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Recent advances in high-throughput sequencing and novel culture techniques have revolutionized our understanding of the human microbiota. However, most studies primarily focused on bacterial communities, often overlooking the fungal component. Building upon our previous metagenomic analysis of the Inuit oropharyngeal microbiome 1, this study used culturomics to provide a more comprehensive view of both bacterial and fungal communities. We analyzed oropharyngeal swabs from the Qanuilirpitaa? 2017 Inuit Health Survey 2, demonstrating the complementarity of metagenomic and culturomic approaches. Our findings highlight the importance of culturomics in revealing low-abundance microorganisms, particularly fungi, which are often underrepresented in metagenomics data. Moreover, we designed an approach to isolate previously uncultivated species. We described two Pauljensenia sp., and provided insights into the phylogenetic relationship between Schaalia and Pauljensenia genera. This study underscores the necessity of a holistic approach to microbiome research, combining multiple techniques to fully elucidate microbial diversity in unique populations like the Inuit.

Toxoplasma gondii is a widespread human pathogen that has multiple, clinically relevant stages in its complex life cycle, including fast-replicating tachyzoites and latent bradyzoites. Bradyzoite differentiation is triggered by stress responses that lead to changes in transcription, translation, and metabolism. Two aspects of this process are addressed in this report: first, whether proteins that play roles in bradyzoite differentiation are specific to T. gondii and other bradyzoite-forming parasites of the Sarcocystidae family, and second, whether new bradyzoite differentiation proteins can be identified in T. gondii. To answer these questions, a phylogenetic approach was used, comparing proteomes of select members of the Sarcocystidae family that form morphologically different bradyzoite cysts and members of the Eimeriidae family that do not form cysts. This approach resulted in 8 distinct clusters of T. gondii proteins that reflected different conservation patterns; for example, one cluster showed conservation among all organisms, while another showed conservation in bradyzoite cyst-forming organisms. Known T. gondii proteins involved in bradyzoite differentiation were found in all clusters, indicating that this process uses both highly conserved pathways as well as bradyzoite-specific pathways. Importantly, the cluster containing proteins that are conserved in bradyzoite-forming organisms contained several known regulators of bradyzoites, and will be a source for identifying novel T. gondii proteins that are involved in bradyzoite differentiation.

Bacteria use diverse mechanisms to interact with each other and with eukaryotic hosts, thereby shaping microbiome composition and influencing host health. One of these mechanisms is the production of outer membrane vesicles (OMVs), nanoscale structures that bud off from bacterial cells into the extracellular space. OMVs can deliver bioactive cargoes, including enzymes, RNA and DNA, enabling functions such as cell-to-cell communication, nutrient acquisition and immunomodulation. However, the role of OMVs in beneficial host-associated microbiomes remains unclear. Here, we investigated OMV production in the gut bacteria of the western honey bee (Apis mellifera), which forms a highly conserved and stable microbial community. Using electron microscopy, fluorescence labelling, and nanoparticle tracking analysis, we detected OMV production in every gram-negative species of the normal bee microbiota that we investigated. Vesicles were observed in gut contents of wild and laboratory-inoculated bees, but absent in bees lacking a microbiota. OMVs contained nucleic acids, with more RNA than DNA. Bacterial strains varied in OMV properties, including abundance, size, and zeta potential. These findings indicate that OMVs are likely significant mediators of interbacterial and host-microbe interactions in the bee gut.

Emerging infectious diseases and antimicrobial resistance (AMR) have surfaced as two major public health threats over the past two decades. Consequently, integrative surveillance systems capable of detecting both emerging pathogens and resistance-carrying bacteria are crucial. With advances in next-generation sequencing, simultaneous detection of pathogens and AMR is increasingly feasible. In this study, we used short-read metatranscriptomics complemented by total 16S rRNA metagenomic long-read sequencing to analyze paired oral and plasma samples from a cohort of febrile individuals at two locations in Senegal. Oral microbiomes differed in community composition between locations, and reduced diversity and richness were significantly associated with high fever. We identified at least one known pathogen in 15.33 % (23/150) of samples, with Borrelia crocidurae as the most frequently detected pathogen. We detected both pathogenic and non-pathogenic viruses in oral (10/72) and plasma (09/78) samples. Finally, we observed a high frequency of genes associated with resistance and virulence: 10% of samples expressed at least one AMR gene (ARG), and 24% expressed virulence factor genes. Resistance to widely used beta-lactam antibiotics was the most prevalent. Our findings provide critical data on oral and plasma microbiomes in the context of acute febrile illness in Senegal while expanding understanding of circulating ARGs.

Background: Mucormycosis is a rapidly progressive and often fatal invasive fungal infection caused by moulds in the order, Mucorales. Early diagnosis is essential for effective clinical management; however, conventional diagnostic approaches such as culture and histopathology are slow, insensitive, and require specialist mycological expertise. Although molecular methods are available for disease detection, they are not widely accessible. At present, no enzyme immunoassay (EIA) exists for the detection of mucormycosis. Methods: A murine IgG1 monoclonal antibody (mAb), FH12, was generated against extracellular polysaccharides (EPSs) produced by Mucorales pathogens during active growth. The antibody was characterised for specificity, epitope stability, and antigen localisation using ELISA, immunoblotting, and immunofluorescence techniques. The mAb was incorporated into a Sandwich-ELISA and evaluated using culture filtrates, purified EPSs spiked into human serum, and tissue homogenates from a patient with cutaneous mucormycosis caused by Lichtheimia ramosa. Results: mAb FH12 demonstrated pan-Mucorales specificity and no cross-reactivity with other clinically relevant yeasts and moulds. The epitope recognised by FH12 is periodate-insensitive and moderately heat-stable. The Sandwich-ELISA detected EPS antigens in human serum with limits of detection ranging from pg/mL to low ng/mL levels, and successfully identified the EPS biomarker in patient tissue homogenates. Conclusion: The FH12-based Sandwich-ELISA shows high sensitivity and specificity, and has the potential to be used as a laboratory-based adjunct diagnostic test for the detection of mucormycosis in humans.

Background and AimsThe rising incidence of Crohns disease (CD) in Westernized countries has been linked to changes in diet and increased consumption of food additives, yet the mechanisms by which these factors fuel intestinal inflammation remain unclear. Adherent-invasive Escherichia coli (AIEC), a pathobiont involved in CD pathogenesis, lacks a clear genetic hallmark but exhibits intestinal colonization and virulence traits, raising questions about the evolutionary forces promoting its emergence among select individuals. Here, we investigated how chronic exposure to two common dietary emulsifiers, carboxymethylcellulose (CMC) and polysorbate 80 (P80), along with host inflammation, drives AIEC genomic evolution and pathogenic potential. MethodsWild-type and Il10-deficient mice were monocolonized with AIEC and chronically exposed to CMC, P80, or water. Bacterial isolates were collected and analyzed for genomic diversification, mutations, and phenotype both in vitro and in vivo. ResultsEmulsifiers accelerated AIEC genomic diversification and selected for mutations linked to increased motility, invasion, and pro-inflammatory activity. Moreover, dietary emulsifier-evolved strains displayed a marked fitness advantage in vivo, outcompeting their counterparts in murine hosts, with the greatest advantage observed when evolution occurred under inflammatory conditions. Notably, evolutionary pathways and phenotypic outcomes were shaped by both emulsifier and the hosts inflammatory status, highlighting synergy between diet and host genetics in fostering pro-inflammatory pathobionts. ConclusionThese findings provide an evolutionary framework connecting modern dietary habits to the emergence of pathogenic AIEC strains, and underscore the importance of dietary interventions in individuals at risk for inflammatory bowel disease.

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

This study investigates the seasonal dynamics of the microbiome within the marine sponge Halichondria panicea from Baltic coastal waters, focusing on its symbiotic relationship with Candidatus Halichondribacter symbioticus. Over 16 months, we observed distinct summer and winter microbial communities, transitioning rapidly between these states during spring and fall. Marine sponges host complex microbiomes composed of diverse microbial taxa that play critical roles in host metabolism and nutrient cycling within marine ecosystems. While our understanding of sponge microbiomes has traditionally been based on static characterizations, the temporal dynamics of these associations across seasonal cycles remain poorly understood. In this study, we investigated temporal variation in bacterial symbionts of Halichondria panicea over 16 months in Baltic coastal waters using high-throughput amplicon sequencing of bacterial 16S rRNA gene sequences. The microbiota of H. panicea exhibited host-specific structure and a high degree of stability across seasons, despite fluctuations in environmental factors such as temperature, salinity, photoperiod intensity, and inorganic nutrient availability. In contrast, bacterial communities in surrounding seawater displayed large seasonal shifts which potentially mix with the sponge bacterial community, suggesting that different degrees of ecological pressures act on free-living and symbiotic marine bacteria. These findings establish an empirical baseline for identifying abnormal shifts in symbiont communities, which could be indicative of environmental stress or biological disturbance events.

Shotgun metagenomics has become a cornerstone of microbiome research, yet the complexity of existing workflows remains a major barrier for life scientists without dedicated bioinformatics support. Manual database setup, detailed sample sheet preparation, and management of software dependencies can make routine analysis difficult and time-consuming. Cross-study comparisons are further hampered by inconsistent processing pipelines, database versions, and profiling strategies, limiting reproducibility and the potential for large-scale meta-analyses. We present OpusTaxa, an open-source Snakemake workflow that provides end-to-end processing of short paired-end shotgun metagenomic data with minimal configuration. Users provide either FASTQ files or Sequence Read Archive accessions; OpusTaxa automatically downloads required databases, performs quality control, removes host reads, and executes taxonomic profiling, metagenome assembly, and functional analysis. All analysis modules can be independently toggled, and per-sample outputs are automatically merged into harmonised, cross-sample tables ready for downstream exploration. Across two public datasets, we demonstrate how OpusTaxa can be used to compare consistency across complementary taxonomic profilers and to estimate microbial load in addition to standard metagenomic workflows. AvailabilityOpusTaxa is freely available at https://github.com/yenkaiC/OpusTaxa. Documentation, test data, and example configurations are included in the repository.

Bacillus paranthracis was formally defined as a species in 2017, after decades of carrying the name "emetic B. cereus" based on cereulide production and clustering within B. cereus sensu lato phylogenetic group III. Commonly associated with foodborne intoxication, reports rarely link B. paranthracis to non-foodborne clinical illness. As such, the new taxonomy and close resemblance of the name to the biothreat pathogen Bacillus anthracis cause confusion in diagnostic and public health settings. To address this issue, B. paranthracis clinical strains (n=20) from the CDC collection were tested with microbiological methods used for identification of B. anthracis and antimicrobial susceptibility testing. Some B. paranthracis phenotypes were similar to B. anthracis, however others were inconsistent across strains. Like B. anthracis: 3 strains tested capsule positive, 5 were non-hemolytic on blood agar, and 9 non-motile. All B. paranthracis strains were resistant to gamma phage lysis, which differentiated them from B. anthracis. Treatment regimens for B. paranthracis infections are not well established, as antimicrobial therapy is not indicated for emetic intoxication caused by B. paranthracis. Notably, six B. paranthracis strains had elevated minimal inhibitory concentrations to anthrax-recommended antibiotics: one for ciprofloxacin, three for doxycycline and tetracycline, and two for clindamycin. Rapid MinION sequencing was assessed for antimicrobial resistance detection prediction but had limited value when using PiMA v.1. These microbiological observations and susceptibility profiles of B. paranthracis expand our understanding of this pathogen, strengthening our ability to differentiate this bacterium from B. anthracis to improve diagnosis and patient outcomes. IMPORTANCEThis study describes in vitro characterization of 20 archived clinical strains of B. paranthracis, an opportunistic pathogen identified more frequently in recent reports. Our findings highlight phenotypic differences and similarities between B. paranthracis and B. anthracis using standard microbiological methods and drug susceptibility profiling. We also assess a rapid B. anthracis specific MinION long read genome sequencing workflow with B. paranthracis. This report highlights the overlapping morphological features shared by B. paranthracis and B. anthracis to improve future laboratory diagnosis and strengthen anthrax preparedness. This article will effectively reach an audience of public health professionals and microbiologists strengthening anthrax preparedness.

Monascus spp. are economically important filamentous fungi that have been utilized in the production of beneficial metabolites such as Monascus pigments and monacolin K, as well as in the brewing of some Asian fermented foods. The delimitation of Monascus species has traditionally relied on phenotypic traits; however, this morphological classification approach is susceptible to subjective judgments and variations in cultural conditions and also may not necessarily be related to the actual genetic relationship. Consequently, synonymy and misidentification frequently occur in Monascus taxonomy, highlighting the urgent need for a convenient and reliable classification system for this genus. In this study, a phylogenetic analysis of 82 representative Monascus strains, encompassing all previously recognized species of the genus, was conducted based on the concordance of five gene genealogies (BenA, CaM, ITS, LSU, and RPB2) to clarify species delimitation and resolve phylogenetic relationships within Monascus. The results revealed that the genus Monascus is resolved into 11 species, which are clustered into two sections: Floridani (including M. argentinensis, M. flavipigmentosus, M. floridanus, M. lunisporas, M. mellicola, M. pallens, and M. recifensis) and Rubri (including M. pilosus, M. purpureus, M. ruber, and M. sanguineus). M. pilosus and M. sanguineus were reaffirmed as distinct species due to their well-supported and divergent phylogenetic lineages. Additionally, M. albidulus, M. anka, M. barkeri, and M. fumeus are synonymized with M. pilosus, while M. aurantiacus and M. rutilus are synonyms of M. purpureus. Finally, a comprehensive list of accepted Monascus species along with their corresponding barcode sequence data is provided.

The majority of emerging infectious diseases are zoonotic, having their origin in wildlife before spilling over into the human population. While small mammals are recognized as critical reservoirs for these viruses, their viral diversity remains largely uncharacterized across many African countries. We conducted molecular surveillance of synanthropic rodents and shrews in the Kibera informal settlement in Nairobi and the rural Taita Hills region of Kenya to detect and characterize potential zoonotic viruses. Tissue samples from 228 rodents and shrews were screened for six viral families using PCR assays. Rat hepatitis E virus (HEV) (Rocahepevirus ratti), a rodent-associated virus with potential for human spillover, was identified in Mus musculus and Rattus norvegicus from Kibera. NGS was conducted for the HEV positive samples, and we obtained two near-complete HEV genomes from Rattus norvegicus, which clustered within rodent-associated HEV genotypes in the phylogenetic analysis. The two sequences from the Rattus norvegicus cluster together, indicating a close genetic relationship. Paramyxoviruses belonging to the genera Jeilongvirus and Parahenipavirus were detected both from Taita and Kibera in nine different samples from Rattus norvegicus, Mus minutoides, Crocidura sp and Acomys ignitus. One paramyxovirus positive sample (Acomys ignitus) from Taita was selected for further sequencing with NGS, and a complete genome of a new jeilongvirus was assembled. Phylogenetic analysis of the detected viruses confirmed the close relation to previously known rodent-borne jeilongviruses but also revealed potentially novel jeilong- and parahenipavirus species. Our findings highlight the circulation of potentially zoonotic viruses in both urban and rural small mammals in Kenya. It emphasizes the necessity of continued genomic surveillance of zoonotic viruses to mitigate risks of their spillover into human populations. HighlightsO_LISurveillance reveals diverse rodent-borne viruses circulating in Kenya. C_LIO_LIRat-HEV was detected in Rattus norvegicus and Mus musculus from an urban low-income area. C_LIO_LIParamyxoviruses were detected across multiple rodent and shrew species, including novel Acomys ignitus jeilongvirus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/719784v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@194e81eorg.highwire.dtl.DTLVardef@11342cdorg.highwire.dtl.DTLVardef@186ad97org.highwire.dtl.DTLVardef@eeb516_HPS_FORMAT_FIGEXP M_FIG C_FIG

Microbial communities are critical to the functioning of ecosystems and shape the ecology and evolution of host organisms. However, we have a limited understanding of how host-associated and free-living microbes differ in their structure and biogeography. Here, we test whether host-associated (fish gut) and free-living (lake bacterioplankton) microbes exhibit different metacommunity structure, spatial turnover, and consistency with neutral expectations using two independent lake systems. We characterized microbial communities in lake water (Vancouver Island and Sierra Nevada) and guts in two fish species (stickleback and brook trout) using 16S amplicon sequencing. We compared alpha and beta diversity within lakes, quantified spatial turnover (distance-decay), and tested for departure from neutral abundance-occurrence expectations between bacterioplankton and fish gut microbiomes. Fish microbiomes had lower alpha diversity compared to bacterioplankton, but higher beta diversity within lakes. Bacterioplankton were more similar across lakes yet showed stronger patterns of spatial turnover with distance than fish gut microbiomes. A neutral model explained a substantial proportion of abundance-occurrence relationships in bacterioplankton communities but performed poorly for fish-associated microbes. Our study indicates that host-associated and free-living microbes have disparate patterns of metacommunity structure and spatial turnover consistent with differences in the strength of neutral ecological processes. Fish microbiomes were less diverse at the local scale but more variable across space and time than bacterioplankton communities, suggestive of potentially strong local selection and/or reduced microbial exchange among hosts compared to environmental communities. Importantly, we observed highly consistent patterns across both lake systems despite differences in host species, sampling design, and region, demonstrating that differences in the distribution of host and environmental microbes are potentially widespread. This study demonstrates how host association fundamentally alters the diversity and spatial distribution of microbes, emphasizing the need to incorporate hosts into broader frameworks of microbial biogeography.

Diet is an important ecological modulator of the oral microbiome, yet population-level evidence on a broader spectrum of food components remains limited. This cross-sectional study investigated associations among dietary intake, oral rinse microbiome, and oral disease conditions in a nationally representative sample of United States adults from the National Health and Nutrition Examination Survey. A total of 3,254 participants with oral rinse microbiome sequencing data were included, with oral conditions classified as oral health, caries-only, periodontitis-only, or co-existing disease. Dietary intake was assessed using 24-hour dietary recalls and summarized as dietary indices and energy-adjusted food components. Associations between diet and the oral microbiome were evaluated using community-level analyses, regression models, mediation analyses, and unsupervised clustering, while accounting for oral conditions. This study found that dietary intake, as a combined variable set, explained 3.6% of the variance in oral rinse microbial community structure; this was comparable to oral disease status or smoking and larger than sociodemographic factors. Healthier dietary profiles, including higher health-associated dietary index scores and greater vegetable and fruit intake, were associated with taxa commonly linked to oral health (e.g., Neisseria, Cardiobacterium and Lautropia). In contrast, added sugars, alcoholic drinks, cured meat, potatoes, dairy products, and higher dietary inflammatory index scores showed opposite association patterns. Mediation analyses suggested that coordinated microbial groups may partly link dietary exposures with oral disease outcomes, particularly for vegetables and added sugars. Additionally, three population-level dietary patterns were identified, among which the plant-rich pattern was associated with more favorable oral health and microbial profiles enriched in nitrate-reducing commensals, including Neisseria and Haemophilus. Overall, dietary intake was associated with oral microbiota composition and oral health conditions, supporting ecological influences of dietary components beyond sugar on oral bacteria and dental diseases. Longitudinal studies are needed to clarify the direction and causality of these relationships.

O_LIWD40 is a highly conserved protein domain in eukaryotes, playing a critical role in various cellular process. C_LIO_LIWe conducted genome-wide functional analysis of WD40 genes in Fusarium graminearum--a phytopathogenic fungus that causes severe yield loss and mycotoxin contamination in major cereal crops. C_LIO_LIComprehensive phenome analysis of 119 WD40 gene deletion mutants across 22 distinct phenotypic traits revealed phenotypic divergence within the phenome, establishing a strong correlation between virulence and sexual reproduction. Notably, 21 "core WD40 genes" were identified, offering valuable insights into divergent biological processes. C_LIO_LIPilot interactome studies of Fgwd101 and Fgwd133 provided further insights into their potential pathobiological functions. Our investigation contributes to broadening our knowledge of the biological mechanisms underlying fungal pathogenesis and may assist in the identification of targets for antifungal agents. C_LI

We previously reported that the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) recognizes hepatitis B virus (HBV) X protein (HBx) to activate the NF-E2-related factor 2 (Nrf2) signaling pathway, thereby inhibiting HBV replication, and that HBx promotes K6-linked polyubiquitylation of Nrf2. However, the molecular mechanism remains unclear. Here, we investigated the role of HECT, UBA, and WWE domain-containing E3 ubiquitin ligase 1 (HUWE1) in HBx-mediated K6-linked polyubiquitylation of Nrf2 and its impact on HBV replication. Cell-based ubiquitylation assays demonstrated that HUWE1 knockdown reduced HBx-mediated K6-linked polyubiquitylation of Nrf2, while overexpression of wild-type HUWE1, but not the catalytically inactive HUWE1(C4341A) mutant, enhanced it, indicating that HUWE1 E3 ligase activity is required. Coimmunoprecipitation and proximity ligation assays demonstrated that HUWE1 interacts with HBx in the cytoplasm and binds Nrf2 only in the presence of HBx, suggesting that HBx bridges HUWE1 and Nrf2 into a ternary complex. Cycloheximide chase assays demonstrated that HUWE1 knockdown destabilized Nrf2 in HBx-expressing cells, supporting a role for HUWE1 in Nrf2 stabilization via K6-linked polyubiquitylation. Furthermore, HUWE1 knockdown or treatment with the HUWE1 inhibitor BI8626 significantly increased HBV RNA and pgRNA levels in HBV-infected cells. Collectively, these results demonstrate that HUWE1 promotes K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner to inhibit HBV replication. IMPORTANCEHepatitis B virus (HBV) chronically infects approximately 254 million people worldwide, yet host mechanisms that restrict viral replication remain incompletely understood. The Kelch-like ECH-associated protein 1 (Keap1)/ NF-E2-related factor 2 (Nrf2) signaling pathway is a central defense against oxidative stress. Under basal conditions, Nrf2 is degraded via Keap1/Cullin3-mediated K48-linked polyubiquitylation. We previously demonstrated HBV infection promotes Nrf2 stability through non-canonical K6-linked polyubiquitylation. Here, we identify the E3 ubiquitin ligase HUWE1 as the enzyme responsible for K6-linked polyubiquitylation of Nrf2. HBV X protein (HBx) recruits HUWE1 to Nrf2, forming a HUWE1/HBx/Nrf2 complex that switches Nrf2 ubiquitylation from K48 to K6, stabilizing Nrf2 and suppressing HBV replication. These findings reveal a novel antiviral mechanism exploiting a non-canonical ubiquitin code and highlight HUWE1 as a potential therapeutic target against chronic HBV infection.

This pilot study assessed the composition and diversity of the urinary microbiome from clinically confirmed UTI samples using 16S rRNA sequencing, whilst also exploring inter-individual variability of microbial community structure. We examined ten urine samples from patients with culture-positive UTIs. Demographic and clinical metadata, including age, sex, body mass index (BMI), diabetes status and recent antibiotic exposure was recorded per sample. Metagenomic DNA was extracted from microbial samples and sequenced to generate genus-level taxonomic profiling through 16S rRNA gene sequencing. Relative abundance tables were generated for each of the samples to identify dominant bacterial genera within each sample and summarize cohort level microbial patterns. To evaluate within-sample richness and evenness, alpha diversity indices (Shannon, Simpson, observed features and Chao1) were computed; beta diversity was measured using Bray-Curtis dissimilarity with principal coordinates analysis (PCoA) for graphical representation. The studys findings revealed the sex and moderate clinical diversity of the study sample; all samples were confirmed as having been taken from a UTI patient and exhibited a wide level of heterogeneity regarding the microbial composition of each urine sample. Overall, Pseudomonas was the dominant genus present, however, specific samples had approximately 50% of their microbiomes composed of Klebsiella, Proteus, and Escherichia species as well as approximately 25% of their total microbes were made up of Burkholderia spp., which are closely related to the genus of interest used during the course of this study. The observed alpha diversity of each sample displayed considerable variation for the included samples with a continuum of samples ranging from a single dominant microbe to a highly diverse mixed population producing a highly diverse polymicrobial population/bacterial composition, with some ratios of individual taxa to collective taxa of many samples repeatedly illustrating the exact nature of the specimen. Furthermore, a significant degree of Beta diversity was found between the patients, providing compelling evidence of identifiable differences among urinary microbiomes between patients with UTI. This pilot project provides a clear indication of the diversity and overall heterogeneity of urinary microbiota found in the UTI patients studied. In addition, the results of this study support the notion that the ecological complexities present within a urinary microbiome cannot necessarily be established through conventional culture methods, and that combined with molecular techniques such as 16S rRNA sequencing of bacterial DNA could be used to quantify and characterize the ecologic condition of urinary microbiota separate from the traditional high prevalence of identifiable uropathogens.

ObjectivesAntimicrobial resistance (AMR) in Gram-negative pathogens is driven by complex and coordinated molecular mechanisms that remain incompletely characterized. This study integrated phenotypic, genomic, and quantitative proteomic analyses to characterize multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria circulating in an intensive care unit (ICU) in Northeastern Brazil. MethodsA total of 259 Gram-negative isolates collected between 2019 and 2021 underwent species identification, antimicrobial susceptibility testing, and targeted qPCR for resistance genes. Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa representing susceptible, MDR, and XDR phenotypes were selected for whole-genome sequencing and label-free quantitative proteomics. Differential protein abundance was assessed using Limma with |log2FC| > 1 and p < 0.05. ResultsK. pneumoniae (47%), A. baumannii (24%), and P. aeruginosa (21%) predominated. Carbapenem resistance reached 44%, 93%, and 61%, respectively, and MDR/XDR phenotypes occurred in >30% of isolates. Genomic analyses revealed dense resistomes with coexisting {beta}-lactamases (blaKPC, blaNDM, blaCTX-M, OXA) and widespread efflux systems. Proteomic profiling demonstrated phenotype-associated differences in outer membrane proteins, transport systems, regulatory proteins, and metabolic pathways. XDR isolates showed additional enrichment of envelope remodeling proteins, stress response mechanisms, and proteostasis-associated factors. ConclusionsMDR and XDR Gram-negative ICU pathogens exhibit coordinated resistance architecture characterized by accumulation of resistance genes and adaptive proteomic remodeling. Integrated multi-omics approaches provide mechanistic insight into antimicrobial resistance and support improved surveillance and therapeutic strategies. What is known?O_LIAntimicrobial resistance is a priority and a serious problem in global health, resulting in high rates of morbidity and mortality. C_LIO_LIKlebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are on the World Health Organizations (WHO) priority list as major causes of morbidity and mortality worldwide. C_LIO_LIClassical characterization of susceptibility and resistance phenotypes does not capture the complexity of antimicrobial resistance and hampers effective control measures and actions to minimize the evolutionary dynamics of resistance in these bacteria. C_LI What is new?O_LIThe study characterizes the phenotypic pattern of antimicrobial susceptibility, the presence and sequencing of the resistome and virulome, and analyzes the label-free quantitative proteome of susceptible, MDR, and XDR phenotypes in strains of K. pneumoniae, A. baumannii, and P. aeruginosa circulating in hospital ICUs in Brazil. C_LIO_LIMDR and XDR gram-negative phenotypes are associated with a dense resistome, with widespread dissemination of beta-lactamase genes (bla_KPC, bla_NDM, bla_CTX-M, and OXA) and RND-type (MEXs) and acrAB-tolC efflux pumps, without changes in virulence genes. C_LIO_LIProteomic analysis demonstrated increased production of beta-lactamases, components of efflux pump systems, outer membrane protein synthesis, protection for oxidative stress mechanisms, proteins for iron acquisition, and systemic regulators. XDR strains additionally showed enhanced remodeling of the cell envelope, activation of proteostasis, and metabolic adaptation. C_LI

The human gut microbiome plays a critical role in host health, yet its functional organization in disease remains poorly understood. Most studies focus on taxonomic composition or pathway abundance, which fail to capture higher-order interactions governing system-level behavior. Here, we investigated microbiome functional organization in inflammatory bowel disease (IBD), including Crohns disease (CD), ulcerative colitis (UC), and healthy controls (HC), using a network-based framework across 60 metagenomic samples. Functional pathway profiles were used to construct correlation-based interaction networks, followed by analysis of network topology, functional redundancy, keystone pathway architecture, and system robustness. Disease-associated networks (CD and UC) exhibited reduced global connectivity, increased modular fragmentation, and centralization of keystone pathways, indicating a shift from distributed organization to more fragmented and fragile network structures compared to healthy controls. Notably, machine learning models demonstrated that network-derived features achieved higher classification performance (accuracy up to 0.824) compared to redundancy-based measures. These findings reveal that microbiome dysfunction in IBD is driven by large-scale reorganization of functional interaction networks rather than loss of functional capacity. This study highlights the importance of network-level analysis in understanding microbiome-associated disease and provides a systems-level framework for future research.

Microbial degradation of suspended and sinking organic carbon regulates long-term oceanic carbon storage by controlling the efficiency of the biological pump. Yet microbial controls on carbon export and remineralization remain poorly constrained, limiting predictions of how ocean carbon cycling will respond to climate change. Here, we combined in situ sampling with ship-based incubations to quantify prokaryote-driven removal rates of suspended and sinking total organic carbon (TOC). Samples were collected below the mixed layer during three stages of a spring Phaeocystis pouchetii bloom in the Labrador Sea. Phaeocystis blooms can dominate regional phytoplankton biomass and are expected to increase under future climate. Removal rates were used as a proxy for carbon lability and combined with 16S rRNA metabarcoding and carbon composition analyses to link microbial community structure with substrate characteristics. Removal rates of sinking particles (0.02-0.06 d-1) were an order of magnitude higher than those of suspended TOC (0.002 d-1) during bloom-decline and non-bloom. In contrast, during late-bloom, suspended carbon exhibited rates of 0.01 d-1, comparable to sinking particles, and was enriched in exopolymer-rich colonies. Prokaryotic community composition varied primarily among bloom stages rather than carbon fractions, indicating that bloom stage-- and thus particle origin and composition--was the dominant control on bacterial degradation and assembly. Bacterial diversity peaked where carbon was refractory and originated from mixed phytoplankton. Together, these results demonstrate that suspended Phaeocystis-derived carbon can be rapidly remineralized when blooms produce exopolymer-rich colonies and highlight bloom stage as key regulator of microbial carbon processing and biological pump efficiency.