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bioRxiv2025

Deep-learning-based interpolation of longitudinal microbiome data powers biologically informative discovery

The human microbiome is a foundational and dynamic foundation for several health-related functions and disease processes. Advances in microbiome sequencing have enabled the characterization of microbial communities in several niches. Longitudinal microbiome studies further strive to discover clinically informative microbial community trajectories. However, these data are fraught with dropout events, high noise, and irregular sampling that limit and prevent the use of many available longitudinal analysis tools. To address these challenges, we introduce Bidirectional GRU-ODE-Bayes (BGOB), a deep learning framework developed for longitudinal microbiome interpolation. BGOB combines bidirectional information flow and ODE-based continuous modeling to jointly interpolate and smoothen trends across individual participants, providing uniform, denoised time intervals across patients. BGOB enables vastly improved performance in differential abundance testing and time-to-event analysis, and makes possible longitudinal analyses requiring uniformity, such as lead-lag detection and temporal clustering. After interpolation, previously low-powered datasets are able to broadly recapitulate known microbiology and elucidate interacting microbial communities. We highlight several associations between microbial taxa and disease, including novel species associated with Early Childhood Caries and disruption of key healthy gut microbiota in Inflammatory Bowel Disease. The BGOB package is publicly available at https://github.com/Rachel-Lyu/BGOB_n_test.

microbiology↗

bioRxiv2025

Measurement Quality Metrics to Improve Absolute Microbial Cell Counting

Total and viable microbial cell counts are increasingly important for applications including live biotherapeutic products, food safety, and probiotics. In microbiology, cells are quantified using methods such as colony forming unit (CFU), flow cytometry, and polymerase chain reaction (PCR), but different methods measure different aspects of the cells (measurands), and results may not be directly comparable across methods. In the absence of a ground-truth reference material for cell count, one cannot quantify the accuracy of any cell counting method, which limits method performance assessments and comparisons. Herein, a modified analysis of cell counting methods based on the ISO 20391-2:2019 standard was developed and demonstrated for microbial cell samples diluted over a log-scale range of concentrations. Escherichia coli samples ranging in concentration from approximately 5 x 105 cells/mL to 2 x 107 cells/mL were quantified using CFU, Coulter principle, fluorescence flow cytometry, and impedance flow cytometry. Quality metrics modified from the ISO standard were calculated for each method and shown to be repeatable across replicate experiments. The quality metrics illustrate large differences in proportionality and variability across methods, with total cell counts in good agreement and viable cell count having more variability. As the ISO standard is meant to guide fit-for-purpose method selection, interpretation of the results and quality metrics can drive method choice and optimization. The framework introduced here will help researchers select fit-for-purpose counting methods for quantification of microbial total and viable cells across a range of applications.

microbiology↗

bioRxiv2025

Polyphasic discrimination of Shewanella seohaensis from closely related species and a whole-genome multilocus (wgMLST) scheme for the evaluation of diversity within this Shewanella clade

Shewanella is an environmentally ubiquitous genus with significant roles in bioelectrochemical applications and human infections. However, identification problems involving Shewanella putrefaciens, Shewanella xiamenensis, and Shewanella seohaensis, have been reported, potentially hindering research progress in these areas. In this study, we explored how to discriminate between these species. By comparing the genomes of Shewanella spp. available in public databases with that of the newly sequenced strain DSM9451, we showed that this strain is a member of the species S. seohaensis. Of the eight public genomes associated with this species, only two were correctly identified in public databases. Phenotypic analysis revealed distinct features of S. seohaensis with respect to S. putrefaciens and Shewanella decolorationis. However, only differences in the intensity of biochemical reactions were observed between S. xiamenensis and S. seohaensis. To discriminate between these closely related species and explore their diversity, a whole-genome multilocus sequence typing scheme was developed. The scheme distinguished related species and revealed significant diversity among S. seohaensis and S. xiamenensis isolates, suggesting that both species may harbor isolates with significantly different metabolic properties. These differences are so wide within the clade that may explain why these two species are difficult to distinguish. The identification of exclusive genes of each species allowed the design of a simple molecular method to differentiate S. seohaensis from closely related species, which will help in clarifying its role in human infections and environmental processes. ImportanceMisidentification within the Shewanella genus, particularly between S. putrefaciens, S. seohaensis, and S. xiamenensis, has been reported and misperceives scientific research on Shewanella spp. in diverse fields, including both biotechnological applications and human infections. Near-complete 16sRNA gene sequencing fails to correctly classify many Shewanella species, and MALDI-TOF systems used in clinical microbiology laboratories are suboptimal species-level identification. In this study, we identified phenotypic characteristics that can guide differentiation and classification, and building upon the identification of species-specific genes, we suggest an accurate and cost-effective molecular test as an alternative to genome sequencing. The proposed wgMLST scheme allows the exploration of species and strain diversity, highlighting the limitations of generalizing results from studies of a single strain. As an emergent pathogen and biotechnological candidate, the proper identification by a single molecular test will enhance the insights about these species towards biotechnology development and public health safety.

microbiology↗

bioRxiv2025

Historic mine waste contains diverse microbial communities that reflect waste type and geochemistry

Waste rock and tailings left behind by historic mining operations can contain substantial critical mineral resources. However, over the decades and centuries since these deposits were emplaced, microbial communities developed that can catalyze rock weathering and elemental cycling, which could have impacted the economic resources but also might be harnessed for future biomining or other metal recovery efforts. Here we combined cell counting, rRNA gene and transcript sequencing, and whole rock geochemistry to compare the composition and abundance of microbial communities from five inactive mine sites in South-Central New Mexico that contain critical minerals. While acidic seeps and adits at the sites contained organisms commonly found in acid rock drainage and bioleaching operations, these organisms were only present at very low abundance in the waste rock and tailings, which were instead dominated by bacteria and archaea that mostly represent inorganic nitrogen- and organic carbon-oxidizing populations. Generally, rRNA transcript libraries contained many of the same organisms as rRNA gene libraries, indicating that most of these microbial populations are active. Differences among total and active communities correspond to waste rock geochemistry, including concentrations of sulfur, iron, and other variables such as copper, lead, and rare earth elements. Nevertheless, many of the rRNA gene and transcript sequences in these deposits were from groups without cultured representatives, and these unknown microorganisms are likely important for biogeochemical cycling over the long lifetime of these waste deposits. We also discuss recommendations for microbiological assessment of similar large historic mine waste deposits. IMPORTANCENew Mexico has a long history of mining, with hundreds of mining districts across the state, many of which contain inactive operations with historic tailings and waste rock. Because metallurgical processing was in its infancy when most of these mines were active, they contain substantial metal resources in tailings and waste rock that could be used to support domestic demand for critical minerals. We found that microbial communities associated with these deposits do not represent typical bioleaching communities, and instead are dominated by taxa not typically associated with mine wastes. However, the deposits do contain rare iron and sulfur-cycling taxa that could catalyze metal mobilization, as well as active populations of novel microorganisms that are likely important for biogeochemical cycling. These mine waste-associated microbial communities could represent important resources for bioremediation and other biotechnological applications to recover valuable elements from these and other historic mine waste deposits.

microbiology↗

bioRxiv2025

Detection of Paracoccus yeei in Spontaneous Bacterial Peritonitis using Rapid, Long Read Sequencing

Ascites is a common complication in patients with decompensated liver cirrhosis. Spontaneous bacterial peritonitis (SBP) is the most frequent infection, affecting up to 30% of hospitalized patients with ascites. Multidrug-resistant bacteria in patients with liver cirrhosis are becoming more common, particularly in nosocomial infections. With early detection and adequate antibiotic therapy, the mortality rate of SBP can be reduced from over 90% to 15-20%. However, the etiologic agent remains undetected by conventional pathogen diagnostics from ascites in more than half of the patients. Here, we describe a robust workflow combining long read metagenome sequencing with best practices for low microbial biomass sample processing and bioinformatic analytics, to detect and characterize pathogens from the cirrhotic ascites of SBP patients, as a complement to routine microbiological diagnostics. This approach identified Paracoccus yeei as a likely etiologic agent for a patient who developed a fever after successful treatment of an Escherichia coli infection. Analysis of the genome of P. yeei recovered directly from ascites delineated pathogenicity-related features and an antimicrobial resistance profile consistent with the patients treatment. Pangenome phylogenetics placed the P. yeei strain closest to isolates associated with abdominal infections. Our study underscores the utility of metagenomics for a rapid and comprehensive assessment of infectious agents, suggesting a novel and rarely reported pathogen contributing to SBP with potential implications for diagnostics and therapeutic strategies. ImportanceOur study demonstrates the clinical utility of real-time, long-read metagenomics in identifying elusive pathogens in Spontaneous Bacterial Peritonitis (SBP), a condition with high mortality and frequently inconclusive conventional diagnostics. By uncovering Paracoccus yeei as a likely secondary etiologic agent and characterizing its pathogenicity and AMR profile, our findings expand the known etiology of SBP and emphasize the potential of metagenomics to improve diagnostic accuracy, guide targeted treatment, and ultimately reduce mortality rates in infectious diseases. This approach could shape future clinical practice for managing complex infections.

microbiology↗

bioRxiv2025

Type VI secretion system activity at lethal antibiotic concentrations leads to overestimation of weapon potency

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Timelapse microscopy reveals T6SS-armed Acinteobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes especially where target survival is low. We also show that incubating A. baylyi / E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

microbiology↗

bioRxiv2025

Community and functional stability in a working bioreactor degrading 1,4-dioxane at the Lowry Landfill Superfund Site

1,4-dioxane (dioxane) is an emerging contaminant that poses risks to human and environmental health. Bacterial dioxane degradation is increasingly being studied as a method to remove dioxane from contaminated water. However, there is a lack of studies on microbial community structures and functions within efficient, large scale, biodegradation-based remediation technologies. The Lowry Landfill Superfund Site (Colorado, USA) uses an on-site, pump-and-treat facility to remove dioxane from contaminated groundwater by biodegradation. Here, 16S rRNA and shotgun metagenomic sequencing were used to describe microbial community composition, soluble di-iron monooxygenase (SDIMO) alpha hydroxylases, and potential for dioxane degradation and horizontal gene transfer in bioreactor support media from the facility. Support media showed diverse microbial communities dominated by Nitrospiraceae, Nitrososphaeraceae, and Nitrosomonadaceae. Pseudonocardia were also detected, suggesting a potential presence of known dioxane-degraders. Candidate SDIMOs belonged mostly to Group V, followed by Groups IV, II, and I (based on read depth). The most abundant Group V clade contained 38 proteins that were phylogenetically related to DxmA-like proteins, including that of Pseudonocardia dioxanivorans CB1190 (a known dioxane degrader). Seventeen Lowry contigs containing DxmA-like proteins contained protein-coding genes potentially involved in chemical degradation, transcriptional regulation, and chemical transport. Interestingly, these contigs also contained evidence of potential horizontal gene transfer, including toxin-antitoxin proteins, phage integrase proteins, putative transposases, and putative miniature inverted-repeat transposable elements. These findings improve our understanding of potential dioxane biodegradation mechanisms in a functioning remediation system. Further studies are needed to definitively confirm microbial activity and enzymatic activity towards dioxane removal in this site. IMPORTANCEAs an environmental contaminant, 1,4-dioxane poses risks for water quality and human health. Used as a solvent and chemical stabilizer in a variety of manufacturing and industrial applications, microbiological methods of detoxification and mitigation are of interest. The degradation of 1,4-dioxane by the bacterium Pseudonocardia spp. is the best understood example; however, these studies are largely based on single isolate, bench-scale, or in silico experiments. Consequently, a knowledge gap exists on bacterial degradation of 1,4-dioxane at environmentally relevant concentrations using functioning remediation technologies at scale. This study addresses this gap directly by describing microbial taxa, enzymes, and potential horizontal gene transfer mechanisms associated with an active treatment plant located on a 1,4-dioxane-impacted U.S. Environmental Protection Agency (EPA) superfund site. As 1,4-dioxane contamination gains more attention, these findings may prove useful for future facilities aiming to promote and optimize removal by biodegradation.

microbiology↗

bioRxiv2025

Evaluation of Probiotic Bacteria for the Reduction of Urea, p-Cresol, and Indole Levels

IntroductionEnd-stage kidney disease (ESKD) is associated with the accumulation of uremic toxins such as urea, p-cresol, and indole, which significantly contribute to systemic complications such as inflammation, oxidative stress, and gut dysbiosis. Probiotics have demonstrated potential in modulating gut microbiota and reducing these toxins. We evaluated the in vitro efficacy of multiple probiotic strains of Lactobacillus, Bifidobacterium, Sterptococcus, Bacillus and others in degrading urea, p-cresol, and indole. MethodsThe probiotic strains were initially trained on toxin media prior to their evaluation of their toxin breakdown efficiency. Additionally microbiological methods using selective Christiansons broth/agar and Stuarts broth/agar were applied to assess urea breakdown. Liquid Chromatography-Mass Spectrometry (LC-MS) was utilized to quantify p-cresol and indole degradation. ResultsThe results indicated that all probiotic strains exhibited significant activity in reducing urea, p-cresol, and indole concentrations in the culture media. In urea breakdown we observed an oscillation urea/ammonia ratio at 24 hr intervals, although a complete elimination of the ammonium byproduct was not feasible. Among all tested probiotics, Lactobacillus species showed the highest efficiency in urea breakdown. Furthermore, the toxin removing efficacy of the probiotics was evaluated in a simulated gut environment using the TNO invitro gut model. ConclusionCumulatively, the findings suggest that probiotics could offer a promising strategy for the reduction of uremic toxins and their associated complications in ESKD patients.

microbiology↗

bioRxiv2025

Echoes of 1816: Microbial Footprints in Heritage Artifacts From Argentina's Museum of Independence

Historical artifacts preserved in museums are invaluable cultural treasures, yet they are often vulnerable to biodeterioration caused by microbial colonization. This study presents the first comprehensive investigation of microbial communities inhabiting heritage artifacts from Argentinas Museum of Independence. By integrating advanced microscopy with phenotypic and genomic characterization, we analyzed samples collected from wooden objects, textiles, architectural elements, and exterior walls. Scanning electron microscopy revealed diverse and well-structured biofilms, with intricate three-dimensional arrangements embedded in extracellular polymeric substances. A total of 49 bacterial strains were isolated and identified via VITEK MALDI-TOF mass spectrometry, with a predominance of Gram-positive genera such as Bacillus, Micrococcus, and Kocuria. Remarkably, the 19th-century albumen print photograph emerged as the most biodiverse artifact, yielding 21 distinct strains, including Streptomyces, Oceanobacillus, and thermophilic Caldibacillus thermoamylovorans. The protein-rich and halophilic environment of the albumen layer likely facilitated microbial colonization and persistence. Pseudomonas species were exclusively associated with this photographic substrate, further underscoring its niche specificity. Human-associated taxa like Staphylococcus epidermidis and Staphylococcus equorum were prevalent in high-contact areas, while exterior surfaces displayed distinct microbial signatures, including potential pathogens linked to environmental exposure. These findings reveal a complex and artifact-specific microbial landscape, emphasizing the need for tailored, bio-informed conservation strategies. This work advances the field of heritage microbiology and supports efforts to safeguard culturally significant objects through a deeper understanding of their microbial ecosystems.

microbiology↗

bioRxiv2025

Nanopore direct RNA sequencing (DRS) of MS2 bacteriophages in E.coli throughout its life cycle reveals a complex transcriptional activity to control and maintain its growth

The RNA bacteriophage MS2 is an RNA phage that infects the bacterium E. coli and is one of the most studied and prototypical model phages in molecular biology and microbiology. Previous research revealed complex translational control and fine-tuning for MS2 replication. However, the dynamics of its transcriptional activity and replication during the life cycles within the bacteria remain elusive. Here, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout multiple life cycles. We discovered that MS2 phages sustain a high level of transcriptional activity required for replication. We found large amounts of subgenomic small transcripts from RNA degradation, Nanopore DRS bias, and transcripts containing the coat-encoding region, required for virion assembly. We found hybrid reads due to the error-prone activity of the MS2 replicase complex by the template-switching mechanism. We evidenced that RNA modification is conserved throughout the entire life cycle in full-length transcripts without the acquisition of new modifications, whereas small transcripts did acquire newly modified sites. The conserved sequence and secondary structure (U-rich hairpin) of {Psi} installation sites were the most amenable to RNA modification, and one site aligned well with host RluA-mediated installation. Overall, our investigation reveals a more complex transcriptional dynamics of MS2 phages than anticipated within E. coli to maintain its growth and replicate under host pressure.

microbiology↗

bioRxiv2025

Common biological features of Mycobacterium tuberculosis MmpL3 inhibitors

MmpL3 is a promising new target for antitubercular drugs, but the microbiological properties of MmpL3 inhibitors are not fully understood. We compared the activity and mode of action of 11 structurally diverse compound series that target MmpL3. We confirmed activity was via MmpL3 using strains with differential expression of MmpL3. MmpL3 inhibitors had potent activity against replicating M. tuberculosis, with increased activity against intramacrophage bacilli and were rapidly bactericidal. MmpL3 inhibition induced cell wall stress concomitantly with a boost in ATP levels in M. tuberculosis. Mutation in MmpL3 conferred resistance to all series at different levels. Molecules did not negatively impact membrane potential, pH homeostasis or induce reactive oxygen species and were inactive against starved bacilli. Our study revealed common features related to the chemical inhibition of MmpL3, enabling the identification of off-target effects and highlighting the potential of such compounds as future drug candidates.

microbiology↗

bioRxiv2025

Rapid Detection of Carbapenemase-Producing Organisms Using a Luminescent Biosensor

Carbapenemase-producing organisms (CPOs) pose an urgent global health threat due to their ability to inactivate carbapenems, a group of last resort antibiotics. Infections caused by these pathogens are associated with poor patient outcomes, high mortality rates, and added burden to infection prevention and control programs, making early detection vital to ensure optimal antimicrobial therapy and appropriate implementation of infection control practices. In this study, we report the application of a luminescent whole-cell biosensor for the rapid detection of CPOs. This biosensor provides positive test results within 2.5 hours, inclusive of setup time, and has been validated with a panel of laboratory and clinical isolates producing a diverse range of carbapenemases (KPC, NDM, IMP, VIM and OXA-48-like). The assay successfully identified all CPO isolates tested with a sensitivity of 100%, including strains producing weak OXA-48-like carbapenemases which are sometimes missed by currently used detection methods. The assay also demonstrated a specificity of 100 %, with all non-CPO clinical isolates testing negative under the optimized assay conditions. Due to the rapid time-to-positivity, minimal setup requirements, and high sensitivity, this test could serve as an attractive alternative to CPO detection methods currently employed by clinical microbiology labs, and could also facilitate CPO screening in other settings (e.g., environmental, agricultural).

microbiology↗

bioRxiv2025

Enhancing DNA recovery in low-biomass snow algae samples: a comparative study of extraction methods and their effect on community composition

High-throughput sequencing is a powerful tool for environmental microbiology and can be particularly important for examining community structure and function for organisms that are difficult to culture or environments that are difficult to mimic like snow algae. DNA extraction significantly impacts these analyses, often introducing more variation than PCR or sequencing. Snow algae are widespread on mountain and polar snowfields where they contribute to biogeochemical cycling and accelerate melt. Despite increasing research on snow algae, inconsistencies in DNA extraction remain a major challenge, and no recommended method exists for assessing their community composition and richness. Here, we compared three common extraction methods (DNeasy PowerSoil Pro, DNeasy PowerWater, and phenol-chloroform) alongside ultrasonication in samples with varying snow algae abundance. The extraction method strongly influenced resulting microbial profiles assessed by amplicon sequencing of rRNA genes. Ultrasonication improved DNA yield in low-biomass samples and enhanced the recovery of resilient cells, including mature-phase snow algae likely due to improved cell lysis step. This is the first systematic comparison of DNA extraction methods for snow algae, highlighting how methodological choices affect microbial community analyses. Our findings provide insights to improve standardization, enhancing the reliability of future studies in snow and ice environments.

microbiology↗

bioRxiv2025

Genomic and Functional Characterization of Priestia megaterium MOD5IV isolated from the rhizosphere of Caesalina spinosa (Mol.): Enhancing Phytoremediation Potential in Multi-Metal Contaminated Soils

AimsMetal contamination poses a global threat due to its widespread occurrence and the high toxicity of these elements. Phytoremediation has emerged as a preferred approach for the bioremediation of metal-contaminated soils. The search for microorganisms facilitating phytoremediation, especially plant-growth-promoting bacteria (PGPB), has become critical to advance ecosystem remediation efforts. This research aimed to characterize in-depth a Priestia megaterium strain isolated from multimetal contaminated soils located at the Atacama Desert, showing potential for bacteria-assisted phytoremediation. Methods and ResultsThe strain MOD5IV exhibited notable PGPB features: phosphate and potassium solubilization, nitrogen fixation, phytohormone production, and growth promotion of Arabidopsis thaliana. Genomic analysis revealed a 5,254,635 bp chromosome plus nine plasmids, hosting over 6,000 genes. Functional annotation identified genes associated with resistance to copper, cadmium, lead, mercury, zinc, and cobalt. Also, genes linked to PGPB capabilities as siderophore-production, nutrient-solubilization, IAA-synthesis, and nitrogen-fixation. Accordingly, MOD5IV exhibited robust tolerance to multiple metals and enhanced the phytoremediation potential of Caesalpinia Spinosa (Mol.) in laboratory trials. ConclusionsMOD5IV proved to have promising traits for microbe-assisted phytoremediation of metal-contaminated soils. Impact StatementThis study contributed to the characterization of new native multi-metal-resistant PGPR bacteria for phytoremediation of metal-contaminated soils. Increasing the evidence of the Atacama Desert as a source of microbiological solutions for climate adaptation and environmental remediation.

microbiology↗

bioRxiv2025

Viability Differentiation Improves the Diagnostic Potential of 16S rRNA Gene Sequencing in Ready-to-Eat Meat Manufacturing

Molecular-based microbiological approaches have become valuable tools for the food industry. However, even the most advanced molecular techniques are limited in their ability to differentiate based on viability creating the potential for biased results when applied to the food industry. The objective of this study was to generate a viable microbial bio-map of a commercial ready-to-eat (RTE) meat manufacturing process and assess its utility as a diagnostic tool. Product samples were collected from a commercial RTE meat manufacturing facility at various locations throughout processing. Samples were homogenized and aliquoted for culture-based microbial isolation and 16S rRNA gene sequencing. Homogenates were split into pairs and subject to either no treatment (Control) or treated with 25 M PMAxx (PMA) to remove free and non-viable cellular DNA. Overall, PMA treatment resulted in a less rich microbial community compared to Control samples. Paired analysis revealed that the impact of PMA varied by location with the greatest effects being observed at the beginning and end of manufacturing. Both Control and PMA treated samples identified a shift in the microbial population after thermal processing; however, only PMA treated samples identified a secondary shift in the microbial population occurring after slicing. Taxonomic analysis identified Lactobacillus as a predominant genera in sliced and packaged products. These results were further confirmed by the identification of Lactobacillus sakei on packaged product using a culture-based approach. These results suggest PMA treatment provides a higher level of sequencing resolution by removing background DNA. ImportanceMicrobial bio-mapping is a valuable tool for the meat and poultry industry to assess process control and evaluate the efficacy of intervention systems. In recent years it has become more common to incorporate the use of molecular techniques, such as qPCR and 16S rRNA, to quantitatively track target pathogens and gain a more holistic understanding of the microbial community throughout processing. One major limitation we face when applying these DNA-based techniques to the food industry is their inability to differentiate between DNA from viable versus non-viable cells, which may result in the false identification of pathogenic or spoilage microorganisms and bias microbiota results. To practically apply this technology in a ready-to-eat meat manufacturing setting, it is crucial to develop and validate strategies that are capable of differentiating between viable and non-viable cellular DNA.

microbiology↗

bioRxiv2025

A refined low-dose murine model of Mycobacterium ulcerans infection to assess integrated immune networks in Buruli ulcer pathogenesis

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is a slow-growing zoonotic pathogen with distinctive pathogenesis linked primarily to its toxin mycolactone. Recent research has shown that the M. ulcerans infectious dose is very low (<10 colony forming units [CFU]). Buruli ulcer animal infection models traditionally use bacterial challenge doses in the range 104 - 106 CFU; a range orders of magnitude higher than natural infection. These large doses represent an unrealistic challenge for vaccine trials and studies of immunity. Here, we address this issue and describe a murine tail infection model in two genetically distinct mouse strains (BALB/c and C57BL/6) using quality-controlled, M. ulcerans challenge doses (10 - 20 CFU and 100 CFU). Over 24-weeks, we assessed host responses to infection by measuring >70 clinical, immunological and microbiological parameters. Principal findings included a 100% infection rate even at the lowest bacterial challenge, but with a dose-dependent delay in lesion onset and disease progression for both mouse strains. Bacterial growth kinetics were similar between mouse strains. There was a difference in immune profiles between mouse strains and between low (10 CFU) versus high (100 CFU) bacterial challenge doses. C57BL/6 mice exhibited more robust systemic cellular responses and more rapid lesion onset compared to BALB/c mice. There were dose-dependent cytokine and chemokine differences in C57BL/6 mice, while BALB/c mice displayed similar responses across both doses. Antibody responses were only detected late in the infection and were associated with the high-dose inoculum in both strains. Machine learning and other statistical analyses highlighted the importance of activated CD8+ T cells and dendritic cells in the immune response to low-dose infection in C57BL/6 mice. Murine low-dose M. ulcerans infection models provide confidence for future human Buruli ulcer challenge trials and will inform the development of effective vaccines and therapeutics.

microbiology↗

bioRxiv2025

Metabolic and Population Profiles of Active Subseafloor Autotrophs in Young Oceanic Crust at Deep-Sea Hydrothermal Vents

At deep-sea hydrothermal vents, magmatically driven rock-water reactions in the crust generate gases and other reduced compounds that subseafloor microorganisms use for chemolithoautotrophy. In this study, microbial autotrophs from three diffuse flow hydrothermal vents at Axial Seamount in 2013 and 2014 were isotopically labeled using RNA Stable Isotope Probing (RNA-SIP), targeting subseafloor autotrophic mesophiles (30{degrees}C), thermophiles (55{degrees}C), and hyperthermophiles (80{degrees}C). We constructed taxonomic and functional profiles of active chemolithoautotrophs, examined population distributions across sites, and linked primary producers to their specific metabolic strategies within the subseafloor community. Dominant autotrophs exhibited hydrogen-dependent dissimilatory metabolisms such as sulfur and nitrate reduction and methanogenesis, as well as microaerophilic sulfide oxidation even at 80{degrees}C, consistent with fluid chemistries at each site. While hydrogenotrophic methanogenic archaea such as Methanothermococcus were restricted in their distribution and activity, hydrogenotrophic sulfur and nitrate reducers from the Aquificota (Thermovibrio), Campylobacterota (Nautiliaceae, Hydrogenimonas, and Desulfurobacteriaceae) were consistently active and present at all sites and years at both the population and community levels. Hydrogenase transcripts were significantly differentially expressed, and diverse hydrogenases were found in metagenome-assembled genomes of Aquificota members, highlighting the importance and versatility of their hydrogen utilization strategies which likely contribute to their cosmopolitan distribution across geochemically disparate subseafloor sites. Together, this study provides new insights into the functional dynamics and distribution of key subseafloor autotrophic microbial communities in young oceanic crust at deep-sea hydrothermal vents. IMPORTANCEDeep-sea hydrothermal vents are hotspots for life in the dark ocean, where rich animal ecosystems are supported by microbial primary producers utilizing the abundant chemical energy supplied by high-temperature water-rock reactions. Despite increasing knowledge about the geochemistry and microbiology of deep-sea hydrothermal vents, there is still a gap in our understanding of the key microbial players who fix much of the carbon at these sites, especially in the productive subseafloor. In this study, stable isotope probing was used to label active microbial autotrophs in diffuse flow venting fluids from three sites over two years and was combined with metatranscriptomic sequencing to identify their specific metabolic strategies. This research highlights the microbial community composition, function, gene regulation, and population dynamics that enable hydrothermal ecosystems to persist.

microbiology↗

bioRxiv2025

Competition with Pseudomonas aeruginosa induces Staphylococcus aureus in an antibiotic-tolerant viable but non culturable state

Interactions between different species of pathogenic microbes often affect clinical outcome by altering the virulence or antibiotic resistance of individual microbes. By investigating the interactions between Staphylococcus aureus and Pseudomonas aeruginosa at the population, single-cell and molecular level we discovered that a sub-population of S. aureus enters in a viable non-culturable state that it is not detected via standard microbiology assays. In the presence of P. aeruginosa, S. aureus adopts a survival lifestyle similar to previously described intracellular S. aureus persisters, downregulating nitrogen metabolism and amino acid biosynthesis, while upregulating protein maturation processes. Entrance in a viable but non culturable state is the primary survival strategy of S. aureus in response to vancomycin treatment, whereas only a minority of the S. aureus population survive ciprofloxacin treatment while in a viable but non culturable state. These bacterial interactions may shape the evolution of resistance traits of co-infecting pathogens. Manipulating these interspecies adaptations could provide new opportunities for early therapeutic interventions.

microbiology↗