starting from whole cells, cell lysates, or crude bacterial extracts (2, 3, 5, 6). The proof of principle of MALDI-TOF MS for bacterial species identification was shown a decade ago (2, 5, 6); however, due to low reproducibility, it has not been widely adopted in clinical microbiology. We have recently shown that use of a larger mass range for detection (2,000 to 20,000 Da), dedicated analysis software for spectral pattern matching, and a highquality reference database of spectra generated from qualitycontrolled culture collection strains resulted in accurate species identifications, with high intralaboratory reproducibility (7). For interlaboratory reproducibility, there are only very limited data available (8, 10). We therefore evaluated the interlaboratory reproducibility for MALDI-TOF MS-based species identification in a multicenter study, applying the above-described MALDI-TOF MS improvements.(