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bioRxiv2026

AMRgen: an R package for antimicrobial resistance genotype-phenotype analysis

Microbial whole-genome sequence data is now generated at scale, including to support antimicrobial resistance (AMR) surveillance and understand resistance mechanisms, yet analytical infrastructure for systematically linking AMR genotypes to measured phenotypes remains fragmented. Here we present AMRgen, an R package to support systematic AMR genotype-phenotype analysis. AMRgen imports and harmonises genotypic data from common bioinformatics tools, alongside phenotypic data from automated antimicrobial susceptibility testing instruments and public repositories. It supports common analyses linking data to reference distributions, modelling associations, quantifying concordance, and producing publication-ready visualisations including UpSet plots that jointly display genotypic marker combination frequencies and associated phenotypic distributions. We demonstrate AMRgens utility using publicly available surveillance data for World Health Organization priority AMR pathogens, Neisseria gonorrhoeae, Klebsiella pneumoniae, Escherichia coli and Salmonella enterica. AMRgen, available free and open-source at https://AMRgen.org, provides a reproducible end-to-end foundation for genotype-phenotype research in AMR genomics, clinical microbiology, and public health surveillance.

microbiology↗

bioRxiv2026

Identification of antibiotic resistance genes in fecal microbiota selected donors during the establishment of a biobank in the south of Brazil

Fecal microbiota transplantation (FMT) is an effective therapy for recurrent Clostridioides difficile infection and is increasingly explored for other dysbiosis-related disorders. However, its implementation as a regulated therapeutic strategy still requires robust donor screening, biosafety frameworks, and standardized processing workflows. Here, we describe the establishment of the first fecal microbiota biobank in the south of Brazil and evaluate the incorporation of metagenomic sequencing as a complementary layer of donor safety assessment. A structured donor selection pipeline based on international guidelines was implemented, integrating clinical screening, biochemical and serological testing, and microbiological analyses. Of 100 screened candidates, only four donors met all eligibility criteria and were included in the biobank, highlighting the stringency of the selection process. Shotgun metagenomic sequencing revealed a diverse resistome across all donors, including a shared core set of resistance-related genes alongside marked interindividual variability. Dominant antibiotic resistance genes included tetracycline-associated determinants, as well as ermF, CfxA-type {beta}-lactamases, and aminoglycoside-modifying enzymes, each linked to specific gut taxa. Notably, the relatively high abundance of tetW and ermF in Bacteroides fragilis suggests that this dominant commensal species may act as a reservoir for tetracycline and multidrug resistance determinants within the intestinal microbiota. Rather than serving as exclusion criteria, such determinants highlight the importance of integrating functional genomic profiling into donor characterization. Overall, this study provides a framework for microbiota biobank implementation and supports the use of metagenomics as a complementary strategy to improve biosafety and functional assessment in FMT.

microbiology↗

bioRxiv2026

Serial-passage assessment shows no confirmed resistance development to Norway spruce (Picea abies) resin in bacterial species relevant to wound infection

The search for antimicrobials with a low propensity to select resistance has intensified in response to the global antimicrobial resistance crisis. Norway spruce resin (Picea abies) has long been used in Northern European wound care traditions and has shown broad antimicrobial activity in earlier microbiological studies. In the present study, we evaluated whether prolonged exposure to medical-grade spruce resin promotes reduced susceptibility in clinically relevant bacterial species. A 20-day serial-passage experiment was performed with Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis using sub-inhibitory resin concentrations and broth microdilution readouts at baseline, day 10, and day 20. Resistance development was predefined as a [≥]4-fold increase in inhibitory concentration. Baseline inhibitory concentrations were 1.25% for S. aureus, 5.0% for P. aeruginosa, and 2.5% for E. faecalis. After 20 days, inhibitory concentrations were 2.5%, 10.0%, and 2.5%, respectively, corresponding to at most 2-fold changes and remaining below the predefined threshold for resistance development. Validation and vehicle-control arms indicated that these shifts were not attributable to medium transfer or solvent-related bias. These findings suggest that medical-grade Norway spruce resin has a low short-term tendency to select for reduced susceptibility under serial-passage conditions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=134 SRC="FIGDIR/small/723837v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@160479forg.highwire.dtl.DTLVardef@1fe1e95org.highwire.dtl.DTLVardef@89dec3org.highwire.dtl.DTLVardef@17ff134_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2026

Student-Driven Microbiome Exploration: A Low-Cost 16S rRNA Sequencing Curriculum for Undergraduate Biology Education

Microbiome science is increasingly important in modern biology education because microbial communities influence human health, ecosystems, and environmental processes. However, undergraduate microbiome instruction is often limited by the high cost and technical complexity of sequencing-based workflows, restricting opportunities for authentic student-driven research. To address this challenge, we developed a low-cost, inquiry-based curriculum that enables undergraduate students to conduct complete microbiome studies using 16S rRNA gene sequencing. The module integrates project design, environmental sample collection, microbial cell processing, PCR amplification, sequencing, and bioinformatic analysis using open-source tools such as QIIME 2. Cost-reduction strategies included centrifugation-based cell collection and a surfactant-assisted direct PCR workflow that eliminated the need for commercial DNA extraction kits. Students designed independent research projects investigating microbial communities in local environments, including campus water sources and gym equipment surfaces. Assessment data from post-course surveys, knowledge checks, and student research products demonstrated strong learning gains in microbiome concepts, molecular biology techniques, scientific communication, and computational analysis. Students reported high confidence in PCR, experimental design, and microbiome interpretation, while also identifying bioinformatics as the most challenging yet rewarding component of the curriculum. All participants expressed increased interest in future research in microbiology or bioinformatics. Overall, this curriculum provides an accessible, scalable framework for integrating next-generation sequencing into undergraduate education while promoting inquiry-driven learning, student ownership, and engagement in authentic scientific research.

microbiology↗

bioRxiv2026

Oligonucleotide genetics for Pseudomonas aeruginosa enables high throughput hypomorph screening

The continual advancement of genetic tools has been critical to our modern understanding of bacteria, with transposons, plasmids, and homologous recombination becoming workhorses of molecular microbiology. However, precisely specified reverse genetic approaches remain painstakingly slow and inaccessible, particularly in non-model strains. This reality is exemplified by the opportunistic pathogen, Pseudomonas aeruginosa (Pa), where conventional allelic exchange remains the dominant reverse genetic method. Here, we adapt a rapid genetic toolkit for use in Pa, relying directly on commercially available oligonucleotides (120 bases) to create precise genomic mutations through homologous recombination (i.e. oligo recombineering). Oligo Recombineering followed by Bxb-1 Integrase Targeting (ORBIT) uses a short attachment site for an integrating plasmid, which provides traditional antibiotic selection and can also carry flexible cargo. We establish Pa ORBIT works effectively for gene deletion without off target mutations, optimize protocol parameters (e.g. oligo length, electroporation), and demonstrate markerless and clean deletions. Importantly, our toolkit works well in clinical Pa strains as demonstrated by constructing efflux pump deletions in three different isolates. To test the high throughput capabilities of Pa ORBIT, we created over 160 degron-based hypomorphs (i.e. knockdowns) across 43 essential proteins in a pooled mutant library. Upon screening this library with and without antibiotics, we identify highly vulnerable essential proteins and hypomorphs that display synergy with clinical drugs. Therefore, ORBIT can be used for cutting edge low and high throughput investigations in this priority pathogen, setting the stage for answering critical basic and clinical science questions. SignificanceTo understand bacterial genomes, researchers need access to rapid, flexible, precise and high throughput genetic perturbation tools. Here we present an oligonucleotide-based method that satisfies these requirements for use in the opportunistic pathogen, Pseudomonas aeruginosa. By relying on oligos to encode genomic homology arms, no molecular cloning is required - making these tools rapid, robust, and scalable. We benchmark gene deletions in both lab and clinical strains, opening the possibility of rapid genetic studies across the P. aeruginosa pangenomic space. At high throughput, we use an oligo pool to create a mutant library of degron tagged essential proteins. These knockdowns (i.e. hypomorphs) show certain essential genes are highly vulnerable and others are synergistic with clinical drugs, providing insight into future antibiotic and co-therapy development.

microbiology↗

bioRxiv2026

CULTURE AND ISOLATION OF BACTERIA ASSOCIATED WITH MEDITERRANEAN CORALS

Corals harbor a diverse bacterial community that facilitates adaptation and sustains their health. In coral holobiont research, culture-independent approaches have transformed the existing paradigm. Molecular techniques, such as metabarcoding, revealed a high diversity of previously unrecognized bacterial symbionts. Coral microbiota characterization has relied on these techniques over the last decade, but relying solely on them does not provide a detailed understanding of the dynamics of the coral holobiont complex. Returning to classic microbiological methods and in vitro experimentation can yield novel insights into symbiont roles, physiology, and interactions within the holobiont. Under this premise, we aimed to isolate and culture bacteria from four Mediterranean corals. The recovery of 84 pure bacterial isolates and their initial classification based on the 16S rRNA gene revealed substantial diversity among symbionts amenable to culture. Several isolates represent novel species within relevant genera, such as Vibrio, underscoring the value of culture-based studies. All cultures were cryopreserved to guarantee long-term accessibility for future projects. This represents a key step towards describing the roles of bacteria within the coral holobiont, as cultures enable in-depth morphological and physiological characterization of the symbionts and experimental ecology studies.

microbiology↗

bioRxiv2026

Short-term oxycodone exposure produces delayed and persistent gut microbiome disruption in mice

The gut microbiome is a critical part of host homeostasis, yet its resilience following opioid exposure remains poorly understood. While opioid-induced short-term dysbiosis is well documented, the long-term recovery dynamics following oxycodone remain unclear. This study characterized the temporal dynamics of the fecal microbiota in male C57BL/6J mice following a brief 3-day oxycodone regimen (5mg/kg, BID). 16S rRNA gene sequencing was performed at baseline, day 3, 10, 17, and 70. While acute post-treatment phases (day 3 to 10) showed subtle taxonomic shifts in Clostridium_sensu_stricto_1 and Romboutsia, significant community disruption emerged later. By day 17, beta diversity significantly differed from saline controls (P =0.002). At day 70, both alpha diversity (p=0.02) and beta diversity (P=0.007) remained significantly altered, characterized by enriched Akkermansia and Marvinbryantia alongside depleted Eubacterium_xylanophilum. These findings demonstrate that even brief oxycodone exposure triggers persistent, non-recovering dysbiosis that became detectable only after treatment cessation and persisted through day 70. This suggests that the window for microbiome recovery exceeds two months in mice (equivalent to several human years), highlighting a potential long-term risk for patients prescribed short-term opioid courses. ImportanceShort-term opioid exposure is generally assumed to cause only transient disruption of the gut microbiome. However, the duration of microbiome recovery following clinically relevant opioid treatment remains poorly defined. In this study, we show that a brief three-day course of oxycodone in mice resulted in delayed and persistent alterations in gut microbial community structure that remained detectable for at least 70 days after treatment cessation. Notably, significant divergence in microbial composition emerged weeks after exposure rather than immediately following treatment, suggesting that short-term opioid use may initiate longer-lasting remodeling of the gut microbiome than previously appreciated. These findings highlight the importance of considering extended recovery timelines when evaluating the microbiological consequences of opioid exposure.

microbiology↗

bioRxiv2026

Pathogen and host biomarkers to aid early diagnosis and prognosis of tuberculous meningitis

BackgroundTuberculous meningitis (TBM) is the most sinister form of extrapulmonary tuberculosis (EPTB), associated with high mortality due to delayed diagnosis and limited sensitivity of conventional and molecular tests. Current study evaluated the diagnostic utility of Lipoarabinomannan antigen (LAM) detection in CSF and urine and explored host inflammatory biomarkers for diagnosis and prognosis of TBM. MethodsThis prospective observational study enrolled 80 patients with presumptive TBM at a tertiary care centre. CSF samples were subjected to AFB microscopy, liquid culture(MGIT-960), GeneXpert MTB/RIF (GX), and LAM lateral flow assay. Urine LAM was performed at baseline. Serum and CSF levels of IL-1{beta}, IL-6, TNF-, IFN-{gamma}, IL-17A, and IP-10 were measured at baseline and after 1 month treatment. ResultsAmong 80 participants, 23 (28.7%) had definite TBM and 46 (57.5%) had probable TBM. CSF LAM sensitivity and specificity against microbiological reference standards was 43.5% and 80.7%, while urine LAM sensitivity (60.9%) and specificity 82.5% was higher. Against composite reference standards, both CSF and urine LAM showed reduced sensitivity but achieved 100% specificity. Serum IL-1{beta} showed the best diagnostic performance (AUC 0.943; sensitivity 88.9%, specificity 90.9%). Elevated serum and CSF IP-10 levels were associated with poor outcomes, whereas declining IL-6 and TNF- levels correlated with treatment response. ConclusionLAM detection in CSF and urine may serve as a highly specific, rapid rule-in test for TBM. Host inflammatory biomarkers, especially IL-1{beta} and IP-10, show additional diagnostic and prognostic value. Combining LAM testing with cytokine biomarkers may improve early diagnosis and efficient clinical management of TBM.

microbiology↗

bioRxiv2026

Southeastern Australian Montane Fens Harbour Distinct Microbial Communities Rich in Novel Diversity

Montane fens are rare and microbiologically poorly characterised wetland ecosystems in south-eastern Australia, and their microbial communities remain virtually unexplored. Here, we profile the microbiomes of Victorian montane fens using 16S rRNA metabarcoding of 12 soil cores collected along a 12-m transect and sampled across four depth horizons. Surface soils exhibited slightly higher alpha diversity than deeper layers, but the most pronounced differences occurred in community composition, with surface microbiomes significantly distinct from all subsurface depths. To contextualise these communities within global environmental diversity, we compared them with 9662 Earth Microbiome Project samples spanning 24 environmental materials processed using comparable methods. Montane fen microbiomes were one of the most diverse environmental materials analysed and compositionally distinct from all comparator biomes. Overrepresentation analysis identified signature microbial taxa, including archaeal lineages from the Crenarchaeota and Methanomicrobia and bacterial phyla such as Acidobacteria, highlighting taxa involved in ecological processes associated with acidic, saturated, and organic-rich soils. Notably, the majority of sOTUs detected in montane fens were unique to this environment - the highest proportion of source-specific taxa among all biomes analysed. Together, these findings demonstrate that southeastern Australian montane fens harbour a highly distinctive and largely uncharacterised microbial community, underscoring their ecological uniqueness and the importance of conserving these rare alpine wetlands. Data SummarySequencing data is available in SRA BioProject PRJNA1398590; accessions SRR36684598 through SRR36684645. Metadata and accessions for collected montane fen samples are included in Table S1 and metadata for Earth Microbiome Project samples included in this study are listed in Table S4. Impact StatementWetland ecosystems are increasingly recognised as important reservoirs of microbial diversity, yet many remain poorly characterised in global microbiome surveys. In this study, we provide the first characterisation of microbial communities inhabiting montane fens in southeastern Australia and place them in a global context using publicly available environmental microbiome data. We show that these fens harbour exceptionally diverse microbial communities that are compositionally distinct from other environmental sources processed using comparable methods, with a high proportion of taxa that are not present in any other sample in an existing reference dataset. By extending global comparisons to an under-sampled wetland type, this work adds to the growing body of evidence that significant microbial diversity remains undocumented in geographically and ecologically restricted environments. The findings are relevant to researchers working in microbial ecology, environmental genomics, and biogeography, as well as those interested in wetland function and conservation. While largely descriptive, this study represents an important step in expanding environmental genome catalogues and provides a baseline framework for future genomic, functional, and mechanistic investigations of montane wetland microbiomes.

microbiology↗

bioRxiv2026

Persistent Staphylococcus aureus in human musculoskeletal tissues despite targeted antibiotic therapy

Staphylococcus aureus is a leading cause of musculoskeletal infection with frequent recurrence despite surgery and antibiotic therapy. Its persistence in human tissues under antibiotic pressure remains unclear. We performed longitudinal in situ analyses of matched tissue samples before and during therapy from patients undergoing repeated surgery using advanced imaging and tissue-based metagenomics. S. aureus remained detectable in all samples--including culture-negative specimens--and whole-genome sequencing confirmed persistence of the infecting strain. Antibiotic initiation reduced bacterial burden by 1.0Slog10 bacteria/mm3 but residual loads stabilized thereafter. Quantitative imaging identified a culture detection threshold ([~]103bacteria/mm3), indicating that culture negativity reflects diagnostic sensitivity limits rather than true bacterial clearance. Persistence under antibiotic therapy was associated with high baseline burden and intracellular sequestration, whereas long-term persistence was characterized by predominantly extracellular bacteria capable of driving relapse. These findings reveal sustained tissue persistence despite apparent microbiological clearance and guide precision strategies to target resilient bacterial reservoirs.

microbiology↗

bioRxiv2026

Longitudinal RNA Seq analyses reveal the prominent role of Vagococcus in broiler meat spoilage microbiome

BackgroundRaw broiler meat products are highly perishable, and microbial activity is the main factor limiting their shelf lives. The spoilage microbiomes of broiler meat products have been studied mainly using traditional culturing methods and 16S rRNA gene amplicon sequencing, neither of which can show the activity of whole microbiome. Previous metatranscriptomic research of broiler spoilage has also remained limited to specific spoilage organisms rather than entire microbiomes. ResultsOur longitudinal study of broiler meat spoilage discovered the successions of active bacterial microbiomes and metabolic pathway activities of spoilers at 4{degrees}C and 6{degrees}C. Samples taken daily were subjected to metatranscriptomic analyses in combination with non-targeted metabolomic, traditional microbiology, and sensory analyses until advanced spoilage took place. Carnobacterium divergens, Carnobacterium maltaromaticum and Vagococcus proximus were the most active species at both temperatures. Carnobacteria are known poultry spoilers whereas Vagococcus proximus, a species recently described, played an unexpected active role in the microbiome. It became dominant in samples stored at 6{degrees}C and its activity increased also at 4{degrees}C after the use-by date. Central carbohydrate metabolism was the most common KEGG orthology pathway module of the microbiome at both temperatures. C. divergens and C. maltaromaticum showed stable metabolic profiles during the spoilage process, whereas V. proximus displayed a shift from high ATP synthesis activity to increased fatty acid and carbohydrate metabolism when spoilage advanced in samples stored at 4{degrees}C. Non-targeted metabolomics showed similar metabolomic trends across both temperatures. At 6{degrees}C, time-dependent changes were generally more pronounced, and the spoilage markers tyramine and spermidine showed greater accumulation. ConclusionsAs expected, the rate of spoilage is higher at 6 than 4{degrees}C, however we did not anticipate a similar overall trajectory of the spoilage processes. Our results link V. proximus as a key active spoiler in broiler meat and demonstrate the efficacy of using RNA-seq together with metabolomics to decode the function of a meat spoilage microbiome. This demonstrates that spoilage microbiomes consist of active species we have been neglecting due to the technological limitations of the standard methods. Future studies targeting to the metabolism and detecting of Vagococcus are warranted.

microbiology↗

bioRxiv2026

Microfluidic-based high-throughput isolation enhances the recovery of novel strains and diversity from Arctic soil microbiome

Microbial cultivation remains essential for understanding the physiology, ecology, and biotechnological potential of environmental microbes, yet conventional plate-based methods (CPM) recover only a minute fraction of the environmental microbiome. Polar regions, particularly Arctic soils, represent unique reservoirs of "microbial dark matter" that remain challenging to cultivate, owing to oligotrophic conditions, low temperatures and freeze-thaw cycles that impose severe physiological constraints on microbial growth. Here, we report the first systematic application of microfluidic droplet technology (MDT) to Arctic active-layer soil microbiota and benchmark its performance against CPM using identical starting cell numbers, R2A medium, and incubation at 15{degrees}C. MDT achieved 6.5- to 8.1-fold higher recovery rates than CPM and improved isolation throughput by >180-fold. Near-full-length 16S rRNA gene sequencing (PacBio) revealed that MDT recovered significantly higher taxonomic richness across all taxonomic levels, with 256 genera detected in the high-cell-input group (DropAS_H) versus 211 in the corresponding plate group (PlateAS_H). Notably, MDT yielded a more even community distribution, significantly reducing the dominance of fast-growing copiotrophs such as Pseudomonas and Flavobacterium. Moreover, approximately 50% of sequences from MDT were affiliated with potential novel species (<98.46% identity to type strains), and 27% with potential novel genera (<95% identity). Strain verification by Sanger sequencing confirmed 12 of 17 isolates as candidate novel species, among which one strain represented a potential novel genus within Devosiaceae. This study demonstrates that MDT is a powerful platform for accessing the uncultured majority of polar soil microbiota and establishes a pipeline for high-throughput isolation of novel cold-adapted bacteria. IMPORTANCEArctic soils harbor a vast reservoir of microbial diversity that remains largely inaccessible due to the extreme oligotrophic conditions and low temperatures characteristic of polar environments, leading to slow growth rates and extended lag phases in most microbes. Conventional plate-based methods (CPM) inherently favor fast-growing copiotrophs while suppressing rare or slow-growing lineages. Here we demonstrate that microfluidic droplet technology (MDT) overcomes these fundamental constraints, representing its first systematic application to polar microbiology. By physically isolating individual cells into nanoliter-scale bioreactors, MDT mitigates interspecific competition, thereby releasing slow-growing and oligotrophic taxa that are otherwise outcompeted in bulk cultures. The water-in-oil emulsion format further enables extended low-temperature incubation without evaporative loss or airborne fungal contamination, issues that frequently compromise long-term plate-based cultivation of Arctic samples. Relative to CPM, MDT increased recovery rates by >6-fold and isolation throughput by >180-fold, while markedly enhanced both taxonomic richness and evenness. Exclusively recovered by MDT, the oligotrophic genus Caulobacter and numerous cold-adapted genera underscore that MDT accesses physiologically distinct fractions of the cryospheric microbiome. Furthermore, the integration of near-full-length 16S rRNA gene sequencing with MDT cultivation assessment provided substantially improved phylogenetic resolution for species-level identification and novel taxon delineation. Collectively, these findings establish MDT as a transformative platform for cryospheric culturomics, accelerating the construction of comprehensive polar strain collections essential for understanding cold-adaptation mechanisms and exploiting the biotechnological potential of Earths frozen microbiomes.

microbiology↗

bioRxiv2026

Genomic architecture and adaptive plasticity of Enterococcus lactis strains isolated from extreme Semi-arid environments

The presence of Enterococcus lactis in semi-arid "resource islands" the remarkable ecological plasticity of a species often associated with host-related environments. Characterizing the genomic mechanisms that facilitate its persistence in extreme edaphic niches is crucial for exploring its biotechnological potential in arid agriculture. This study characterized the genomic architecture, abiotic stress tolerance, and plant growth-promoting (PGP) capabilities of six E. lactis strains isolated from the rhizosphere of Pithecellobium dulce and Haematoxylum brasiletto in La Guajira, Colombia. We compared the pangenomes of the isolates with clinical and environmental reference strains. Genomic predictions were validated through in vitro assays for thermal, saline, and pH stress, PGP traits, and biosafety (hemolysis, biofilm formation). Analysis revealed a pangenome with a conserved 2,113-gene core and a highly plastic 3,134-gene accessory genome. The core genome encodes robust machinery for osmotic stress (e.g., opuA-C operons) and DNA repair (uvrC), while the accessory genome is heavily shaped by Horizontal Gene Transfer, containing abundant Mobile Genetic Elements (6.3%-16.4%). Phenotypically, strains exhibited high resilience to heat (50{degrees}C), salinity (5% NaCl), and alkalinity (pH 12). Adaptation in these isolates favors metabolic parsimony: rather than complex phytohormone synthesis, the strains prioritize inorganic phosphate solubilization (conserved pst system) and harbor a complete 2,3-butanediol cluster for volatile-mediated plant interaction. Notably, strain IS_B39 produced siderophores and carried a specific RiPP-like biosynthetic cluster, indicating niche-specific functional diversification. Genomic and phenotypic screening confirmed a safe profile, lacking key virulence factors. These findings define a robust, low-risk genomic toolkit, supporting the potential of E. lactis as a tailored bioinoculant for sustainable agriculture in extreme, water-limited environments. ImportanceEnterococcus species are traditionally studied as clinical pathogens or dairy-associated bacteria, leaving their ecological role in natural, non-host environments largely overlooked. This study challenges conventional paradigms by exploring Enterococcus lactis strains naturally persisting in the extreme, water-limited soils of semi-arid "resource islands" in La Guajira, Colombia. Through functional genomics and laboratory validation, we demonstrated how these bacteria utilize a specialized genetic toolkit to withstand extreme heat and alkalinity, while actively promoting plant resilience. Rather than relying on complex hormone production, they optimize vital nutrient uptake like phosphorus. These findings significantly advance environmental microbiology by uncovering the hidden survival strategies of lactic acid bacteria in arid lands, showcasing their immense potential as sustainable bioinoculants to support global dryland agriculture under climate change stress.

microbiology↗

bioRxiv2026

Divergent specificity of PatA, GabT, and IlvE defines the branched transamination of Nε-carboxymethyllysine and its metabolite Nε-carboxymethylcadaverine in Escherichia coli

Thermal food processing generates N{varepsilon}-carboxymethyllysine (CML), a key advanced glycation end product (AGE) and marker of the Maillard reaction in food. Escherichia coli utilizes CML as a nitrogen source. While SpeC initiates degradation by decarboxylating CML to N-carboxymethylcadaverine (CM-Cad), the enzymes liberating the nitrogen remained unknown. Here, we identify PatA, GabT, and IlvE as the glutamate-dependent transaminases responsible for CML and CM-Cad transamination. Our results reveal a branched metabolic network rather than a linear pathway: PatA shows specificity towards both substrates, while GabT and IlvE selectively process CM-Cad and CML, respectively. We further demonstrate that the carboxymethyl piperideinium ion (CM-Pip) is formed spontaneously following CM-Cad transamination and reveal the previously unknown carboxymethyl-tetrahydropicolinic acid (CM-THPA) as novel metabolite in CML metabolism. Combining molecular microbiology, biochemistry, and analytical chemistry, we demonstrate that these transaminases are essential for integrating dietary CML into bacterial nitrogen metabolism, providing a model for microbial AGE processing via underground metabolism.

microbiology↗

bioRxiv2026

The ultrasmall ocean microbiome: a reservoir of microbial diversity and nitrogen fixation.

Marine microbiology has long considered ultrasmall (<0.2 {micro}m) size fractions as virus-dominated, yet emerging evidence suggests potential prokaryotic activity with implications for global biogeochemical cycles. Here, we present the first comprehensive genome-resolved survey of this fraction, built on 4,058 metagenome-assembled genomes (MAGs) representing 1,152 species across Bacteria (90.7%) and Archaea (9.3%), dominated by Pseudomonadota, Bacteroidota, and Nanoarchaeota. Over 490 non-redundant MAGs occurred exclusively in the ultrasmall fraction, with 66.8% representing novel taxa, particularly abundant in Arctic surface waters. Beyond known ultrasmall organisms (e.g., DPANN archaea and Patescibacteria), which show expected genomic and metabolic reduction, functional annotation revealed diverse metabolic potential across nitrogen, sulfur, and carbon cycling. Focusing on nitrogen fixation, we identified 13 non-cyanobacterial diazotroph species with complete nitrogenase operons spanning four bacterial classes. These resolved into two ecotypes: cosmopolitan low-abundance taxa, and Arctic ultrasmall-restricted strains reaching up to 16.7% relative abundance. Two lineages (50-400-T64 and Novosphingobium) showed ultrasmall exclusive bipolar distributions across Arctic and Southern Oceans. Beyond nitrogen fixation, these diazotrophs encoded pathways for denitrification, DNRA, sulfur oxidation, and carbon fixation. Our findings reveal an overlooked reservoir of microbial diversity including diazotrophs in the ultrasmall ocean microbiome, with significant implications for polar nitrogen budgets and global biogeochemical models.

microbiology↗

bioRxiv2026

Incremental AMR acquisition driving successive genotype replacements and the rise of extensively drug resistant (XDR) Shigella sonnei in Australia over 20 years.

BackgroundIn Australia, the burden of shigellosis is predominantly in returning travellers or in men who have sex with men (MSM). Here, we combine genomic data with comprehensive epidemiological data on sexual exposure and international travel to explore population dynamics of Shigella sonnei and the expansion of multi-drug resistant (MDR) and extensively drug-resistant (XDR) sub-lineages. MethodsA population-level study of all cultured Shigella sonnei isolates in the state of Victoria, Australia, was undertaken between January 2002 and December 2024. Antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatic analyses of 1,305 Shigella sonnei isolates were performed at the Microbiological Diagnostic Unit Public Health Laboratory. Enhanced metadata on source attribution including travel and sexual exposure were collected through surveillance forms or by interviews. ResultsThis study highlights significant shifts in Shigella sonnei cases in Victoria from sensitive strains to MDR and then XDR, particularly in the MSM-associated groups but also associated with a large point source outbreak. We describe an historical pattern of shifting genotype prevalence, and replacement to more varied and higher proportions of antimicrobial resistance over the last decade, resulting in the establishment of two distinct but highly concerning XDR sub-lineages within Victoria. ConclusionsOur genomic-epidemiological analyses highlight that drug-resistant Shigella sonnei remains an ongoing public health threat, and the importance of ongoing surveillance. We determined local evolutionary trajectories and identified expanding sub-lineages that informed shifts in clinical management and antimicrobial recommendations over time, including the use of azithromycin and carbapenems. Placing these local dynamics within the broader global epidemiology, we link how regional evolution interconnects with international dissemination, proving valuable context for guiding local, national and global strategies for prevent outbreaks and antimicrobial resistance.

microbiology↗

bioRxiv2026

Evidence that the Kuenenia stuttgartiensis encapsulin does not protect against NO damage

A paradigm shift is underway in microbiology: many prokaryotes, long considered to lack the compartmentalization present in all eukaryotic life, have been found to possess a great diversity of protein based intracellular compartments. Notably, the genomes of many marine and freshwater anaerobic ammonium oxidizing (anammox) bacteria encode one of these compartmentalization strategies; encapsulin nanocompartments. These systems structure suggests a role for anammox encapsulins in the anammox metabolism, a process of global biogeochemical significance, which results in the loss of biologically available nitrogen from aquatic environments. Here we test if the most common anammox encapsulin architecture could provide a mechanism to detoxify NO, one of the reactive intermediates produced in the core anammox metabolism. Through experiments in which the Kuenenia stuttgartiensis encapsulin was heterologously expressed by an inducible plasmid in E. coli, we show evidence that suggests the K. stuttgartiensis encapsulin provides no protection from NO.

microbiology↗

bioRxiv2026

Industrialization restructures the domestic dog gut microbiome while preserving host specificity

Industrialization has reshaped human gut microbiomes, but its effects on other human-associated mammals remain poorly understood. Domestic dogs provide an informative comparative system because they have shared human environments and food systems for millennia yet retain distinct host biology. However, most canine microbiome studies have focused on industrialized companion animals, limiting our understanding of the ecological range of the domestic dog gut microbiome. We analyzed fecal 16S rRNA gene profiles from 261 dogs sampled across Nepal, Thailand, the United Arab Emirates, and the United States, spanning forager, agrarian, pastoralist, urban, and industrialized lifestyles; 257 dogs remained after excluding recent antibiotic exposure. Lifestyle was the strongest measured correlate of canine gut microbiome composition, and this structure persisted in restricted analyses of mature, non-shelter dogs sampled from temperate climate regions. Industrialized dogs differed from non-industrialized dogs through directional genus-level turnover, restructuring of VANISH- and BloSSUM-like microbial guilds, and shifts in predicted functional potential. Non-industrialized dogs were not microbiologically uniform: pastoralist dogs carried non-industrialized microbiome profiles but diverged from a simple forager-to-industrialized continuum. Cross-species comparisons with humans sampled across matched lifestyle categories showed parallel lifestyle-associated restructuring in both hosts, but host species remained the dominant axis of variation and the genera responding to industrialization were largely host-specific. These findings expand the ecological baseline for the domestic dog gut microbiome and identify industrialization as a major axis of microbiome restructuring in a long-term human-associated mammal. More broadly, they show that shared lifestyle transitions can impose parallel ecological pressures across host species without overriding host-specific community assembly.

microbiology↗