MicrobiologyOpen

Bacteria require rapid adaptation under fluctuating environmental conditions. Commonly recognized global regulators enable bacteria to respond promptly to external changes, though they are either restricted to specific bacterial taxonomies or physiological statuses, suggesting that additional regulators are required for adaptation. DNA methylation is a reversible modification affecting bacterial gene regulation. However, conventional methods can only detect one DNA methylation form each round, leaving the understanding of DNA methylation in bacterial adaptation mostly unknown. This study aimed to identify genome-wide DNA methylation variation (N6-methyladenine, N4-methylcytosine, and 5-methylcytosine) in Escherichia coli under different culture conditions using Oxford Nanopore sequencing. DNA samples from six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure) during the exponential and stationary phases were extracted. When culture conditions were compared to the normal condition, E. coli exhibited more differentially methylated sites during the exponential phase than in the stationary phase. During the exponential phase, the genes differentially methylated in all conditions were involved in cellular activities, such as cellular and metabolic processes. During the stationary phase, universally differentially methylated genes were associated with oxidation responses. Subsequent analysis found that although DNA methylation analysis was affected by batch effects, some genes (e.g. rpoS) showed consistently differential methylation across datasets. Our findings suggest that the E. coli DNA methylation profile was affected by growth phases and conditions, and DNA methylation profiling by Oxford Nanopore sequencing could be a potential approach for gene activity estimation in environmental samples. ImportanceBacterial DNA methylation is a reversible genetic modification affecting gene regulation, enabling rapid adaptation. Three major forms in bacteria are N6-methyladenine, N4-methylcytosine, and 5-methylcytosine. Using Oxford Nanopore sequencing, we characterized genome-wide variation in these methylation types in Escherichia coli under six conditions (normal, low oxygen, low pH, high temperature, high salt, and recovery after low pH exposure). DNA methylation signatures in E. coli varied with growth conditions. Using the normal condition as a baseline, E. coli during the exponential phase exhibited more differentially methylated genomic loci under stress conditions compared to the stationary phase. Under stress conditions, genes with differential methylation were associated with cellular processes or oxidative responses, depending on the growth phase. Our findings reveal that the DNA methylation signature in E. coli was affected by growth phases and conditions, and Oxford Nanopore-based DNA methylation profiling could be a potential approach for gene activity estimation in environmental samples.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven cyanine monomer and dimer dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms.

Metabarcoding is a powerful tool for biodiversity comparisons, where standard-size DNA barcodes (>500 bases) offer better taxonomic resolution than shorter ones. Still, the choice of sequencing platforms and bioinformatics pipelines may strongly affect inferred diversity due to various technical biases. We assessed the relative performance of Illumina MiSeq i100 (2x500 paired-end), PacBio Revio and Oxford Nanopore MinION sequencing and bioinformatics pipelines, using full-length ITS amplicon sequencing datasets from a 103-species mock community and 45 composite soil samples. Despite numerous low-quality reads, PacBio yielded the lowest overall error rate and highest number of taxa. Illumina revealed the highest proportion of chimeric and index-switched reads, along with a strong bias towards shorter amplicons. MinION data analysed using PRONAME and Minovar - a bioinformatics pipeline presented here - had the largest proportion of low-quality data, and rare taxa were lost during data filtering and read polishing steps. Although Minovar enabled amplicon sequence variant (ASV) level precision for common taxa, we recommend clustering ASVs into OTUs. For PacBio, standard filtering approaches outperformed the ASV approach because they retained rare taxa. For Illumina, a stringent ASV approach or removal of rare OTUs would limit artefacts. Across all platforms, excess PCR cycles promoted chimeric and low-quality reads and lost quantitativity in biodiversity assessments. With moderate differences in effect sizes, all analytical approaches supported the conclusion that sampling design determines how we see soil biodiversity responses to land use. For biodiversity surveys based on the full-length ITS metabarcoding, we recommend using PacBio sequencing with standard, non-ASV pipelines.

Listeria monocytogenes can grow as a saprophyte on decaying plant material, but can also switch to a pathogenic lifestyle. This switch is mediated by the virulence regulator PrfA, which activates the expression of most virulence genes. PrfA activity is tightly regulated by several mechanisms to ensure that virulence genes are only expressed within the host. One of these regulatory mechanisms is the sugar-dependent repression. In the presence of readily metabolizable sugars, which are imported via phosphotransferase systems (PTS) such as cellobiose, PrfA is repressed; however, the precise mechanism is still unknown. Using a sugar screen, trehalose was identified as the first PTS-dependent sugar that supports growth of L. monocytogenes, but does not seem to impact PrfA activity. We demonstrated that the PTS permease TreB is the sole trehalose importer. After import, trehalose-6-phosphate is cleaved by the phosphotrehalase TreA; however, loss of TreA does not fully abolish growth on trehalose suggesting that L. monocytogenes encodes an additional phosphotrehalase. 13C-Labeling experiments revealed that trehalose metabolism is repressed in the presence of glucose, while it can be metabolized in the presence of glycerol. Additionally, these experiments provided evidence that trehalose and cellobiose are metabolized via identical pathways, including glycolysis and the incomplete TCA cycle, although trehalose has a slower uptake and/or metabolization rate. We therefore hypothesize that sugar-dependent PrfA repression correlates with sugar transport and/or consumption rates, potentially due to varying availability of phosphoenolpyruvate (PEP), which serves as both a metabolic intermediate and phosphate donor for PTS-dependent transport.

Naphthenic acids are amphipathic compounds whose toxicity has primarily been attributed to narcosis toxicity to cell membranes. However, few methods exist that specifically study the membrane disruption and toxicity of this complex family of cyclic, polycyclic and acyclic alkyl-substituted carboxylic acids. Here we describe a whole cell biosensor approach that relies on the ability of Pseudomonas aeruginosa, a ubiquitous environmental organism and opportunistic pathogen, to sense membrane damage (narcosis) and induce protective genes to repair and protect the outer membrane. Many classes of membrane disrupting antimicrobials induce the expression of two operons that encode protective defense systems against outer membrane (OM) damage, including antimicrobial peptides, chelators, and detergents. We demonstrate that the pmrF and spdE2 transcriptional lux reporters are induced by exposure to individual NA compounds with diverse structures, as well as mixtures and naphthenic acid fraction compounds (NAFCs). To further support the narcosis hypothesis, we demonstrated that NA permeabilizes the outer membrane to assist in lysozyme killing, and disrupts the inner membrane integrity, allowing uptake of the DNA binding dye propidium iodide. The conventional OM permeability assay that measures NPN fluorescence is not applicable to study NAs, because they stimulate NPN fluorescence in the absence of cells. This narcosis biosensor approach constitutes a rapid and simple method to measure narcosis and could be developed as a novel toxicity indicator of oil sands tailings.

Antimicrobial resistance (AMR) is a major global public health threat, and Enterobacterales producing extended-spectrum {beta}-lactamases (ESBLs) represent some of the most common and concerning pathogens in clinical settings. Importantly, the dissemination of these resistance mechanisms is largely driven by mobile genetic elements (MGEs), particularly plasmids. Advancing our understanding of AMR evolution through experimentation requires moving beyond domesticated laboratory strains and towards clinically relevant isolates. However, despite the abundance of genomic data in public repositories, there is a lack of well-characterised clinical collections available for experimental work. Here, we characterise the RyC collection, which includes 50 multidrug-resistant, ESBL-producing Escherichia coli and Klebsiella spp. strains isolated from the gut microbiota of hospitalised patients at Hospital Universitario Ramon y Cajal (Madrid, Spain). We generated high-quality genome assemblies for all strains using a combination of short- and long-read sequencing technologies. From these data, we performed a comprehensive characterisation of the pangenome, mobilome, resistome and defensome of the collection. We present the RyC collection as a robust and experimentally tractable resource to study AMR evolution and MGEs dynamics in clinically relevant bacterial backgrounds. Impact statementAntimicrobial resistance (AMR) is a growing global health threat driven by the rapid dissemination of resistance genes among clinically relevant bacteria. A major challenge in studying AMR evolution is the reliance on domesticated laboratory strains, which poorly represent the complexity of pathogens circulating in hospitals. Here, we introduce the RyC collection, a set of well-characterised, multidrug-resistant Enterobacterales isolates obtained from hospitalised patients. By combining high-quality genome sequencing with detailed analyses of their gene content and mobile genetic elements (MGEs), this collection provides a realistic and experimentally tractable system to study how resistance evolves and spreads. The RyC collection will facilitate research on AMR dynamics, plasmid biology and host-MGEs interactions, ultimately contributing to the development of more effective strategies to combat antibiotic-resistant infections.

Infections caused by multi-drug-resistant organisms (MDROs) pose a significant public health threat, responsible for over 2 million hospitalizations and 23,000 deaths annually in the United States. Microbiome dysbiosis (imbalance) is considered one of the main causes for MDRO colonization and the resulting infections. Rapid detection and intervention of MDRO outbreaks are crucial to alleviating strain on patients and healthcare facilities. Current diagnostic methods for MDRO detection are too slow and costly to provide the rapid MDRO detection necessary for patient care facilities. Here we present a rapid, accurate and cost-effective electrochemical sensor capable of MDRO detection down to [~]104 colony forming units (CFU)/g in mice and human stool samples. Our novel sensor utilizes probe-modified Screen-Printed Electrodes (SPEs) capable of hybridizing target gene sequences associated with MDROs. The resulting probe/target complex generates a unique and highly sensitive signal detectable down to 10 atto molar or 10 CFU/mL of target TEM-1 gene. The use of these pre-functionalized SPEs reduces individual sample analysis time to less than an hour. Several target sequences from two chromosomal target genes (AmpC and AcrB found in E. coli) have been identified and successfully detected in clinical stool samples with results comparable to the standard quantitative PCR method. Additional target genes associated with antibiotic resistance (TEM-1, VanA, KPC and SHV) have also been successfully detected in vitro and are ready for clinical evaluation. Future development includes multiplexing the sensor to simultaneously detect up to three MDROs target genes, including {beta}-lactamases that hydrolyze {beta}-lactams, the most commonly used antibiotics in clinical settings. This novel sensor platform will be a rapid, economical, point-of-care device with little requirement of reagent handling or technical training.

Hydrogenophilus thermoluteolus TH-1 is a thermophilic hydrogen-oxidizing bacterium capable of producing poly(3-hydroxybutyrate) (PHB) from CO2. To redirect carbon flux for producing other useful biomaterials, we disrupted the acetoacetyl-CoA reductase genes (phaB1 and phaB2), which are central to the primary PHB synthesis pathway. Unexpectedly, the resulting {Delta}phaB1B2 mutant still accumulated PHB under autotrophic conditions, reaching approximately 25-35 % of the wild-type level. Furthermore, PHB accumulation in the mutant was significantly restored when fatty acids (butyrate and oleate) were used as carbon sources, whereas acetate and malate resulted in reduced accumulation. These results suggest the existence of a PhaB-independent PHB synthesis pathway. We propose that intermediates from the {beta}-oxidation of fatty acids are converted to (R)-3-hydroxybutyryl-CoA, bypassing the disrupted PhaB enzymes. Additionally, the basal PHB production from non-fatty acid sources implies the involvement of a reverse {beta}-oxidation pathway. This study highlights the metabolic versatility of strain TH-1 for future metabolic engineering.

The fish skin microbiome serves as a protective barrier, influencing host health and facilitating interactions between the host and its environment. While several studies have characterised the composition and roles of the fish skin microbiome, there remains a paucity of data on how environmental variation influences these microbes in natural populations. Here, we used 16S rRNA gene sequencing to characterise the skin microbiome of wild three-spined stickleback populations and examine how environmental factors influence microbial diversity and community composition across 17 freshwater lochs on the island of North Uist, Scotland. Analysis of 239 samples revealed a set of dominant bacterial genera commonly associated with other fish species, including Janthinobacterium, Pseudomonas, Acinetobacter, and Psychrobacter, that constituted a core skin microbiota across lochs. Microbiome composition was primarily shaped by environmental variables, particularly habitat, water pH, conductivity, and metal concentrations, with pH emerging as a key driver of community structure. Host sex also influenced microbiome variation, with several taxa differing in relative abundance between males and females. Alpha-diversity was higher among stickleback fish from lochs with a neutral pH compared with those from alkaline and acidic environments. Differential abundance analyses identified 27 and 24 amplicon sequence variants (ASVs), respectfully, associated with variations in pH and host sex, including members of Psychrobacter, Sphingobacterium, Carnobacterium, Chryseobacterium, and Arthrobacter, highlighting the combined influence of environmental and host factors on microbiome composition in wild fish populations in freshwater environments.

Chitosan is an oligosaccharide derived from chitin that is protonated at acidic pH to form a polycation. Its positive charge promotes the interaction with negatively charged components of the yeast cell surface, which has been associated with increased cell permeability and growth inhibition. In this study, we investigated the interaction of chitosan with the cell surface and its permeabilizing capacity in three yeast species displaying distinct susceptibility profiles, Saccharomyces cerevisiae, Candida albicans and Debaryomyces hansenii. We evaluated the correlation between differential susceptibility and chitosan association at the cell surface, as well as cell permeabilization, by integrating growth analyses with surface-binding assays, including FITC-conjugated chitosan to monitor surface association and cellular integration over time, and ultrastructural examination by transmission electron microscopy (TEM). Our results showed that chitosan exhibited varying effects on the growth and permeability of each yeast strain, with D. hansenii being the most susceptible. Furthermore, we observed the incorporation of chitosan onto the cell surface and confirmed its role as a permeabilizing agent. Finally, we used chitosan-induced permeabilization as a method to measure the activity of selected enzymes in situ, demonstrating its potential for studying metabolic functions in permeabilized yeast cells. Overall, our findings establish chitosan as a strain-dependent antifungal agent and a useful tool for functional biochemical analyses in yeast.

BackgroundBarcoding of isogenic strains is a powerful approach to assess pathogen population dynamics during infection. Here, we adapted WISH-barcoding to Salmonella Typhimurium ATCC14028s to evaluate its suitability for pooled infection experiments in streptomycin-pretreated mouse models. ResultsWISH-barcoded wild-type pools showed pronounced population instability, characterized by stochastic strain loss and segregation into high- and low-fitness subpopulations. Whole-genome sequencing identified recurrent mutations in the methyltransferase rsmG and loss of the P3 plasmid carrying streptomycin resistance in low-fitness strains; neither was observed in {Delta}invG or {Delta}ssaV pools. We propose that rsmG mutations were enriched during strain construction carried out under streptomycin selection, following loss of the P3 plasmid. Control experiments demonstrated that rsmG mutations and P3 loss are counter-selected in vivo and attenuate gut-luminal colonization in streptomycin-pretreated mice. ConclusionWhile population dynamics experiments with ATCC14028s are feasible in principle, wild-type strains are prone to acquiring fitness-altering mutations during in vitro construction when using the P3 plasmid and streptomycin, highlighting the need for careful pool validation prior to use.

Microbial ammonia oxidation, the first and rate-limiting step of nitrification, plays a central role in soil nitrogen cycling. It is most relevant in agricultural soils as nitrifiers compete with crops for ammonia-based fertilizers. Therefore, synthetic nitrification inhibitors are widely used alongside fertilizers to reduce the activities of dominant drivers of this process, i.e. ammonia-oxidizing archaea (AOA) and bacteria (AOB). However, the physiological responses of ammonia oxidizers remain poorly resolved. Here the response of the AOA Nitrososphaera viennensis to the nitrification inhibitors 3,4-dimethylpyrazole phosphate (DMPP) and allylthiourea (ATU) were investigated using a combination of functional genomics, physiological assays, and relief experiments. The results overturn earlier assumptions that DMPP and ATU act by chelating free copper. Both compounds affected ammonia oxidation and triggered broader shifts in energy metabolism and stress-response pathways, which diverged markedly between the two inhibitors. We propose a competitive inhibition of the ammonia monooxygenase complex with DMPP as it can be alleviated by additional ammonia and elicits activation of urea acquisition, while ATU acted as a non-competitive inhibitor generally inducing quiescence. Both modes of inhibition were associated with clear transcriptomic and proteomic signals that will be advantageous for the identification of mechanisms of other nitrification inhibitors in the future. Key word: Ammonia-oxidizing archaea, nitrification, nitrification inhibitors, archaea, nitrogen cycle

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.

The prevalent transcriptional repressor NrdR binds to highly conserved prokaryotic sequences in the promoter regions of operons encoding the essential enzyme ribonucleotide reductase. The NrdR binding sites consist of two partially palindromic 16 bp sequences (NrdR boxes) separated by a 15-16 bp linker sequence. We have assessed the requirement of both boxes for binding, the propensity of different NrdRs to bind to heterologous binding sites, and that the linker sequence is only limited to length and not sequence conservation. As we have observed several deviations from the conserved sequences of the NrdR boxes, we here test the conservation requirements of individual basepairs in the NrdR boxes using a synthetic DNA fragment (Synt DNA) to which the NrdR proteins from the actinomycete Streptomyces coelicolor and the gammaproteobacterium Escherichia coli bind equally well as to their homologous binding sites. By introducing isolated mutations to Synt DNA and testing the binding capacity of NrdR from S. coelicolor and E. coli we expand our understanding of what criteria are needed to build a functional binding site for the NrdR repressor.

Manual colony counting remains the rate-limiting, operator-dependent step in culture-based food microbiology quality control (QC). Automated colony analysis using machine learning (ML) offers the potential to standardise, accelerate, and improve the traceability of this process. However, systematic multi-method validation data for AI-based platforms against recognised international standards remain scarce. We conducted a prospective, multi-study validation of the Reshape Smart Incubator which is an automated imaging and ML-based colony analysis system, across eight ISO microbiological reference methods. In total, 887 plates were analysed, spanning qualitative (presence/absence) detection of Listeria spp. (ISO 11290-1) and Salmonella spp. (ISO 6579), and quantitative enumeration of total viable count (ISO 4833), Bacillus cereus (ISO 7932), Enterobacteriaceae (ISO 21528), coagulase-positive Staphylococci (ISO 6888), yeasts and moulds (ISO 21527), and lactic acid bacteria (ISO 15214). Automated results were benchmarked against the consensus of three or more trained technicians. The platform achieved 100% agreement with manual assessment for all both qualitative detection methods (ISO 11290-1, ISO 6579) with zero false positives and zero false negatives. For quantitative enumeration, agreement ranged from 92.97% (ISO 15214, n=122, using ISO-aligned {+/-}10%/>30 CFU thresholds) to 98.46% (ISO 21528, n=130). Where discrepancies occurred, they largely coincided with plates showing high inter-technician variability. Precision testing demonstrated a coefficient of variation of 5.88% and a mean standard deviation of 0.44 CFU for low-count plates. This study presents a comprehensive multi-ISO validation of an AI-based colony analysis system to date. The AI models demonstrated performance comparable to or exceeding that of trained human technicians across a broad range of microbiological targets, agar types, and colony morphologies, thereby supporting their use as a validated and traceable alternative to manual plate reading in accredited food microbiology quality control laboratories.

BackgroundExtended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae are among the WHOs highest-priority antibiotic-resistant pathogens. Plasmids are the main drivers of ESBL dissemination, yet their reservoirs and transmission dynamics remain poorly understood in low- and middle-income countries such as Vietnam, where infections caused by ESBL-producing bacteria are prevalent. MethodsHere, we characterised the genetic structure of 68 ESBL-encoding conjugative plasmids isolated from the human gut microbiome (HGM) of healthy Vietnamese children. We further examined the extent of ESBL plasmid transfer between the HGM and human disease-causing Enterobacteriaceae pathogens (including Shigella sonnei, non-typhoidal Salmonella, extraintestinal pathogenic Escherichia coli ST131), as well as E. coli isolated from animals. ResultsThe dominant plasmid Inc groups found in cephalosporin-resistant human gut bacteria were IncF, IncB/O/K/Z and IncI1, carrying mainly blaCTX-M-14, blaCTX-M-15, blaCTX-M-27 and blaCTX-M-55. These plasmids from the HGM, rather than from animal E. coli, share higher genetic similarity to plasmids in human pathogens, suggesting that human gut is the main reservoir for clinically relevant ESBL plasmids. We also found that widespread ESBL plasmid variants exhibited higher conjugation frequencies, facilitating broader geographical and host dissemination. In contrast, less mobile plasmids persisted mainly through clonal expansion of their bacterial hosts. ConclusionsThese findings highlight the central role of the human gut as a reservoir for ESBL plasmids and provide insights into the biological factors contributing to their successful spread among pathogenic Enterobacteriaceae. Our work underscores the need for targeted interventions to reduce colonization and transmission of ESBL-producing bacteria within the human gut.

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.

Fluorescent reporters cover a wide range of applications in both basic and applied research. Whether a study involves microscopic imaging to study (co)-localization of proteins, FRET, biosensing, or quantifying gene expression, fluorophores are attractive reporter candidates due to their relatively straightforward in vivo readout. For microbiological applications, a wide variety of fluorescent proteins with varying excitation and emission wavelengths, brightness levels, and maturation times are available. Careful consideration is required when selecting from this large suite of proteins, especially when choosing multiple fluorophores. This is further complicated in phototrophic organisms, which exhibit strong autofluorescence, especially towards the red part of the spectrum, effectively eliminating common candidates such as mCherry. In this study, the specific properties and performance of a selection of fluorescent proteins are systematically evaluated against the background of photosynthetic pigment-derived autofluorescence in the cyanobacterium Synechocystis sp. PCC 6803. Specific readouts of different combinations of fluorescent proteins are also analyzed using high-throughput methods, namely plate reader fluorescent scans and single-cell flow cytometry to quantify fluorescence. The ultimate goal is to assess each fluorescent protein with regard to: 1.) Its ability to be discerned from cyanobacterial autofluorescence. 2.) Its compatibility with other fluorophores in this context. 3.) Its overall suitability in cyanobacterial research. Several highly suitable fluorescent proteins for use in cyanobacteria are identified, including mTagBFP2, mNeonGreen and mScarlet-I and suitable combinations, covering nearly the whole spectrum of visible light. This study expands the knowledge and toolset for current and future researchers and uncovers a whole spectrum of possibilities for fluorescent protein selection in cyanobacterial cell biology.

Campylobacter jejuni is a major cause of food-borne gastroenteritis and is responsible for substantial mortality and economic losses in meat and dairy production. Detecting C. jejuni in contaminated food samples remains difficult because current assays are culture-based, slow, and can yield false positives. As a result, contamination may not be identified for several days, limiting detection at the point of production. Developing improved assays has also been challenging because Campylobacter genetics and the biology of clinical isolates remain poorly understood. Here, we expand the C. jejuni genetic toolbox by sequencing two strains, HC1 and RM1164, derived from patient and food samples. We identified two cryptic plasmids in HC1, one potentially capable of conjugation and another conferring tetracycline resistance. We also engineered a mobilizable plasmid carrying an OriT sequence that can be transferred from Escherichia coli donor strains to C. jejuni RM1164 by conjugation. Together, these clinical isolates and the plasmid system expand the genetic tools available for C. jejuni.