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 investigate the effects of mannanoligosaccharides (MOS) on the in vitro fermentation characteristics and composition of intestinal microbiota in weaned piglets by using an in vitro microbial fermentation technique, with fructooligosaccharides (FOS) taken as the control. Methods Using microbial inocula derived from the jejunal and colonic chyme of piglets, with FOS and MOS as respective substrates, this study measured microbial gas production and fermentation broth pH at five time points (0, 6, 12, 24, and 48 h), and collected fermentation broth samples at each time point for subsequent microbial analysis. Results In the in vitro jejunal microbial fermentation system, both the fermentation broth pH and gas production in the MOS group were significantly higher than those in the FOS group (P<0.05). At 24 h of fermentation, compared with FOS, MOS significantly increased the concentrations of acetate, propionate, and total short-chain fatty acids (SCFAs) (P<0.01). The formate production in the MOS group was significantly lower than that in the FOS group (P<0.01). At 48 h of fermentation, the lactate concentration in the MOS group was significantly lower than that in the FOS group (P<0.01). In the in vitro colonic microbial fermentation system, gas production in the MOS group was significantly higher than that in the FOS group (P<0.05). At 48 h of fermentation, the production of formate, acetate, butyrate, SCFAs, and lactate in the MOS group was significantly higher than that in the FOS group (P<0.01). The 16S rRNA gene sequencing results of the jejunal fermentation broth revealed that at 48 h of fermentation, both the Shannon and Simpson indices in the MOS group were significantly higher than those in the FOS group (P<0.01). Furthermore, the microbiota composition exhibited disparity between the MOS and FOS groups. Moreover, the relative abundances of Bifidobacterium, Limosilactobacillus, and Megasphaera were significantly higher in the MOS group than in the FOS group (P<0.05). Conclusion Compared to FOS, MOS enabled the microbiota in the small intestine of piglets to significantly improve the microbial community structure, increase the abundance of beneficial bacteria such as Bifidobacterium, enhance gas production, and promote the generation of acetate and other SCFAs. These findings suggested that MOS held potential for modulating microecology in the small intestine of weaned piglets.

Understanding the source, colonization rules, and dynamic evolution process of the gut microbiota is of significant importance for regulating host health, given its crucial role in nutrient metabolism, immune regulation, and intestinal barrier function. This article comprehensively reviews the composition and functions of the gut microbiota and explores the origins and transmission pathways, with a particular focus on the effects of maternal-infant transmission and paternal inheritance on the structure and functions of the gut microbiota. We chart microbial assembly across pivotal life stages, distill the driving factors involved in the community assembly process, and critically appraise the advances in metagenomic and source-tracking toolkits. The review provides an integrated framework for microbiome-targeted strategies aimed at reconstructing a health-oriented gut ecosystem.

Objective To identify the key amino acid residues of the TetR family transcription factor BcPDR1 in Botrytis cinerea, thereby laying a foundation for elucidating the mechanism by which BcPDR1 regulates the growth, development, and pathogenicity of this pathogen. Methods The key amino acid sites of BcPDR1 were analyzed by bioinformatics methods, and four conserved regions (32-34 aa, 76-95 aa, 140-150 aa, and 189 aa) were selected for site-directed mutagenesis. On the basis of the knockout mutant ΔBcpdr1, the mutants BcPDR1-M1 (Δ32-34), BcPDR1-M2 (Δ76-95), BcPDR1-M3 (Δ140-150), and BcPDR1-M4 (mutation of Ile to Lys at 189 aa) were constructed. A comparative analysis of the phenotypic characteristics and pathogenicity was conducted on the four aforementioned mutants and the wild-type strain of B. cinerea, ΔBcpdr1, the complemented strain CE. Results The colony morphology, mycelial morphology, and growth rates of BcPDR1-M1, BcPDR1-M2, BcPDR1-M3, and BcPDR1-M4 were similar to those of ΔBcpdr1, but significantly different from those of BC22 and CE. These mutants could form lesions on tomato fruits and tobacco leaves, while their lesion areas were significantly smaller than those of BC22 and CE. Conclusion The regions 32-34, 76-95, 140-150, and the 189th amino acid are the regulatory sites for BcPDR1 to exert its functions.

Pasteurella multocida (Pm) and Mannheimia haemolytica (Mh) are major bacterial pathogens responsible for bovine respiratory diseases. However, the diversity of these two pathogens in transported calf populations remains poorly understood, which severely hinders the effective prevention and control of their infections. Objective To investigate the diversity of Pm and Mh in a group of fattening calves purchased from a calf trading market in Inner Mongolia and transported to a breeding farm in Hechuan, Chongqing. Methods After arrival, nasal swabs were collected from calves showing respiratory symptoms at four time points for bacterial isolation and culture. Suspected Pm and Mh colonies were selected based on colony morphology, hemolytic characteristics, and Wright-Giemsa staining results, followed by PCR identification and 16S rRNA gene sequencing for confirmation. Furthermore, the serotypes, biochemical and antibiotic resistance profiles, virulence genes, and resistance genes of the isolates were analyzed. Results A total of 23 strains of Pm serotype A, 10 strains of Mhserotype A6, and 1 strain of Mhserotype A2 were isolated from 68 nasal swabs collected at 4 different time points, and only 1 nasal swab harbored both Pm and Mh. Some isolates exhibited diversity in biochemical and antibiotic resistance profiles, which had no significant correlation with sampling time points. Antimicrobial susceptibility testing revealed that Pm and Mh isolates were resistant to most penicillins, aminoglycosides, and lincosamides but remained sensitive to cephalosporins and quinolones. Resistance gene detection showed that β-lactamase resistance (bla_TEM ) genes were detected in 73.91% of Pm isolates and 90.91% of Mh isolates, while sulfonamide resistance (sul2) genes were found in 69.57% of Pm isolates and 18.18% of Mh isolates. Only one Mh isolate carried aminoglycoside resistance genes (aadA25 and aadB). Discrepancies were observed between resistance phenotype and the presence of selected resistance genes. All Pm and Mh isolates were pathogenic. Virulence gene analysis confirmed that Pm isolates consistently carried tonB, hsf-1, nanB, oma87, and tbpA, while Mh isolates showed the detection rates of 100% for gapA and dnaN, 82% for lktA, plpB, and tbpB, and 0 for ptfA. Conclusion These findings suggest that calves purchased from trading markets and transported over long distances to new farms harbor Pm and Mh strains exhibiting diversity in biochemical characteristics and drug resistance, which pose challenges for effective infection control. This study provides critical insights for developing prevention and control strategies against Pm and Mh infections in transported calves.

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.

Objective To construct high-yield engineering strains of Halomonas campaniensis XH26 by introducing five recombinant plasmids (pHX01-pHX05), each carrying the P _tac promoter and combinations of the genes asd, lysC, ectA, ectB, and ectC. This metabolic engineering strategy was coupled with the response surface methodology (RSM) for optimization of the culture conditions, thereby enhancing ectoine accumulation. Methods The recombinant plasmids were conjugally transferred from Escherichia coli S17-1(λ-pir) into H. campaniensis XH26, with positive transconjugants selected via gentamicin (50 μg/mL). Recombinant strains were induced with 0.2 mmol/L IPTG, and ectoine accumulation was quantified by HPLC. Critical nutritional variables—NaCl, peptone, l-glutamate, and glucose—were optimized through one-factor-at-a-time experiments, Plackett-Burman design, and Box-Behnken design. Results Five recombinant strains (XH26/pHX01-XH26/pHX05) were successfully constructed. Culture in the MG medium revealed that strain XH26/pHX04 (overexpressing asd-lysC-ectA-ectB) achieved the highest ectoine titer of (1.32±0.04) g/L. Strains XH26/pHX05 and XH26/pHX03 achieved the ectoine titer of (1.19±0.07) g/L and (1.07±0.08) g/L, respectively, while XH26/pHX02 yielded a lower titer of (1.02±0.14) g/L. The medium composition optimized by RSM was composed of 116.08 g/L NaCl, 16.30 g/L peptone, 169.57 g/L l-glutamate, and 15.53 g/L glucose. Under these optimized conditions, the titer of ectoine produced by XH26/pHX04 increased to (1.81±0.02) g/L, representing a significant increase of 301.56% compared with that of the wild-type strain XH26. Conclusion This study demonstrates that using H. campaniensis as a chassis and overexpressing a key gene combination (asd, lysC, ectA, ectB) under a strong promoter, synergized with culture medium optimization via RSM, can significantly boost the ectoine yield of recombinant strains. The findings provide a robust technical framework for the subsequent industrial production of ectoine.

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.

Coronaviruses pose a serious threat to human and animal health. Their main protease (Mpro) plays a critical role in both the viral life cycle and host immune regulation, serving as a key target for the development of broad-spectrum anti-coronavirus drugs. The core function of Mpro lies in its specific cleavage of viral polyproteins pp1a and pp1ab to release functional non-structural proteins (NSPs), thereby driving the assembly of the viral replication/transcription complex. Additionally, Mpro can target and cleave key molecules in host immune signaling pathways, facilitating viral immune evasion. Given its dual roles and highly conserved catalytic center, the research on Mpro has become a major focus in the field. This review systematically outlines the structural features and functional diversity of Mpro, with an emphasis on its catalytic mechanism in the viral replication cycle and its role in mediating immune suppression. Furthermore, this article details the screening methods and design strategies for Mpro inhibitors, aiming to offer theoretical foundations and novel insights for the development of anti-coronavirus drugs targeting this critical protein.

Objective To screen indigenous rhizobia with high salt tolerance and plant growth-promoting traits from alfalfa nodules and clarify their phylogenetic status and functional potential, thereby providing strain resources and a theoretical basis for developing localized, efficient alfalfa symbiosis and further addressing the constraints of saline soil on alfalfa production in Inner Mongolia. Methods Indigenous alfalfa rhizobia were collected from six saline-alkali sites in Inner Mongolia via a trapping method. Following isolation and purification, the taxonomic status of the strains was determined by 16S rRNA gene sequencing. The plant growth-promoting functions were evaluated through nodulation tests, along with assays for nitrogen fixation, phosphorus solubilization, potassium solubilization, indole-3-acetic acid (IAA) production, and exopolysaccharide (EPS) content. Seed germination and pot experiments were carried out to evaluate the salt stress-alleviating effect of the target strain. Results A total of 250 strains were isolated, with Sinorhizobium meliloti being predominant (227 strains, 90.8%). Eight efficient nodulating strains (e.g., 1B1Y and 1B2Y) were screened out, all of which possessed nitrogen-fixing and potassium-solubilizing capabilities. Some strains (e.g., 2B3Y and 9B3Y) could solubilize organic phosphorus and produce siderophores. Strain 9B3Y secreted the highest amount of IAA (67.5 mg/L), while 16C1Y produced the highest EPS content (2.684 g/L). Under salt stress (0.4% NaCl), the aboveground fresh weight of alfalfa inoculated with strain 2B3Y increased by 29% compared with that of the saline control, and the strain significantly alleviated the inhibition on seed germination (notably increasing the root length). Conclusion The strains screened out, particularly strain 2B3Y, can effectively mitigate the inhibitory effects of salt stress on alfalfa through nitrogen fixation and the secretion of IAA and EPS. These strains show promise for application in alfalfa production and soil improvement in saline regions of Inner Mongolia.