Objective To isolate the Streptomyces hebeiensis strain JL9001 with significant biocontrol potential against tomato Fusarium wilt from Pseudostellaria heterophylla roots, elucidate the complete genome sequence and functional annotation of the strain, and extract genetic data pertaining to its secondary metabolites, thus offering a valuable microbial resource and a theoretical foundation for the biological management of tomato Fusarium wilt. Methods The colony morphology on various media was examined via the plate streaking technique. The antagonistic properties of strain JL9001 against Fusarium oxysporum were evaluated through the plate confrontation assay. The activities of metabolites (crude fermentation extract) against F. oxysporum were assessed via the microdilution method. The effectiveness of strain JL9001 in managing tomato Fusarium wilt was evaluated through root drenching with the fermentation broth. Whole genome sequencing of strain JL9001 was conducted, and the sequencing data were analyzed by appropriate software for species identification, gene prediction, functional annotation, and prediction of secondary metabolite biosynthesis gene clusters. Results Strain JL9001 demonstrated optimal spore production on the SIM medium. Antagonistic assays indicated that it inhibited the mycelial growth of F. oxysporum by 40.18%. Furthermore, the crude fermentation extract at a concentration of 1 000 μg/mL completely inhibited F. oxysporum. Pot trials revealed that irrigation with the fermentation broth of JL9001 resulted in a 51.61% reduction in tomato Fusarium wilt on day 13. The genome of strain JL9001 comprised 7 700 822 base pairs with the G+C content of 71.46%, encompassing 6 589 genes. Analysis predicted the presence of 27 biosynthetic gene clusters for secondary metabolites including terpenoids, polyketides, and siderophores, which may possess antimicrobial properties. Conclusion This study elucidates, through antagonistic and pot experiments, that strain JL9001 effectively mitigates the incidence of tomato Fusarium wilt. The analysis of the genomic composition and functional gene information of strain JL9001 provides a basis for exploring the antimicrobial mechanisms of natural products, examining secondary metabolite biosynthetic gene clusters, and assessing the potential of Streptomyces-derived secondary metabolites.

Objective Tad pili are widely distributed in Gram-negative bacteria and are associated with the virulence of various pathogens. However, the studies about the Tad pili in Vibrio parahaemolyticus remain limited. This study aimed to elucidate the role of the Tad pilus secretin CpaC (VP2419) in the biological functions of V. parahaemolyticus. Methods The cpaC-deleted mutant (ΔcpaC) and complemented strain (CΔcpaC) were constructed from the wild-type (WT) strain (SH112) of V. parahaemolyticus. The strains were compared in terms of biofilm formation, competitiveness, swarming and swimming motility, cell adhesion, cytotoxicity, as well as virulence, tissue colonization, and pathology in mice. Results Regarding environmental adaptation, compared with the WT strain, ΔcpaC exhibited significantly decreased competitiveness, motility, and biofilm formation. In terms of pathogenicity, ΔcpaC demonstrated significantly reduced cell adhesion, cytotoxicity, as well as attenuated virulence, tissue colonization, and pathological damage in mice, compared with the WT strain. Conclusion As the Tad pilus secretin in V. parahaemolyticus, CpaC participates in multiple functions related to environmental adaptation and pathogenicity, including competitiveness, biofilm formation, motility, cell adhesion, cytotoxicity, and tissue colonization. These findings provide important insights for a deeper understanding of the biological functions of Tad pili.

Objective To systematically isolate and purify the polysaccharide from the mycelium of Streptomyces rochei D74 (SRP), elucidate its fine structure, and evaluate the effect of the purified polysaccharide fraction on the growth of Salvia miltiorrhiza hairy roots and the biosynthesis of tanshinones, along with the underlying mechanism. Methods The crude polysaccharide was extracted using hot water, which was followed by ethanol precipitation and deproteinization via the Sevag method. Further purification was performed using DEAE-52 anion-exchange chromatography and Sephadex G-100 gel filtration chromatography. The physicochemical properties and structural features of the main active fraction, SRP-W-2, were systematically characterized by Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography-mass spectrometry (HPLC-MS), and nuclear magnetic resonance (NMR). The effects of SRP-W-2 on hairy root growth and the biosynthesis of tanshinones were assessed by measuring biomass, tanshinone content, and the expression levels of key biosynthetic genes. Results SRP-W-2 was obtained with a yield of 2.41%. It was primarily composed of glucose and galactose at a molar ratio of 12.53:1. Structural analysis revealed that the backbone of SRP-W-2 consisted of →4)-α-d-Glcp-(1→ and →4)-α-d-Galp-(1→ residues, with branching points at →4,6)-α-d-Glcp-(1→ and →4,6)-α-d-Galp-(1→. The side chain was identified as α-d-Glcp-(1→4)-α-d-Glcp-(1→. Bioactivity assays demonstrated that SRP-W-2 significantly enhanced both the biomass of S. miltiorrhiza hairy roots and the accumulation of tanshinones. After 15 d of treatment with 50 mg/L SRP-W-2, the dry weight of the hairy roots increased by 37.52%. Meanwhile, the content of cryptotanshinone (CT), dihydrotanshinone I (DT-I), tanshinone I (T-I), and tanshinone IIA (T-IIA) was increased by 19.0-fold, 6.4-fold, 2.8-fold, and 4.8-fold, respectively. Gene expression analysis further indicated that SRP-W-2 up-regulated key genes involved in the tanshinone biosynthetic pathway, including HMGR, DXS, DXR, and GGPPS. Conclusion The polysaccharide fraction SRP-W-2 from S. rochei D74 simultaneously promoted the growth of S. miltiorrhiza hairy roots and the biosynthesis of tanshinones, demonstrating its potential as an effective elicitor. This study provided a new strategy for the utilization and development of S. miltiorrhiza resources.

Objective To perform biological characterization and genomic analysis for a multidrug-resistant Streptococcus strain isolated from the tonsils of a dead piglet, thus elucidating its phenotypic and genotypic characteristics. Methods The tonsillar tissue homogenate from the dead piglet was inoculated into the Streptococcus enrichment medium for enrichment culture. The enriched culture was then streaked onto Columbia blood agar medium for Streptococcus isolation. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a biochemical identification system, and 16S rRNA gene sequencing were employed for strain identification. The minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Whole-genome sequencing was conducted via the Illumina platform. The ResFinder database was used to analyze the resistance genes, and mutations in the genes encoding penicillin-binding proteins (PBPs) were investigated through multiple sequence alignment. Results The phylogenetic analysis based on 16S rRNA gene sequences identified the isolate as Streptococcus orisratti. Antimicrobial susceptibility testing revealed that the strain was resistant to penicillin G, erythromycin, clindamycin, and compound sulfamethoxazole, and it exhibited reduced susceptibility to linezolid. Whole-genome analysis identified the presence of the macrolide and lincoamide resistance gene ermB and the oxazolidinone resistance gene optrA. Furthermore, multiple mutations were detected in the genes encoding PBPs. Conclusion This study reports the first isolate of S. orisratti harboring both ermB and optrA and exhibiting resistance to penicillin. It highlights the capacity of porcine-derived streptococcal strains to acquire multidrug resistance genes, underscoring the need for increased vigilance regarding the resistance traits and transmission risks of animal-derived streptococci.

Objective To determine the efficacy of Cucurbita pepo cv Dayangua (CPD) in alleviating hypoxia and explore the potential mechanisms involving the modulation of the gut microbiota and its metabolites. Methods Male Kunming mice were randomly assigned into two groups: a control group (normoxia ddH₂O, ND) and a CPD intervention group (normoxia CPD, CPD). The CPD group received a dose of 800 mg/(kg·d) of CPD, while the ND group received an equal volume of ddH₂O for 15 consecutive days. One hour after the final administration, mice from each group were placed in wide-mouth bottles, and the survival time was observed and recorded. Fecal samples collected prior to the last administration were subjected to 16S rRNA gene amplicon sequencing and targeted metabolomics analysis. Correlation analysis between gut microbiota and metabolites was subsequently performed. Results CPD intervention significantly prolonged the survival time of mice under hypoxic conditions compared to the ND group. CPD altered the structural composition of the gut microbiota in mice. Linear discriminant analysis effect size (LEfSe) revealed significantly different bacterial taxa between the ND group and the CPD group, with higher relative abundance of Bacillota, Lactobacillus, and Alistipes in the CPD group. Microbial genera, including Paraprevotella and Lactobacillus, showed a positive correlation with survival time. Targeted metabolomics identified 9 upregulated and 31 downregulated metabolites in the CPD group. Notably, metabolites such as palmitoleic acid, glyoxylic acid, hendecanoic acid, l-aspartic acid, O-succinylhomoserine, and allantoic acid were significantly enriched and positively correlated with the survival time of mice after CPD intervention. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of differential metabolites showed the highest enrichment in the tryptophan metabolism and glycine, serine, and threonine metabolism pathways. Conclusion CPD intervention significantly prolonged the survival time of hypoxic mice. CPD intervention enriched beneficial microorganisms, including Lactobacillus, and elevated the levels of beneficial metabolites such as choline and allantoic acid. These findings suggest that modulating the “gut microbiota-metabolite” axis may be one mechanism through which CPD enhances host hypoxia tolerance, providing a theoretical basis and potential targets for developing microecological intervention strategies against hypoxia-related diseases.

Biochar serves as an excellent carrier for non-symbiotic nitrogen (N)-fixing bacteria, enhancing their microbial activity and functions. However, the coupling mechanism between non-symbiotic N-fixing bacteria and biochar remains unclear. Objective To explore the effects of different biochar materials on the colonization pattern and N fixation efficiency of non-symbiotic N-fixing bacteria. Methods Non-symbiotic N-fixing bacteria were inoculated onto biochar samples derived from maize straw and wood chips, the particle sizes of which were >2.00 mm, 0.25-2.00 mm, and <0.25 mm. We compared the porosity and specific surface area of different biochar samples. Throughout the incubation period, the dynamic changes in nitrogenase activity and the number of N-fixing bacteria, pH, dissolved organic carbon (DOC), dissolved organic nitrogen (DON), microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) were monitored. Results N-fixing bacteria exhibited more uniform colonization and higher N fixation activity on straw biochar than on woody biochar, especially in the 0.25-2.00 mm group. The straw biochar with the particle size of 0.25-2.00 mm and inoculated with strains showed increases of 82.33%-160.55% and 231.46%-356.08% in the average MBC content and nitrogenase activity, respectively, compared with woody biochar. Moreover, significantly higher content of DOC and DON were maintained in all straw biochar groups, which provided a richer pool of available nutrients for microbial growth. The correlation heatmap indicated that pH significantly affected bacterial colonization and nitrogenase activity. Furthermore, nitrogenase activity showed strong positive correlations with DOC and MBC (P<0.001), which suggested that a carbon-rich environment was a key factor for the growth and N fixation of N-fixing bacteria. Conclusion Straw biochar with the particle size of 0.25-2.00 mm serves as an optimal carrier for non-symbiotic N-fixing bacteria. It provides a favorable microenvironment for the N fixation and some other functions of the bacteria.

Probiotic additives for feed play a crucial role in maintaining the health and improving the production performance of livestock and poultry. However, the application of most probiotics is limited by their sensitivity to environmental stresses (e.g., acid, bile salt, and temperature) in the animal intestinal tract, and microencapsulation serves as a key approach to enhance their stability. Objective This study constructed a metal-polyphenol-prebiotic (Fe-TA-GN) composite coating system for the probiotic strain Enterococcus faecium PL84 isolated by us and verified its protective effect on PL84, aiming to provide technical support for the industrial application of the strain. Methods Coating parameters (Fe³⁺-TA molar ratio and GN concentration) were optimized. Scanning electron microscopy (SEM) and the CCK-8 assay were employed to evaluate the effects of coating materials on the viability of mouse intestinal epithelial cells (IEC-6). The protective effect of the coating system was assessed through in vitro tolerance tests under acidic, bile salt, thermal conditions, as well as in simulated gastrointestinal fluids. Results E. faecium PL84 exhibited the highest cell viability during the logarithmic growth phase, being suitable for microencapsulation. When the molar ratio of Fe³⁺ to TA was 1:3, the PL84-Fe-TA composite particles showed the smallest nanoscale particle size and formed a dense metal-polyphenol network. At a GN concentration of 0.4 mg/mL, the Fe-TA-GN coating layer achieved the highest zeta potential and optimal structural stability. SEM revealed a uniform and continuous surface coating layer of PL84-Fe-TA-GN. In vitro tolerance assays demonstrated that the survival rate of PL84-Fe-TA-GN was higher than that of uncoated PL84 under conditions of pH 3.0 and 0.6% bile salt (P<0.01). After treatment at 60 ℃, the survival rate of the coated strain increased by 16.29% compared with that of uncoated PL84. Additionally, the survival rates of PL84-Fe-TA-GN in simulated gastric fluid and simulated intestinal fluid improved by 20.8% and 13.53%, respectively. The coating materials (Fe-TA, GN, and Fe-TA-GN) had no significant effect on the viability of PL84 (P>0.05). Conclusion When the molar ratio of Fe³⁺ to TA is 1:3 and the GN concentration is 0.4 mg/mL, the metal-polyphenol-prebiotic composite coating system is stable and can significantly enhance the environmental tolerance of E. faecium PL84. Moreover, the coating materials possess good biocompatibility, laying a solid technical foundation for the industrial application of E. faecium PL84.

Objective To investigate the mechanism by which the microbial fermentation product of the traditional Chinese medicine YA3D3 (YA3D3-MHF) improves cognitive function in the APP/PS1 transgenic mouse model of Alzheimer’s disease (AD) via the microbiota-gut-brain axis. Methods APP/PS1 mice were administered either the water extract of YA3D3 (YA3D3-HF) or YA3D3-MHF for 90 days. The gut microbiota structure was analyzed by 16S rRNA gene sequencing, and the fecal levels of short-chain fatty acids (SCFAs) were assessed by GC-MS. The neurotransmitter content in the brain tissue was measured via ELISA, and cognitive function was assessed via the Morris water maze. Network pharmacology and mass spectrometry were employed to identify active components and changes in chemical composition. Results Compared with the model group and the YA3D3-HF group, YA3D3-MHF significantly ameliorated cognitive impairment in mice. The Morris water maze test showed that the high-dose YA3D3-MHF (MH) group had the shortest escape latency and the highest number of platform crossings, approaching the performance of the normal control group. ELISA confirmed that the MH group had the highest levels of 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (GABA), and glutamate (GLU) in the brain. The results of 16S rRNA gene sequencing revealed that the MH group exhibited the highest alpha diversity (Shannon index≈3.2) of gut microbiota and the highest abundance of beneficial bacteria, along with the lowest abundance of pro-inflammatory bacteria. GC-MS analysis indicated that the MH group had the highest levels of total SCFAs, acetate, and butyrate. MS demonstrated that YA3D3-MHF components exhibited reduced polarity and the emergence of new high-activity peaks. Conclusion YA3D3-MHF improves cognitive function in AD mice by modulating the gut microbiota-SCFAs-neurotransmitter axis, outperforming YA3D3-HF. This study provides experimental evidence for AD intervention targeting the gut-brain axis.

Objective To evaluate the in vitro antifungal activity of quercetin against Trichosporon asahii and investigate its molecular mechanism of inducing fungal apoptosis. Methods According to the CLSI M27-A3 protocol, the inhibitory effects of quercetin on planktonic cells and biofilm formation of nine T. asahii strains were determined. On this basis, changes in intracellular reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and cysteinyl aspartate-specific proteinase 3 (Caspase-3) activity were measured following quercetin intervention. Subsequently, transcriptome sequencing was utilized to verify and analyze the differentially expressed genes. Results The minimum inhibitory concentrations (MICs) of quercetin against T. asahii ranged from 8 to 32 μg/mL, and quercetin effectively inhibited biofilm formation. Cellular experiments indicated that quercetin triggered apoptosis by inducing ROS accumulation, reducing MMP, and activating Caspase-3. Transcriptomic data further confirmed the aforementioned mechanisms at the gene expression level. Conclusion Quercetin exerts antifungal activity against T. asahii primarily by inducing oxidative stress-mediated apoptosis.

Objective Mixotrophy that combines phototrophic autotrophy and phagotrophic heterotrophy is widespread among unicellular eukaryotic microalgae and plays a key ecological role in energy flow within food webs and in elemental biogeochemical cycles. However, identifying and characterizing mixotrophic microalgae in natural waters remains technically challenging. Improving current approaches to accurately reveal the diversity of mixotrophic microalgae is an urgent task in this field. Methods Fluorescently labeled prey surrogates and feeding experiments were employed to trace phagotrophic microalgae within plankton communities. Target organisms were captured at the single-cell level through fluorescence-activated cell sorting (FACS), followed by multiple-displacement amplification (MDA) and 18S rRNA gene sequencing for taxonomic identification. On the basis of this FACS-MDA workflow, we established a methodological framework for studying the functional groups of microalgae. Results Applying this approach to multiple freshwater and seawater samples from China, we identified twenty phagotrophic microalgal species belonging to six classes and twelve genera, as well as heterotrophic consumers representing one class and three genera, demonstrating the robustness and broad applicability of this method. Conclusion This study applies the combined FACS-MDA technology to the identification of functional groups of microalgae in natural water bodies. The established technology has broad application prospects in microbial ecology. It enables deeper insights into the functional diversity and in situ feeding activities of environmental microalgae.