BMC Microbiology

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

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.

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.

Pseudomonas aeruginosa relies on the redox cofactor pyrroloquinoline quinone (PQQ) for efficient glucose and ethanol metabolism via periplasmic dehydrogenases (Gcd and ExaA). While PQQ biosynthesis is well-characterized, its uptake mechanisms remain unclear. Here, we identify PA2289 (PqqU), a TonB-dependent transporter, as the primary PQQ importer in P. aeruginosa. Growth assays with PQQ-deficient mutants ({Delta}pqqABCDEH) demonstrated that PqqU is essential for exogenous PQQ uptake, rescuing growth on glucose and ethanol. Genomic analysis across 210 P. aeruginosa and 263 Pseudomonas strains revealed high conservation of PQQ biosynthesis and utilization genes, while PqqU showed lower prevalence (47.7%) in the genus. Transcriptional analyses using fluorescent reporters and qRT-PCR demonstrated that PqqU expression remains unchanged in response to PQQ, varying carbon sources, or iron availability, suggesting constitutive regulation. Comparative proteomics between wild-type and {Delta}pqqABCDEH strains, cultured on glucose or ethanol, uncovered extensive proteomic shifts, underscoring P. aeruginosas metabolic adaptability. Additionally, PQQ-dependent metabolic pathways appear to indirectly influence iron homeostasis, most likely through environmental acidification. Together, these results emphasize the critical role of PqqU in PQQ uptake and its broader significance in shaping the metabolic and environmental versatility of Pseudomonas.

This study investigates the relationship between the gut microbiota and specific diseases. Data was collected from the Human Gut Microbiome Atlas, which examines regional variations across 20 countries on five continents, categorizing microbial species by taxonomy, from genus to species. The Atlas provides color-coded phylum classifications, numerical species counts within the same genus, and an analysis of dysbiosis-related associations with 23 diseases, as well as region-enriched species. The data stratified samples into distinct categories such as westernized, non-westernized, cancerous, and non-cancerous. The findings demonstrate that tree-based ensemble methods, such as Bagging and Boosting prediction methods, achieved the highest accuracies across all categories due to their robustness in handling the complex, high-dimensional data. The XGBoost model yielded the strongest predictive performance, achieving 91% accuracy for westernized cancer-associated samples, 84% accuracy for non-westernized cancer-associated samples, 92% accuracy for westernized samples, and 78% for non-westernized samples. Additionally, advanced topological data analysis was used to assess the global structure and underlying patterns within the dataset. ImportanceThis research aims to connect gut microbiome composition to diseases using global datasets from the Human Gut Microbiome Atlas. The goal was to evaluate how accurately different machine learning algorithms could classify microbiota species and diseases and predict disease associations by comparing westernized and non-westernized populations, including both cancerous and noncancerous groups. These findings can contribute to the future creation of population-specific and disease-specific microbial models.

Bile acids modulate the intestinal microbiota and serve as key signaling molecules in host physiology. Bile acid dysregulation has been implicated in nutritional and inflammatory diseases; however, data on the pool of bile acids present in stunted children or children suffering of environmental enteric dysfunction (EED) is limited, particularly in the upper intestinal compartment where disease phenotypes are most relevant. In this study, we performed a targeted metabolomics approach on 75 bile acids and their derivatives, including gastric and duodenal aspirates and fecal samples from almost 1000 children from two Sub-Saharan cities. We found that levels of secondary bile acids, especially lithocholic acid, are significantly lower in the feces of stunted and EED children, while ursocholic acid and its derivatives are significantly higher. Levels of primary and sulfated bile acids are also increased in the feces of children with EED. Microbiota sequencing revealed that high lithocholic acid levels are positively associated with butyrate-producing bacteria, while negatively associated with oral taxa like Streptococcus and Veillonella. In vitro tests on a panel of reference strains showed that oral bacteria bioaccumulate and are inhibited by a variety of bile acids, while lithocholic and chenodeoxycholic acids modulate the growth of several butyrate-producing bacteria. This effect was even stronger with tauro- or glycol-conjugated bile acids. Exposing stool-derived in vitro communities from children in Afribiota to these bile acids confirmed their positive impact on butyrate producers and negative effect on overgrowing oral taxa. Our findings suggest that secondary bile acids, reduced in stunting and EED, modulate the growth of butyrate-producing bacteria while suppressing harmful oral taxa, highlighting their potential as tools to modulate microbiota composition.

Aminopeptidases are widely distributed in bacteria, but outside of a few model strains, their function is largely unexplored. Focussing on beta-alanine aminopeptidase activity, a new series of selectively-activatable, caged fluorescent probes were designed and synthesised. A beta alanine amino acid was coupled to resorufin or 7-hydroxycoumarin via a self-imolative linker, such that amino acid removal led to gain of fluorescence. These were used to probe selectivity and specificity of probe activation, against a range of priority drug-resistant pathogens. When added to bacterial growth curves run in Muller Hinton broth, these probes allowed essentially real time fluorescence measurement of activation by bacterial species, modelled on the standard microbroth dilution method. Activation was observed for all Pseudomonas aeruginosa and Burkholderia spp strains tested. Selective activation was seen for Ochrabactrum species, with the probe activated by O.anthropii (2/4 strains) but not O.intermedium and strain-specific activation was seen for some isolates of Serratia marcescens (2/4 strains). No activation was observed in any isolates of Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii or Staphylococcus aureus or Eneterocccus faecium/faecalis PAO1 transposon mutants in the putative beta-alanine aminopeptidase gene (annotated as bapF or dmpA; PW3678) showed no activation of the probe in growth assays, confirming the specificity of the probe for beta-alanine aminopeptidase. Transposon mutants in other aminopeptidase genes, including those encoded by pepN, PepP and the prolyl aminopeptidase gene had no effect on probe activation in PAO1. Based on the operon structure in PA01, transposon mutants in two adjacent genes were also tested for probe activation. Mutants in both a putative transcriptional regulator (PW3674) and a predicted amino acid permease (PW3676) retained their ability to activate the beta-alanine probes with activation significantly higher than the wild type, when assessed by the total fluorescence yield after 10 hours growth. This points to both redundancy in permease function and perhaps the presence of a feedback regulatory mechanism controlling beta alanine aminopeptidase activity in P.aeruginosa. Given that the operon structure is conserved in other species, this may point to a common mechanism of beta alanine aminopeptidase function, perhaps related to exploiting beta-alanine containing peptides in certain environmental niches.

BackgroundSegatella copri is characterised as a prominent glycolytic plant-based fiber utiliser within the human gut. My recent work has introduced a new interpretation of the positive impacts of plant-based polysaccharides on S. copri, as a significant negative relationship between S. copri and Blautia spp. was identified. The high rate of electron donor consumption by Blautia spp. indicated that competition for the electron donors, formate, ferredoxin and fumarate, could be the route of the negative relationship but would also explain the positive relationship with plant-based polysaccharides, as they are products of fiber fermentation intestinally. MethodsForty two genomes of S. copri were annotated via Prokka to identify alternative PEP pathways. Phylogenetics allowed effectively classification of the S. copri isolates into species and subspecies clusters. The sequence homology of nucleotides and proteins were analysed against the control, S. copri DSM 18205, to determine the level of conservation in the alternative phosphoenolpyruvate synthesis pathways. ResultsEnolase (eno) was not identified in the S. copri strains; JCM 13468, LKV-178-WT-2C, RHA03, RHA01 and RHA02, and the whole genome phylogenetic grouping of these strains, has proposed the existence of an eno(-) subspecies of S. copri. This work furthered this idea by identifying alternative PEP pathways from formate, ferredoxin and fumarate, which were the most conserved in the eno(-) S. copri genomes. ConclusionThis work has provided rationale to why enolase may not be present within the eno(-) S. copri isolates and have shown that these alternative PEP synthesis pathways could negate the requirement of enolase in cells and may be factor in evolution of S. copri metabolism.

IntroductionAcute myeloid leukemia (AML) is a hematological malignancy associated with muscle wasting. As the relative abundance of Lachnospira eligens was reduced in patients with AML compared to healthy individuals and correlated positively with muscle strength, we hypothesized that L. eligens positively impacts the muscle through the production of small metabolites reaching the systemic circulation. MethodsL. eligens levels were analyzed in two additional independent cohorts. Six L. eligens isolates were characterized through whole-genome sequencing to select clinically relevant strains. The composition of their culture supernatant was analyzed by metabolomics. The impact of L. eligens supernatant on dexamethasone- and interleukin-6-atrophied murine myotubes was assessed. Bioactive metabolites and their production mechanism were identified using among others bioactivity-guided fractionation. The underlying mechanism was also explored on the host side through myotubes transcriptome analysis and metabolic flux analysis. The relevance of bioactive metabolites and their production mechanism was evaluated through clinical data and samples analyses and in a mouse model of leukemia. ResultsThe levels of L. eligens are reduced in independent cohorts of patients with AML and its supernatant counteracts myotube atrophy. This anti-atrophic effect, conserved between strains of the same species, depends on the occurrence of mixed acid fermentation (MAF) in anoxic culture conditions and the presence of its acid end-products acetate, formate and D-lactate. Consistent with those results, blood levels of acetate are decreased and the relative abundance of fecal bacteria capable of performing aerobic respiration is increased in patients with AML. However, bacterial supernatant failed to prevent muscle atrophy and weakness in leukemic mice, likely due to insufficient sustained elevation of acid end-products in the blood. ConclusionThis work reveals the anti-atrophic effect of MAF end-products on myotubes and suggests the importance of considering gut electron acceptor levels (e.g. O2) in disorders affecting muscle health. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/729780v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@112529eorg.highwire.dtl.DTLVardef@1ee1929org.highwire.dtl.DTLVardef@b5f389org.highwire.dtl.DTLVardef@187bc40_HPS_FORMAT_FIGEXP M_FIG Mixed acid fermentation products from Lachnospira eligens counteract myotube atrophy. Our study suggests that gut anaerobiosis is disrupted in treatment-naive patients with acute myeloid leukemia (AML), leading to decreased circulating acetate levels and a reduced relative abundance of L. eligens, which significantly correlated with muscle strength. In line with this framework, in vitro experiments demonstrate that the culture supernatant of L. eligens, which contains mixed acid fermentation (MAF) end-products such as acetate, effectively counteracts C2C12 myotube atrophy in the presence of pro-atrophying stimuli. Further mechanistic experiments indicate a causal role for MAF end-products in this anti-atrophying effect. Created with BioRender.com. Legend: solid frames: experimental results; dashed frames: hypothetical conclusions derived from results; black solid arrow: established correlation; black dashed arrows: hypothetical causation. C_FIG

Species-level Candida identification can inform antifungal management, but reliable identification platforms remain inaccessible in many clinical microbiology laboratories, whereas phase contrast microscopy -- a common feature of routine laboratory microscopes -- is widely available. We asked whether this ubiquitous optical tool, combined with a consumer smartphone and deep transfer learning, could provide a feasible low-cost approach for preliminary Candida species discrimination. Fifteen clinical isolates of four species (C. albicans, C. glabrata, C. tropicalis, C. krusei) were collected from a single clinical microbiology laboratory and imaged using a consumer-grade smartphone coupled to a standard phase contrast microscope. Suspensions in human serum were imaged immediately after preparation (T0) and after 2-hour incubation at 37{degrees}C (T2). Pretrained vision backbone architectures were evaluated as fixed feature extractors under strict Leave-One-Strain-Out cross-validation. The best-performing model -- EfficientNet-B0 embeddings with a Linear Support Vector Machine applied to T2 images -- achieved an apparent internally cross-validated strain-level balanced accuracy of 0.833 and an overall strain accuracy of 86.7% (13/15 strains correctly classified). C. albicans, C. glabrata, and C. tropicalis were each identified with 100% recall. Both misclassified strains belonged to C. krusei -- the species with the smallest panel representation (n=3 strains) -- with misclassification attributable to limited strain diversity and suboptimal image quality. These findings demonstrate promising feasibility for preliminary image-based Candida species discrimination from smartphone-acquired phase contrast microscopy images, and support further evaluation in larger, externally validated strain collections.

BackgroundCystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to chronic infection and mucus retention in the lungs, with lung function gradually deteriorating through recurrent pulmonary exacerbations (PEx). Virulence factors (VFs) of Pseudomonas aeruginosa and Staphylococcus aureus are thought to contribute to pulmonary exacerbations. Our study objective was to identify VF genes related to PEx, high Pseudomonas abundance, and high Staphylococcus abundance in persons with CF (pwCF). MethodsThis was an ancillary study of pwCF treated with IV antibiotics for PEx between 2016-2020 at Childrens National Hospital. Using shotgun metagenomics and ShortBRED, we identified bacterial VF genes and used DESeq2 to determine differential expression of VF genes across comparators. ResultsTwenty-two PwCF experienced 43 PEx. The study cohort had a mean age of 14.6 years, 41% female, 59% white, 36% Hispanic, and 45% had an F508del homozygous CFTR mutation. Minimal differences in VF gene abundance were identified across clinical state. The most differentially increased VF genes found in Pseudomonas high samples were associated with an aminotransferase (log2FC 25.9), flagellar biosynthesis (log2FC 8.3), and type VI secretion systems (log2FC 8.2). The most differentially increased VF genes found in Staphylococcus high samples were an exotoxin (log2FC 26.7), macrolide phosphotransferase (log2FC 25.8), pathogenicity island proteins (log2FC 25.2 and 24.7), and VOC family proteins (log2FC 24.8). ConclusionsThese findings demonstrate that specific VFs associated with immune modulation, motility secretion systems, bacterial motility, and antibiotic resistance are related to P. aeruginosa and S. aureus abundance, providing potential targets for more personalized antimicrobial interventions.

The human gut, containing 100 trillion microbes, is also considered the "second brain," having control over the different functions of the physiological system. With advancements in bioinformatics and the development of sequencing technologies, researchers are able to explore the diversity and functional implications of gut microbiota (GM), which have become strongly associated with a variety of diseases. Microbial imbalance, or dysbiosis, acts as a biomarker for early detection and prognosis of a disease. Artificial Intelligence and Machine Learning (AI/ML) methods, although extensively used in predicting GM associated diseases, are seldom translated to having practical real-world outcomes, necessitating the design of robust AI/ML models applicable in real-world scenario. We have therefore come up with designing stacking-based ensemble architectures (EM1 and EM2), developed by integrating multiple ML-based learning algorithms for improving disease prediction accuracy. The GM datasets, after split into training and test sets, were eventually fed into the proposed two-layer ensemble models, which combines the output from standardized base learners via a meta-classifier, strengthening classification robustness as well as ensuring consistency in optimized performance across diverse datasets. Both the proposed hybrid ensemble models have emerged to be superior performers over all baseline and deep learning models, with an average accuracy of 0.87 and 0.84 respectively. By combining multiple learners, the proposed ensemble models outperform traditional single-algorithm-based approaches to attain higher accuracy and robustness on complex GM datasets. Key messagesO_LIDevelopment of stacking-based hybrid ensemble models (EM), which can be employed to integrate different AI/ML algorithms with better prediction accuracy of gut microbiome (GM)-associated diseases. C_LIO_LIUse of independent GM datasets with preprocessing methods such as SMOTE and PCA to address class imbalance and high dimensionality. C_LIO_LIAll the proposed EM architectures are mostly superior to the existing state-of-the-art AI/ML methods (highest prediction accuracy: 0.87 and 0.84 with EM1 and EM2 models respectively) for GM diseases predictions. C_LIO_LIThe cross-cohort validation demonstrates high prediction accuracy and robustness, (AUC values close to 0.98 and 0.99, for EM1 and EM2). C_LIO_LIThese therefore demonstrate the effectiveness of EM frameworks for GM associated disease prediction, paving the way for corresponding applications in precision medicine. C_LI

Microbial fuel cells (MFCs) technology offers sustainable electricity production. Current research largely focuses on few select model organisms, therefore the true prevalence of exoelectrogenesis amongst bacteria remaining largely unknown. We present a broad-scale survey of monomicrobial electricity production among environmental bacterial isolates inoculated in MFCs, using model organism Shewanella oneidensis MR-1 as a benchmark. Of the assessed taxa, 11-22% displayed exoelectrogenic activity, exceeding current predictions and identifying a further three novel exoelectrogenic species. Phylogenetic analysis based on the 16S sequences enabled the evolutionary relationship between isolates to be visualised, revealing that exoelectrogenesis is non-randomly distributed and phylogenetically conserved. Polarisation studies were implemented, revealing that numerous electron transfer mechanism were being utilised to perform exoelectrogenesis. The results of this study imply that bacterial electricity production is more widespread amongst culturable bacteria than previously estimated, with implications for bioprospecting novel exoelectrogens and predicting electrogenic activity in diverse microbial communities.

Herbivore gut microbiomes may contain a diversity of anaerobic gut fungi (AGF, phylum Neocallimastigomycota), important for fibre degradation. To perform functional studies and elucidate niches of different AGF species, representative fungal isolates must be obtained into axenic culture, which is a resource-intensive process. Here we leverage the integration of morphological and functional assessments of AGF isolates with a newly developed PCR-RFLP strategy, to distinguish and identify isolates of interest from faecal samples from zoo-housed animals. In silico prediction of PCR-RFLP profiles of cultured genera, followed by experimental validation, confirmed that LSU-based PCR-RFLP with AluI and Hyp188I digestion was effective in identification of fungi of distinct genera. Together our workflow resulted in isolation of a so far uncultured Piromyces (NY08) species and Neocallimastix cameroonii from nyala samples, as well as Feramyces austinii from giraffe and Khoyollomyces ramosus from zebra. Amplicon sequencing confirmed that these species dominated AGF communities in their hosts, likely benefiting isolation success, and we identified enrichment conditions which also affected cultivability. The workflow developed here aids efficient AGF isolations, which are instrumental in expanding opportunities for functional studies that provide insight into the physiology and ecology of these fungi and help realise applications in white and green biotechnology. One sentence summaryA validated PCR-RFLP strategy, developed based on genetic diversity data from Neocallimastigomycota, enables efficient identification of isolates of these anaerobic gut fungi from environmental samples, as demonstrated via targeted enrichment of anaerobic gut fungi common in faeces of giraffe, zebra and nyala, resulting in isolation of species of genera Feramyces, Neocallimastix, Khoyollomyces, and a novel Piromyces/NY08 species.

The aim of this study was to evaluate the impact of two sustainable dietary protein sources on the structure and composition of the gut microbiota in European sea bass (Dicentrarchus labrax) juveniles. These protein sources were incorporated to the aquafeeds containing (a) Lupinus albus meal, treated with either exogenous enzymes (Solid state hydrolysis-SSH) or fermented with Saccharomyces cerevisiae (Solid state fermentation, SSF) and (b) Lagocephalus sceleratus meal. In the first case (a), the control aquafeed simulated a standard commercial diet, containing soybean meal whereas in the rest of the diets soybean meal was partially or totally replaced by hydrolysed or fermented Lupin meal. In the second case (b) the fish were fed Lagocephalus sceleratus unprocessed fishmeal as well as treated at different temperatures to deactivate tetrodotoxin (TTX). A control diet with 30% commercial fish meal was also fed as a reference diet. Both diets in all inclusion levels did not cause any significant gut microbiota change, suggesting their neutral role in this aspect. However, the gut bacterial communities of the fish fed with 12.5% lupin meal inclusion, had increased amino acid biosynthetic pathways suggesting a beneficial effect.

Background: Urinary tract infections (UTIs) rank among the most common infections globally, with many linked to indwelling urinary catheters. Our prior culture-based longitudinal evaluation of long-term catheterized nursing home residents revealed persistent asymptomatic colonization by pathogens and demonstrated that CAUTI onset was not necessarily due to new pathogen acquisition. In this study, we optimized metagenomics methods to examine the ecological structure underlying persistent colonization and the transition to infection. Results: We present a comprehensive longitudinal metagenomic analysis of catheterized urine specimens, revealing colonization dynamics of 69 microbial species across 198 samples from 9 individuals. Descriptive ecological metrics were combined with Bayesian mixed-effects models that accounted for repeated within-participant sampling to identify clusters of co-occurring species, determine the impact of perturbations such as antibiotic exposure and catheter changes on community structure, and identify taxa predictive of infection sign and symptom onset. Longitudinal specimens clustered into three main ecological phenotypes: 1) moderate diversity, unstable communities (3 participants); 2) high diversity, stable communities that resisted disruption even after multiple catheter changes (3 participants); and 3) low diversity, pathogen-dominated communities (3 participants). Catheter changes alone did not significantly disrupt community composition, while antibiotic exposures induced major shifts often followed by re-colonization with the same genera within subsequent weeks. Six clusters of species were identified for which relative abundances correlated across perturbations to the microbial community, including a mutually exclusive Enterobacterales cluster and fastidious-anaerobe group cluster. 24 species were found to correlate with onset of signs and symptoms of infection, 11 of which were missed by standard urine culture. Conclusions. The catheterized urinary tract represents a novel ecosystem that is resilient to disruption by catheter changes but susceptible to antibiotic perturbation. Antibiotic exposure did deplete all species associated with signs and symptoms but also depleted potentially benign microbes. Our findings have direct implications for catheter management protocols and antibiotic stewardship in long-term catheterized patients. Prospective evaluation using this framework in a larger cohort can help translate these ecological insights into clinical decision-making tools.

BackgroundMetabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent pediatric disorder with limited treatment options, primarily due to an incomplete understanding of its molecular drivers. Recent research underscores the role of microbial guilds in metabolic health, but the mechanisms by which dysbiosis driven by core species and co-abundant symbionts disrupt metabolic homeostasis in pediatric MASLD remain unclear. ResultsHere, we conducted integrated metagenomic and metabolomic analyses on 285 pediatric subjects including MASLD patients, obese and healthy controls. The gut dysbiosis in MASLD was characterized by a depletion of Phocaeicola vulgatus, Bacteroides uniformis, Parabacteroides distasonis, and Bacteroides thetaiotaomicron. Co-abundance network analysis, integrating our cohort with four public datasets, identified these species as core guild members associated with MASLD. Microbial enrichment analysis showed significant disruptions in carbohydrate metabolism, particularly the downregulation of the tricarboxylic acid (TCA) cycle, fructose and sucrose metabolism, and pentose and glucuronate interconversions. P. vulgatus and B. uniformis were identified as dominant species linked to the downregulation of KEGG orthologs (KOs) in these disrupted pathways that were inversely correlated with hepatic injury biomarkers. CAZyme database analysis further emphasized P. vulgatus as the primary contributor to glycoside hydrolases involved in monosaccharide utilization. Finally, both untargeted and targeted metabolomics analysis validated a disrupted metabolic network centered on the TCA cycle and monosaccharide metabolism in pediatric MASLD. ConclusionOur findings suggest the core guild species P. vulgatus and B. uniformis may serve as critical regulators of carbohydrate metabolism in pediatric MASLD, offering potential mechanistic targets for gut microbiome-based interventions.

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

BackgroundFolate deficiency is a global nutritional problem associated with multiple adverse health outcomes, including impaired one-carbon metabolism and elevated homocysteine levels (hyperhomocysteinemia). Gut microbiota-mediated folate biosynthesis has emerged as a promising strategy for improving host folate status. This study aimed to isolate folate-producing probiotic strains, clarify their folate synthesis mechanisms, and evaluate their regulatory effects on folate metabolism and gut microbiota in folate-deficient mice. MethodsHigh-throughput cultivation and screening were performed to isolate folate-producing probiotics. Whole-genome sequencing, pathway reconstruction, and metabolite profiling in fermented milk were used to explore folate biosynthesis pathways and potential microbial cross-feeding interactions. A folate-deficient mouse model was established to evaluate the effects of a probiotic cocktail on serum folate, homocysteine (Hcy) levels, and gut microbiota composition using microbiological assays, biochemical analyses, qPCR, 16S rRNA gene sequencing, alpha diversity analysis, principal coordinates analysis (PCoA), and Linear discriminant analysis Effect Size (LEfSe) analysis. ResultsOver 1,000 bacterial isolates were obtained, and over 10 strains, mainly belonging to Lactobacillus, Bifidobacterium, and Bacillus, showed folate production levels above 100 ng/mL. Genomic analysis revealed that most selected probiotic strains lacked genes involved in para-aminobenzoic acid (pABA) biosynthesis but retained downstream folate synthesis modules, suggesting a potential dependence on pABA-producing gut commensals for precursor supply through microbial cross-feeding. In fermented milk, probiotic strains mainly produced bioactive folates (5-MeTHF and THF), with strain-specific production capacities; L. plantarum, W. coagulans, and B. animalis subsp. lactis significantly increased 5-MeTHF levels in fermented milk. In vivo, high-dose probiotic intervention significantly elevated serum folate (p<0.01) and reduced Hcy (p<0.05) in folate-deficient mice, while medium-dose intervention showed no significant effects. The probiotic strains colonized the mouse gut in a dose-dependent manner: high-dose group exhibited >4,000-fold increase in relative abundance (Bifidobacteriaceae and Bacillaceae enriched), medium-dose group only enriched Bacillaceae, and low-dose group showed no effective colonization. High dose probiotic treatment enhanced gut microbial species diversity (increased Shannon index) and restored folate deficiency-induced gut microbiota dysbiosis (PCoA clustering closer to normal group). ConclusionThis study screened high folate-producing probiotic strains and demonstrated their ability to synthesize active 5-MeTHF, which may rely on microbial cross-feeding in gut microbiota. Furthermore, we demonstrated that folate-producing probiotic intervention significantly improves folate status and Hcy metabolism and restores gut microbiota homeostasis in folate-deficient mice. These findings suggensted that such probiotics could serve as a safer, more physiological intervention for folate deficiency and hyperhomocysteinemia, especially in populations with MTHFR polymorphisms.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen, with antibiotic resistance mediated by the vanA and vanB operons. Rapid and accurate detection of antibiotic resistance is critical for the timely treatment of bacteremia and sepsis. Although imaging-based approaches using fluorescence in situ hybridization (FISH) provide a potential diagnostic solution, detecting mRNAs of antibiotic resistance genes (ARGs) in individual cells remains particularly challenging due to their low copy number and transient expression. Here, we present a peptide nucleic acid (PNA)-FISH method for direct detection of vanA- and vanB-associated resistance in individual VREfm cells. A universal probe targeting the conserved region across vancomycin resistance genes and a set of probes exclusively targeting the vanB gene were designed. The universal probe showed increased fluorescence in the vanA-genotype strain upon vancomycin or teicoplanin treatment, and in the vanB-genotype strain upon vancomycin treatment. In contrast, vanB-specific probes showed increased fluorescence exclusively from the vanB-genotype strain upon vancomycin treatment, confirming their specificity to the vanB gene. Efficient cellular penetration and strong hybridization of PNA probes enabled efficient and accurate detection of antibiotic-resistant bacterial cells, even under a wide-field fluorescence microscope. No detectable signals above background were observed in other major bacterial species associated with bacteremia and sepsis. These findings demonstrate robust detection of antibiotic-resistant cells in mixed microbial populations. When integrated with microbe-capturing techniques, this method may support culture-free detection of antibiotic resistance without nucleic acid amplification or sequencing, with the potential to reduce diagnostic turnaround time.