[Objective] The human intestinal tract is rich in microbial resources, which play a significant role in the host’s digestion, absorption, growth, development, etc. Currently, culturomics is widely used in the isolation of beneficial intestinal microorganisms. However, different culture media have preferences, and a single medium is difficult to comprehensively isolate the culturable microorganisms in the intestinal tract. [Methods] We used six reported culture media [brain heart infusion (BHI), Wilkins_Chalgren anaerobe broth (WCBM), (Man-Rogosa-Sharpe) MRS, reinforced clostridial medium (RCM), mucin medium (MM) and modified mucin medium (MMM)] to isolate the microorganisms in the feces of 58 volunteers, with the aim of clarifying the diversity of culturable microorganisms in the intestinal tract and obtaining potential beneficial bacterial strains in the intestinal tract. [Results] A total of 1 052 bacterial strains were isolated from 58 samples, and they were identified as 101 species belonging to 39 genera of 5 phyla. The BHI medium isolated the most bacterial species (50, 49.50%), while the MMM medium isolated the fewest bacterial species (24, 23.76%). Except the MM medium, each of other media could isolate unique genera, and BHI and MMM isolated the most unique genera (5 each). Among the isolated strains, 466 strains were reported to have probiotic effects, including Bacteroides fragilis, Lactiplantibacillus plantarum, Pediococcus acidilactici, and Bifidobacterium bifidum. BHI and MRS media could isolate more beneficial microbial species (10/16, 62.50%). [Conclusion] We explored the diversity of beneficial bacteria in the human intestinal tract from the perspective of pure culture by using multiple culture media, providing rich strain resources for the development of intestinal beneficial microorganisms.

[Objective] The type VI secretion system (T6SS) is a novel virulence factor of Edwardsiella tarda. E. tarda virulence protein P (EvpP) is an effector of the T6SS. To date, the functional mechanisms of EvpP are still poorly understood. This study aimed to comprehensively investigate the biological functions of EvpP and elucidate the roles of T6SS in the pathogenicity of E. tarda. [Methods] We constructed the evpP-deleted mutant (ΔevpP) and complemented strain (ΔevpP-C) to study the effects of evpP deletion on the biological characteristics of E. tarda and infection in macrophages. [Results] No significant differences were observed in the growth curves or physiological and biochemical properties among the wild-type (WT), ΔevpP, and ΔevpP-C. However, compared with WT, ΔevpP exhibited significantly reduced motility, biofilm formation, adhesion rate to RAW264.7 macrophages, intracellular proliferation capacity, and ability to induce host cell autophagy, while triggering increased secretion of tumor necrosis factor-α (TNF-α) by macrophages. The complementation of evpP-C did not fully restore the intracellular proliferation capability, but completely rescued the other phenotypic defects. [Conclusion] EvpP does not affect the growth or physiological and biochemical properties of E. tarda. However, it could enhance the bacterial motility, biofilm formation, adhesion to macrophages, intracellular proliferation, and autophagy, while suppressing TNF-α secretion in E. tarda-infected macrophages. These findings confirm that EvpP plays a critical role in the pathogenicity of E. tarda.

As a subterranean, soil-dwelling insect, Odontotermes formosanus is susceptible to infection by diverse soil-borne pathogenic fungi. Its symbiotic actinobacteria produce bioactive compounds to combat these pathogens, thereby maintaining a stable symbiotic system of O. formosanus with Termitomyces spp. [Objective] To screen and characterize antifungal metabolites from symbiotic actinobacteria of O. formosanus and to elucidate their defensive role in the termite-fungus symbiotic system. [Methods] Actinobacteria were isolated from O. formosanus-associated samples via diverse culture media. Antifungal strains were screened via dual-culture confrontation assays. The active strain OFGS46 was selected for whole-genome sequencing. Biosynthetic gene clusters (BGCs) were predicted by antiSMASH, and secondary metabolites were analyzed by HPLC-MS. [Results] Twenty-three actinobacteria strains were isolated. Strain OFGS46 exhibited inhibition rates of (62.05±0.98)% against Xylaria sp. and (79.99±0.58)% against Talaromyces sp. Whole-genome sequencing on the Illumina NovaSeq platform yielded approximately 1 Gb of high-quality data. De novo assembly generated a draft genome of 8 470 479 bp. CheckM evaluation demonstrated high assembly quality with 99.91% completeness and 4.63% contamination. HPLC-MS results revealed that strain OFGS46 was capable of producing multiple bioactive secondary metabolites, including enduracidin A, WS9326A, kitacinnamycin A, WAP-8294A2, skyllamycin A, cahuitamycin A, pentamycin, scabichelin, coprisamide C, and frankobactin A1. [Conclusion] This study systematically reveals that the symbiotic actinobacterium OFGS46 from O. formosanus suppresses nest pathogens through the production of diverse antimicrobial compounds. These findings not only elucidate, from a perspective of chemical ecology, the role of symbiotic bacteria in host immune defenses through suppressing multiple pathogenic fungi, but also provide a scientific basis for developing novel antimicrobial resources derived from the termite symbiotic system.

[Objective] Citric acid is the main metabolite of Aspergillus niger at pH≤5.0, while l-malic acid becomes the main metabolite at pH 6.0. In this study, we employed transcriptomics to analyze the differences in the expression of key genes in metabolic pathways, aiming to explore the biosynthesis mechanisms of the two organic acids. [Methods] The cells at 48 h and 72 h of the fermentation processes for citric acid and l-malic acid production were selected for transcriptomics analysis. [Results] The transcriptome data of 72 h and 48 h were compared. GO enrichment analysis showed that the upregulated genes related to the synthesis of citric acid were concentrated in carbohydrate metabolism, while those related to the synthesis of l-malic acid were concentrated in ion transport process. The acid protease genes ANI_1_62014 (aspergillin II) and ANI_1_654124 (aspartic protease pepA) showed extremely high transcription levels during citric acid synthesis, while the key genes ANI_1_2494074 [3-oxoacyl-(acyl carrier protein) synthase] and ANI_1_2488074 (biosynthetic fatty acid synthase subunit β) essential for fatty acid chain synthesis showed extremely high transcription levels in the l-malic acid synthesis pathway. The transcription level of zinc cluster transcription factor [Zn(II)₂Cys₆ transcription factor] was higher in the synthesis process of l-malic acid. HacA, AP-1, and AtfA in the bZIP family showed higher transcriptional levels in response to environmental low pH stress during citric acid synthesis. Compared with l-malic acid synthesis, citric acid synthesis was accompanied by upregulated transcription levels of ANI_1_66114 (hexokinase), ANI_1_2950014 (citrate synthase), and ANI_1_478154 (citrate transporter) and a downregulated transcription level of ANI_1_3136024 (isocitrate dehydrogenase). Efficient glycolysis, citric acid synthesis, and citric acid transport capacity and low isocitrate dehydrogenase level were the key factors for citric acid production. In the process of l-malic acid synthesis, cytoplasmic ANI_1_440184 (pyruvate carboxylase), cytoplasmic ANI_1_12134 (malate dehydrogenase), ANI_1_914104 (isocitrate lyase), and ANI_1_2040144 (malate transporter) showed upregulated transcriptional levels. The cytoplasmic rTCA pathway and glyoxylic acid carboxylation pathway were thereby determined to be the main pathways for l-malic acid synthesis. [Conclusion] This study inferred the key differential metabolic pathways for the production of citric acid and l-malic acid by analyzing integrated transcriptomic data, and screened significant differentially expressed core genes, transcription factors, and potential transporters. These results provide important clues and a theoretical basis for elucidating the regulatory mechanisms of citric acid and l-malic acid synthesis.

The lined seahorse (Hippocampus erectus) is a major cultured seahorse species with significant economic value in China. Bacterial diseases frequently occur in intensive aquaculture environments, among which skin ulceration is one of the most detrimental diseases affecting H. erectus farming. Skin ulceration is mainly caused by Vibrio spp., while the pathogen complexity and diversity remain unclear. [Objective] This study identified dominant bacterial strains from ulcerative lesions of H. erectus in Zhangzhou, Fujian and characterized their pathogenicity, antibiotic resistance profiles, and virulence traits, aiming to provide a scientific basis for disease prevention and control. [Methods] Bacteria were isolated from ulcerated and internal tissue samples of diseased seahorses. Species identification was performed via morphological observation, physiological-biochemical tests, 16S rRNA gene phylogenetic analysis, and reinfection of seahorses. The isolates were cultured for the measurement of hemolytic activity, caseinase production, and salinity tolerance. Ten virulence genes were detected by PCR. The susceptibility of the isolates to 30 antibiotics was tested via the disk diffusion method. Artificial infection was performed with zebrafish as a model to determine the median lethal dose (LD₅₀). [Results] Fifteen dominant strains were isolated from various tissue samples of diseased seahorses. Among them, three strains (HCE003, HCE070, and HCE098) exhibited β-hemolysis and high overall antibiotic resistance rates (50.0%-56.7%). HCE003 carried vvh, pPHDD1, and hlyAch, while both HCE070 and HCE098 carried hlyA, trh, hlyAch, and vhh. HCE003, HCE070, and HCE098 were preliminarily identified as highly pathogenic strains and were further characterized as Citrobacter freundii, Shewanella algae, and Vibrio rotiferianus, respectively. The three strains were capable of growing normally at the salinity of 15‰. Artificial challenge tests demonstrated that they could induce skin ulceration in H. erectus upon reinfection. The median lethal doses of HCE003, HCE070, and HCE098 in zebrafish were 1.71×10⁵ CFU/mL, 3.68×10⁵ CFU/mL, and 2.51×10⁶ CFU/mL, respectively. [Conclusion] This study is the first to report the isolation of multidrug-resistant and highly virulent C. freundii and S. algae from H. erectus with skin ulceration, indicating that non-Vibrio pathogens can also contribute to skin ulceration in seahorses. These findings provide scientific support for the development of targeted disease management strategies and therapeutic agents in seahorse aquaculture.

[Objective] To investigate the regulatory effect of the cAMP receptor protein (CRP), a transcription factor in Acinetobacter baumannii, on the cas3 gene. [Methods] CRP was expressed and purified via a prokaryotic expression system. EMSA was employed to examine CRP binding to the cas3 promoter. qPCR was conducted to evaluate the regulatory effect of CRP on cas3 expression. To further confirm the regulatory function of CRP, we constructed a mutant strain Δcrp. The impact of crp deletion on A. baumannii virulence was then analyzed via the biofilm formation assay, adhesion and invasion assays with A549 cells, a Galleria mellonella model, and a murine model of bacterial infection. [Results] EMSA demonstrated that CRP specifically bound to the cas3 promoter. The qPCR results showed that cas3 transcription was downregulated (P<0.001) in Δcrp. Compared with the wild-type strain, Δcrp exhibited no significant difference in growth capacity but enhanced biofilm formation (P<0.001) as well as strengthened adhesion (P=0.003<0.050) and invasion (P<0.001) in A549 cells. Furthermore, Δcrp demonstrated a markedly increased lethality rate in G. mellonella within 72 h. Furthermore, the murine infection experiment revealed that Δcrp possessed higher colonization capacity in the lungs than the wild-type strain (P<0.001). [Conclusion] CRP acts as a transcriptional activator that directly binds to the cas3 promoter to activate its transcription, thereby attenuating the virulence and pathogenicity of A. baumannii.

[Objective] To evaluate the edible safety of Penicillium polonicum CK2023-1, a key strain responsible for flavor formation of traditional bacon in Chengkou, thus providing a scientific basis for developing microbial starter for bacon fermentation. [Methods] The genomic DNA sequence of P. polonicum CK2023-1 was determined via third-generation sequencing combined with second-generation sequencing and annotated via GO, KEGG, and antiSMASH databases. The spore suspension (1×10⁹ spores/kg) of CK2023-1was applied to cured meat surfaces, followed by 26 d local smoking in Chengkou. Non-volatile metabolites were analyzed via LC-MS/MS. Acute oral toxicity (7 d) and subacute oral toxicity (28 d) tests in mice were conducted to assess pathogenicity. [Results] The genome of CK2023-1 was 33.27 Mb, with the N₅₀ of 5 236 196 bp and the G+C content of 52.91%, containing 10 901 predicted coding genes (49.02% of the genome). Eighty-three biosynthetic gene clusters (BGCs) for secondary metabolites were identified, including known mycotoxin (verrucosidin) BGC. Compared with that in the toxigenic strain P. polonicum X6, the BGC in CK2023-1 exhibited an inversion. No mycotoxins were detected in fermented bacon, and neither acute nor subacute toxicity tests caused morbidity or mortality in mice. [Conclusion] P. polonicum CK2023-1 has a well-defined genomic structure, produces no mycotoxins, and is non-pathogenic, meeting the safety criteria for food-grade microbial strains.

[Objective] To develop a low-cost and highly sensitive endotoxin detection reagent and detection method with recombinant horseshoe crab factor C enzymogen (rFC). [Methods] The Bac-to-Bac baculovirus expression system was used to express rFC in Sf9 cells and the activity of rFC was measured by the end-point fluorescence assay with endotoxin. The conditions of protein expression were optimized, and ion exchange was used for crude enzyme separation. An endotoxin detection method with rFC based on end-point fluorescence assay was established after the reaction conditions were optimized. Furthermore, the established method was compared with the conventional limulus amebocyte lysate (LAL). [Results] The expression level of rFC was 110.42 mg/L, increasing by 4.75 times. The linear range of endotoxin detection was 0.005-1.000 EU/mL in 1 h, with a good linearity and the limit of detection being 0.005 EU/mL. The applicability rate of this method for actual samples was 92.45%. The consistency of the detection results was 83.67%, and 89.80% of the samples had consistent detection limits with LAL. [Conclusion] This study achieves the efficient expression of rFC and establishes an endotoxin detection method with higher sensitivity than LAL, which has great potential for application.

[Objective] To establish a method of semi-thin section preparation and scanning electron microscopy (SEM) observation with a simple operation, a short cycle, and a low technical threshold, which can be used as a rapid pre-screening strategy for the complex process of transmission electron microscopy (TEM) ultra-thin sections, in response to the requirements of rapid identification and dynamic observation of typical subcellular structures (such as cell wall, cytoplasm, spore, and vacuole) of microbial samples in the process of fermentation production. [Methods] Bacillus subtilis, Escherichia coli, and Pichia pastoris, taken as the research objects, were embedded in Embed 812 epoxy resin to prepare semi-thin sections with different thicknesses (200, 500, and 1 000 nm) and resin embedding blocks (> 1 000 nm) with samples. After platinum coating by ion sputtering, the subcellular structure was observed by SEM. Ultra-thin sections (70 nm) of the above microbial samples were also prepared, stained with lead and uranium, and imaged by TEM. The imaging effect and operation efficiency of the above two methods were compared. [Results] The 200 nm semi-thin section can clearly and completely display various kinds of microbial subcellular structures under a scanning electron microscope. The image quality was significantly better than that of 500 and 1 000 nm sections and resin block samples. Its resolution was close to the TEM observation level, and it can save about 6.5 h. [Conclusion] The method of SEM combined with 200 nm semi-thin sections was successfully applied to the high-resolution imaging of microbial subcellular structure for the first time, which can clearly identify the typical ultrastructural morphology, and has the advantages of a simple operation, a short cycle, a low cost, and high safety. It has strong versatility and promotion value, and provides a new and effective solution for the biological electron microscopy technology system.

To systematically analyze the current status and research trends and identify the key research hotspots in the field of gut microbiota and metabolic syndrome (MetS) from 2005 to 2024, thus providing references for future research and intervention strategies. Relevant literature on gut microbiota and MetS was retrieved and screened from the Web of Science Core Collection. Bibliometric tools such as VOSviewer, CiteSpace, and the R package bibliometrix were used to analyze the publication trends, countries and institutions, research themes, and emerging hotspots. A total of 4 210 relevant publications were included. The annual number of publications showed an increasing trend, which was particular rapid after 2010. China and the United States led in publication output, and major research findings were published in journals such as Nutrients, Gut, and Nature. Research hotspots primarily covered the fields of nutrition and diet, biochemistry and molecular biology, and microbiology. Keyword evolution analysis revealed a shift from early descriptive studies on gut microbiota composition to mechanism investigations focusing on dysbiosis-related pathways such as energy metabolism, inflammatory responses, and gut-organ axes. Co-occurrence analysis further indicated that key microbial metabolites (e.g., short-chain fatty acids and bile acids) and microbiota-targeted interventions (e.g., probiotics and fecal microbiota transplantation) had become focal points in recent studies. This bibliometric study comprehensively summarizes the research landscape of gut microbiota and MetS and highlights emerging trends and directions. Given the limitations of conventional therapies in terms of targeting specificity, patient adherence, and long-term safety, microbiota-based interventions offer a promising breakthrough for the prevention and treatment of MetS, providing valuable theoretical support for future precision medicine.