Microbiology spectrum最新文献

Dermatophagoides farinae (DFA), the most prevalent aeroallergen in allergic asthma, releases extracellular vesicles (EVs) containing dfa-miR-276-3p, which plays an important role in DFA sensitization. In this study, we utilized AAV-dfa-miR-276-3p to create a mouse model with dfa-miR-276-3p overexpression in lungs. Using this model, we demonstrated that dfa-miR-276-3p acted as a priming factor that exacerbated DFA extract (DFE)-induced airway inflammation in mice. We also confirmed that dfa-miR-276-3p functioned as a priming pro-inflammatory factor in human bronchial epithelial BEAS-2B cells, enhancing the release of inflammatory cytokines induced by DFE. Furthermore, we found that dfa-miR-276-3p regulated stanniocalcin 1 (STC1) in a targeted manner, leading to increased inflammatory cytokine secretion and activation of the reactive oxygen species (ROS)/nuclear factor kappa B (NF-κB) pathway. Notably, the addition of recombinant human STC1 alleviated the inflammatory effects of dfa-miR-276-3p, reducing airway inflammation and dampening ROS/NF-κB signaling. Parallel findings in mouse models confirmed that dfa-miR-276-3p drove DFA-induced airway inflammation through STC1-dependent regulation of the ROS/NF-κB pathway. Our study reveals a cross-kingdom regulatory role for DFA-derived miRNAs in the pathogenesis of allergic asthma, highlighting dfa-miR-276-3p as a crucial priming factor in the process of allergic airway inflammation induced by DFA.IMPORTANCEWe demonstrated that dfa-miR-276-3p acted as a priming factor that exacerbated Dermatophagoides farinae (DFA) extract-induced airway inflammation in mice, and functioned as a priming pro-inflammatory factor in human bronchial epithelial BEAS-2B cells. In addition, dfa-miR-276-3p was found to regulate stanniocalcin 1 (STC1) in a targeted manner, leading to increased secretion of inflammatory cytokines and activation of the reactive oxygen species (ROS)/nuclear factor kappa B (NF-κB) pathway, and addition of recombinant human STC1 alleviated the inflammatory effects of dfa-miR-276-3p, reducing airway inflammation and dampening ROS/NF-κB signaling. Parallel findings in mouse models confirmed that dfa-miR-276-3p drove DFA-induced airway inflammation through STC1-dependent regulation of the ROS/NF-κB pathway. Our findings provide new insights into the role of DFA-derived miRNAs in the development of allergic asthma and propose an alternative pathway for DFA sensitization that may have significant clinical implications for allergy prevention and treatment.

The Enterobacter cloacae complex (ECC) is one of the major causes of hospital-acquired infections. However, achieving accurate species identification and comprehensive resistance profiling remains difficult in clinical practice, with a limited understanding of species-specific resistance patterns. ECC isolates were collected from patients with bloodstream infections at Peking Union Medical College Hospital between 2015 and 2020. Whole-genome sequencing (WGS) was performed to identify species, analyze antimicrobial resistance genes, and explore genomic variation in serial isolates. Multi-locus sequence typing (MLST) profiles were extracted from the WGS data. Phylogenetic analysis was conducted based on hsp60 sequences. Eleven hsp60 clusters were identified, with cluster VIII being the most prevalent (28/108). Average nucleotide identity (ANI)-based species classification showed Enterobacter hormaechei (31.5%) and Enterobacter xiangfangensis (15.7%) were dominant species. Five clade-cluster pairs (B-VIII, A-VI, G-XI, D-III, R-IX) accounted for 74% of isolates. A total of 90 sequence types (STs) were detected, including 29 novel STs. Resistance gene analysis revealed a high prevalence of bla_ACT, with distinct distribution patterns observed among different species. Twenty isolates were carbapenem-resistant, with three carrying bla_NDM-1/5. Enterobacter roggenkampii was the most common species (5/20) among all carbapenem-resistant isolates, and 83.3% of the isolates showed resistance to both carbapenems and colistin. Comparative genomics of longitudinal isolates from individual patients revealed adaptive single-nucleotide polymorphisms (SNPs) in pco genes. This study provides a detailed genomic characterization of ECC isolates from bloodstream infections, highlighting species diversity, resistance gene distribution, and potential within-host evolution. These insights advocate genome-based surveillance in managing ECC infections and understanding resistance evolution in clinical contexts.IMPORTANCEEnterobacter cloacae complex (ECC) is a major cause of hospital-acquired bloodstream infections, yet species-level identification and resistance profiling remain challenging. As one of the largest whole-genome sequencing (WGS)-based studies of ECC bloodstream isolates in northern China to date, we performed whole-genome sequencing of 108 ECC isolates, revealing high genetic diversity and identifying 29 novel sequence types. We clarified the correspondence between species, clades, and clusters and highlighted Enterobacter roggenkampii as a potential high-risk species linked to carbapenem and colistin resistance. Our findings not only improve the understanding of ECC population structure and resistance evolution in China but also provide valuable genomic data for future epidemiological surveillance and species-level diagnostics.

Clostridium butyricum exhibits a dual role, acting not only as a probiotic but also as an opportunistic pathogen associated with neonatal necrotizing enterocolitis (NEC) and infant botulism. We aimed to establish high-resolution genotyping frameworks to improve molecular surveillance and outbreak investigations. We analyzed 297 C. butyricum genomes, including 200 isolates from preterm neonates across 13 French neonatal intensive care units over a 20-year period and 97 publicly available genomes. A core-genome multilocus sequence typing (cgMLST) scheme was developed using chewBBACA, defining 2,621 loci, and applied to genomes with ≥95% locus presence. Core-genome single-nucleotide polymorphism (cgSNP) analysis was performed for complementary resolution. Phylogenetic cgMLST classified isolates into nine major clades. Some clinical strains displayed clonal relationships, whereas others were geographically and temporally unrelated. All botulinum neurotoxin type E-producing strains were grouped within a single clade. NEC-associated isolates showed geographic and temporal clustering, but no clade was uniquely linked to NEC. cgSNP analysis identified 11 clusters with overall discriminatory power similar to cgMLST while providing finer resolution for NEC-related strains. We propose robust cgMLST and cgSNP schemes for C. butyricum, enabling high-resolution genotyping and supporting epidemiological surveillance and outbreak investigation of this emerging opportunistic pathogen in neonatal settings.

Importance: Clostridium butyricum has been identified in fecal samples from both asymptomatic neonates and cases of necrotizing enterocolitis (NEC). Using a large collection of strains from different origins and spatiotemporal contexts, we developed and established a cgMLST scheme for the molecular typing of C. butyricum. Our results show that most C. butyricum strains cluster independently of origin and spatiotemporal context factors. However, specific cgMLST clades of C. butyricum were found for plant and botulinum neurotoxin type E strains. Clonal strains were also identified. No specific cgMLST clade was found to be genetically associated with NEC. cgSNP showed higher discriminatory power compared to cgMLST. Importantly, cgSNP provided better discriminatory power for strain relatedness with respect to strains isolated from NEC patients.

Biofertilizers offer a promising avenue to reduce phosphate fertilizer reliance. However, inconsistent lab-to-field conversion undermines confidence in the technology. Increasingly, initial screens of bacteria are conducted in silico, based on the presence of a widely accepted set of phosphate solubilization-associated "canon" genes. However, these genes capture only a subset of the mechanisms observed experimentally in microbe-mediated solubilization. This study uses a transcriptomic systems approach to compare the transcriptional response of three novel Pantoea rara strains with divergent phosphate solubilization indices (PSI): a wild type (PSI = 1.74), an enhanced efficiency mutant (PSI = 4.13), and a null mutant (PSI = 0), which are indistinguishable using canonical gene markers alone, to phosphate limitation. Soluble phosphate limitation triggered robust but transient transcriptional responses across 80 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Despite its presence, the gcd gene, the most commonly used marker for phosphate solubilization functionality, was not upregulated in either of the solubilizing strains. Instead, each strain appeared to rely on a unique combination of organic acids, including acetic, citric, formic, and malic acid. Notably, the divergent phenotypes of both mutants were linked to a rewiring of the phosphate limitation response, namely a heightened limitation response in the enhanced mutant and an apparent "phosphate blindness" in the null mutant. These findings suggest that system-level characterization of phosphate solubilizers can reveal auxiliary pathways and genes overlooked by current annotation frameworks. Integration of auxiliary contributors with the existing canon could improve the predictive power of pre-field phosphate solubilization screens and subsequently help bridge the lab-to-field gap in biofertilizer development.IMPORTANCEDespite the potential benefits to the productivity of agricultural systems and the health of local ecosystems, enthusiasm for phosphate-solubilizing biofertilizers has been dampened by a significant performance gap that occurs when candidates are transitioned from the lab to the field. This inconsistency is partially attributable to narrow screening strategies that rely on simplified in vitro assays and the broad use of a narrow set of canonical genes, such as gcd, as functional proxies. This study leveraged a rare set of three Pantoea rara strains that differ phenotypically in their phosphate solubilization capacity but are identical using standard genetic markers. This system enabled the identification of overlooked genetic and regulatory contributors. These findings reveal limitations in current screening methods and underscore the need for a more comprehensive molecular framework to guide biofertilizer discovery.

The QuantiFERON-TB Gold Plus (QFT-Plus; Qiagen, Hilden, Germany) assay is widely used for latent TB infection (LTBI) screening; however, borderline results often show variability during follow-up. We investigated the longitudinal variability of borderline TB1 and TB2 results throughout the follow-up period. A total of 770 individuals with initial borderline results (0.2-0.7 IU/mL) in either the TB1 or TB2 tube were retrospectively collected over a 5-year period. Agreement and correlation between the initial TB1 and TB2 results were analyzed. Trends in reversion and conversion were evaluated separately for each tube and for combined results among individuals with available follow-up tests. Initial TB1 and TB2 borderline results showed weak agreement (Cohen's κ = 0.441, 95% CI 0.401-0.480) and moderate correlation (Spearman's ρ = 0.640, 95% CI 0.596-0.680; P < 0.001). TB2 exhibited the highest variability during the first follow-up (33.8%, 47/139), which decreased with subsequent testing. Among those with ≥2 follow-ups, 30.2% (13/43) showed result changes. Cases with borderline values in both TB1 and TB2 showed higher rates of reversion and conversion, whereas having a low-negative result (<0.2 IU/mL) in at least one tube was associated with minimal variability. The QFT-Plus assay provides separate TB1 and TB2 values, and integrating these results may improve interpretation. Follow-up testing is particularly warranted when borderline results occur in both tubes. Further studies are needed to define appropriate criteria and optimal retesting intervals.

Importance: This is the first study to longitudinally assess borderline QuantiFERON-TB Gold Plus (QFT-Plus) results in an intermediate TB burden setting, highlighting the need for defined criteria and retesting intervals.

Calcific aortic valve disease (CAVD) is a common disorder associated with substantial morbidity and mortality. Although the gut microbiome has complex associations with cardiovascular disease, its variation across the calcification spectrum in CAVD remains poorly defined. We profiled aortic-valve transcriptomes from 31 patients spanning graded calcification and paired these with matched stool microbiome profiles. We identified subtle yet widespread transcriptional changes in mild CAVD (m-CAVD), consistent with a progressive relationship between calcification burden and gene-expression remodeling. At the community level, the gut microbiome in m-CAVD exhibited an intermediate configuration between non- and higher-calcification profiles, suggesting an early shift in the gut ecosystem along the disease continuum. At the genus level, we identified 11 taxa associated with stage; notably, Anaerococcus increased with calcification burden, whereas Rheinheimera declined across stages. These results refine the pathophysiology landscape of CAVD by connecting stage-dependent valvular transcriptional changes with coordinated shifts in the gut microbiome and indicate that early, microbiome-targeted interventions may be promising.

Importance: Calcific aortic valve disease is a common valvular heart disease. Due to the difficulty in sampling arterial calcified tissues, research on the interaction between their gene expression and the gut has been limited. In this study, by analyzing the transcriptional profiles of calcified aortic valve tissues from patients with different levels of calcification and the characteristics of their corresponding gut microbiota, we identified consistent features between lesion gene expression and gut microbiota variation. This provides important evidence for the association between the gut microbiota and disease development stages, offering a new perspective for understanding disease progression and early intervention.

Bifidobacterium represents a diverse genus of commensal gut bacteria with key roles in human health, from metabolizing indigestible fibers to protecting against pathogens. While metagenomic studies have highlighted significant strain diversity for Bifidobacterium species within individuals, their systematic isolation and phenotypic characterization can be hampered by the significant effort and biases inherent in traditional culturomics. Here, we explored the utility of a high-throughput single-cell dispensing system (B.SIGHT)-based workflow for accelerating the process of isolating diverse Bifidobacterium strains from fecal samples. Systematic assessment of this workflow revealed a high single-cell dispensing frequency (>88%) and the ability to preserve species diversity when a pool of Bifidobacterium strains was dispensed. Culturing-related factors including the use of an effective selection medium, such as the Bifidus Selective Medium supplemented with mupirocin, and the length of pre-dispensing incubation were found to be critical in determining isolation success. Leveraging this workflow, we obtained a total of 622 viable isolates from five Singaporean fecal samples, of which >98% were found to be from Bifidobacterium species. Whole-genome sequencing of 96 isolates identified six different Bifidobacterium species with both inter- and intra-subject strain and lineage diversity, and the majority (>66%) were novel relative to large public genomic databases. Our findings highlight the ability of this high-throughput culturomics workflow to accelerate the recovery of diverse and novel Bifidobacterium strains, enabling further interrogation of their functional characteristics and advancing our understanding of important bacterial species in the gut microbiome.

Importance: The field of high-throughput microbial culturomics is still in its early stages. Enhancing our ability to isolate and phenotypically test bacterial strains from complex communities is crucial for advancing microbiome research and healthcare development. Given the time and cost inefficiencies of traditional culturing methods, a more efficient, high-throughput approach to obtain isolates is needed. In the present study, we assessed a single-cell dispensing platform and developed a workflow to isolate diverse Bifidobacterium strains from fecal samples. We demonstrated here the capability of this novel technology to efficiently obtain hundreds of isolates of a targeted group, covering both species and strain diversities. This generalizable and scalable method can potentially allow for the high-throughput recovery of microbes from other taxonomic groups, providing a fundamental step in improving the culturomics framework to complement metagenomic approaches and enable isolate-level functional studies of important microbes.

Wolbachia induces female-biased sex ratios in host populations through male-specific killing, thereby enhancing its spread via maternal transmission. The prophage-associated gene wmk has been proposed as a key effector underlying this male-killing (MK) phenotype. Interestingly, wmk homologs are found across diverse Wolbachia strains, regardless of the presence of male killing, and show extensive sequence divergence. However, the functional implications of this sequence variation-particularly among distant homologs-remain poorly understood. Here, we analyzed wmk homologs from 18 Wolbachia genomes, including 17 publicly available genomes and one de novo assembled genome from the parthenium beetle. We identified a highly divergent wmk homolog in the latter and predicted its protein structure using AlphaFold2, followed by molecular dynamics simulations to characterize its molecular features. Our analyses reveal that wmk-encoded protein exhibits a modular architecture comprising two helix-turn-helix (HTH) domains and an additional accessory domain not previously described. Modeling of inter-domain interactions further supports the functional relevance of this modular organization. Comparative analyses across all homologs distinguish divergent wmk from other canonical variants based on sequence composition, structural organization, and phylogenetic clustering. Notably, the structural features that differentiate these homologs also distinguish wmk from other known HTH-type regulators. Together, our findings provide new molecular insights into the architecture and evolution of wmk, offering a framework to understand the mechanistic basis of the MK phenotype in Wolbachia.IMPORTANCEWolbachia-induced male killing presents a promising strategy for the biocontrol of vector and pest populations. The wmk gene has been identified as a candidate underlying this phenotype. Yet, the significance of its sequence variation-particularly between highly divergent homologs-remains unclear. Here, we characterize a divergent wmk homolog from a novel Wolbachia strain. Then, we compare sequence and structural features of wmk homologs across a total of 18 Wolbachia strains using AlphaFold2 and molecular dynamics simulations. Our results highlight key molecular features in divergent variants and provide new insights into wmk evolution, laying a basis for exploring its functional diversity across Wolbachia lineages.

The objective of this study is to establish a droplet digital PCR (ddPCR) method for the detection of human parvovirus B19 (B19V) and provide accurate and reliable technical support for molecular biological diagnosis and epidemiological investigation of the virus. Specific primers and a TaqMan probe targeting the NS1 region of the B19V genome were designed, and a reaction system based on ddPCR was constructed and optimized. The methodology was validated through sensitivity, specificity, and repeatability tests. Subsequently, the method was applied to eight B19V-positive samples to evaluate its practical applicability. Methodological validation experiments demonstrated excellent sensitivity with a good linear relationship (R² = 0.9974) and a minimum detection limit of 1.013 × 10⁻¹ copies/μL. No cross-reactivity was observed with 11 respiratory pathogens, 5 pathogens presenting similar clinical manifestations, and 4 blood-borne pathogens. The inter- and intra-assay coefficients of variation for high-, medium-, and low-concentration samples were all ≤10%. Furthermore, this method was successfully applied to clinical samples, with stable detection of B19V in five high-concentration throat swab samples and three low-concentration blood samples. The ddPCR method established in this study exhibits high sensitivity, specificity, and repeatability, offering a robust tool for molecular diagnosis and epidemiological monitoring of B19V infection.

Importance: Early human parvovirus B19 (B19V) infections were sporadic or occurred in small clusters, attracting little attention. Since late 2023, nine European Union/European Economic Area (EU/EEA) countries have reported a significant increase in B19 infections. Moreover, the virus has robust physicochemical tolerance and the potential to resist pathogen removal processes such as filtration, inactivation, and pasteurization, which have raised the close attention and vigilance of international organizations, governments, and the public. Despite existing qPCR and antigen/antibody tests, the growing number of infections in multiple countries highlights the need for a more accurate and efficient detection system. Based on this, our team carried out related research and built a system configuration based on the third-generation droplet digital polymerase chain reaction platform, with a view to updating the B19V detection method.