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.
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 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.
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.
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.
Rhodococcus equi is an intracellular pathogen that causes pyogranulomatous pneumonia in foals through replication within macrophages. Protective immunity is generally attributed to T helper 1 (Th1)-type cellular responses, although the mechanisms underlying effective immunity remain incompletely defined. In this study, we assessed the immunological responses to various R. equi-derived antigen preparations in a murine model. Immunization with live R. equi protected against bacterial proliferation following challenge, whereas immunization with formalin-killed bacteria increased the bacterial burden. Similarly, immunization with bacterial components or secreted products lacking defined antigen specificity resulted in enhanced susceptibility to infection and increased mortality. Antibody analysis revealed that IgG derived from immunized mice significantly enhanced intracellular R. equi proliferation in vitro. These findings suggest that antibody-mediated uptake may contribute to intracellular survival and replication of R. equi. These observations are consistent with an antibody-dependent enhancement (ADE)-like phenomenon in this experimental model. Conversely, live immunization induced a CD8+ T cell-skewed response following infection. Collectively, these results indicate that antibody-associated humoral immunity is associated with disease exacerbation, whereas Th1-type cellular immunity correlates with protective immunity against R. equi infection.IMPORTANCERhodococcus equi is a major cause of severe pneumonia in foals, and effective vaccines remain unavailable. Recent studies have suggested that antibody responses can have complex effects during bacterial infections; however, their roles in R. equi infection remain poorly understood. In this study, we demonstrated that immunization with nonviable R. equi antigens induces antibody responses associated with increased bacterial burden and disease severity. These antibodies increased intracellular bacterial loads of R. equi in vitro, suggesting that antibody-mediated uptake may facilitate intracellular survival and replication in this experimental model. Contrarily, protective immunity induced by live bacteria was associated with T helper 1 (Th1)-biased responses and early CD8+ T cell expansion. These findings highlight the importance of considering the balance between humoral and cellular immunity in vaccine development and underscore the need for strategies that elicit protective Th1-driven cellular immunity against R. equi infection.
Fast and reliable antimicrobial susceptibility testing (AST) is essential for timely optimization of therapy in patients with bloodstream infections. This multicenter study assessed the performance of the bioMérieux VITEK REVEAL (VITEK REVEAL) system in comparison with the Beckman Coulter MicroScan (MicroScan) using positive blood culture (PBC) isolates. A total of 553 gram-negative PBCs were obtained from six clinical sites, with Enterobacterales representing the majority (91.1%, 504/553). Overall, 7,945 antimicrobial/organism combinations were evaluated to compare AST performance between VITEK REVEAL and MicroScan. VITEK REVEAL demonstrated strong concordance with MicroScan, achieving 98.1% essential agreement (EA) and 96.0% categorical agreement (CA). High agreement rates were observed for most antimicrobials, including amikacin, ceftazidime-avibactam, and meropenem-vaborbactam, each showing 100% EA and CA. Across all evaluations, discrepancies included 38 very major discrepancies (VMDs), 64 major discrepancies (MDs), and 218 minor discrepancies. Reference broth microdilution discrepant testing resolved most VMDs in favor of VITEK REVEAL, whereas most MDs aligned with MicroScan results. Species-level performance demonstrated strong agreement: Escherichia coli (98.1% EA, 95.6% CA), Klebsiella pneumoniae (99.2% EA, 97.7% CA), and Pseudomonas aeruginosa (97.4% EA, 95.1% CA). Time-to-result (TTR) analysis highlighted a major advantage of the VITEK REVEAL system. The mean overall TTR was 7.9 h (SD 0.4 h); sequential reporting enabled earlier results with a mean TTR of 6.5 h (SD 1.1 h). On average, VITEK REVEAL provided AST results 26-32 h (includes 18-24 h subculture for MicroScan) earlier than MicroScan, supporting its utility for fast, accurate AST reporting and potentially improving early antimicrobial decision-making.IMPORTANCEThis study represents one of the largest evaluations of VITEK REVEAL for fast antimicrobial susceptibility testing (AST) systems, including 553 gram-negative positive blood cultures across six clinical sites in the United States. The findings provide critical evidence for laboratories considering the adoption of fast AST, demonstrating that VITEK REVEAL delivers a strong combination of essential and categorical agreement (98.1% essential agreement [EA], 96.0% categorical agreement [CA] across 7,945 antimicrobial/organism combinations) and markedly reduced time-to-result (TTR). VITEK REVEAL performed consistently across key Enterobacterales and non-fermenters, including resistant phenotypes and β-lactam/β-lactamase inhibitor agents, underscoring its utility in diverse clinical scenarios. By providing susceptibility results 26-32 h earlier than MicroScan, this system may contribute to nimbler clinical decision-making, allowing for better antimicrobial stewardship practices.
Rapid antimicrobial susceptibility testing (AST) is crucial for combating antimicrobial resistance and guiding effective therapy. Conventional phenotypic AST methods are often time-consuming, requiring 18-72 h for results, while genotypic approaches often depend on pre-existing knowledge of resistance markers. Although nucleic acid-based phenotypic AST methods have reduced turnaround time, attempts to shorten antibiotic exposure often produce only small differences between susceptible and resistant bacteria. Consequently, most loop-mediated isothermal amplification (LAMP)-based phenotypic AST assays still require prolonged antibiotic exposure (up to 4 h) or rely on sophisticated high-resolution nucleic acid detection, such as digital LAMP, to accurately detect these subtle changes, limiting their simplicity. Therefore, there is a critical need to develop a rapid assay with both short antibiotic exposure times and a simple readout. To address this unmet need, we developed PMAxx dye-assisted colorimetric Dual DNAzyme-LAMP (PD-cDDLAMP), a rapid nucleic acid-based phenotypic AST method that enhances suppression of nucleic acid amplification from antibiotic-susceptible bacteria through the selective binding of photoreactive DNA-crosslinker dye (PMAxx) to the DNA of membrane-compromised cells. This strategy enables reliable discrimination between antibiotic-resistant bacteria ("signal-on") and antibiotic-susceptible bacteria ("signal-off") after only 30 min of antibiotic exposure using a user-friendly colorimetric DNAzyme-LAMP readout system. PD-cDDLAMP achieved 91.7% sensitivity, 83.3% specificity, and 87.5% accuracy in detecting Escherichia coli exposed to ampicillin and tetracycline, outperforming current methods by reducing antibiotic exposure time to just 30 min. The method was further validated in spiked milk samples, demonstrating its potential for on-site AST in dairy farming. PD-cDDLAMP's simplicity, speed, and reduced workflow complexity make it a promising tool for rapid AST. IMPORTANCE:Rapid, reliable antimicrobial susceptibility testing (AST) is essential for guiding targeted therapy and combating the escalating threat of antimicrobial resistance (AMR). However, current phenotypic ASTs are too slow for urgent decision-making, and genotypic assays cannot fully capture emerging or unknown resistance mechanisms. The PMAxx dye-assisted colorimetric dual DNAzyme-loop-mediated isothermal amplification (PD-cDDLAMP) assay introduced in this work provides a rapid nucleic acid-based phenotypic AST capable of distinguishing antibiotic-resistant from susceptible bacteria within 30 min of antibiotic exposure. By combining PMAxx-mediated suppression of nucleic acid amplification in susceptible cells with equipment-free colorimetric DNAzyme-LAMP readout, PD-cDDLAMP bridges the gap between speed and phenotypic accuracy. Its high diagnostic performance, compatibility with complex matrices such as milk, and minimal instrumentation requirements position this method as a promising point-of-care tool for clinical microbiology laboratories and on-site testing environments. PD-cDDLAMP has the potential to significantly shorten time-to-result while maintaining reliability, thereby supporting antimicrobial stewardship.
Highly sensitive molecular assays are increasingly used for malaria diagnosis but are not recommended for follow-up, as Plasmodium falciparum DNA detection may persist after effective antimalarial treatment, complicating these tests' clinical interpretation. The biological basis of this persistence remains poorly defined. We investigated the potential role of gametocytes in the context of treated infections. In a prospective cohort of 100 successfully treated patients, we analyzed 379 blood samples collected up to day 28 (D28) post-treatment. We used quantitative PCR (qPCR) to detect parasite DNA targeting PfvarATS, a mainly asexual-stage marker, and 18S ribosomal RNA (18S rRNA), a pan-stage marker. To differentiate residual nucleic acid detection from ongoing parasite transcriptional activity among patients' samples that remained DNA-positive at D28, we used RT-qPCR targeting PfvarATS, 18S rRNA, and gametocyte-specific transcripts (PfAP2-G, Pfs16, PfMGET, and PfCCP4). After anti-malarial treatment, 61% of samples remained qPCR positive at D28. PfvarATS transcripts declined sharply (12% positivity rate at D28), while 18S rRNA transcripts persisted longer (60% at D28). Gametocyte-specific markers were detected in a subset: Pfs16 (immature stages) was frequent early (100% at D0 and 94% at D3), while mature gametocyte markers (PfCCP4/PfMGET) peaked at D7 (33%) and were still detectable in 12% of samples at D28. Therefore, most D28 PCR positivity (88%) occurred without detectable mature gametocyte transcripts. Persistent P. falciparum DNA detection post-treatment in a non-endemic setting predominantly reflects non-viable parasites or residual genetic material and a minority of circulating mature gametocytes. This highlights a key diagnostic pitfall: post-treatment PCR positivity does not equate to treatment failure. Results must be interpreted with clinical context, timing, and assay targets.IMPORTANCEThis study confirmed that persistent P. falciparum DNA detection after effective antimalarial treatment mainly reflects residual parasite material, challenging the use of PCR in treatment efficacy follow-up. By excluding mature gametocytes as the primary source of persistent DNA detection, this study advances our understanding of post-treatment parasite nucleic acid kinetics in the organism and supports the importance of investigating alternative biological mechanisms.
Noroviruses (family Caliciviridae) are important enteric pathogens in humans and animals. The closely related GIV and GVI noroviruses in cats and dogs suggest interspecies circulation; however, their diversity and host restrictions have not been fully elucidated. In this study, we retrospectively investigated noroviruses in diarrheic cats in Hungary, determined the complete genomes of representative strains (n = 3), and performed comparative sequence and structural analyses of all available feline and canine GIV and GVI sequences. Fecal samples (n = 90; November 2024-January 2025) from stray and free-roaming cats were screened using RT-PCR. Capsid sequences and complete genomes were determined using 3'/5'RACE/RT-PCR, NovaSeq+ (Illumina), and Sanger sequencing techniques. Analyses included phylogenetic trees, p-distance comparison plots, recombination detection, VP1 P-domain dimer, and complete VP1-based capsid modeling. Eight (8.9%) samples were norovirus positive. All known feline genotypes (n = 4 GIV.2, n = 1 GVI.1, n = 1 GVI.2) were identified, each with the (GVI.P1) P-type. Nearly identical GIV.2 strains were found in the Budapest area (November 2024), representing the first non-shelter-associated case accumulation (a potential outbreak) of norovirus in cats. One complete genome of each genotype was determined. Multiple host-specific substitutions separate feline and canine noroviruses across viral proteins, including the surface-exposed VP1 P-domains and a feline-specific 10- or 12-amino-acid-long deletion in the GVI.2 P2 subdomain of currently unknown importance. Lower p-distance boundaries for inter-/intra-genogroups and inter-P-types were also established. Recombination at the ORF1/ORF2 junction was observed in two feline lineages, indicating that recombination and host adaptation jointly drive the evolution of carnivore noroviruses.IMPORTANCEDespite the major public health relevance of noroviruses, research has predominantly focused on human strains, while noroviruses of carnivores, particularly feline strains, remain poorly characterized. This study provides genetic, phylogenetic, and structural insights into feline noroviruses circulating in Hungary, including indications of the first reported large, non-shelter-associated potential outbreak of feline GVI.2 norovirus among free-living cats in Budapest and its surrounding areas, highlighting the natural epidemic potential of these viruses. Comparative genomic analyses demonstrated consistent host-based separation of closely related feline and canine GIV and GVI noroviruses across multiple genomic regions, supporting ongoing host-specific evolution. Structural analyses identified characteristic deletions within the highly variable surface-exposed P2 subdomain of feline GVI.2 capsid proteins, suggesting potential roles in interactions of receptors/attachment factors and/or immune evasion. Additionally, this study demonstrates the utility of pairwise p-distance analyses for recombinant strain recognition and for refining genetic demarcation criteria within feline and canine norovirus genogroups and P-types.
The onset of the chronic stage of infection by bacteria in the Burkholderia cepacia complex depends on the controlled transition to the biofilm-associated lifestyle. In this study, we systematically investigated the effect of all five hns genes of Burkholderia cenocepacia H111 on the motile-to-biofilm lifestyle switch and pathogenicity. The hns genes are distributed across the three replicons (hns1.1 and hns1.2 on chromosome 1, hns2 on chromosome 2, and hns3.1 and hns3.2 on the megaplasmid pC3). We showed that deletion of either hns1.1 or hns1.2 affects cell growth and promotes the cell transition to the biofilm, with the major impact observed in cells lacking hns1.2. According to a comparative transcriptome analysis, the motile-to-biofilm switch linked to the absence of hns1.2 is supported by downregulation of flagellar and chemotaxis genes and upregulation of several genes involved in biofilm formation. In contrast, biofilm-related genes were downregulated; biofilm formation was reduced; and motility was increased in cells lacking hns3.1. An opposite impact of hns1.2 and hns3.1 on the motile-to-biofilm transition was also supported by using cells expressing these hns genes at high levels. Furthermore, hns3.1 and hns1.1 were shown to play a role in bacterial virulence in the Galleria mellonella infection model. Therefore, this study uncovers hns genes as new players of the motile-to-biofilm transition in B. cenocepacia. IMPORTANCE:Several opportunistic pathogenic bacteria can thrive as motile, free-living cells in soil and water, as well as in biofilm in their hosts. This includes the closely related species comprising the Burkholderia cepacia complex, which chronically colonize the airway in individuals with cystic fibrosis. Over the last two decades, the number of genes involved in the control of the motile-to-biofilm lifestyle switch has increased in this bacterial group, particularly in Burkholderia cenocepacia. In the present study, we uncover three hns genes that participate in the motile-to-biofilm lifestyle switch in B. cenocepacia H111 under the conditions tested. While two chromosomally encoded hns genes work to maintain cells at the motile stage, a megaplasmid-encoded hns gene facilitates the cell transition to biofilm. By systematically investigating all five hns genes, we considerably expand our understanding of the control of the motile-to-biofilm lifestyle switch in the Burkholderia cepacia complex.
Microsporidia MB, a recently discovered endosymbiont Anopheles arabiensis mosquitoes, shows great potential as a malaria-blocking tool due to its Plasmodium-blocking phenotype. The symbiont utilizes vertical (mother-to-offspring) and horizontal (sexual) transmission routes to propagate across anopheline populations. Microsporidia MB occurs throughout the Anopheles life cycle, with no evidence to date of the existence of a secondary host. In this study, we conducted a comprehensive molecular screening of Microsporidia MB in aquatic macrofauna associated with Anopheles larval habitats to determine whether they serve as secondary hosts for Microsporidia MB. We collected aquatic macrofauna from anopheline breeding sites along the Ahero irrigation scheme, including insects from the orders Hemiptera (families Naucoridae, Micronectidae, Nepidae, and Notonectidae), Coleoptera (families Staphylinidae, Noteridae, Hydrophilidae, and Dytiscidae), Diptera (anopheline and non-anopheline larvae and pupae), Orthoptera, and Ephemeroptera; snails (Hygrophila); crustaceans (Podocopida); and frogs (Anura). We detected Microsporidia MB in 15.04% of anopheline larvae, but not in non-anopheline mosquito larvae, pupae, or any other aquatic macrofauna, despite their continuous interaction with anopheline mosquitoes in the larval habitats. The absence of Microsporidia MB detection across the diverse assemblage of aquatic macrofauna surveyed in this study suggests that these taxa are unlikely to constitute major secondary hosts or reservoirs of the symbiont. While broader surveys across additional taxa are needed to fully exclude alternative hosts, the consistent detection of Microsporidia MB at high infection intensities in Anopheles mosquitoes supports a strong host association. This reinforces its potential as a targeted, species-specific, and environmentally safe symbiont-based intervention for malaria control.IMPORTANCEMalaria continues to pose a significant global health burden, particularly in sub-Saharan Africa, and causes thousands of deaths annually. Microsporidia MB is an anopheline-associated symbiont that is vertically and horizontally transmitted in Anopheles mosquitoes and shows great promise as a complementary malaria control tool due to its Plasmodium blocking phenotype. However, its lifecycle is not fully understood. Understanding its lifecycle and host range is important for assessing the symbiont's ecological safety and feasibility for field release. This study found no evidence of Microsporidia MB in a wide range of aquatic macrofauna using sensitive molecular screening, despite their frequent contact with infected mosquito larvae. While non-detection does not rule out rare or transient infections, the results suggest that these organisms are unlikely to serve as major hosts of the symbiont. This apparent host specificity reduces concerns about spreading to non-target species and supports the development of Microsporidia MB as a targeted malaria-control intervention.
Invasive infections caused by Candida spp. are associated with high morbidity and mortality, particularly in immunocompromised patients, and are increasingly difficult to treat due to rising antifungal resistance. In this work, we further investigate the antifungal properties of a previously reported nickel N-heterocyclic biscarbene derived from caffeine (compound 5) and describe the synthesis of an analogous nickel N-heterocyclic biscarbene based on benzimidazole (compound 8), designed to evaluate the impact of the biscarbene and xanthine frameworks on activity and toxicity. Compound 5 displayed selective activity against Nakaseomyces glabratus (Candida glabrata), inhibited biofilm formation, and showed greater cellular accumulation in this species. In a Galleria mellonella infection model, compound 5 significantly reduced fungal burden while exhibiting lower cytotoxicity than the benzimidazole analog. Importantly, although compound 5 and fluconazole are individually fungistatic, their combination was fungicidal against N. glabratus. In the presence of compound 5, the minimal inhibitory concentration of fluconazole decreased against both a fluconazole-resistant petite mutant and a respiratory-competent N. glabratus strain evolved in vitro to fluconazole resistance. Compound 5 increased the frequency of petite mutants, suggesting an effect on mitochondrial function; however, its retained activity against these mutants and its synergism with fluconazole in the petite background indicate an additional mechanism of action. These findings identify compound 5 as a promising antifungal adjuvant.IMPORTANCEInvasive infections caused by N. glabratus are increasingly hard to treat because this pathogen is often tolerant to azoles and is developing resistance to echinocandins, leaving clinicians with few effective options. Our work explores a nickel-caffeine complex that shows selective activity against N. glabratus and low toxicity, features that make it attractive as a promising candidate for further preclinical evaluation. Notably, this compound enhances the activity of fluconazole, turning a commonly used fungistatic drug into a fungicidal combination against both susceptible and resistant N. glabratus. By also affecting mitochondrial function yet remaining active in respiratory-deficient mutants, the compound appears to act through mechanisms distinct from existing antifungals. These properties suggest that metal-based xanthine complexes could be developed as adjuvants to enhance azole efficacy against difficult-to-treat N. glabratus infections, addressing an important priority identified for fungal pathogens.
Dengue virus (DENV) remains a critical global health threat, necessitating rapid pan-detection and precise serotyping. In this study, we developed a multiplex real-time RT-qPCR assay for simultaneous detection and identification of DENV1-4. Guided by a systematic genomic conservation analysis of 4,754 sequences, we targeted the highly conserved 5' untranslated region (5' UTR) to design one pan-DENV and four serotype-specific primer-probe sets. The assay demonstrated robust analytical performance, with the limit of detection (LOD) ranging from 15.8 to 1.58 × 103 copies/mL and the lowest LOD (15.8 copies/mL) observed for serotype I with specific primer-probe sets. There was no cross-reactivity with other common pathogens of the Orthoflavivirus or Alphavirus genus pathogens, and clinical specificity was further validated as 100% using a negative cohort of non-dengue febrile samples. Validation using 53 clinical DENV-positive samples showed 100% concordance with a reference diagnostic kit for serotype identification. While the pan-DENV set served as a high-specificity screening tool with broad reactivity, it exhibited a clinical sensitivity of 88.7%, particularly missing low-titer samples (Ct > 30). This discrepancy, coupled with the superior sensitivity of the serotype-specific sets, suggests the presence of incomplete genomic fragments in acute-phase sera, highlighting the advantage of our 5' UTR-targeted approach in assessing viral genomic integrity. This dual-layered multiplex assay provides a robust, rapid, and cost-effective tool for clinical diagnosis and epidemiological surveillance.IMPORTANCEDengue fever is a leading cause of systemic viral disease worldwide. Co-circulation of four distinct serotypes complicates clinical management and increases severe disease risk. Here, we presented a novel multiplex RT-qPCR assay enabling simultaneous pan-DENV detection and serotype identification in two tubes, comprising six reactions including an internal control (IC), thereby streamlining dengue diagnosis. A key innovation is the systematic selection of the 5' UTR as the diagnostic target, guided by big-data genomic analysis. Unlike assays targeting the 3' UTR, which may overrepresent viral loads by capturing redundant subgenomic orthoflavivirus RNA (sfRNA), our 5' UTR-targeted design provides a more stringent measure of genomic integrity. Furthermore, the modular performance of our assay combining broad screening via a pan-DENV set with high-sensitivity subtyping which offers a dual-layer diagnostic framework. This work provides a high-performance tool for timely clinical intervention and enhances our molecular understanding of DENV fragments in clinical specimens.
