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In this study, we used single-cell sequencing to analyze the gut microbiome of an adult male patient with acute cerebral hemorrhage undergoing antibiotic treatment. We identified 92 bacterial species, including 23 Firmicutes and one archaeon from Methanobacteriota, along with 69 unclassified strains. Single-cell sequencing effectively detected bacteria carrying antibiotic resistance genes (ARGs), particularly in unclassified species, and traced the evolution of these genes across diverse bacterial taxa. Notably, the cfr(C) gene was detected in 11 bacterial species following antimicrobial treatment, with mutation patterns characterized in Enterococcus faecalis, Klebsiella pneumoniae, Ruthenibacterium UN-1, and four unclassified species. In total, 29 ARG subtypes across eight types were identified in 13 known, five unknown, and 18 unclassified species, allowing us to trace their evolution routes. In addition, we detected a total of 309 horizontal gene transfer (HGT) events, in which several genes like folE and queE were frequently involved. The products of these genes are known to enhance the ability of the recipient bacterial strains to repair DNA damage and maintain genomic stability, especially following prolonged antibiotic treatment. Comparison between isolated strain genomes (IS-KP1) and single-cell analysis confirmed the presence of at least two K. pneumoniae strains in the patient, with one exhibiting a larger extent of involvement in ARG co-evolution. This strain was found to contain the cfr(C) and fosXCC genes, which were absent in IS-KP1. Klebsiella strains were also found to participate actively in HGT events. In conclusion, the study identified a wide range of ARGs and HGT events within the microbiome. The detection of K. pneumoniae strains with distinct ARG evolution patterns underscores the gut microbiome's adaptability to environmental changes. These findings facilitate the development of novel antimicrobial strategies by fine-tuning the gut microbiome composition.IMPORTANCEThis study highlights the power of single-cell sequencing to unravel the diversity and dynamics of the gut microbiome during antibiotic treatment in a patient with acute cerebral hemorrhage. By identifying antibiotic resistance genes (ARGs) in both known and unclassified bacterial species, we reveal the intricate evolution and horizontal transfer of resistance traits across taxa. The discovery of distinct ARG patterns, including the emergence of the cfr(C) gene in multiple species and its co-evolution in K. pneumoniae, underscores the gut microbiome's adaptability to antimicrobial pressures. These findings provide critical insights into the mechanisms driving resistance dissemination and offer potential pathways for developing precision microbiome-based therapies to combat antibiotic resistance.

Inflammatory bowel disease (IBD) is a major precursor to colorectal cancer (CRC). Our previous study demonstrated that combined administration of the probiotics Clostridium butyricum (CB) and Akkermansia muciniphila (AKK) significantly alleviated IBD and CRC symptoms in mice. Increasing evidence suggests that probiotic metabolites (postbiotics) offer significant advantages in disease prevention and treatment without the stability and safety concerns associated with live bacterial therapies. To further explore the therapeutic potential of CB- and AKK-fermented metabolites against IBD and CRC, we established a DSS-induced IBD model and DSS/AOM-induced CRC orthotopic models in mice and evaluated the effects of CB and AKK metabolites on alleviating IBD and CRC. The results revealed that the fermented metabolites of CB and AKK (designated as SupCB and SupAKK, respectively) exhibited significant synergistic effects. Mixed fermented metabolites (designated as SupCBAKK) outperformed individual metabolites, significantly alleviating IBD and CRC symptoms by modulating immune responses, repairing the mucosal barrier, and ameliorating gut dysbiosis. Notably, SupCBAKK synergized with the immune checkpoint inhibitor anti-PD-L1 (aPD-L1), enhancing tumor sensitivity to immunotherapy and amplifying antitumor immune responses. These findings underscore the potential of SupCBAKK as a novel postbiotic formulation for mitigating IBD and CRC progression and offer innovative strategies for developing CB- and AKK-based therapeutic interventions.

Importance: This study highlights the therapeutic potential of SupCBAKK, a novel postbiotic formulation derived from the combined fermentation metabolites of CB and AKK, IBD, and colitis-associated colorectal cancer through the modulation of gut microbiota and immunometabolism.

Mosquitoes harbor diverse microbial communities that influence their potential to transmit pathogens. However, the ecological drivers shaping these microbiomes, particularly in under-sampled regions like Africa, remain poorly resolved. We conducted a large-scale metatranscriptomic survey of 3,940 Aedes and Culex mosquitoes from diverse ecological zones across Kenya. Our analyses revealed that viruses dominated the overall transcriptome, while bacteria exhibited the greatest taxonomic richness. Geographic location emerged as the primary driver of microbial community structure, whereas host genus identity shaped virome diversity at local or city-level scales. Culex mosquitoes harbored higher viral richness, particularly in coastal regions, while Aedes supported more diverse bacterial assemblages. Microbial co-occurrence networks exhibited distinct topologies across hosts: Culex networks featured cross-domain interactions and viral keystone taxa, whereas Aedes networks were more cohesive and robust, centered on bacterial hubs. We identified 102 distinct viruses from 24 families, including 31 putative novel RNA viruses. Segment-resolved phylogenies revealed cryptic clades within Bunyavirales, Picornavirales, and other lineages. Collectively, our findings highlight the scale-dependent influences of geography and host identity on mosquito microbiomes in East Africa and demonstrate the utility of metatranscriptomics in uncovering hidden microbial diversity and ecological interactions. These insights provide a foundation for ecologically informed arthropod vector surveillance and microbiome-based intervention strategies.IMPORTANCEMosquitoes are more than just flying syringes; they are complex ecosystems hosting a variety of microbes. Understanding what shapes this microbial world inside mosquitoes is key to developing new control strategies. Our study of nearly 4,000 mosquitoes from Kenya reveals that where a mosquito lives matters most for its overall microbial makeup, but its genus dictates which viruses it carries. We discovered that different mosquito types have distinct microbial social networks: one type has a fragile network centered on viruses, while the other has a resilient network built around bacteria. This means that strategies to disrupt disease transmission by targeting mosquito microbes may need to be tailored to a specific mosquito genus. Our work provides a map of these microbial ecosystems, highlighting potential new viruses and offering insights for future public health surveillance and interventions.

Soil microorganisms perform biogeochemical processes fundamental to soil functions. Bulk respiration, or microbial metabolic emission of CO₂, is the classic indicator of soil biological activity. However, among the millions of microbes per gram of soil, only 0.1%-2.0% are metabolically active at any given time. Understanding the relationship between bulk soil respiration and microbial activity is complicated by microbes potentially awakening from quiescent states during incubation test periods. Here, we investigated this relationship through parallel measurements of substrate-induced respiration and translationally active cell counts using bioorthogonal non-canonical amino acid tagging (BONCAT). After a 6-h incubation of agricultural soil with glucose, galactose, or only water, active cell counts were positively correlated with respiration rates. As hypothesized, active cell numbers increased rapidly compared to total cell numbers after a 6-h incubation with glucose, suggesting newly activated cells. Additionally, carbon-amended soils respired more than water-only soils with similar active cell counts. This suggested that cells in carbon-rich environments were turning over freshly added carbon faster or metabolizing it less efficiently than those exposed to native substrates only. This study distinguishes for the first time microbial activation in the soil matrix using a translation signal, providing evidence that respiration rates reflect active cell numbers and varied metabolic responses, decoupled from cell growth upon soil wetting and carbon addition. We propose BONCAT as a useful tool to gain mechanistic insights into microbial activation and recommend combining it with tracking substrate incorporation and phylogenetics.IMPORTANCEMany critical ecosystem services provided by soils rely on active microbes, even though most soil microbes are known to be quiescent or dormant much of the time. This study demonstrates that microbes become translationally active within hours after substrate addition and that the correlation between active cell numbers and soil respiration rates varies with the type of substrate. Advancing knowledge in this area will enable better interpretation of bulk soil respiration tests by land managers and inform modeling efforts that relate soil microbial respiration to global carbon dynamics.

The National Institute of Allergy and Infectious Diseases (NIAID) Data Ecosystem Discovery Portal (https://data.niaid.nih.gov) provides a unified search interface for over 4 million data sets relevant to infectious and immune-mediated disease (IID) research. Integrating metadata from domain-specific and generalist repositories, the Portal enables researchers to identify and access data sets using user-friendly filters or advanced queries, without requiring technical expertise. The Portal supports discovery of a wide range of resources, including epidemiological, clinical, and multi-omic data sets and is designed to accommodate exploratory browsing and precise searches. The Portal provides filters, prebuilt queries, and data set collections to simplify the discovery process for users. The Portal additionally provides documentation and an API for programmatic access to harmonized metadata. By easing access barriers to important biomedical data sets, the NIAID Data Ecosystem Discovery Portal serves as an entry point for researchers working to understand, diagnose, or treat IID.IMPORTANCEValuable data sets are often overlooked because they are difficult to locate. The NIAID Data Ecosystem Discovery Portal fills this gap by providing a centralized, searchable interface that empowers users with varying levels of technical expertise to find and reuse data. By standardizing key metadata fields and harmonizing heterogeneous formats, the Portal improves data findability, accessibility, and reusability. This resource supports hypothesis generation, comparative analysis, and secondary use of public data by the IID research community, including those funded by NIAID. The Portal supports data sharing by standardizing metadata and linking to source repositories and maximizes the impact of public investment in research data by supporting scientific advancement via secondary use.

Necrotizing soft tissue infections (NSTIs) are rapidly progressive and life-threatening diseases caused by diverse bacterial pathogens. While classical virulence factors, such as toxins and secretion systems, have been extensively characterized, the role of metabolic fitness in supporting bacterial survival within the nutrient-restricted host environment remains underexplored. Edwardsiella tarda, a human-pathogenic bacterium implicated in NSTIs, represents an emerging model for studying non-canonical pathogenic strategies. Here, we employed transposon-directed insertion site sequencing (TraDIS) to identify genes critical for E. tarda survival in a murine soft tissue infection model. A genome-wide screen revealed 41 genes significantly depleted during the infection, including those involved in iron and zinc acquisition (fetB, zupT), vitamin biosynthesis (pdxK, cobA), and polyamine metabolism (speB). Functional assays using defined minimal media demonstrated that supplementation with vitamin B6 or putrescine enhanced bacterial growth, validating their contribution to fitness under nutrient-limited conditions. Our findings indicate that E. tarda pathogenesis is driven not solely by classical virulence factors but also by its ability to acquire essential nutrients and adapt metabolically to host-imposed nutritional stress. This study provides the first genome-wide fitness map for E. tarda during soft tissue infection and reveals new targets for therapeutic intervention that disrupt nutrient acquisition systems. These results also emphasize the broader relevance of metabolic adaptation as a determinant of virulence in invasive bacterial infections.IMPORTANCENecrotizing soft tissue infections (NSTIs) are severe, rapidly progressing bacterial infections with high morbidity and mortality. Although classical virulence factors such as toxins have been widely studied, much less is known about how pathogens adapt metabolically to survive within the nutrient-restricted environment in host tissues. This study uses Edwardsiella tarda, an emerging NSTI pathogen, as a model to identify genes required for in vivo fitness using transposon insertion sequencing. By revealing the critical roles of nutrient acquisition and metabolic adaptation, rather than toxin production alone, this work challenges conventional paradigms of bacterial virulence. Our findings suggest that targeting bacterial nutrient acquisition pathways may offer a novel therapeutic approach to control invasive infections. Furthermore, this study provides the first genome-wide fitness map of E. tarda during soft tissue infection, offering a valuable resource for future research into polymicrobial wound infections and host-pathogen nutrient competition.

Mobile genetic elements (MGEs) are major drivers of horizontal gene transfer, including the spread of antimicrobial resistance (AMR) genes. However, determining the microbial host of an MGE in complex microbiomes remains challenging. Here, we spike a niche-aspecific Bacillus velezensis strain carrying a plasmid and linear phage-plasmid into a batch bioreactor simulating the human gut, and use it as a spike-in control to assess the performance of Hi-C sequencing and Oxford Nanopore Technologies (ONT)-enabled DNA methylation detection to identify MGE-host pairs. To improve recovery of low-abundance genomes, we used a novel ONT adaptive sampling (AS) strategy that depletes de novo assembled, sample-specific high-abundance contigs, rather than relying on reference genomes. This approach led to an approximately twofold enrichment of low-abundance replicons, including the spike-in strain. Methylation-based host assignment failed for the B. velezensis MGEs, likely due to the absence of DNA methylation. In contrast, Hi-C successfully linked the phage-plasmid to its host, but not the plasmid, likely due to non-intact cells, and only after removing artefactual signals through bioinformatic processing. For a native Escherichia coli strain, Hi-C and methylation data linked it to two plasmids. Selective isolation and whole-genome sequencing of both the native E. coli and spike-in B. velezensis then confirmed the metagenomic observations. Our results highlight that Hi-C and methylation data can provide powerful insights into MGE-host associations, but their interpretation requires careful computational analysis and biological validation. Moreover, our AS strategy offers a cost-efficient method to boost coverage of low-abundance genomes, improving metagenomic investigation of MGEs in complex microbiomes.

Importance: Mobile genetic elements are important contributors to horizontal gene transfer, including of antimicrobial resistance genes. Understanding which microbes carry these mobile elements is vital to assess the spread of resistance. Here, we use a nanopore adaptive sampling approach to increase detection of low-abundance bacteria and mobile elements and use DNA methylation detection and Hi-C sequencing to determine mobile element hosts. By introducing a known bacterium and isolating a native strain, we could evaluate the performance of these methods, indicating that although powerful, they require careful experimental design, interpretation, and validation. However, when combined, these approaches enable a comprehensive investigation of mobile elements and gene transfer dynamics in complex environments.

Endoplasmic reticulum (ER) stress-related mucin depletion could be involved in the pathogenesis of ulcerative colitis (UC). Akkermansia muciniphila (A. muciniphila) uses mucin as its sole energy source and shows potential in the treatment of colitis. However, the effects and underlying mechanisms of A. muciniphila on colonic epithelial ER stress in colitis are largely unknown. Colitis was induced by adding 2.5% dextran sulfate sodium (DSS) in drinking water. Mice were orally administered A. muciniphila (3*10^{^}7, 3*10^{^}8 cfu/day) once daily for 10 days during DSS intervention. Ultra high performance liquid chromatography q-exactive orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap-HRMS)-based metabolomic analyses were performed on feces. 16S rRNA sequencing was used to quantify and characterize the gut microbiota of mice. Metabolomic analysis showed that P-hydroxyphenyl acetic acid (p-HPAA), the metabolite with the highest variable importance in projection (VIP) score that was elevated by A. muciniphila, was negatively correlated with acetic acid levels and exhibited a potential inhibitory effect on ER stress. Additionally, A. muciniphila supplementation decreases the abundance of Parasutterella, a genus implicated in bile acid homeostasis. By restoring the levels of deoxycholic (DCA) and ursodeoxycholic acid (UDCA), A. muciniphila administration normalized the bile acid pool size and composition altered by colitis. A. muciniphila supplementation protected colon shortening and histological injury in wild-type (WT) mice, but not in farnesoid X receptor-null (FXR^-/-) mice. Mechanistically, our results demonstrate that A. muciniphila alleviates DSS-induced colitis by targeting inositol requiring enzyme 1α(IRE1α) and unspliced XBP1 (XBP1u) within the ER stress pathway, with the regulation of XBP1u being FXR-dependent. Supplementation with A. muciniphila at appropriate doses may, thus, offer a promising therapeutic strategy for Ulcerative colitis (UC).

Importance: UC is a chronic inflammatory disease in which inflammation begins in the rectum and extends proximally throughout the colon. A.muciniphia is significantly reduced in UC patients and shows promise as a next-generation probiotic. However, the mechanisms behind its protective effects are not fully understood. Our study reveals that A. muciniphila alleviates experimental colitis by reshaping the gut microbiome and correcting imbalances in bile acid metabolism. Crucially, we identify a novel mechanism where A. muciniphila acts through the host bile acid receptor FXR to suppress a specific ER stress pathway (XBP1u) in colon cells, thereby helping to restore the intestinal barrier. These findings provide a scientific basis for using A. muciniphila as a targeted therapeutic strategy for UC.

Methane is an end product of plant biomass digestion by gut microbiota, though the amount produced and/or released varies between hosts. On a per-unit-of-feed basis, macropodid marsupials (e.g., kangaroos) have been reported to emit less methane than ruminant livestock, despite a similar diet, although measurements exist for only a subset of macropodid species. Competition for hydrogen within the gut microbiome, particularly through alternative hydrogen sinks to methanogenesis, influences methane production; therefore, characterizing hydrogen management strategies within a host system can provide insights into methane emission profiles. In this study, we analyzed 33 fecal microbiomes of 14 marsupial species (predominantly captive animals) to provide the first systematic characterization of methanogen types and hydrogen-cycling genetic capacity across marsupial gut microbiomes. We recovered 1,394 metagenome-assembled genomes and identified host-associated bacterial signatures that varied significantly between marsupial species. Comparative analysis with fecal microbiomes from high- and low-methane-emitting mammals revealed that marsupials display heterogeneous hydrogen management strategies: some harbor elevated methanogenesis genes (mcrA, methanogen-specific hydrogenases), while others show enrichment of bacterial hydrogen-uptake hydrogenases and alternative electron acceptor pathways (nitrate/nitrite reduction, sulfite reduction). This predicted functional variation occurs both between and within marsupial families and gut types, suggesting that hydrogen management capacity may differ within taxonomic and anatomical classifications. These results demonstrate that marsupial gut microbiomes cannot be treated as a functionally homogenous group regarding methane emissions and highlight the need for species-specific measurements to accurately assess their methanogenic potential and inform ecological models of greenhouse gas production.IMPORTANCEHerbivorous marsupials such as kangaroos and wallabies have been reported to produce significantly lower methane emissions than ruminant livestock despite eating a similar diet, yet the microbial mechanisms underlying this difference remain poorly understood. Here, we conduct a comparative study of fecal microbiomes of 14 marsupial species to provide the first investigation of hydrogen-cycling genetic capacity across these animals. Through comparative analysis with fecal microbiomes of high- and low-methane-producing animals, we identify enrichment of bacterial genes for alternative hydrogen uptake and disposal pathways in some marsupials, supporting competition for hydrogen playing a role in the level of methane production. These data also indicate variation in hydrogen management between marsupials, including within species, suggesting methane emission capacity may vary at the level of the individual.