Objective To determine the cause of death of an adult crocodile in the Alligator sinensis Management Center in Anhui Province. Methods Bacteria were isolated from the heart, liver, lung, and spleen via the culture method, and the isolates were identified by morphological observation, biochemical tests, and molecular biological methods. Furthermore, the mucus phenotype was determined by means of the string test. Multilocus sequence typing (MLST) was conducted on the basis of seven housekeeping loci. Virulence gene analysis, pathogenicity test, drug resistance gene analysis, and antimicrobial susceptibility testing were conducted to clarify the pathogenicity and drug resistance of the isolates. Results The pathogenic bacteria isolated from the four organs were morphologically consistent Gram-negative bacilli. Through biochemical tests and 16S rRNA gene and khe sequencing, the isolates were identified as Klebsiella pneumoniae YZE01, capsular serotype K2. String test showed that the strain was hypermucinous K. pneumoniae, and MLST analysis showed that the strain belonged to sequence type 25 (ST25). The strain carried six virulence genes: fimH, entB, rmpA, rmpA2, mrkD, and wabG. Pathogenicity tests showed that some of the tested mice died within 24 h after infection with YZE01, and the same strain was isolated from the heart, liver, lung, and spleen. The lung tissue of infected mice showed hemorrhage and congestion lesions to different degrees. In addition, RT-qPCR revealed that the transcript levels of IL-1β, IL-6, IL-8, and TNF-α in the lung peaked at 12 h post-infection and then declined. The strain carried three drug resistance genes (blaSHV, armA, and ermB), and it was not sensitive to cephalexin, cefazolin, ampicillin, streptomycin, gentamicin, erythromycin, roxithromycin, and clindamycin. Conclusion The isolated strain K. pneumoniae YZE01 carries a variety of virulence genes and has strong pathogenicity and drug resistance. It is considered as a major cause of death in A. sinensis. The findings are conducive to the prevention and control of diseases in A. sinensis.

Objective Fusarium wilt caused by Fusariumoxysporum f. sp. nivum is a typical soil-borne disease in watermelon production, posing significant threats. This study investigates the microbial community structures in the rhizosphere soil of healthy and Fusarium wilt-affected watermelon plants to clarify the regulatory effects of this disease on the physicochemical properties and microbial communities of rhizosphere soil. It aims to reveal the interactions between pathogen enrichment, beneficial microbial decline, and soil environmental factors, providing theoretical support for the green control of Fusarium wilt in watermelon plants by rhizosphere microbiome regulation. Methods Rhizosphere soil samples were collected from healthy plants (HT group) and Fusarium wilt-infected plants (FT group) of the watermelon variety ‘Xiaoyu No. 5’ in Shaoyang, Hunan. Physicochemical indicators including total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), and available potassium (AK) were measured. Illumina high-throughput sequencing was employed to analyze the structures and diversity of microbial communities in the rhizosphere soil of healthy and disease-infected plants. Results The FT group had lower content of TP, AP, and AK in the rhizosphere soil than the HT group (P<0.05). The TN, organic matter (OM), and pH in the FT group were lower without significant differences than the HT group. The FT group had higher fungal ACE and Chao1 indices (P<0.05), higher bacterial ACE and Chao1 indices (P>0.05), and higher fungal and bacterial Simpson indices (evenness) (P<0.05) than the HT group. The abundance of Bacillota was significantly higher in the HT group than in the FT group, whereas that of Ascomycota was significantly higher in the FT group. At the genus level, the abundance of beneficial bacteria such as Neobacillus and Bacillus decreased in the FT group, while that of the pathogenic genus Fusarium increased sharply from 0.06% to 2.40%. The redundancy analysis (RDA) indicated that TN, TP, and OM were key drivers of bacterial community changes, whereas TN, OM, and AK were core regulators of fungal communities. Functional prediction suggested enhanced functions such as stress responses and energy metabolism of bacteria, alongside increased potential for functions such as plant cell wall degradation of fungi, in the diseased rhizosphere. Conclusion The occurrence of Fusarium wilt in watermelon plants leads to depletion of phosphorus and potassium in the rhizosphere soil and disrupts microbiome balance. This is manifested by the enrichment of Fusarium and the decline of beneficial bacteria (e.g., Neobacillus and Bacillus). Soil TN, OM, and AK are key environmental factors regulating this imbalance, with AK deficiency potentially serving as a pivotal link between soil environmental degradation and disease intensification. These findings provide crucial theoretical support for developing eco-friendly control strategies-potassium supplementation and stabilization alongside the targeted cultivation of beneficial microbial communities-targeting Fusarium wilt in watermelon plants.

Objective The immunoinflammatory response induced by spinal cord injury is a key factor hindering the recovery of neurological functions. Recent studies have shown that gut microbiota dysbiosis can participate in the immune regulation of the central nervous system through the gut-spinal cord axis. This study aims to explore whether curcumin can exert its protective effect on spinal cord injury by reshaping the gut microbiota and thereby regulating the local Treg/Th17 balance in the spinal cord. Methods Female Sprague-Dawley rats weighing 200‒220 g were randomly assigned into the sham operation group, spinal cord injury group, curcumin group, fecal microbiota transplantation group, fecal microbiota transplantation+ curcumin group, and fecal microbiota transplantation+curcumin+GPR inhibitor group. Neurological function recovery was evaluated based on the Basso-Beattie-Bresnahan motor function score and gait analysis. Histopathological changes in the injured area were observed via hematoxylin-eosin staining, Nissl staining, and Luxol Fast Blue staining. RT-qPCR, ELISA, and Western blotting were employed to quantify the expression levels of key transcription factor forkhead box protein 3 (FOXP3) for Treg cells, anti-inflammatory cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β1, as well as key transcription factor retinoic acid receptor-related orphan receptor gamma t (RORγt) for Th17 cells and pro-inflammatory cytokines IL-17 and IL-6 in the spinal cord of each group. Results Compared with the spinal cord injury group and fecal microbiota transplantation group, the curcumin group and fecal microbiota transplantation+ curcumin group showed the most significant improvement in neurological function, specifically manifested by significant increases in BBB motor function scores and gait coordination, along with a marked reduction in the scope of spinal cord injury. At the molecular level, the two groups showed significantly upregulated gene and protein levels of FOXP3, IL-10, and TGF-β1 and significantly inhibited expression of RORγt, IL-17A, and IL-6 in the spinal cord tissue. This suggests that after curcumin intervention in the gut microbiota, the immune balance shifted toward a Treg-dominated anti-inflammatory state. Notably, the aforementioned beneficial effects of curcumin-modified gut microbiota were reversed after combined use of the GPR inhibitor. Conclusion This study indicates that curcumin can act on the gut microbiota to promote the recovery of motor function after spinal cord injury. Curcumin may exert the effect by activating the GPR signaling pathway, thereby upregulating Treg viability, inhibiting Th17 differentiation, and ultimately correcting the Treg/Th17 imbalance. This provides new experimental evidence and application value for using curcumin and its modified gut microbiota as an adjuvant therapeutic strategy for spinal cord injury.

Objective With the widespread use and promotion of plastic film mulching on the Qinghai-Xizang Plateau, a series of issues caused by its application have also emerged. Given the fragile eco-environment of the plateau, it is necessary to investigate the effects of different types of plastic film mulching on the soil microbial community structures in farmland ecosystems. Methods Three treatments—pre-planting soil (ZQ), soil covered with conventional polyethylene mulch (CMPs), and soil covered with biodegradable mulch (BMPs)—were established. Soil physicochemical properties were measured, and high-throughput sequencing of the 16S rRNA gene and ITS region was employed to analyze microbial diversity, community structure, and their associations with environmental factors, on the basis of which the impacts of mulch types on soil microorganisms were evaluated. Results Differences in soil physicochemical factors were observed among different treatments (P<0.05). There were no significant differences in alpha diversity indices for both bacteria and fungi among the treatments, indicating that short-term plastic film mulching did not significantly alter the richness and diversity of microbial communities. The dominant bacterial phyla were Pseudomonadota, Actinomycetota, Acidobacteriota, and Chloroflexota, with most dominant genera being unclassified. The dominant fungal phyla were Ascomycota, Basidiomycota, Mortierellomycota, with dominant genera including Mortierella and Solicoccozyma. Network analysis revealed that the main drivers of bacterial and fungal community structures were pH and microplastic (MP) content, respectively. This result reflected functional differences of fungi and bacteria. Fungi, as primary decomposers, were more sensitive to MP pollution, whereas bacterial community structure was more closely related to soil pH. Functional prediction showed that, in bacteria, only the metabolism pathway within the KEGG level 1 showed a positive correlation with the mulching treatment, and no significant differences in COG functions were observed between treatments. In fungi, saprotrophic functions predominated, and their relative abundance changed significantly among treatments. Conclusion Short-term plastic mulching does not significantly affect microbial alpha diversity, but alters the community structure. Compared with conventional PE mulch, biodegradable mulch shows greater potential in enhancing soil nitrogen and organic carbon pools. However, it leads to more severe short-term MP accumulation, accompanied by the risk of pathogenic fungal enrichment. Therefore, its long-term ecological effects require further assessment.

Trees can form mutualistic symbionts with mycorrhizal fungi. Different mycorrhizal types affect the community structure of endophytic fungi by regulating tree physiology and root microenvironment, thus becoming a key link driving the interaction network between soil and microorganisms in tropical forests. However, the mechanisms by which different mycorrhizal types regulate the diversity and community composition of endophytic fungi in tropical tree roots are still not fully understood. Objective To explore the effects of different mycorrhizal types on the diversity and community structure of root endophytic fungi in tropical trees, as well as their key driving factors, systematically clarifying how mycorrhizal types affect the composition and diversity of endophytic fungal communities by regulating root traits and rhizosphere environment, and identifying the key driving factors. Methods On the basis of 3 773 sets of soil and root data collected from three research sites of Chinese Ecosystem Research Network (CERN) in Xishuangbanna tropical forest, China, we integrated and constructed a dataset at the tree species level. This dataset encompassed data of the root traits, soil physical and chemical properties, and the operational taxonomic unit (OTU) abundance of endophytic fungi in the roots of 119 trees (54 species) with arbuscular mycorrhizas (AM) and 31 trees (12 species) with ectomycorrhizas (ECM), and it was then used for the research. Results The alpha diversity of endophytic fungi in the roots of AM trees was higher than that of ECM trees (P<0.05). Mycorrhizal types affected the dominant groups of root endophytic fungi. Ascomycota had the highest relative abundance (43.17%) in the roots of AM trees, and Basidiomycota had the highest relative abundance (65.17%) in the roots of ECM trees. The co-occurrence network analysis showed that the endophytic fungal network was denser in the roots of AM trees and more modular in roots of ECM trees. Soil properties were the dominant driving factors for the endophytic fungal communities in the roots of AM trees, while the endophytic fungal communities in the roots of ECM trees were regulated jointly by root traits and soil properties. Soil phosphorus was a key factor affecting the endophytic fungal communities in the roots of AM and ECM trees. Conclusion In tropical forest ecosystems, AM drives trees to form species-rich and closely interacting endophytic fungal communities in the roots, and the assembly process is mainly regulated by soil factors. ECM trees form a specialized symbiotic fungal system, whose construction is regulated by both root traits and soil factors. In addition, soil phosphorus is the core factor driving the formation of endophytic fungal communities in the roots of the two types of trees.

Objective The engineering of the reductive glycine pathway (rGlyP) in Komagataella phaffii (syn. Pichia pastoris) represents a promising strategy for the co-utilization of methanol and CO₂. However, the efficiency of this pathway is constrained by the insufficient supply of intracellular reduced nicotinamide adenine dinucleotide (NADH), as the native alcohol oxidase (AOX) pathway generates hydrogen peroxide rather than NADH, leading to energy loss and oxidative stress. To overcome this bottleneck, this study reconstructed the methanol oxidation pathway and employed a subcellular compartmentalization strategy to optimize the carbon flux and energy metabolism. Methods Five different sources of NAD⁺-dependent methanol dehydrogenase (MDH) were screened in an aox1/aox2-deficient strain by using the growth curve and methanol utilization rate as indicators to determine the optimal MDH, and the methanol induction concentration was optimized. Subsequently, a compartmentalization strategy was employed by fusing the peroxisomal targeting signal 1 (PTS1) to MDHN1T, which targeted the enzyme to the peroxisome to spatially couple methanol oxidation with formaldehyde detoxification. Results The MDHN1T derived from Cupriavidus necator had the best catalytic performance, and the optimum methanol induction concentration was optimized to be 0.6%. Under co-utilization conditions, the engineered strain achieved a methanol consumption rate of 28.98 mg/d, with the total intracellular NAD_total pool, NADH/NAD⁺ ratio, and biomass being 1.3, 1.2, and 2.2 folds, respectively, of those in the parental strain. Conclusion This study successfully alleviates the redox cofactor imbalance in the rGlyP and enhances co-utilization of methanol and CO₂ in K. phaffii, providing a robust chassis and a theoretical basis for the development of microbial cell factories utilizing one-carbon resources.

Objective To construct a recombinant Escherichia coli strain for the expression of the bacteriophage-derived lytic enzyme Lys162, an efficient and broad-spectrum recombinant enzyme, thus providing a technological foundation for developing novel antimicrobial agents. Methods On the basis of the whole-genome sequencing data of bacteriophage pEC.M2929.1AR.1, the protein structure was predicted via bioinformatics tools, and molecular docking analysis was performed to evaluate the substrate-binding affinity. The expression vector pET28a(+)-Lys162 and the engineered E. coli BL21(DE3) expression system were constructed. Lys162 was further assessed for its environmental stability, in vitro antibacterial activity, and lytic spectrum. Results Structural analysis predicted that Lys162 was an N-acetylmuramidase-type lytic enzyme containing a conserved catalytic domain. Molecular docking confirmed its high-affinity binding to peptidoglycan. The enzyme was expressed in a soluble form in E. coli BL21(DE3) and purified to reach a concentration of 1.89 mg/mL. In vitro assays demonstrated that Lys162 at 125 μg/mL exhibited significant lytic activity against E. coli M2929.1AR, along with potent lytic effects against multiple pathogenic bacteria including Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp. The enzyme retained stable activity within a pH range of 4.0-11.0 and at temperatures between 4 ℃ and 60 ℃. Conclusion Lys162 transcends the host specificity of its parental phage, demonstrating broad-spectrum antimicrobial activity and considerable environmental adaptability. Its synergistic effect with EDTA suggests a practical strategy for performance optimization. These results establish a foundation for developing novel enzymatic antimicrobials to address challenges associated with bacterial antibiotic resistance.

Objective Earthworm intestines, rich in carbohydrates and organic acids, are considered potential hotspots for the horizontal transfer of antibiotic resistance genes (ARGs). However, direct evidence is lacking regarding whether reactive oxygen species (ROS) are produced under anaerobic conditions in earthworm intestines and how ROS regulate plasmid conjugation. This study aimed to investigate the contribution of organic matter metabolism to ROS generation in earthworm intestines and how ROS affected the conjugative transfer of plasmids. Methods Pheretimaguillelmi was used as a model organism to establish the anaerobic microcosm systems simulating in-situ substrate concentrations of earthworm intestines. Four treatments with glucose, lactate, acetate, and amino acids as sole carbon sources were set up. The role of ROS was verified by adding ROS scavengers. Using the fluorescent probe technology, ion chromatography, and qPCR, we determined the production levels of •OH, O₂^•-, and H₂O₂, the consumption of organic substrates, and the abundance changes of the conjugation-related genes gfp, mCherry, trfA, and trbB, respectively. Results ROS was detected in all the treatments. The glucose group showed the highest •OH, O₂^•-, and H₂O₂ yields (0.684, 0.988, and 6.371 μmol/L, respectively) on day 2, which were significantly higher than those in other groups, while the acetate group showed the lowest yields. The substrate consumption rate followed the trend of glucose>lactate>amino acids>acetate, which was consistent with the ROS generation trend. Correspondingly, the glucose group exhibited the highest abundance of gfp, trfA, and trbB (3.47×10⁶, 6.73×10⁶, and 7.86×10⁶ copies/μg DNA) and conjugation frequency (8.9×10^-4), which were the lowest in the acetate group. After ROS scavenging, the conjugation frequencies in all the treatments significantly decreased by 73%‒92%. Mantel analysis revealed that hydroxyl radical showed the most significant correlation with conjugation frequency and abundance of trfA and trbB, indicating that •OH was the core ROS driving conjugative transfer. Unclassified Enterobacteriaceae and Clostridiumsensu stricto 10 were identified as the core microbial taxa coupling ROS generation and conjugation. Conclusion Organic matter metabolism in the anaerobic earthworm intestine can significantly promote ROS generation. ROS further regulates the conjugative transfer of ARGs among microbial strains by altering the abundance of conjugation-related genes.

Objective To investigate the function of ring finger protein 31 (RNF31) in the replication of foot-and-mouth disease virus (FMDV) and to provide a theoretical basis for the research on the molecular mechanism by which the host protein RNF31 regulates FMDV replication. Methods CRISPR/Cas9 gene editing was employed to design two sgRNA sequences in the exon segment of RNF31, and recombinant plasmids were constructed by ligation with the pX459-puro vector. The recombinant plasmids pX459-RNF31-sgRNA were transfected into PK-15 cells, followed by screening under the action of puromycin to obtain the cell lines with RNF31 gene knockout. The effect of RNF31 gene knockout on FMDV replication was detected by Western blotting, RT-qPCR, and TCID₅₀ methods. Results Compared with wild-type cells, the knockout of RNF31 significantly increased the protein level, mRNA level, and virus titer of FMDV. Conclusion We successfully construct the cell lines with RNF31 gene knockout and prove that RNF31 plays a key role in the replication of FMDV. This result provides data support for further research on the mechanism by which RNF31 inhibits FMDV replication.

Colorectal cancer (CRC), one of the most common malignancies of the digestive system, is characterized by complex pathogenic mechanisms and an overall poor prognosis. The gut microbiota and its metabolites play a dual role in CRC by modulating various forms of programmed cell death (PCD), either promoting or inhibiting tumorigenesis and influencing the tumor responses to chemotherapy and immunotherapy. This review systematically summarizes recent advances in understanding how the gut microbiota regulates CRC initiation, progression, and responses to therapies through the modulation of apoptosis, autophagy, ferroptosis, and pyroptosis. Furthermore, it discusses the potential clinical-translational implications of these findings, aiming to provide a theoretical foundation for elucidating CRC pathogenesis and developing novel therapeutic strategies targeting the gut microbiota.