As a result of climate change, increased flooding events have been recorded across the globe. The role of soil microbiomes in soil health and fertility is well recognized, as these microbial communities drive essential processes such as nutrient cycling, organic matter decomposition, and plant health. The composition and function of these communities can be highly sensitive to environmental changes. The aim of this study is to identify the impacts of flooding on soil microbiome composition and diversity. Paired flooded and non-flooded agricultural soils were collected from Germany, Ireland, and Portugal, and characterized using full-length 16S rRNA gene and ITS amplicon sequencing. Following false-discovery-rate correction, flooding was not associated with significant differences in bacterial or fungal alpha diversity. In contrast, paired PERMANOVA detected small overall effects of flooding on bacterial Bray-Curtis, Jaccard, and unweighted UniFrac distances, and on fungal Bray-Curtis and Jaccard distances, although community composition was structured more strongly by country. At the taxonomic level, Thermodesulfobacteriota was more highly represented in flooded soils, whereas the fungal phyla Ascomycota and Mortierellomycota were less represented. Eight bacterial genus-level assignments differed between conditions after correction, whereas no fungal genus-level differences remained significant. Flooding was associated with some geographically dependent restructuring of bacterial and fungal communities rather than a consistent loss of microbial diversity. These findings demonstrate the importance of regional context when evaluating microbiome responses to flooding and identify the need for future studies that integrate microbial data with measured soil physicochemical characteristics.IMPORTANCEFlooding is an increasingly important disturbance in agricultural soils, yet its effects on bacterial and fungal communities remain incompletely understood. By comparing matched flooded and non-flooded soils across Germany, Ireland, and Portugal, this study shows that flooding was associated with small shifts in community composition without a consistent reduction in alpha diversity. Geographic location explained the dominant patterns in community structure, while a limited number of bacterial and fungal taxa differed between flooding conditions. These findings indicate that microbiome responses to flooding are context-dependent and are expressed primarily as changes in community composition rather than uniform losses of richness or evenness. The study provides a cross-country baseline for future work linking flood-associated microbial changes to measured soil chemistry, flooding history, and agricultural recovery.
The recent report by T. Murillo, L. E. Enrique Chaves-González, S. Temmam, S. Bermúdez, et al. (Microbiol Spectr 14:e04078-25, 2026, https://doi.org/10.1128/spectrum.04078-25) expands the known geographic and ecological range of Jingmen tick virus (JMTV) by detecting the virus in Amblyomma mixtum ticks collected from horses in Costa Rica. This is an important finding because A. mixtum can feed on wildlife, domestic animals, and humans, creating a possible interface for virus movement across various hosts. The study also places the Costa Rican virus in a wider phylogenetic context, linking it to JMTV diversity reported from other regions. However, the detection of viral RNA in ticks should not be interpreted as proof of local disease, human infection, or active transmission, especially in the absence of supporting results. Instead, it reflects an important signal for careful viral surveillance. Here, I discuss how JMTV illustrates the need for ensemble approaches that combine field sampling, phylogeny, segment-level genome analysis, serology, experimental validation, and data-driven virus discovery tools.
Mold contamination causes economic losses and poses risks to human and animal health. Tobacco leaves, as a high-value agricultural product, are particularly susceptible to mold contamination during post-harvest handling. In this study, the antifungal performance of Bacillus velezensis ZD-F13 was evaluated on tobacco leaves. In laboratory-scale assays, ZD-F13 treatment reduced the fungal colony count by 84% relative to the Penicillium group. Electronic-nose and gas chromatography-ion mobility spectrometry analyses showed that the volatile profile of the Penicillium + ZD-F13 group was closer to that of the normal leaves group than to that of the Penicillium group. To further evaluate its practical applicability, B. velezensis ZD-F13 was tested on 5 kg of tobacco leaves and reduced the fungal colony count by 50% relative to the untreated group. Microbial community analysis revealed changes in bacterial and fungal community composition, including an increase in the relative abundance of Bacillus from 0.09% to 48.1%. These findings support the potential application of B. velezensis ZD-F13 for controlling tobacco leaf mold and providing a basis for further development of postharvest biocontrol strategies.IMPORTANCEMold contamination of tobacco leaves causes substantial economic losses and can compromise product quality and safety during postharvest storage and processing. Effective control strategies remain limited, particularly those capable of suppressing mold without disturbing the natural microbial community or altering quality-related characteristics. This study showed that ZD-F13 treatment reduced fungal colony counts under controlled laboratory conditions and was associated with a lower fungal colony count in the 5-kg practical trial. ZD-F13 treatment also shifted the volatile profile toward that of healthy leaves without causing a statistically detectable loss of Shannon diversity under the tested conditions. These findings suggest that ZD-F13 has potential as a postharvest biocontrol agent for tobacco leaves.
Internal transcribed spacer (ITS) sequencing has been increasingly used for the diagnosis of fungal infection. However, data on its real-world clinical utility are limited. We retrospectively evaluated the diagnostic yield from direct clinical specimens using the final diagnosis as the gold standard, and studied the impact of ITS sequencing on antifungal management in a university hospital with an unrestricted order setting. A total of 315 specimens were collected from 227 patients between November 2023 and April 2024. Of these, 59 (18.7%) were from fungal infection cases. ITS sequencing demonstrated a sensitivity of 35.6% and specificity of 96.9%. Agreement with fungal culture was 79.7% (κ = 0.583). In our setting, the median turnaround time of the positive results was 12 (12-14) days. ITS sequencing altered clinical management in only 2 patients (0.9%). The culture-negative but ITS sequencing-positive specimens mostly represented colonizers from bronchoalveolar lavage fluid. In an unrestricted setting, ITS sequencing showed low sensitivity and minimal impact on clinical decision-making. To maximize diagnostic benefit and utility, implementation of diagnostic stewardship is necessary. IMPORTANCE:This study provides a real-life evaluation of internal transcribed spacer (ITS) sequencing in an unrestricted clinical setting. We demonstrated fair sensitivity and minimal impact on antifungal alteration. We identified critical implementation barriers, including prolonged turnaround times and the frequent detection of colonizers, which fail to guide acute decision-making. These findings are vital for informing hospital policy and advocating for strict diagnostic stewardship to maximize clinical benefit.
The increasing resistance of Candida albicans to conventional antifungal agents poses a major clinical challenge, underscoring the urgent need for novel therapeutic strategies. Drug combination therapy has emerged as a promising approach to overcome antifungal resistance. In this study, we evaluated the synergistic antifungal activity of dehydronuciferine (DHN), a natural aporphine alkaloid, in combination with fluconazole (FLC) against six clinically drug-resistant C. albicans isolates. Strong synergism was confirmed by checkerboard microdilution assay, with fractional inhibitory concentration index (FICI) values ranging from 0.063 to 0.265. Notably, for strain CA10, the minimum inhibitory concentration of FLC was reduced from >512 to 0.25 μg/mL in the presence of DHN (FICI = 0.125), representing a >2,000-fold reversal of resistance. In a Galleria mellonella infection model, the DHN + FLC combination significantly improved larval survival compared with either monotherapy (P < 0.01). Phenotypic assays demonstrated that the combination markedly suppressed key virulence traits, including hyphal growth, biofilm formation, and epithelial adhesion. Transcriptomic profiling revealed that these phenotypic changes were associated with the downregulation of hypha- and adhesion-related genes and the ergosterol biosynthesis pathway. Together, these findings identify dehydronuciferine as a promising antifungal chemosensitizer that enhances fluconazole efficacy against drug-resistant C. albicans through multitarget perturbation of resistance- and virulence-associated processes. Further mechanistic and preclinical studies are warranted to evaluate its translational potential.IMPORTANCEDrug-resistant Candida albicans infections are increasingly difficult to treat, and the pipeline for new antifungal drugs remains limited. In this study, we show that the natural aporphine alkaloid dehydronuciferine (DHN) acts synergistically with fluconazole to potently inhibit drug-resistant C. albicans by simultaneously suppressing hyphal growth, biofilm formation, and epithelial adhesion. In a Galleria mellonella infection model, the DHN-fluconazole combination significantly improved survival compared with either agent alone. This combination strategy, which pairs an approved antifungal with a natural compound, offers a promising approach to combat antifungal resistance and may accelerate the development of alternative therapeutic options without the need for entirely new drug classes.
Toxoplasma gondii infection remodels the gut microbiome, yet its impact on microbial vitamin biosynthetic potential and host redox metabolism remains unclear. Here, we integrated mouse gut metagenomes with publicly available metagenome-assembled genomes (MAGs) to construct a genome-resolved atlas of B-vitamin and vitamin K2 biosynthesis. From 45,697 MAGs, we curated 4,771 representative genomes, of which 2,682 met high-quality criteria (completeness ≥90%, contamination <5%). Functional annotation identified 229,717 vitamin-related genes corresponding to 177 Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologs across de novo pathways for eight B vitamins, thiamine (B1), riboflavin (B2), niacin (B3), pantothenate (B5), pyridoxine (B6), biotin (B7), folate (B9), cobalamin (B12), and vitamin K2. Among the high-quality genomes, 1,665 encoded complete de novo pathways for at least one vitamin, highlighting functional specialization and community-level complementarity. Transcripts per million-normalized metagenomic read counts revealed significant differences in KEGG ortholog abundances across six of the nine vitamin pathways. Reanalysis of metagenomic data from infected mice (acute, chronic, and control; n = 10 per group) revealed a stage-dependent reduction in α-diversity of vitamin biosynthesis pathways during acute infection, and a clear β-diversity separation from chronic and control groups. Core niacin biosynthesis genes (nadB, nadA, nadC) displayed phylum-specific redistribution, indicating selective remodeling of microbial NAD+ precursor production under infection-induced metabolic stress. These results suggest that T. gondii infection disrupts cooperative vitamin biosynthetic networks while specifically modulating niacin pathways linked to host NAD+ metabolism. IMPORTANCE:Gut microbes can synthesize essential vitamins, but how infection alters this function is poorly understood. By integrating mouse gut metagenomes with genome-resolved microbial data, we show that Toxoplasma gondii infection reshapes the vitamin biosynthetic potential of the gut microbiome in a stage-dependent manner. Acute infection reduces the diversity of vitamin biosynthesis pathways and shifts the taxonomic distribution of key niacin biosynthesis genes involved in microbial NAD+ precursor production. These findings identify vitamin metabolism, especially niacin-related pathways, as a sensitive functional axis of microbiome remodeling during infection. Our work links microbial taxonomic changes to functional metabolic consequences and suggests that microbiome-mediated regulation of NAD+-related metabolism may contribute to host redox adaptation during T. gondii infection.
Acute myeloid leukemia therapy is accompanied by antibiotic exposure and marked gut microbiome disruption. We analyzed 508 longitudinal stool 16S rRNA gene profiles from 67 patients in PRJNA451154. We summarized the depletion of putative short-chain fatty acid (SCFA)-associated genera, pathobiont expansion, and domination with an ecological composite score (ECS). Disruption peaked descriptively during days 15-28 (D15-D28). In a patient-clustered generalized estimating equation model jointly including treatment window and antibiotic exposure during the preceding 0-14 days, the D15-D28 ECS contrast was attenuated (coefficient, 0.251; 95% confidence interval [CI], -0.372 to 0.874), whereas recent antibiotic exposure remained associated with higher ECS (0.722 per 7 antibiotic-days; 95% CI, 0.529 to 0.915). The D1-D7 to D15-D28 increase persisted with patient-level fivefold cross-fitted score construction (paired difference, 1.279; bootstrap 95% CI, 0.861 to 1.758). Treatment-window-adjusted models retained associations of serum butyrate/isobutyrate with the putative SCFA-associated genus signal (0.476; 95% CI, 0.309 to 0.643) and fixed ECS (-0.372; 95% CI, -0.516 to -0.227). ECS did not add stable information beyond Shannon loss and maximum pathobiont abundance for clinical events during the subsequent 7 or 14 days. Frozen ecological formulas produced similar rankings across two external data sets but did not provide clinical validation. Thus, D15-D28 was a descriptive window strongly coupled to recent antibiotic exposure, and ECS functioned as an integrative ecological summary rather than a validated predictor.IMPORTANCEAcute myeloid leukemia therapy creates a clinically important perturbation of gut bacterial communities, yet microbiome injury is often summarized by diversity alone or by isolated taxa. We integrated the depletion of genera associated with short-chain fatty acid production and expansion of potential pathogens into a longitudinal ecological score. The apparent disruption peak during days 15-28 was largely attenuated after accounting for recent antibiotic exposure, whereas patient-level resampling supported the stability of the ecological contrast. Associations with serum metabolites persisted after adjustment for treatment period, but amplicon sequencing cannot establish microbial function or metabolite production. The composite score did not add stable information beyond diversity loss and maximum potential-pathogen abundance for near-term clinical events. These findings define a reproducible framework and a high-yield sampling window for prospective studies while avoiding causal or predictive claims.
Extra-intestinal pathogenic Escherichia coli (ExPEC) is a significant cause of invasive infections, including urinary tract infections, neonatal meningitis, and bacteremia. The emergence of multidrug-resistant ExPEC strains has intensified the need for effective prophylactic interventions. Johnson & Johnson has developed ExPEC10V, a multivalent vaccine targeting ten distinct O-antigens associated with pathogenic ExPEC strains. To assess the humoral immune response elicited by ExPEC10V, an initial enzyme-linked immunosorbent assay (ELISA) targeting E. coli O-polysaccharide (O-LPS) antigens was employed. However, given the vaccine's multivalent antigenic composition, and to improve throughput and efficiency, a multiplex electrochemiluminescent (ECL) immunoassay was developed, qualified, and validated to enable simultaneous determination of IgG antibody titers against all 10 O-antigens, and the carrier protein (EPA) in human serum. The ECL assay was validated for relative accuracy, precision, and linearity using serum samples from an ongoing clinical trial. Precision of incurred samples was systematically assessed across multiple runs to ensure reliable detection of baseline antibody levels. Specificity was validated through competitive inhibition using homologous and heterologous O-LPS antigens. Assay performance was further confirmed by assessing total error, linearity, and dilutional linearity across a broad concentration range. Robustness was tested by varying critical incubation times, while ruggedness was demonstrated using two different ECL plate readers. Sample stability was verified under multiple freeze/thaw cycles and prolonged storage at 4-8°C. The assay demonstrated acceptable precision, relative accuracy, linearity, and robustness across all tested antigens. While most antigens met ruggedness and specificity criteria, O6A failed during simultaneous heterologous competition; however, acceptable specificity was achieved when tested with individual heterologous antigens. To further investigate the O6A outcome, various antigen coating concentrations were evaluated; no improvement in performance was observed at either higher or lower concentrations. Sample handling and storage conditions were comparable to reference conditions, and pre-coated plates remained stable for up to 3 years. Validation of the ECL-based immunoassay for determining IgG antibodies titers against E. coli O-antigens and the EPA protein in human serum was successfully completed, with all parameters meeting predefined acceptance criteria. The assay offers a robust, sensitive, and efficient platform for evaluating vaccine-induced immune responses, supporting both clinical development and future research applications. IMPORTANCE:Extraintestinal pathogenic E. coli (ExPEC) is a major cause of serious infections, and growing antibiotic resistance makes these illnesses harder to treat. Vaccines such as ExPEC10V could help prevent these infections before they start, reducing the need for antibiotics and improving patient outcomes. To understand how well this vaccine stimulates protective antibodies, scientists need reliable tools to measure immune responses. Traditional tests could only evaluate one bacterial target at a time, which was slow and labor‑intensive for a vaccine containing 10 components. By developing a new multiplex electrochemiluminescent assay, we can now measure antibodies to all 10 ExPEC targets simultaneously using a very small amount of sample. This improvement increases efficiency, supports large clinical studies, and accelerates progress toward a preventive vaccine for an urgent global health need.
Ciprofloxacin resistance in Salmonella Typhimurium is a significant public health concern, and the mechanisms by which the resistance evolves are poorly defined. Here, by serial passaging under antibiotic selection, we isolated ciprofloxacin-resistant S. Typhimurium mutants and subjected them to whole-genome sequencing to reveal the major mutations associated with resistance. The Low CipR mutant acquired four chromosomal mutations in ramR, icdA, lipB, and gyrA, and the High CipR mutant gained additional mutations in gyrB, yaiC, and corA. Functional characterization determined that mutations in ramR resulted in efflux pump upregulation, while disruptions in the TCA cycle caused by mutations in icdA and lipB led to metabolic alterations. These changes indirectly enhanced resistance by increasing the expression of the global regulator MarA and reducing OmpF-dependent membrane permeability. Despite the observation of the G105A substitution in GyrA, enzymatic assays confirmed the failure to support resistance to ciprofloxacin, possibly because the structural alteration remained minimal. GyrB488-489dup was associated with maintained supercoiling under ciprofloxacin and enhanced fluoroquinolone resistance, suggesting a major role in resistance evolution. Other mutations in yaiC impaired biofilm and, in corA, intracellular accumulation of magnesium, possibly stabilizing the bacterial cell envelope under antibiotic pressure. The findings provide novel explanations for the multifaceted mechanisms leading to ciprofloxacin resistance in Salmonella and suggest targets to combat antimicrobial resistance.IMPORTANCEAntibiotic resistance in Salmonella Typhimurium is an increasing public health concern, yet the genetic changes that allow bacteria to become resistant are not fully understood. In this study, we evolved ciprofloxacin-resistant Salmonella in the laboratory and identified the mutations that arise during resistance development. We found that resistance does not result from a single change but from multiple adaptations affecting drug efflux, metabolism, and the antibiotic target. Some mutations increased the activity of pumps that remove antibiotics from the cell, while others altered bacterial metabolism and reduced membrane permeability, making it harder for the drug to enter. A duplication in the DNA gyrase subunit GyrB played a particularly important role in maintaining DNA function under antibiotic stress. Together, these results reveal how diverse genetic changes cooperate to generate ciprofloxacin resistance and provide insights that may help guide strategies to combat drug-resistant Salmonella infections.
The livestock sector is standing out for its substantial use of antimicrobials but is also investing in the development of mitigation strategies. Supranutritional concentrations of zinc (Zn) are served to piglets to prevent post-weaning diarrhea. This practice is detrimental to the piglet's assimilation of nutrients and can impact the environment through the excretion of unassimilated Zn, contributing to the persistence of antibiotic resistance by co-selection. In this study, the ileum and colon microbiome of piglets fed low (175 ppm), moderate (953 ppm), or high (2,991 ppm) concentrations of zinc oxide for 21 days after weaning were investigated using metagenomics and 16S rRNA amplicon sequencing. The pH and volatile fatty acids (VFA) were measured in cecum and colon. The colon resistome was investigated using metagenomics and PCR. The high Zn supplementation led to an increase in pH and a decrease in the VFA in both the cecum and colon. The bacterial diversity in colon was negatively impacted by the high Zn diet, with thriving Clostridiaceae and depletion of beneficial populations (e.g., Lactobacillaceae and Selenomonadaceae). Of concern, higher abundances of blaCTX-M and intI1 were observed in piglets fed with the moderate Zn diet. The findings from this study support the limitation of Zn to low concentrations. IMPORTANCE:Developing alternatives to antibiotics is imperative to mitigate the global antimicrobial resistance crisis, particularly within the livestock sector, the predominant consumer of these agents. Yet, interventions introduced without due consideration may precipitate unintended consequences. In North American swine production, supranutritional zinc supplementation in pig feed has been adopted to prevent post-weaning diarrhea and sustain profitability. However, excessive zinc disrupts iron and copper metabolism and perturbs mitochondrial homeostasis, impairing energy metabolism, antioxidant defenses, and innate immunity. Moreover, high dietary zinc increases the excretion of this heavy metal, contributing to environmental pollution and fostering antibiotic-resistant bacteria through co-selection. This study provides further evidence supporting the restriction of zinc supplementation to nutritional needs, as supranutritional diets compromised the fermentative capacity of the pig gut microbiome and increased the frequency of clinically relevant antimicrobial resistance markers.
Amplicon sequencing investigations of surface microbiota in food facilities often report the relative abundance of bacteria and fungi. However, physiological differences among cell types can result in variable cell recovery and DNA yields, thereby skewing relative abundance estimates. Here, we evaluated (i) variations in cell recovery among different bacterial and fungal species after surface swabbing and (ii) the impact of DNA extraction protocols on relative abundance estimates from artificially inoculated stainless steel surfaces. Our results showed that Escherichia coli (Gram-negative cell), Listeria monocytogenes (Gram-positive), Bacillus cereus (bacterial spore), Alicyclobacillus suci (bacterial spore), Exophiala phaeomuriformis (fungal cell), Aspergillus fischeri (fungal spore) differed significantly (P < 0.05) in their recovery rates from stainless steel surfaces. Vegetative cells (E. coli and L. monocytogenes) exhibited lower average recovery rates from surface swabbing (2.9%-6.6%) than spores (35.2%-94.9%). Extending the bead-beating step in DNA extraction by 10 min generally improved yields though the impact varied by organism. For example, DNA yields of E. coli increased from 70 to 84 ng/mL while that of L. monocytogenes increased only from 23.2 to 29.2 ng/mL. Cell recovery and DNA extraction impacted relative abundance estimates from amplicon sequencing. Starting off at equal relative abundances of 25%, L. monocytogenes was underestimated (9%-17%) in downstream calculations, while B. cereus was overestimated (36%-44%). These results underscore the limitations of amplicon sequencing for microbiota characterization on food facility surfaces and highlight the need to improve current swabbing and DNA extraction methods. IMPORTANCE:Amplicon sequencing has been used to characterize microbial communities on facility surfaces. However, few studies have evaluated the accuracy of the amplicon sequencing workflow for quantifying spoilage and pathogenic organisms in these microbial communities. Here, we assessed the accuracy of amplicon sequencing to evaluate the relative abundance of spoilage and pathogenic organisms commonly found in food-processing environments. The results revealed biases in relative abundances due to limitations in cell recovery and DNA extraction methods. These findings revealed the potential biases in surface microbiota characterization in food facilities and the need to refine current recovery and extraction methods to enhance the accuracy of microbiota characterization.
Myxobacteria are fascinating and important prokaryotes with remarkable multicellular behaviors, which make them a model system for studying prokaryotic development and cooperation. Although there have been sporadic discoveries of myxobacterial species unable to fruit, it is unclear whether the non-fruiting characteristic is due to taxon-specific genetic deficiency or suboptimal cultivation conditions. Aggregicoccus is a non-fruiting myxobacterial genus typified by a single validly published species, Ag. edonensis. In this study, we report five novel Aggregicoccus strains, which are classified into three novel type species, Ag. lacus, Ag. agri, and Ag. guangxiensis, based on polyphasic taxonomic analysis. All the Aggregicoccus strains are unable to produce fruiting bodies, but can still sporulate. We compared the genome differences between Aggregicoccus and Myxococcus; both genera belong to the Myxococcaceae family, and all the genomes are of similar sizes. The results showed that the Aggregicoccus strains are inherently deficient in the fruiting body-associated genomic information (FAGI). We propose an assessment of FAGI for the classification of non-fruiting myxobacterial species.IMPORTANCEFruiting body formation is traditionally regarded as a defining trait of myxobacteria. Here, we report that Aggregicoccus spp., including six strains of four species, can sporulate but are deficient in the fruiting body-associated genomic information (FAGI). This demonstrates that the non-fruiting characteristic in Aggregicoccus stems from inherent genetic deficiencies rather than suboptimal cultivation. Our findings highlight the need to assess FAGI presence in classifying non-fruiting lineages, innovate the isolation method, and refine our understanding of the diversity and evolution of the myxobacteria.
Ultraviolet B (UVB) radiation is a major environmental stressor that induces oxidative stress, inflammation, and skin barrier dysfunction. Although Staphylococcus epidermidis (S. epidermidis) is a beneficial skin commensal, current evidence on its protective effects against UVB-induced skin damage has primarily focused on reference strains or a limited number of individual metabolites. The broader photoprotective potential of postbiotics derived from newly isolated skin commensal strains remains insufficiently characterized. Here, we investigated the anti-photodamage activity and underlying mechanisms of the cell-free supernatant of S. epidermidis CCSM0287 (SE 287-CFS), a multifunctional skin commensal isolated from healthy skin, in UVB-induced HaCaT cells. SE 287-CFS significantly reduced intracellular reactive oxygen species (ROS), malondialdehyde (MDA), and pro-inflammatory cytokines, while enhancing antioxidant enzyme activity and interleukin-10 (IL-10) secretion. Mechanistically, SE 287-CFS inhibited p65 nuclear translocation and suppressed activation of the ROS/MAPK/NF-κB signaling pathway. In addition, SE 287-CFS increased transepithelial electrical resistance (TEER) and promoted keratinocyte proliferation and migration, indicating improved skin barrier function. Non-targeted metabolomics combined with correlation analysis suggested that butyric and adipic acids were primarily associated with antioxidative and anti-inflammatory effects. Other organic acids, including succinic, acetic, and propanoic acids, may contribute to skin barrier repair. Together, this study demonstrates that SE 287-CFS protects against UVB-induced photodamage by modulating oxidative stress, suppressing inflammation, and improving skin barrier function. It also highlights the potential of postbiotics derived from skin commensals as candidates for microbiome-based skin photoprotection.IMPORTANCEStaphylococcus epidermidis is a dominant commensal skin bacterium that plays a crucial role in maintaining skin homeostasis and defending against various external stressors. UV radiation is a primary environmental factor that induces oxidative stress and inflammatory responses, resulting in photodamage and impaired skin barrier function. This study demonstrated the significant protective effect of SE 287-CFS in counteracting ultraviolet B (UVB)-induced skin damage. By modulating the reactive oxygen species (ROS)/MAPK/NF-κB signaling pathways, SE 287-CFS effectively alleviated oxidative stress, reduced inflammatory cytokine production, and enhanced skin barrier function. This research highlighted the potential of using skin commensal bacteria as a novel therapeutic strategy to enhance skin protection against UVB-induced damage, providing a promising approach to improve skin health under environmental stressors like UV radiation.
Bacterial endophthalmitis, an intraocular infection and inflammation, often progresses rapidly and leads to irreversible vision loss, especially in culture-negative cases where the diagnosis is delayed. We profiled infection-associated metabolomic and lipidomic alterations in the vitreous of patients with microbiological and clinically confirmed bacterial endophthalmitis to understand pathogenesis and identify distinct markers that drive infection and retinal injury. Untargeted metabolomic and lipidomic profiling of vitreous samples from affected patients and non-infectious retinal controls was performed using liquid chromatography coupled to tandem mass spectrometry. Metabolites and lipids with P-value < 0.05 were considered for further pathway-specific analysis. Metabolomic profiling distinctly segregated infected from control samples, revealing dysregulation in purine metabolism, amino acid turnover, polyamine synthesis, redox regulation, and vitamin pathways. Key metabolites, xanthine, hypoxanthine, spermine, seryl-valine, seryl-isoleucine, thymine, O-methyltyramine, and phenyltrimethylammonium, were significantly elevated. These elevations were accompanied by enhanced nucleotide degradation and increased proteolytic activity. Markers of immune activation and oxidative stress were concurrently upregulated, reflecting the broader biochemical disruption characteristic of bacterial endophthalmitis. Antioxidant seleno-L-methionine was downregulated, indicating redox imbalance. Purine, glutathione, vitamin B6, beta-alanine, and arginine-proline metabolism were broadly disrupted. Complementary lipidomics showed extensive vitreous remodeling. Alongside, fatty acids, bile acid derivatives, and membrane lipids were also dysregulated. Bacterial endophthalmitis triggers strong and consistent changes in metabolite and lipid profiles within vitreous, which reflect alterations in immune activation, oxidative stress, tissue breakdown, and metabolic reprogramming. The persistence of these biomolecular signatures in the vitreous highlights host response to infection and subsequent mechanistic elucidation of the specific drivers of retinal inflammation and progressive tissue damage.IMPORTANCEThe study highlights that bacterial endophthalmitis is not only driven by microbial burden but also by host metabolic and lipidomic reprogramming, transforming the understanding of disease pathogenesis. This study reveals metabolite-lipid networks in ocular infections, laying a critical foundation for precision medicine strategies in endophthalmitis management. Specifically, we found significant increases in key metabolites including xanthine, hypoxanthine, spermine, seryl-valine, seryl-isoleucine, thymine, O-methyltyramine, and phenyltrimethylammonium, together reflecting increased nucleotide degradation, proteolytic activity, and immune metabolic reprogramming. These molecular signatures not only provide deeper mechanistic insights into ocular infection but also provide potential biomarkers that could aid in diagnosis and monitoring of infection when conventional microbiological methods are inconclusive. The results highlight the clinical importance of metabolic profiling as a powerful tool to optimize visual outcomes, guide adjunctive therapeutic decisions, and enhance prognostic evaluation in patients affected by the condition.
Norovirus remains a leading cause of foodborne gastroenteritis globally. While isothermal amplification technologies offer advantages over traditional reverse transcription quantitative PCR (RT-qPCR) in terms of speed and portability, there is a lack of domestically developed isothermal amplification systems for norovirus in China. Using a domestic patented cross-priming amplification (CPA) technology, we established CPA systems targeting the predominant norovirus genotypes GII.3, GII.4, and GII.17. The systems were evaluated using 224 fecal samples with confirmed genotypes via sequencing. Our lab-developed CPA (Lab-CPA) systems demonstrated high sensitivity, detecting 98.0% (49/50) of GII.3, 100% (80/80) of GII.4, and 96.9% (31/32) of GII.17 samples, with 100% specificity across all genotypes. Compared to certified clinical RT-qPCR kits, the total percent agreement was 98.7%, 99.3%, and 96.9% for GII.3, GII.4, and GII.17, respectively, accompanied by a significant linear correlation in threshold times. Crucially, the Lab-CPA systems reduced processing time by 44-60 min compared to RT-qPCR. Successful integration into the commercial EasyNAT platform using all-in-one cartridges further streamlined the workflow while maintaining accuracy. Our CPA systems offer a rapid, cost-effective, and streamlined diagnostic solution, ideally suited for point-of-care testing in resource-limited primary healthcare environments.IMPORTANCENorovirus remains a critical public health threat, driving demand for rapid, deployable diagnostics. While isothermal amplification offers theoretical advantages over reverse transcription quantitative PCR, the absence of domestically developed systems in China has created a reliance on imported assays, limiting point-of-care testing capabilities. This study addresses that gap by establishing a novel cross-priming amplification system tailored to China's predominant norovirus genotypes. Achieving high sensitivity/specificity and significantly reducing processing time, this work validates a robust, field-adaptable alternative to conventional methods. Furthermore, successful integration with the commercial EasyNAT platform demonstrates immediate translational potential. By providing a cost-effective, high-performance domestic solution, these findings significantly enhance decentralized surveillance and emergency response capacity for norovirus infections in resource-limited settings.
Widespread prevalence of fluoroquinolone-resistant Campylobacter spp. in meat chicken is a major concern for public health. We examined the prevalence and antimicrobial resistance of thermotolerant Campylobacter spp. in 14 German organic meat chicken farms rearing slow-growing broilers, male layer hybrids, or dual-purpose cockerels in a longitudinal study. Most farms participated with four subsequent flocks. Each flock was sampled three times: approximately 1 month after hatching (before outdoor access), 2 weeks after first outdoor access, and at the end of the fattening period. Strikingly, most flocks were already Campylobacter-positive before first outdoor access. At the end of the fattening period, 98% of all flocks were positive, with C. jejuni being the predominant species. Antimicrobial resistance of 405 C. jejuni and 79 C. coli revealed overall high levels of ciprofloxacin and tetracycline resistance but absence of macrolide resistance. Ertapenem resistance was observed in individual farms. Generalized linear mixed models with random factors for farm and flocks within farm were used to assess predictors for the occurrence of ciprofloxacin- and tetracycline-resistant C. jejuni. Season, fattening type, and the interaction of sampling time point and fattening type were significant fixed effects. Results for flocks within farms showed clustering effects (ICCCIP = 0.447; ICCTET = 0.412). Whole-genome sequencing of representative isolates confirmed great overall genetic variety, with farm- or fattening type-dependent dissemination of certain multi-locus sequence types. ST21 clonal complex for sequenced C. jejuni and ST828 clonal complex for sequenced C. coli were most commonly detected.IMPORTANCECampylobacter spp. are major foodborne pathogens associated with the consumption and handling of chicken meat. This longitudinal study provides further insight into the prevalence and antimicrobial resistance of thermotolerant C. jejuni and C. coli in German organic meat chicken farms, a growing sector with strict limitations on antibiotic treatment. We included multiple flocks of farms fattening either slow-growing broilers, male layer hybrids, or dual-purpose cockerels to account for a variety of fattening types. Consequently, we were able to demonstrate significant differences in the occurrence of ciprofloxacin- and tetracycline-resistant C. jejuni based on fattening type, sampling time point, and season. Our findings highlight the successful spread and persistence of ciprofloxacin-, tetracycline-, and ertapenem-resistant C. jejuni and C. coli among all three types of organic meat chicken regardless of the lack of antibiotic treatment.
The opportunistic mold Aspergillus fumigatus, a WHO-priority fungal pathogen, causes life-threatening invasive infections in immunocompromised patients. A major antifungal drug target is the ergosterol biosynthesis pathway, primarily regulated by the transcription factors SrbA and AtrR. Recently, we identified RttA as an additional regulator of sterol homeostasis that mediates the activation of the sterol C24-methyltransferase-encoding gene erg6. Here, we demonstrate that overexpression of rttA leads to elevated production of the secondary metabolite fumicycline via induction of fccR, coding for the principal transcriptional activator of fumicycline biosynthesis. Lack of fccR blocked activation of fumicycline biosynthetic genes (fccA-fccE) during rttA overexpression, highlighting the indispensable role of FccR for activation of this cluster. In contrast, rttA inactivation did not abolish fumicycline production during fccR overexpression or during co-cultivation with Streptomyces rapamycinicus. In addition to fccA-fccE, our findings reveal a crucial role of FccR for the upregulation of a gene located adjacent to the fumicycline biosynthesis cluster, here termed fccF. Deletion of fccF severely diminished the conversion of fumicycline B into fumicycline C, uncovering FccF as fumicycline B O-acetyltransferase. Intriguingly, during fccR overexpression and resulting fumicycline overproduction, inactivation of fccF caused growth defects, likely due to precursor toxicity. Together, these findings broaden our understanding of fungal secondary metabolite regulation and reveal a novel role of RttA in the activation of fccR encoding the cluster-specific regulator that activates fumicycline production, including FccF, the terminal acetyltransferase in the pathway.IMPORTANCEAspergillus fumigatus is a major opportunistic fungal pathogen that causes severe infections in immunocompromised individuals and represents a significant global health threat. Understanding the regulatory networks controlling fungal metabolism is important for identifying potential therapeutic vulnerabilities. Here, we uncover a previously unrecognized role for the sterol homeostasis regulator RttA in activating fumicycline production through induction of the pathway-specific transcription factor FccR. We further show that FccR controls expression of an additional biosynthetic gene, fccF, and identify FccF as the terminal O-acetyltransferase required for conversion of fumicycline B to fumicycline C. Disruption of this final biosynthetic step impairs fungal growth during metabolite overproduction, indicating that accumulation of fumicycline B or related intermediates can negatively affect fungal growth. Together, these findings expand our understanding of fungal secondary metabolite regulation and provide new insights into the organization and function of the fumicycline biosynthetic pathway.
Oxford Nanopore Technology (ONT) enables rapid, portable pathogen identification and antimicrobial resistance (AMR) detection, but the reliability of downstream genomic analyses is highly dependent on DNA extraction quality, particularly in resource-limited settings. This study comparatively evaluated four portable bacterial DNA extraction protocols derived from three commercial kits to determine their impact on nanopore sequencing performance, bioinformatics workflow completion, and field deployability. Six gram-negative bacterial isolates (Escherichia coli, n = 4; Pseudomonas sp., n = 1; and Salmonella sp., n = 1) were processed using four extraction protocols: SwiftX DNA, SwiftX DNA with proteinase K (ProtK), SwiftX ParaBact, and NucleoSpin Microbial. Twenty-four resulting DNA extracts were sequenced on a single multiplexed MinION R10.4.1 flow cell. Sequencing data were analyzed using validated Galaxy-based generic and species-specific pipelines. Workflow completion was defined as successful progression through quality control, assembly, virulence, plasmid, and AMR detection modules. DNA purity varied substantially by extraction protocol and was strongly associated with successful workflow completion (Kruskal-Wallis, P = 0.0006). Accordingly, NucleoSpin Microbial achieved 100% workflow completion, and SwiftX ParaBact achieved 83%, while both SwiftX DNA-based protocols failed to complete full workflows. Importantly, key AMR genes required to classify isolates as multidrug-resistant were consistently detected using both NucleoSpin Microbial and SwiftX ParaBact extractions. However, NucleoSpin Microbial assemblies showed significantly higher contiguity and enabled a broader, more complete detection of virulence factors, pathogenicity islands, plasmid replicons, and accessory AMR genes, reflecting enhanced genomic resolution.IMPORTANCERapid whole-genome sequencing is increasingly used to detect antimicrobial resistance and guide public health responses, but its reliability depends strongly on how bacterial DNA is extracted. In this study, we have shown that DNA extraction method choice has a major impact on Oxford Nanopore sequencing performance across clinically relevant gram-negative bacteria. While silica column-based extraction maximized genomic completeness and analytical depth, paramagnetic bead-based reverse purification offered superior portability with sufficient resolution for frontline AMR surveillance. These findings highlight a practical trade-off between field deployability and high-resolution genomic characterization in low-resource settings.
The anaerobic decolorization of azo dyes by Shewanella species relies on an intricate electron transfer network, yet the full complement of genes involved remains incompletely defined. In this study, we used mini-Tn5 transposon mutagenesis to identify genes required for the decolorization of the azo dye acid yellow 36 in Shewanella putrefaciens CN32. Two genes were uncovered: menD, encoding a key enzyme in menaquinone biosynthesis, and lutB, involved in lactate metabolism. Deletion of menD caused a nearly complete loss of decolorization capacity without affecting growth. Supplementation with vitamin K2 or spent supernatant from the wild-type strain restored decolorization, demonstrating that menaquinone is essential. We systematically identified the menaquinone biosynthesis gene cluster of S. putrefaciens CN32, including menA, menB, menC, menE, menF, menG, menH, and paaI. Deleting most of these genes severely impaired decolorization, whereas deletions of menH, paaI, or an adjacent LysR-family regulator gene had no significant effect. Furthermore, acid yellow 36 induced biofilm formation in a dose-dependent manner, and this induction was abolished in most menaquinone synthesis mutants. Acid yellow 36 did not substantially upregulate transcription of the men gene cluster, suggesting that the dye promotes biofilm formation through a direct mechanism rather than by enhancing menaquinone synthesis. Collectively, our results establish menaquinone as a critical component for both azo dye decolorization and biofilm formation in S. putrefaciens CN32, and they point to the existence of a CymA-independent electron transfer branch in this strain.IMPORTANCEAzo dyes are environmental pollutants that can be effectively removed by Shewanella bacteria through anaerobic respiration. Understanding the genetic basis of azo dye decolorization is essential for improving bioremediation strategies. This study identifies menaquinone, a respiratory chain cofactor, as indispensable for the decolorization of acid yellow 36 in Shewanella putrefaciens CN32. By systematically mapping the menaquinone biosynthesis gene cluster, we show that an intact synthesis pathway is required for both dye reduction and biofilm formation, a lifestyle that enhances bacterial survival and metabolic activity in wastewater treatment systems. Importantly, the severe decolorization defect of menaquinone-deficient mutants contrasts with the relatively mild effect of deleting cymA, the canonical electron hub, suggesting the existence of a CymA-independent electron transfer route from menaquinone to azo dyes. These findings expand the current model of extracellular electron transfer in Shewanella and provide new genetic targets for engineering more efficient azo dye-degrading strains.
Syndromic multiplex panels enable rapid cerebrospinal fluid (CSF) diagnosis of meningitis and encephalitis, but clinically relevant inter-platform discordance persists, and its analytical basis remains incompletely understood. In this retrospective multicenter study, 182 residual CSF specimens from 182 patients at 4 Swiss tertiary-care centers and 10 external quality assessment samples were tested centrally by the QIAstat-Dx Meningitis/Encephalitis panel and compared with routine BioFire FilmArray results. Discordant results were adjudicated using pathogen-specific quantitative laboratory-developed tests (LDTs) calibrated to external standards, and enterovirus-positive specimens underwent molecular typing. Among 182 clinical CSF specimens, concordance was 87.9% across shared targets. Complete agreement was observed for herpes simplex virus type 2, Haemophilus influenzae, Neisseria meningitidis, Streptococcus agalactiae, and Cryptococcus neoformans/gattii. Across the remaining shared targets, positive percent agreement ranged from 50% to 93% and negative percent agreement from 98% to 100%. Enterovirus showed the greatest discordance, with 86% positive percent agreement and 10 FilmArray-only detections. LDT testing confirmed low-level enterovirus positivity in four discordant specimens (100‒700 copies/mL), and discordant viral detections clustered at lower target burden than concordant positives (P < 0.001). Molecular typing identified enterovirus species A, B, and C in both concordant and FilmArray-only positive specimens. In conclusion, FilmArray and QIAstat-Dx showed high agreement for CSF pathogen detection. Residual discordance was largely confined to low-burden viral detections, particularly enterovirus, and was more consistent with near-threshold target abundance than with a major type-restricted detection gap. These findings support routine syndromic CSF testing and emphasize interpretation of borderline positives in conjunction with confirmatory LDT testing and clinical context.IMPORTANCESyndromic cerebrospinal fluid panels are increasingly used as first-line tests for suspected central nervous system infection, yet aggregate agreement metrics can obscure clinically relevant differences near the limit of detection. In this multicenter comparison, most discordance clustered in low-burden enterovirus-positive cerebrospinal fluid (CSF) specimens and showed no clear segregation by enterovirus type, indicating that disagreement largely arose from near-threshold detection rather than a major gap in assay inclusivity. The broader implication is that performance comparisons of syndromic panels should move beyond overall agreement and specifically address how platforms handle borderline-positive results. This matters clinically because low-level viral detections can alter post-test probability, antimicrobial stewardship, CSF resampling, and follow-up microbiologic workup. More generally, the data support laboratory-integrated diagnostic strategies that combine multiplex screening with quantitative adjudication, molecular characterization, and interpretation of semiquantitative signals to strengthen the analytical and clinical utility of syndromic testing for central nervous system infections.
