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bioRxiv2025

''Citrobacter bitternis'' Ko et al., 2015 and ''Kluyvera intestini'' Tetz and Tetz, 2016 are heterotypic synonyms of Phytobacter diazotrophicus Zhang et al., 2017

Members of Phytobacter, a recently described genus within the family Enterobacteriaceae, are still frequently misclassified as species of other genera. Here, we present genomic evidence that the taxa "Citrobacter bitternis" and "Kluyvera intestini", as cited in the literature and listed in NCBI databases, are heterotypic synonyms of Phytobacter diazotrophicus. Comparative analyses of 16S rRNA gene phylogeny, core genome phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) consistently place the type strains of both "C. bitternis" and "K. intestini" within the species P. diazotrophicus. Recognizing these taxa as synonyms will help resolve persistent taxonomic inconsistencies and reduce misidentification in clinical and environmental microbiology.

microbiology↗

bioRxiv2025

Chromoprotein-modified plant pathogenic bacteria: tools for experimental tracking and visualization

Microbiology research often requires tracking of specific bacterial strains within a host infection system or in the environment, as well as differentiation of strains in a co-infection or microbe-microbe interaction scenario. Various tools are used for this purpose including antibiotic resistance marker genes, fluorescent proteins, DNA sequence-based methods, and phenotypic markers. Chromoproteins produce intense pigmentation visible in ambient light, and are a unique option for bacterial tracking that does not require use of antibiotics, specialized equipment, or DNA sequencing. Development of traceable bacterial strains across a wide range of species is important to facilitate the investigation of challenging research questions and expand our understanding of microbial dynamics in complex environments. In this study, different species of plant pathogenic bacteria (Xylella fastidiosa, Pantoea stewartii, Pseudomonas syringae and Xanthomonas campestris) were modified with a set of chromoproteins and tested in plant infection assays to evaluate chromoprotein stability and impact on bacterial pathogenicity. Chromoprotein modification by chromosomal insertion was highly successful in X. fastidiosa, and stable during infection in grapevines. Plasmid-based expression of chromoproteins in P. stewartii, P. syringae, and X. campestris had mixed results depending on the specific species-chromoprotein combination. Overall, these results provide some successful chromoprotein-modified plant pathogen strains for use by the research community, as well as insight into which chromoproteins might be best utilized in different bacterial species. ImportanceInvestigation of challenging research questions in plant health requires availability of the necessary microbial tools. This study adapts chromoprotein-modification for bacterial tracking of plant pathogens during host infection and plant-plant transmission. Successful development of visibly colored strains of Xylella fastidiosa, Pantoea stewartii, Pseudomonas syringae, and Xanthomonas campestris provides a useful resource for research and educational purposes, as well as insight into optimization of chromoprotein expression in a range of plant pathogen species.

microbiology↗

bioRxiv2025

Uncovering the Hidden Yeast Diversity in Fermented Coffee: Insights from a Shotgun Metagenomic Approach

Yeasts play a pivotal role in coffee fermentation, shaping microbial succession and contributing to the development of final flavor profiles. Despite their importance, yeast taxonomy in this context remains poorly resolved. Traditional classification methods often result in misidentifications due to the limited resolution of classical microbiological techniques and the rapidly evolving taxonomic framework driven by advances in phylogenomic. Moreover, the diversity of budding yeasts in coffee fermentations remains underexplored using high-resolution approaches such as metagenomics. To address this gap, we applied a shotgun metagenomic strategy and reconstructed metagenome-assembled genomes (MAGs) from multiple coffee fermentation samples and, using a robust phylogenomic framework based on 832 conserved single-copy genes. We confidently classified 24 yeast MAGs within the subphylum Saccharomycotina. These included well-known taxa such as Pichia kluyveri, Hanseniaspora spp., Torulaspora delbrueckii, and members of the Kurtzmaniella clade. Most MAGs were placed in strongly supported monophyletic groups (ultrafast bootstrap = 100), with short intra-clade branch lengths indicative of intraspecific variation. Pichia kluyveri emerged as the most abundant and widespread species, detected in all analyzed metagenomes, followed by Hanseniaspora spp. Our results underscore the power of high-resolution phylogenomic for classifying yeast MAGs and highlight the ecological importance of Pichia, Hanseniaspora, Torulaspora, and Kurtzmaniella in spontaneous coffee fermentations.

microbiology↗

bioRxiv2025

Genome-wide screening using TraDIS accelerates identification of key adaptive mutations of longer-term evolution experiment in Escherichia coli.

Long term laboratory-based evolution experiments are a powerful tool that are increasingly being used to study fundamental aspects of evolution and to identify genes that contribute to overall fitness under different conditions. However, even with automation, the time that they take to execute limits the extent to which evolution experiments can be used as part of a high throughput approach to understand the links between genotype and phenotype. Mutations that lead to genetic loss of function are frequently selected for in evolution experiments. Thus in principle these experiments could be done more rapidly by starting not with clonal isolates but with dense transposon libraries that will contain loss of function mutations in all non-essential genes. Here, we test this hypothesis by comparing the results of long term (5 month) evolution experiment, in which E. coli was grown with daily transfers in unbuffered LB starting at pH 4.5, with short term (5 and 10 day) experiments on a high density transposon library in the same strain and under the same conditions. We show that there is very significant overlap in the genes and pathways identified using the two methods. Use of this approach thus has the potential to significantly increase the throughput of laboratory-based evolution and enable rapid testing of a wide range of parameters that may have an impact on evolutionary trajectories. Author summaryUnderstanding how bacterial populations adapt to environmental stress is central to microbiology and evolutionary biology. Laboratory evolution experiments are commonly used to uncover the genetic changes that confer increased fitness, but these experiments are often slow and laborious. In this study, we asked whether the results of long-term adaptive laboratory evolution (ALE) could be predicted using a faster method: short-term selection of a dense transposon mutant library, analysed by transposon-directed insertion site sequencing (TraDIS). We evolved E. coli K-12 MG1655 in unbuffered LB at pH 4.5 for 5 months and compared the mutations that arose to those selected after just 10 days of evolution using a high-density transposon library in the same strain and conditions. We observed significant overlap in the genes identified by both approaches, including independent disruptions in shared regulatory pathways. This suggests that short-term selection on a diverse mutant population can uncover many of the same adaptive changes seen in long-term evolution experiments. We also identified and validated the fitness effects of several mutations uniquely found in the transposon approach. Rather than predicting specific mutations from first principles, this method offers a rapid, empirical means of anticipating which genes are likely to be involved in adaptation under defined conditions, helping to guide further mechanistic studies by offering a powerful shortcut for investigating microbial evolution.

microbiology↗

bioRxiv2025

A genotoxin associated with colorectal cancer linked to gut dysbiosis in children with cystic fibrosis

Cystic fibrosis (CF) substantially alters the gastrointestinal microbiome from an early age, leading to significant changes in microbial composition and functionality. This study explores the physiological and microbiological factors contributing to dysbiosis in children with cystic fibrosis (cwCF), characterized by an increase in potentially pathogenic Escherichia coli and a decrease in beneficial anaerobes such as Bacteroides. In this study, we employed an in vitro medium representative of the nutritional environment of the CF colon to test the role of factors including mucin, fat, bile, pH, antibiotics and features associated with inflammation (e.g., nitrate, sulfate, formate, reactive oxygen species) on growth of clinical isolates of E. coli and Bacteroides spp. We further examined interactions between these two microbes under CF-like conditions to understand modulators of microbial competition, and identified glycerol, a surrogate of increased fat, as a significant driver of altered microbial competition. Finally, we investigated genetic determinants influencing these microbial interactions, with the focus on glycerol metabolism, by performing a transposon mutagenesis screen in E. coli. Results of this screen pointed to the role of colibactin production in mediating this microbial competition; colibactin is a DNA-damaging genotoxin associated with the increased risk of colorectal cancer (CRC) in CF populations. This work enhances our understanding of mechanisms of microbial competition in the CF gut, while potentially enhancing our understanding of colorectal cancer risk in persons with CF through the identification of early-life microbial biomarkers. Significance StatementThe risk of CRC development in CF populations is significantly increased. This study examines the interplay of altered intestinal physiology in the microbial dysbiosis common in the CF gut, implicating the high fat environment in a competition-mediated depletion of immune-modulating Bacteroides. This work identifies candidate features of the young CF intestine and gut microbiome that may contribute to advanced development of CRC in these populations, informing potential therapeutic approaches.

microbiology↗

bioRxiv2025

Biogeographical and phylogenetic constraints on horizontal gene transfer and genome evolution in Streptomyces

The role of horizontal gene transfer (HGT) in shaping bacterial genomes is well recognized, but constraints on gene exchange and the degree to which these constraints shape genome evolution remain poorly described. In this study, we sought to determine whether geographic and phylogenetic distance constrains HGT within and between bacterial species. To address this question, we isolated strains (n = 17) of two closely related bacterial species, Streptomyces griseus and Streptomyces pratensis from two ecologically similar sites. We identified homologous recombination events within the core genomes of these species (557 recent and 457 ancient) and determined that patterns of recombination were constrained primarily by phylogeny rather than geography. Notably, shell accessory genes were over three times more likely to be shared between the same species than with non-related geographical neighbors. The richness of secondary metabolite gene clusters is highly variable with an average of 35 - 55 clusters per genome, depending on clade membership. The majority of secondary metabolite gene clusters (60%) are found in all strains, indicating that they were present in the most recent common ancestor of S. griseus and pratensis. We conclude that most HGT in the core and accessory genome is phylogenetically constrained, while HGT of shell genes is more likely constrained by geography. This outcome indicates that the predominant mechanisms of HGT favor high phylogenetic relatedness, and that rapid gene acquisition and loss in the accessory genome could aid with adaptation to contemporary environmental conditions. ImportanceHorizontal gene transfer (HGT) is a vital ecological and evolutionary force in microbiology, but we still lack a precise understanding of how precisely HGT acts on the gene pool of a species or genus. While HGT can complicate phylogenetic analyses and assumptions of homology, its role in adaptation and acquiring secondary metabolites should not be overlooked. Microbial ecologists agree that the pangenome is a shifting collection of genes that can be influenced by both vertical inheritance and ecological factors. This study examines how the Streptomyces pangenome is shaped by these two forces and offers an important quantitative insight into how HGT shapes bacterial genome dynamics.

microbiology↗

bioRxiv2025

Revealing Community Dynamics in Polymicrobial Infections through a Quantitative Framework

Laboratory models provide tractable, reproducible systems that have long served as foundational tools in microbiology. However, the extent to which these models accurately mimic the biological environments they represent remains poorly understood. A quantitative framework was recently introduced to assess how well laboratory models capture microbial physiology in situ. However, applications of this framework have been limited to characterizing the physiology of a single species in human infections, leaving a gap in our understanding of overall microbial community physiology in polymicrobial contexts. Here, we extended this framework to evaluate the accuracy of laboratory model systems in capturing community-level functions in polymicrobial infections. As a proof of concept, we applied the extended framework to a polymicrobial model of human chronic wounds (CW) infection. CWs harbor metabolically diverse bacterial species that engage in a range of microbe-microbe interactions, ultimately impacting community dynamics and disease progression. However, studies on the mechanistic drivers of chronic wound infection have relied on single species or pairwise approaches. Here, we demonstrate that our adapted framework can be used to develop accurate polymicrobial models. Further, we demonstrate that this extended framework can be used to evaluate the occurrence of known microbe-microbe interactions. Building on our prior work in large-scale metagenomic and metatranscriptomic analysis, we propose a highly accurate 6-member synthetic bacterial community model that is representative of the taxonomic and functional complexity of human CW infections. This approach will support the development of ecologically relevant polymicrobial models and the development of better treatment strategies.

microbiology↗

bioRxiv2025

Analyses of protein expression and genetic fitness determinants reveal dynamic pathways active in starved Pseudomonas aeruginosa

Heterotrophic bacteria rapidly deplete essential macronutrients during growth and must navigate subsequent periods of growth arrest imposed by starvation. Nutrient limitations can be dynamic in nature, requiring ongoing regulatory adjustments involving new protein synthesis despite total biosynthetic activities being dramatically lower than during growth. Here, we have characterized the responses of the opportunistic pathogen Pseudomonas aeruginosa to prolonged starvation for carbon or nitrogen sources, and to transitions between these states. We find that most cells survive both types of starvation for more than a week and maintain low but robustly detectable levels of protein synthesis in the absence of growth. Nitrogen-starved cells are larger, make more proteins and retain fewer ribosomes than carbon-starved cells, indicating that distinct physiological strategies are adopted during the two starvation types. We found that the newly synthesized proteomes of each starvation type are distinct, although many of the most highly synthesized proteins are shared between both conditions. Interestingly, we observed a temporary burst of protein synthesis as cells were transitioned between the two starvation conditions, which may reflect active remodelling of the proteome during growth arrest. We also used transposon insertion sequencing to identify genes impacting fitness in both starvation conditions and during transitions between the two and found that a highly overlapping set of global regulators most strongly influenced survival. Combining these datasets, we highlight proteases and chaperones; flagellar motility; and the nitrogen-related phosphotransferase system as key fitness-impacting functions that are actively maintained by growth arrested Pseudomonas aeruginosa. ImportanceMolecular microbiology has traditionally focused on exponential growth in model organisms as the preferred context in which to study bacterial physiology, especially the regulation of new protein synthesis. However, in natural environments, including many infection contexts, Proteobacteria frequently enter growth arrest due to nutrient limitation. The dynamics and regulation of protein synthesis in growth-arrested cells remain poorly understood, especially in pathogens. Furthermore, growth arrest increases tolerance to a variety of stresses, including many clinically used antimicrobials. We have conducted a comprehensive exploration of the proteins being made by growth arrested Pseudomonas aeruginosa during total nitrogen or carbon starvation and at the transition between these two starvation types, and the genes supporting fitness under these conditions. These datasets suggest dynamic redistribution of resources among important cellular functions and will serve as a resource for further investigations of starvation-induced growth arrest, a ubiquitous but understudied physiological state of heterotrophic bacteria.

microbiology↗

bioRxiv2025

Enhancing the biocorrosion resistance and biocompatibility of Aluminium substrates using Graphene Oxide-PEDOT:PSS Hybrid Coating

Biofilm-associated infections on medical devices remain a major clinical challenge due to antimicrobial resistance, biofouling, and biocorrosion, compromising implant longevity and biocompatibility. Conventional biomedical devices utilizing stainless steel and titanium exhibit limited resistance to biofilm-prone physiological environments, necessitating development of next-generation implant materials. While alumina (Al2O3) is favored for its bio-inertness, it raises concerns like leaching and systemic toxicity. Aluminum alloy 1050 (AA1050) offers intrinsic corrosion resistance via a passive oxide layer under dry conditions but remains prone to microbiologically influenced corrosion (MIC) under humid conditions. In the recent years, graphene family of materials have emerged as promising surface coating for metals and alloys due to their strong barrier properties and antimicrobial efficacy. Most graphene derivatives like GO require relatively higher concentrations to achieve antimicrobial activity, however compromising biocompatibility, limiting in vivo human uses. This study addresses these caveats by exploring GO activity at lower concentrations (50-500 {micro}g/ml) on AA1050 to achieve a balance between antimicrobial efficiency and cytocompatibility. Additionally, poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS, 1 {micro}g/ml) was integrated with GO to form a hybrid coating on Al (Al_GO/P), to improve GO adhesion, and corrosion resistance. Biological evaluations against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) demonstrated superior antifouling and antimicrobial efficacy of Al_GO/P substrates compared to GO-coated Al (Al_GO) and bare Al surfaces. Corrosion rate, FESEM, ICP-MS, and cytotoxicity analyses further confirmed reduced biocorrosion, minimal ion leaching, along with enhanced biocompatibility of the hybrid coated surfaces. Al_GO/P containing GO at 100 and 250 {micro}g/ml concentration achieved the optimal balance between antimicrobial activity and biocompatibility and can be used as in vivo implant materials. Hence, this technology can be implemented towards surface modification of biomedical devices to mitigate periprosthetic infections as well as support ex vivo applications requiring durable antimicrobial performance.

microbiology↗

bioRxiv2025

Fluorescence in situ hybridization (FISH) and detection of Bacteria and Archaea cells in fecal samples

Oligonucleotide probes have been used to detect bacteria and archaea that colonize the cattle and pig digestive system. The Fluorescent in situ hybridization (FISH) is a molecular biology technique that uses oligonucleotides of 15 to 25 nucleotides of length associated with a fluorescent molecule. In microbiology, the FISH technique utilizes probes targeting ribosomal RNA (rRNA). It is one amongst others, a staining technique that allows the identification, detection and quantification of microorganisms without prior cultivation by means of epifluorescence and confocal laser scanning microscopy (CLSM). In this study, we describe the usage of the confocal laser scanning microscopy coupled with fluorescence in situ hybridization (FISH), in order to detect and quantify of bacteria and archaea in fecal samples from cattles manure and swine slurry.

microbiology↗

bioRxiv2025

Shifts in ruminant fermentation during inhibition of methanogenesis are reflected in the isotope compositions of volatile fatty acids.

Ruminant animals are a major source of the potent greenhouse gas methane, but they are also a tractable target for climate solutions. Several strategies have been developed to lower methane emissions from ruminants, including feed additives that inhibit methanogenic archaea. Sustainable solutions must eliminate methane emissions without hampering the microbial fermentation of plant material, which the animal host relies on for carbon and energy. However, current tools cannot directly quantify or characterize the metabolic pathways of in vivo ruminant fermentation. To fill this gap, we developed an electrospray (ESI) Orbitrap mass spectrometry technique to measure the stable isotope ratios (13C /12C and 2H/1H) of volatile fatty acids (VFAs) at their natural isotopic abundances directly from rumen fluid. We tested this technique on in vitro incubations of rumen fluid fed three different substrates with and without the additive Asparagopsis taxiformis. We found that the isotope composition of VFAs changed and reflected a remodeling of microbial fermentation pathways. Specifically, acetates{delta} 13C value increased when methanogens were inhibited, suggesting a lower relative rate of acetate synthesis and a lack of acetogenic activity. Furthermore, the{delta} 2H value of propionate decreased, which may indicate a change in the balance between the two pathways of propionate synthesis toward the less energetic acrylate pathway. Both signals were consistent across feed types. Taken together, our results provide evidence that fermentative metabolism is remodeled during methanogenesis inhibition and decreases relative fluxes through ATP-generating pathways. More broadly, this study demonstrates the utility of ESI-Orbitrap-based isotopic analysis for studying rumen microbial ecology. IMPORTANCESlowing methane production from ruminant animals (e.g. cows) is a major target for mitigation of anthropogenic climate change. While strategies that eliminate microorganisms producing methane have been successful, they have cascading impacts on the microbial ecology of the rumen, possibly affecting animal health and productivity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/682381v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1368f20org.highwire.dtl.DTLVardef@ee87baorg.highwire.dtl.DTLVardef@10f714org.highwire.dtl.DTLVardef@1cd4f6a_HPS_FORMAT_FIGEXP M_FIG C_FIG Of particular importance is microbial fermentation, which generates easily digested volatile fatty acids (VFAs) from hard-to-breakdown plant matter. To better understand how fermentation responds to methane mitigation strategies, we measured the isotope composition of VFAs in cow rumen. Our results indicate that fermentation changes pathways when methane production is inhibited to those that generate less energy for the cell. As methane mitigation strategies are developed in the coming decade, isotopic analysis of VFAs may be a useful and accessible contribution to our understanding of rumen microbiology.

microbiology↗

bioRxiv2025

mAIcrobe: an open-source framework for high-throughput bacterial image analysis

Quantitative analysis in bacterial microscopy is often hindered by diverse cell morphologies, population heterogeneity, and the requirement for specialised computational expertise. To address these challenges, mAIcrobe is introduced as an open-source framework that broadens access to advanced bacterial image analysis by integrating a suite of deep learning models. mAIcrobe incorporates multiple segmentation algorithms, including StarDist, CellPose, and U-Net, alongside comprehensive morphological profiling and an adaptable neural network classifier, all within the napari ecosystem. This unified platform enables the analysis of a wide range of bacterial species, from spherical Staphylococcus aureus to rod-shaped Escherichia coli, across various microscopy modalities within a single environment. The biological utility of mAIcrobe is demonstrated through its application to antibiotic phenotyping in E. coli and the identification of cell cycle defects in S. aureus DnaA mutants. The modular design, supported by Jupyter notebooks, facilitates custom model development and extends AI-driven image analysis capabilities to the broader microbiology community. Building upon the foundation established by eHooke, mAIcrobe represents a substantial advancement in automated and reproducible bacterial microscopy.

microbiology↗

bioRxiv2025

Defining the Mycobacterium tuberculosis Pangenome and Suggestions for a New Composite Reference Sequence

Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), a global disease with diverse clinical and microbiological manifestations. Studies into the biological causes of this phenotypic diversity have been largely limited to a few reference strains. A pangenome approach is likely to provide new insights. Pangenomic tuberculosis studies have been limited the availability of only fragmented genome sequences and error-prone reference genomes. We used a de novo assembly pipeline that generates extremely complete and accurate whole genome sequences to generate 50 closed Mtb genomes across all seven major lineages. We identified 3,377 core gene clusters and 379 accessory clusters. Analysis showed multi-copy core clusters were largely due to gene fragmentation (76%), paralogs (12%), nearly identical gene duplications (4%), or combinations (8%). Sixteen hypervariable regions (HVRs) were identified, including novel paralogs and variable PE/PPE genes. We consolidated these findings into a Pangenome Gene Reference Resource (PGRR) for precision alignment. Our study demonstrates the closed nature of the Mtb pangenome, with most variation in accessory genes and HVRs. The PGRR provides a foundation for improved drug/vaccine target discovery and highlights the need to move beyond the commonly used H37Rv strain to study Mtb genetic and phenotypic diversity. IMPORTANCETuberculosis (TB), caused by Mycobacterium tuberculosis, affects millions globally. Genetic differences among Mtb strains have been difficult to resolve due to incomplete genome references. We sequenced and analyzed complete genomes of 50 Mtb strains from all lineages, identifying 16 hypervariable regions and 3,498 core gene clusters whose diversity mostly stemmed from gene fragmentation, paralog duplication and deletion events and differences in the PE/PPE gene family representation. These differences may explain many of the varied clinical manifestations of TB. We created Pangenome Gene Reference Resource to unify genetic data for precise comparison studies to aid in developing new drugs vaccines and other interventions against this disease.

microbiology↗

bioRxiv2025

Unraveling yeast diversity in food fermentation using ITS1-2 amplicon-based metabarcoding

A detailed characterization of the microbial ecosystem involved in the production processes of fermented foods is essential. Although fermented foods are an important part of the human diet and have seen an increasing interest nowadays, some challenges still need to be solved. Specifically, yeast identification through culture-independent methodologies is still limited to the genus level. Unlike for bacterial species identifications, long-read sequencing technologies have barely been used for yeast species identification, and, to the best of the authors knowledge, it has not been validated with mock communities reflecting food fermentation processes yet. Therefore, in the current study, we present an amplicon-based metabarcoding approach targeting the full-length internal transcribed spacer (ITS) region comprising the ITS1, 5.8S rRNA gene, and ITS2 using the PacBio HiFi sequencing platform. This method was validated using mock communities composed of yeast species involved in sourdough, lambic beer, and cocoa fermentation processes. Accurate species-level identification was achieved for most of the species. However, special attention should be given to Saccharomyces-rich niches, as accurate species-level identification for this genus is still challenging. Furthermore, underestimation of the relative abundance of species with short ITS regions, such as Pichia and Brettanomyces, occurred. In addition, the method was successfully applied to describe the yeast diversity present in two sourdough and two lambic beer samples. Overall, the current method provides an unprecedented way of determining the species-level yeast composition of complex ecosystems present in fermented food products. ImportanceTo date, species-level identification of common yeasts present in food fermentation ecosystems has been difficult, if not impossible, when using short-read sequencing methods. However, species-level identification is essential when evaluating and describing the characteristics of fermented food microbiomes. The current study reports on the development and validation of an amplicon-based metabarcoding approach combined with long-read PacBio HiFi sequencing targeting the full ITS region, comprising the ITS1 and ITS2 regions as well as the 5.8S rRNA gene. The described methodology enables species-level identification of the most common yeasts present in food fermentation ecosystems. This new methodology is of importance for all researchers in the field of fermented foods. By extension, researchers in other fields of microbiology can find inspiration in this paper.

microbiology↗

bioRxiv2025

Enterococcus faecium colonization and persistence in a model of diabetic wound infection

Chronic wound infections are a common comorbidity of diabetes mellitus and can progress to amputation if untreated, yet effective strategies to manage these infections are limited. Commensals such as Enterococcus faecium and Staphylococcus epidermidis can transition into opportunistic pathogens when host defenses are compromised, underscoring the complexity of chronic wound microbiology. E. faecium, particularly vancomycin-resistant strains, are an understudied, clinically important cause of chronic diabetic wound infections. Using a low-dose streptozocin-induced diabetic mouse model, we characterized E. faecium wound infection dynamics and identified differences in colonization and clearance compared to non-diabetic animals. At eight hours post infection (hpi), control mice exhibited higher E. faecium wound colony forming units (CFU) than diabetic mice, but cleared the infection more efficiently, resulting in similar CFU by 24 hpi. By contrast, diabetic mice showed impaired clearance, with elevated CFU persisting through 72 hpi. In mixed species infection with S. epidermidis, S. epidermidis CFU increased at 72 hpi while E. faecium CFU remained comparable to single species infection. Despite strong initial cytokine and neutrophil responses, E. faecium persisted in all wounds. Sustained neutrophil recruitment at 72 hpi occurred only in diabetic mice, whereas macrophage accumulation increased from 24 to 72 hpi in all wounds, including sterile controls. Histological analysis showed epithelial hyper-thickening in both groups, indicating that diabetes and E. faecium each contribute to impaired wound healing. This study establishes a diabetic mouse model of E. faecium wound infection and suggests that E. faecium modulates innate immune responses to persist in the wound bed.

microbiology↗

bioRxiv2025

Biological insights and methodological challenges learned from working with a diverse heterotrophic marine bacterial library

BackgroundOrganized collections of bacterial strains can help bridge the gap between studying model organisms and communities, through comparative experiments between genetically and phenotypically diverse strains. We describe the establishment and initial characterization of a library of 62 marine heterotrophic bacteria, selected to represent a significant fraction of the genome-encoded functional diversity and a wide range of known phytoplankton-bacteria interactions. We focus on important but often undiscussed aspects of collecting and maintaining such a library, verifying strain identity, and applying classical microbiological methods across diverse strains. ResultsCultured strains contain up to hundreds of mutations compared with the reference genomes, with non-synonymous mutations in rpoB and/or rpoC genes observed in [~]15% of the cultures. Most strains grow well at 25{degrees}C, but the dependence of growth rate on temperature and the width of the temperature niche vary between strains in a systematic manner. We describe steps towards designing a universal, defined, minimal media for marine bacteria, revealing that growth inhibition on amino acids and peptides by carbohydrates is widespread. Cell counts obtained from flow cytometry and colony plating differ systematically, as do different methods to assess motility. Finally, we discuss traits potentially related to microbial interactions such as hemolysis, biofilm formation, and antibiotic resistance. Gammaproteobacteria such as Alteromonas, Pseudoalteromonas, and Vibrio reveal consistently robust growth, and activity, perhaps explaining why these clades are well-explored. ConclusionExplicitly discussing the insights and challenges of working with strain libraries will pave the way to robust, reproducible, and generalizable mapping of bacterial traits across diversity.

microbiology↗

bioRxiv2025

Feeding your enemy's enemy: Acidifying bacteria inhibit pathogenic bacteria more strongly with increasing glucose

Classical microbiology has focused on directly suppressing pathogens using drugs, ignoring other harmless microbial species living alongside the pathogens. We now have a much better understanding of how species interact and affect one anothers growth within microbial communities, for example through chemical production. Here we capitalize on this understanding to demonstrate how one can manipulate and control the strength of interactions between bacterial species, and combine this with antibiotics to fully suppress and eliminate pathogens. Using experiments and a mathematical model, we first show how Citrobacter freundii can reduce the environmental pH to enhance the effect of ampicillin on the pathogen Pseudomonas aeruginosa. This negative interaction from C. freundii to P. aeruginosa can be strengthened by increasing glucose concentrations. Our proof-of-concept approach also worked against other pathogens: Klebsiella pneumoniae and Agrobacterium tumefaciens, and a different commensal: Lactobacillus plantarum, a common probiotic species. Overall, we show that taking advantage of the community and chemical context in which microbes live can help to develop efficient strategies to control them. In the medical context, this approach can help to eliminate pathogens thereby reducing our reliance on antibiotics.

microbiology↗

bioRxiv2017

Chemotaxis allows bacteria to overcome host-generated reactive oxygen species that constrain gland colonization

The epithelial layer of the gastrointestinal tract contains invaginations, called glands or crypts, which are colonized by symbiotic and pathogenic microorganisms and may function as designated niches for certain species. Factors that control gland colonization are poorly understood, but bacterial chemotaxis aids occupation of these sites. We report here that a Helicobacter pylori cytoplasmic chemoreceptor, TlpD, is required for gland colonization in the stomach. tlpD mutants demonstrate gland colonization defects characterized by a reduction in the percent of glands colonized, but not in number of bacteria per gland. Consistent with TlpDs reported role in reactive oxygen species (ROS) avoidance, tlpD mutants showed hallmarks of exposure to large amounts of ROS. To assess the role of host-generated ROS in TlpD-dependent gland colonization, we utilized mice that lack either the ability to generate epithelial hydrogen peroxide or immune cell superoxide. tlpD gland colonization defects were rescued to wild-type H. pylori levels in both of these mutants. These results suggest that multiple types of innate immune generated ROS production limit gland colonization and that bacteria have evolved specific mechanisms to migrate through this gauntlet to establish in the glands.\n\nClassification: Biological sciences; microbiology\n\nSignificance statementMicrobial colonization of the gastrointestinal tract occurs at distinct sites within the tissue including glandular structures found in the stomach and intestine. Multiple lines of evidence suggest that glands supply niches that promote chronic microbial colonization, a process that is critical for symbiotic and pathogenic bacteria to maintain themselves. In this report, we show that host-produced reactive oxygen species (ROS) constrain gland colonization by the gastric pathogen Helicobacter pylori. A bacterial cytoplasmic chemoreceptor, TlpD, allows H. pylori to avoid ROS and enhances H. pyloris ability to colonize a broad swath of glands. We propose that hosts limit gland access and spread by producing ROS, and bacteria counter with chemotactic responses that allow navigation through this gauntlet.

microbiology↗