[Objective] To isolate and identify a strain of foot-and-mouth disease virus (FMDV) from bovine oropharyngeal fluid (OPF) and subsequently analyze the amino acid mutations in antigenic proteins and the proliferative properties of the isolated virus. [Methods] RT-PCR and sequencing were performed to identify the FMDV-positive OPF samples. The FMDV strain was isolated from bovine OPF samples by cell inoculation and identified by indirect immunofluorescence assay. The whole genome sequence of the strain was obtained by fragment amplification and sequencing. The amino acid sequences were compared to reveal the amino acid mutations in the antigenic proteins of the strain. The proliferative properties of the isolated FMDV strain were analyzed by the plaque forming assay and one-step growth curve. [Results] A FMDV strain O/FMDV/OP/2022/B was successfully isolated from bovine OPF, belonging to serotype O and Ind-2001 lineage. Amino acid mutations were identified in the antigenic proteins VP1, VP2, and VP3 of O/FMDV/OP/2022/B. In addition, the plaque of O/FMDV/OP/2022/B in BHK-21 cells was smaller than that of the vaccine strain O/FMDV/HN/93. The one-step growth curves showed that O/FMDV/OP/2022/B had significantly lower replication titer than O/FMDV/HN/93 in BHK-21 cells. [Conclusion] A strain of FMDV was isolated from bovine OPF, and it displayed amino acid mutations in antigenic proteins and had lower proliferative capacity than the vaccine strain O/FMDV/HN/93. This study contributes to a better understanding of the mechanism of persistent FMDV infection in the bovine oropharynx and provides a reference for the prevention and control of FMD.

[Objective] To extract and purify extracellular polysaccharides (EPS) from Sphingomonas echinoides 1K04342 and investigate the ameliorating effects and mechanisms of the purified EPS on glucose and lipid metabolism disorders induced by oleic acid (OA) in human hepatoma cells (HepG2). [Methods] A response surface methodology was employed to optimize the ultrasound power, ultrasound time, and incubation time for maximizing the EPS yield of S. echinoides 1K04342 from ultrasound-assisted extraction. The crude EPS underwent deproteinization and was further purified through a DEAE-Sepharose Fast Flow anion exchange column. The effects of the purified EPS on the proliferation, glucose uptake, and α-glucosidase inhibition and pancreatic lipase inhibition rates in HepG2 cells were evaluated. The most effective EPS component for mitigating OA-induced glucose and lipid metabolism disorders in IR-HepG2 cells was selected for in-depth physicochemical and bioactivity analyses. [Results] The optimal EPS extraction parameters were as follows: ultrasound power at 59.39 W, extraction for 42.08 s, and incubation for 9.24 h, under which the estimated yield of EPS reached 2.13 g/L. Under the adjusted parameters (60 W, 42 s, and 9.2 h), the EPS yield was 2.10 g/L, which was statistically consistent with the theoretical value (2.13 g/L). The optimal component for ameliorating glucose and lipid metabolism disorders in IR-HepG2 cells was EPS-2, which showed the total sugar content of 74.3%, with a monosaccharide profile comprising fucose, rhamnose, arabinose, galactose, glucose, and mannose. EPS-2 alleviated OA-induced glucose and lipid metabolic disorders in IR-HepG2 cells. EPS-2 reversed OA-induced decreases in cell viability. EPS-2 may activate the IRS-1/PI3K/AKT pathway, up-regulating the protein levels of GSK3β and FoxO1 and down-regulating the mRNA and protein levels of PEPCK and G6Pase, thereby mitigating glucose metabolism disorders. EPS-2 may regulate OA-induced lipid metabolism disorders by activating the AMPK/ACC1/SREBP-1C pathway, lowering the triacylglycerol (TG), total cholesterol (TC), and low-density lipoprotein (LDL) levels, and increasing the high-density lipoprotein (HDL) level. [Conclusion] EPS-2 derived from S. echinoides 1K04342 could effectively ameliorate glucose and lipid metabolism disorders in IR-HepG2 cells.

[Objective] To explore the antifungal activity and inhibitory mechanism and identify the main antagonistic substances of the crude extract of Pseudomonas protegens FD6 in fermentation. [Methods] The plate confrontation test was employed to screen the optimal medium, extractant, and solvent and determine the antifungal activity stability of the crude extract. The main antifungal substances in the crude extract were identified by MALDI-TOF/TOF mass spectrometry, and the antifungal spectrum of the crude extract was explored. The inhibition mechanism of the crude extract against Botrytis cinerea was investigated by mycelial morphology observation, spore germination test, PI staining, and conductivity determination. [Results] The crude extract of FD6 in the potato juice medium without any carbon source displayed the strongest antifungal activity. The methanol-solved crude extract obtained with ethyl acetate as the extractant had the strongest inhibitory activity. The main antifungal substance of the crude extract was identified as a cyclic lipopeptide, orfamide A, based on mass spectrometry data. In addition, the crude extract displayed broad-spectrum inhibitory activities against Phomopsis amygdali, Fusarium graminearum, Colletotrichum gloeosporioides, and Monilinia fructicola. The antifungal activity of the crude extract was stable after treatment at 121 ℃ for 20 min, within the range of pH 4.0-10.0, and with protease K and trypsin. The mycelial morphology of B. cinerea became abnormal after treatment with the crude extract. The treatment with 2.0 mg/mL crude extract completely inhibited the conidial germination of B. cinerea, caused a 50% damage of the cell membrane, increased the conductivity, and led to leakage of intracellular nucleic acids and proteins. [Conclusion] The metabolites produced by P. protegens FD6 have broad-spectrum and stable antifungal activities, serving as lead compounds for exploration of new green fungicides.

[Objective] To investigate the antibiotic resistance and virulence of pet-derived Klebsiella pneumoniae in some areas of Guangdong Province. [Methods] Fecal swabs were collected from dogs and cats for strain isolation, and 16S rRNA and khe genes were amplified by PCR to identify the strains. The sensitivity of K. pneumoniae isolates to 17 antibiotics was determined by the agar diffusion method. PCR was employed to detect resistance genes to β-lactams (bla_SHV, bla_CTX, and bla_TEM), carbapenems (bla_KPC and bla_NDM), aminoglycosides (rmtB), quinolones (qnrS and oqxAB), sulfonamides (Sul1 and Sul2), amphenicols (floR), tetracyclines (tet(A)), and fosfomycin (fosA3) and some virulence genes (rmpA, maga, fimH, mrkD, uge, WabG, kfu, Aerobactin, and ureA). [Results] A total of 126 strains of K. pneumoniae were isolated from 428 fecal samples, with an isolation rate of 29.4%. The 126 isolates had high resistance rates to amoxicillin (75.40%), ampicillin (73.81%), and cotrimoxazole (61.90%). They were moderately sensitive to ceftazidime, amikacin, apramycin, and enrofloxacin, and they were sensitive to tigecycline, colistin, and meropenem. The detection rate of the resistance gene oqxAB was the highest, which was 86.51%, followed by those of the β-lactam resistance gene bla_SHV (73.81%) and the tetracycline resistance gene tet(A) (52.68%). Other resistance genes were detected to varying degrees (0.79%-46.03%) and the carbapenem resistance gene bla_KPC, colistin resistance gene mcr-1, and aminoglycoside resistance gene rmtB were not detected. Among the virulence genes, the urease gene urea, lipopolysaccharide-related gene uge, and fimbria-related gene fimH showed the detection rates of 100.00%, 95.54%, and 91.07%, respectively. Other virulence genes were detected to varying degrees (2.70%-8.90%), while the capsule-related gene magA and the fimbria-related gene mrkD were not detected. [Conclusion] In some areas of Guangdong Province, the antibiotic resistance of pet-derived K. pneumoniae is serious, but its pathogenicity is relatively weak. Monitoring of its drug resistance and pathogenicity should be strengthened. In addition, antibiotics should be strictly managed and rationally used in the clinical practice, so as to avoid the generation and dissemination of multidrug resistant strains of K. pneumoniae.

[Objective] To delineate the fungal diversity characteristics and disparities in the rhizosphere soil of naked barley from fields exhibiting different root rot incidences, thereby informing targeted and effective disease management strategies. [Methods] Rhizosphere soil samples of naked barley were collected from the healthy field and the fields with root rot incidences of 5%, 10%, 15%, and 20%, respectively. The 18S rRNA gene of fungi from the samples was amplified, and high-throughput sequencing was conducted on the Illumina-MiSeq platform. Following quality control, classification, and annotation, the data were analyzed for fungal diversity from various perspectives and taxonomic levels. [Results] The healthy sample and the sample with the root rot incidence of 5% had the highest fungal diversity, while the sample with the incidence of 10% exhibited the lowest diversity. Additionally, co-occurrence network analysis revealed more complex species interactions in the healthy sample and the sample with the incidence of 5%. The root rot incidence had a negative correlation with the relative abundance of Ascomycota but a positive correlation with the relative abundance of Basidiomycota and Glomeromycota. Dominant fungal classes were Agaricomycetes, Chytridiomycetes, and Sordariomycetes. Significant variations in the distribution of fungal groups with high average relative abundance were noted across samples. Specially, the healthy sample had the highest relative abundance of Eurotiales and the lowest relative abundance of Glomerales, which were converse in diseased samples. Arthrodermataceae was significantly enriched in the healthy sample, while Sordariaceae, Myxotrichaceae, and Olpidiaceae were preferentially associated with the samples exhibiting incidences of 10%, 15%, and 20%, respectively. At the genus and species levels, the composition of dominant fungal communities in the healthy sample and the sample with the incidence of 5% was similar, and it was similar in the samples with incidences of 10%, 15%, and 20%. FUNGuild predicted a decrease in the relative abundance of plant pathogens and an increase in the relative abundance of saprotrophs with the increase in disease incidence. [Conclusion] The progression of naked barley root rot is intricately linked to the disruption of the equilibrium within the rhizosphere fungal community. In the context of precision management of naked barley root rot, it is imperative to regulate and sustain the balance of the abundance of dominant fungal taxa in the rhizosphere.

[Objective] Conventional culture methods can merely uncover a fraction of microbial diversity. A vast number of microorganisms remain unculturable under laboratory settings. The advent of in-situ culture technology offers a key solution to this predicament. This study endeavors to innovate the in-situ culture technology, explore microorganisms under hitherto unknown culture conditions, and probe into their potential applications within the realm of uncultured microorganisms. [Methods] PCR tubes were employed as the core of the device, and the idea of sorting prior to culture was introduced. Microorganisms were separated from the environment by means of polymer membranes for independent culture. The efficacy of the device was validated through the pure culture of Escherichia coli. Moreover, this device was applied to in-situ culture in diverse environments such as soil, sewage, and mountain spring water. [Results] The abundance of E. coli increased significantly in the pure-culture device. When the concentration of the polymer membrane reached 15%, the encapsulation effect effectively prevented the escape of E. coli. The co-culture experiments further corroborated this finding. In the in-situ culture experiments, the device designed in this study successfully cultured single-cell microorganisms from sewage, soil, and mountain spring water samples. Sequencing results indicated that this device could culture species recalcitrant to be cultured in the laboratory. Comparison with the NCBI database verified that new species were successfully cultured, which demonstrated the effectiveness of the culture device in various environments. [Conclusion] The culture method designed in this study is suitable for single-cell microbial culture, enrichment of specific microbial communities, and co-culture of multiple microorganisms. The device can isolate and culture richer and more microorganisms than conventional culture methods. This new technology not only isolates and cultures more microorganisms but also manages to culture those previously unculturable under laboratory conditions. It holds great significance for microbiological and ecological research.

[Objective] To study the response relationship of the biological properties of bacterial communities to long-term climate change. [Methods] The loess (YL, PL, and RL) and paleosol (YS, PS, and RS) sequences, which have persisted for approximately 500 000 years at Shimao (Y), Potou (P), and Renjiapo (R), were sampled. The structures and functions of the soil bacterial communities were predicted by bioinformatics approaches, including high-throughput sequencing and FAPROTAX. [Results] The physical and chemical characteristics associated with the loess-paleosol alternation in the three regions reflected the shifts in climatic conditions, including dry, cold, warm, and wet phases during soil development. Over this period, the climate at Renjiapo was characterized by the highest temperatures and precipitation levels, with strong influences from summer winds. Shimao experienced the driest conditions and the lowest temperatures. Potou exhibited the intermediate climatic conditions between Renjiapo and Shimao. The abundance and diversity indices of the bacterial communities in Shimao were higher than those in Potou and Renjiapo. Across all the three regions, the dominant bacterial phyla were Proteobacteria, Actinobacteria, and Acidobacteria although their relative abundance varied significantly. The bacterial communities in the loess and paleosol layers of Shimao showed greater compositional similarity, forming a network structure driven by synergistic interactions. The ecological functions of the bacterial communities in the loess-paleosol sequences were primarily associated with carbon, nitrogen, and sulfur cycling. Carbon and nitrogen cycling was weakly expressed in Shimao, while nitrogen cycling was more prominent in Potou. Renjiapo exhibited the most pronounced carbon cycling. [Conclusion] Soil bacterial communities in warm and humid climates tend to exhibit more complex network structures and greater functional diversity. In contrast, bacterial communities in dry and cold climates are characterized by similar composition and synergistic patterns, which enable these communities to adapt to harsh conditions by increasing their abundance and diversity, compensating for nutrient limitations associated with reduced carbon and nitrogen cycling.

[Objective] Stenotrophomonas can promote the growth of plants and aquatic algae. However, limited studies have focused on the bacteriophages infecting Stenotrophomonas. In this study, we isolated bacteriophages infecting Stenotrophomonas from water and studied their biological and genomic characteristics, aiming to improve our understanding of the ecological functions of Stenotrophomonas and its bacteriophages. [Methods] The double-layer plate method was employed to isolate bacteriophages infecting S. indicatrix EB12 from lake water. The biological characteristics, whole genome sequence characteristics, gene functions, phylogenetic relationship, and protein structures of the isolates were analyzed. [Results] Transmission electron microscopy showed that two isolates Ste-X and Ste-D were short-tailed bacteriophages. The incubation period of both strains was 20 min. Ste-X and Ste-D showed the optimal multiplicities of infection being 1 000 and 10, burst sizes of 28.8 PFU/cell and 131.1 PFU/cell, temperature tolerance ranges of 20-60 °C and 20-70 °C, and pH tolerance ranges of pH 4.0-13.0 and pH 3.0-12.0, respectively. Ste-X and Ste-D were deactivated by UV irradiation for 60 min and 120 min, respectively. Their host ranges were narrow. The genomes of both strains showed the lengths of 39 429 bp, with the G+C content of 57.80% and 57.85%, respectively, and both of them contained 65 open reading frames (ORFs). The genomes of the two strains contained only three different bases. However, their homology with other bacteriophages was low, which suggested that the two strains were novel short-tailed bacteriophages. The structures of the proteins encoded by ORF34 and ORF52 were predicted. Result showed thant the proteins encoded by ORF34 were acidic, stable, and hydrophilic, containing two conserved domains, ChtBD3 and PHA00661, with the three-dimensional structures dominated by random coils. The proteins encoded by ORF52 were alkaline, unstable, hydrophilic, with no conserved domain and the three-dimensional structures dominated by α-helixes. [Conclusion] Two novel strains of short-tailed bacteriophages with high genome similarity but distinct biological characteristics were isolated from water.

[Objective] Using Sphingomonas paucimobilis as the starting strain, a high-yield gellan gum-producing engineered strain was constructed through metabolic engineering, and fermentation process optimization was performed, providing both theoretical support and technical foundations for the efficient biosynthesis of gellan gum with this bacterium. [Methods] A CRISPR-Cas9-based gene editing system was developed for S. paucimobilis, and subsequently employed to genomically integrate two key gellan gum biosynthesis genes: the regulatory protein gene (gelA) and the β-1,4-glucuronosyltransferase gene (gelK), both under the control of constitutive promoters. Building upon this foundation, fermentation parameters including carbon source, nitrogen source, pH, and dissolved oxygen were systematically optimized through single-factor experiments. [Results] The engineered strain FMME-GG08 achieved a gellan gum yield of 10.8 g/L in shake-flask cultivation, representing a 130.2% enhancement over the parental strain. Following fermentation process optimization, the production level reached 20.1 g/L in 15 L scale bioreactors, with a sucrose conversion efficiency of 0.50 g/g. [Conclusion] This study not only successfully constructed a high-yield gellan gum-producing strain and established an efficient fermentation process, providing a reliable technical solution for industrial production, but also developed a genetic editing strategy that serves as an important reference for metabolic engineering of non-model microorganisms to produce other high-value exopolysaccharides.

The vpa1443-vpa1445 gene cluster (mfpABC) encoding the membrane fusion protein (MFP) is hypothesized to be involved in the biofilm formation of Vibrio parahaemolyticus (Vp). The gene mfpA (vpa1445) encodes a Ca²⁺-binding extracellular protein containing a repeats-in-toxin (RTX) domain, while its function is still under exploration. [Objective] To study the influences of mfpA mutation on the biofilm formation and motility of Vp. [Methods] The single mutants of three genes in the mfpABC gene cluster were constructed, and the Vp motility was compared between the three mutants and the wild type. Furthermore, the influences of mfpA mutation on bacterial motility and biofilm formation were analyzed in detail, and the expression of related genes was analyzed by RT-qPCR. Moreover, the cytotoxicity of ΔmfpA to HeLa cells was investigated. [Results] The mutation of mfpA significantly reduced the swimming and swarming motility of Vp. Crystal violet staining and scanning electron microscopy results showed that the mutation of mfpA enhanced the biofilm formation and increased the content of extracellular polysaccharides and proteins. RT-qPCR confirmed that the expression levels of flagellar genes were downregulated, while those of extracellular polysaccharide synthesis-related genes were upregulated in ΔmfpA. The cytotoxicity of ΔmfpA significantly decreased compared with that of wild type. [Conclusion] The mutation of mfpA can affect the expression of genes associated with flagella and extracellular polysaccharides to reduce the motility, enhance the biofilm formation, and attenuate the cytotoxicity of Vp.