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This study aims to characterize the phenotypic behavior and in vivo persistence of a ceftriaxone-tolerant Neisseria gonorrhoeae clinical isolate from a single patient and evaluate the potential role of tolerance in treatment failure. A previously identified ceftriaxone-tolerant vaginal isolate was compared with isogenic and clinical non-tolerant strains. Bacterial growth was assessed in vitro, and tolerance was quantified using the minimum duration required to kill 99% of the population (MDK99), and persistence was evaluated in an in vivo Galleria mellonella infection model. Whole-genome sequencing (WGS) and transcriptomic (RNA-sequencing [RNA-seq]) profiling were performed to identify tolerance-associated genetic and transcriptional signatures. The tolerant strain exhibited prolonged MDK99 values across ceftriaxone concentrations, persisting for up to 24 hours under drug exposure. It also showed delayed early-phase growth, suggesting a fitness cost. In vivo, the tolerant strain remained viable up to 8 hours after treatment, whereas non-tolerant strains were cleared. WGS revealed identical gene content across all isolates, but non-synonymous mutations in pilE_3, a type IV pilin gene, were exclusively present in tolerant strains. RNA-seq analysis showed upregulation of pilin-associated genes and downregulation of zinc-independent ribosomal paralogs (rpmE2 and ykgO), suggesting a combined mechanism of surface remodeling and translational suppression associated with the tolerant phenotype. Ceftriaxone tolerance enables prolonged survival of N. gonorrhoeae despite apparent susceptibility by standard MIC-based testing. This phenotype may contribute to treatment failure, recurrent infection, and ongoing transmission, indicating the need for revised diagnostic and therapeutic strategies.IMPORTANCECeftriaxone remains the last reliable option for gonorrhea therapy, yet recurrent infections can occur despite isolates being classified as susceptible by MIC testing. One possible explanation is antibiotic tolerance, a phenotype that allows survival during drug exposure without changes in MIC. Although tolerance has been described in other pathogens, its role in gonococcal infection has remained poorly defined. In this study, we provide the first detailed characterization of a ceftriaxone-tolerant Neisseria gonorrhoeae clinical isolate associated with repeated treatment failure. By combining in vitro killing assays, an in vivo Galleria mellonella infection model, whole-genome sequencing, and transcriptomic profiling, we demonstrate that tolerance enables prolonged survival under ceftriaxone and is linked to pilin gene variation and ribosomal remodeling. These findings illustrate how a clinically observed phenomenon can be mechanistically dissected and emphasize tolerance as a hidden factor contributing to gonococcal persistence and potential treatment failure.

Bacteria produce a diverse range of specialized metabolites that influence the health and behavior of neighboring cells and, therefore, have potential applications in treating diseases. Deciphering the intended ecological functions of specialized metabolites is challenging due to the small scales at which these interactions occur and the complexity of unraveling simultaneous responses to multiple signals. In this study, we investigated the chemical interactions between two marine bacterial colonies, Vibrio parahaemolyticus PSU5429 and Bacillus pumilus YP001. When the two bacteria were grown in proximity on agar, V. parahaemolyticus exhibited swarming motility toward B. pumilus, but close approach to the B. pumilus colony was impeded by a zone of inhibition. Matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS) suggested that lipopeptides produced by Bacillus induced swarming motility, a finding corroborated by genomic and chemical analyses of YP001. Based on activity and metabolomics guidance, the antibiotic amicoumacin B was found to be responsible for the observed antibiosis, while swarming motility by V. parahaemolyticus was induced by lipopeptides and two lipoamides. In this scenario, lipopeptide production by the Bacillus colony induces the Vibrio colony to swarm toward a lysis zone, resulting in a possible "catch and kill" effect. These results demonstrate the complexity of behaviors and outcomes exhibited by microbes under the simultaneous influence of different allelochemicals, suggesting possible interplays between antibiotics and compounds that induce motility.

Importance: Microbes communicate and compete using small molecules, yet linking specific metabolites to visible behaviors is difficult. We combine imaging mass spectrometry, genomics, analytical chemistry, and bioassays to decode an interaction between a marine Bacillus and the pathogen Vibrio parahaemolyticus. Surfactin-like lipopeptides act at a distance to stimulate Vibrio swarming and draw cells toward the colony. Amicoumacin B accumulates at the interface and halts growth, yielding a simple "catch and kill" outcome. This study shows that the spatial localization of natural products shapes microbial behavior on surfaces and provides a general, scalable workflow that maps chemistry to phenotype. Beyond this case, the approach can be applied broadly to understand and, ultimately, tune microbial interactions relevant to marine ecosystems, aquaculture health, and microbiome engineering.

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.

Staphylococcus aureus clonal complex 59 (CC59) has emerged as a significant public health threat in Asia, yet the mechanisms driving its host adaptation and global evolutionary success remain poorly understood. Here, we performed a comprehensive genomic analysis of 3,994 global CC59 isolates, which included 549 isolates associated with bloodstream infections from China. Our analysis revealed three phylogenetically distinct lineages exhibiting region-specific distribution patterns, tracing their origins to the USA, Australia, and China. Notably, high-risk CC59 clones circulating in Taiwan likely diverged from mainland Chinese strains during the 1940s-1960s, coinciding with historical population migration following the Chinese civil war around 1949. Among China-associated CC59 strains, respiratory tract colonization was related to high cross-source linkage across multiple ecological niches, suggesting its role as a dissemination hub, particularly for bloodstream infection (BSI). Additionally, we observed significant enrichment of Clf-Sdr family proteins in human isolates, especially in BSI cases. Functional characterization using ΔclfB and ΔsdrD knockout strains demonstrated impaired biofilm formation, recapitulating findings in USA300. These findings establish an evolutionary framework for CC59 surveillance and highlight promising potential targets for anti-virulence therapeutics.

Importance: The prevalence and propagation of Staphylococcus aureus clonal complex 59 (CC59) in Asia are serious public health concerns. To understand its adaptation to hosts and worldwide evolutionary success, we analyzed the genomic population structure of all CC59 isolates and traced their evolutionary history. Our research indicates that CC59 lineages developed through unique evolutionary routes that vary across time and space, highlighting their adaptation to diverse ecological environments. This study presents a comprehensive genomic epidemiology framework that integrates extensive metadata analysis with evolutionary assessment. It serves as a model for future S. aureus monitoring and provides insights into potential targets for interventions focused on reducing virulence.

Hundreds of thousands of individual microbe-molecule interactions regulate the flux, transformation, and fate of carbon stored in the climatically important reservoir of marine dissolved organic matter (DOM). While marine microbial communities have been characterized at high resolution for over a decade, observations of the molecules cycled by the microbial-chemical network at similar resolution are limited. In addition, bulk characterizations of DOM can mask the complex network of interactions comprised of rich chemical diversities. Here, we present a three-year, depth-resolved, molecular time-series of DOM and prokaryoplankton at the Bermuda Atlantic Time-series Study (BATS) site. Both time-series exhibited seasonality that was compositionally distinct and primarily endemic to one sampling depth. We also putatively identified four exometabolites (gonyol, glucose-6-sulfate, succinate, and trehalose) that exhibit seasonal accumulation. We hypothesize these patterns result from environmental conditions that alter community composition on a seasonal timescale and thus shift the relative proportions of microbial functions that produce and consume the substrates. Critically, we observed the interannual composition of seasonal DOM molecules to be more stable than the taxonomy of the microbial community. This points to an important role of functional redundancy in regulating DOM composition. We tested this observation by querying metagenomes for pathways that utilize metabolic by-products putatively identified in the DOM time-series. We find that core microbial metabolisms, either those required by all or by a subset of marine microbes, are important predictors of DOM composition. The molecular-level characterization of DOM herein highlights the potential imprint of microbial activity on seasonal DOM composition.IMPORTANCEMarine dissolved organic matter (DOM) is a major carbon reservoir that acts as a critical control on the Earth's climate. DOM dynamics are largely regulated by a complex web of chemical-microbial interactions, but the mechanisms underpinning these processes are not well understood. In a three-year time-series, we found that the identity of the microbes is more likely to change between years than the composition of the DOM molecules. The taxonomic variability suggests that metabolisms shared across taxa, encoded by genes that conduct core microbial functions, are responsible for the more stable composition of DOM. While more than three decades of marine prokaryoplankton time-series are available, a similar reference for DOM molecules was missing. This time-series provides an improved understanding of the different responses of DOM molecules and microbes to seasonal environmental changes.

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.

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.

Tibetan antelopes, native to high-altitude plateau regions, play an important role in the local ecosystem. Their gut harbors antimicrobial-resistant microbes, including potential pathogens. To explore this, we analyzed 33,925 metagenome-assembled genomes (MAGs), including 7,318 from 68 Tibetan antelopes sequenced in our laboratory. We first profiled the composition of antibiotic resistance genes (ARGs) and then examined their associations with virulence factor genes (VFGs). In total, 2,968 ARGs were identified, conferring resistance to 23 antibiotic classes, with elfamycin resistance being most prevalent. Two ARGs were located on phage-derived sequences, though their phage taxonomy could not be resolved. ARGs were significantly correlated with VFGs, particularly genes linked to adherence and effector delivery systems. Given potential dissemination risks, we further assessed associations between ARGs and mobile genetic elements (MGEs), finding that insertion elements accounted for the largest number of ARG-MGE links. Comparative analysis with other plateau animals and humans revealed seven ARGs uniquely present in Tibetan antelopes. In summary, this study provides the first comprehensive overview of ARG composition in Tibetan antelope gut microbiomes, establishing a baseline for future hypothesis-driven studies and antimicrobial resistance surveillance in wildlife.

Importance: Investigating the drug resistance of Tibetan antelope (Pantholops hodgsonii) gut microbiota serves as a critical biological indicator for assessing the impact of human activities (particularly antibiotic contamination) on the fragile ecosystem of the Qinghai-Tibet Plateau. This study untangles the invasion of antibiotic resistance genes (ARGs) into remote conservation areas, suggesting that Tibetan antelopes may act as potential vectors for ARG dissemination across plateau environments. Such findings not only highlight threats to wildlife health but also provide an ecological warning regarding the pervasive environmental risks posed by the global antimicrobial resistance crisis in natural ecosystems.