[Objective] To construct a strain XH02Δlpxtg270 with knockout of the LPXTG motif protein-coding gene from Corynebacterium pseudotuberculosis XH02 and explore the role of lpxtg270 in the growth, biofilm formation, and infection of XH02. [Methods] CRISPR/Cas9 was employed to construct XH02Δlpxtg270. The knockout strain and the wild strain XH02 were compared in terms of biological characteristics, invasion into J774A.1 macrophages, and pathogenicity in mice. [Results] Compared with XH02, XH02Δlpxtg270 did not change significantly in the colony morphology, growth curve, adhesion to J774A.1 macrophages, or intracellular proliferation, while it demonstrated reductions in the biofilm formation and invasion into J774 A.1 cells. Moreover, the release of lactate dehydrogenase and secretion of interleukin-1β from J774 A.1 cells infected with the knockout strain decreased compared with those infected with the wild strain. Compared with XH02, XH02Δlpxtg270 showed weakened pathogenicity in mice and decreased loads in the liver, spleen, kidney, lung, and brain, causing milder pathological changes of above organs in mice. [Conclusion] LPXTG270 of C. pseudotuberculosis is related to the biofilm formation and invasion into macrophages, playing a key role in the pathogenicity of this bacterium in mice.

Currently, the sustainable development of global agriculture is facing multiple challenges, including soil degradation, resource constraints, and environmental pollution. With the continuous growth of the population and the increasing demand for food quality, improving soil health has become a crucial foundation for ensuring food security. Although chemical fertilizers play an important role in maintaining the high yields and high quality of plants, their excessive or unreasonable use can cause environmental problems, such as soil acidification and water eutrophication. Rhizosphere microbial communities play an essential role in plant nutrient acquisition, tolerance to environmental stress, and adaptation to environmental changes. Among them, synthetic microbial communities (SynComs) are designed via the targeted assembly of multiple microorganisms with well-defined functions and clear genetic backgrounds, enabling the achievement of complex functionalities that cannot be accomplished by single strains. They are powerful tools for deciphering the key interface interaction mechanisms among plants, soil, and microorganisms and play a vital role in promoting efficient utilization of plant nutrients, enhancing plant stress resistance, and increasing the efficiency and reducing the application of fertilizers. This study reviews the conceptual evolution, current research trends, and construction principles, methods, and tools of SynComs, and summarizes the role of SynComs in the sustainable development of agriculture from the aspects of promoting plant growth, inhibiting biotic and abiotic stresses, and improving and restoring soil health. Furthermore, this paper makes an outlook on the future research directions and emphasizes the research and development of targeted microbial agents, the application of artificial intelligence (AI) in community assembly, and the performance improvement of SynComs in field applications, aiming to support the coordinated and multi-objective development of food security, efficient resource utilization, and environmental protection through near-natural microbial means, thereby facilitating the green agricultural development of China.

Alcoholic liver disease (ALD) is a liver disease caused by long-term excessive alcohol consumption and is one of the most common chronic liver diseases worldwide. At present, no effective approach is available for preventing or reversing this disease, causing a serious social burden and increasing the pressure for prevention and treatment. In recent years, researchers have found that long-term alcohol abuse can lead to significant changes in the structure and function of the gut microbiota, thereby disrupting the balance of the gut microbiota, which can promote the progression of ALD. Therefore, maintaining gut microbiota balance can provide new targets for the prevention and treatment of ALD. This article reviews the research and intervention progress of the gut microbiota and its metabolites in ALD in recent years, providing reference for future studies on the pathogenesis and treatment of this disease.

Candida auris, as a multidrug-resistant fungus, pose a challenge to clinical treatment because of biofilm formation. Currently, effective intervention measures against its biofilm remain to be developed. [Objective] To explore the antifungal activity and biofilm inhibition mechanism of the Chinese medicine active compound matrine (MT) against C. auris. [Methods] The minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and sessile minimum inhibitory concentration (SMIC) of MT against C. auris were determined by the microdilution method. The time-growth curve and colony morphology of C. auris under the intervention of MT were observed by the plate method. The changes in the hydrolytic enzyme activity of the C. auris biofilm treated with MT were determined. The changes in cell surface hydrophobicity (CSH) of C. auris biofilm treated with MT were observed by the water-hydrocarbon two-phase method. The effects of MT on the metabolic activity and structure of C. auris biofilms were observed by the XTT method, crystal violet method, and confocal laser scanning microscopy (CLSM). The changes in the cell nucleus of C. auris in the biofilm treated with MT were detected by DAPI staining. The protective effect of MT on the host infected with C. auris was observed by the Galleria mellonella larvae infection model. [Results] The MIC of MT against C. auris was 128 μg/mL, while the MFC and SMIC were both 512 μg/mL. The inhibition mechanism of MT against the proliferation of C. auris mainly involved reducing the CSH, inhibiting the mature biofilm formation, significantly decreasing the metabolic activity, and inducing abnormal nuclear morphology. The experiments with G. mellonella larvae further confirmed that MT could alleviate the invasive damage caused by C. auris. [Conclusion] MT has a significant antifungal effect on C. auris and can effectively inhibit the biofilm formation, providing a new candidate drug and potential target for clinical treatment of multidrug-resistant C. auris infections.

Double-crossover homologous recombination using the SacB negative selection system is commonly employed for genome editing in Gram-negative bacteria. However, its negative selection efficiency varies significantly across strains, being frequently compromised by differences in metabolic characteristics or genomic composition. [Objective] To develop a novel counterselection system based on the type VI secretion system (T6SS) effector Txe1 to enhance the efficiency of seamless genome editing. [Methods] We first modified the conventional suicide plasmid pDM4 by introducing a kanamycin resistance gene, generating the derivative plasmid pDM4K. Subsequently, we replaced sacB with an l-arabinose-inducible expression cassette encoding the C-terminal domain of Txe1 (araC-P_BAD::txe1^CTD ), constructing a novel counterselection plasmid pTL1010. Using the virulence gene tssB of Edwardsiella piscicida FC2 as the target, we systematically evaluated and compared the counterselection efficiency of the Txe1 system with that of the conventional SacB system. [Results] Under induction with l-arabinose, the Txe1-based counterselection system achieved the efficiency of 91.1% (false-positive rate of 8.9%), outperforming the SacB system which had a false-positive rate of 100% (P<0.01). [Conclusion] The newly developed Txe1-based counterselection plasmid pTL1010 significantly enhances the efficiency of seamless genome editing in E. piscicida and provides a highly effective tool for precise genetic manipulation in Gram-negative bacteria.

Endometriosis is a chronic, estrogen-dependent gynecological condition often associated with pelvic pain, dyspareunia, difficulties of defecation and urination, and infertility. The disease poses significant treatment challenges and has a high recurrence rate, causing considerable distress to patients and substantial social and economic impacts. Currently, the exact etiology of endometriosis remains unclear, and the lack of specific biomarkers complicates early diagnosis. Advances in high-throughput sequencing technologies have significantly propelled research into the human microbiome, revealing intricate connections between microbiota and diseases. Notably, the microbiota in the female oral cavity, genital tract, peritoneal cavity, and gut are closely linked to the development and progression of gynecological disorders. This article reviews the correlations between the human microbiome and endometriosis and explores the potential applications of microbiota in the diagnosis and treatment of endometriosis.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive method of nucleic acid isothermal amplification, is characterized by high specificity and sensitivity and holds broad application prospects in nucleic acid detection. However, the development of detection schemes based on LAMP encounters issues, such as non-specific and non-template amplification during the amplification process, which can affect the accuracy and specificity of the detection results. This article elaborates on the recent research progress in the strategies avoiding false positives and enhancing detection efficacy of LAMP, which include primer design, optimization of conditions, and introduction of special chemical substances. Finally, this article explores the breakthroughs and innovations of LAMP in the field of detection.

[Objective] To construct a PK15 cell line stably expressing the prolyl oligopeptidase (POP) by using the PiggyBac transposon system and systematically investigate its regulatory role in the proliferation of foot-and-mouth disease virus (FMDV). [Methods] A recombinant PiggyBac vector carrying the POP gene was constructed and transfected into PK15 cells, and the expression of the target protein was detected by Western blotting. The monoclonal cell line stably expressing POP was isolated via the limiting dilution method. The stability of mRNA and protein levels of POP was verified by RT-qPCR and Western blotting, respectively. The viability of the selected cell line was assessed by the Cell Counting Kit-8 (CCK-8) assay. FMDV replication kinetics in the stable cell line were comprehensively analyzed by Western blotting, RT-qPCR, and the 50% tissue culture infective dose (TCID₅₀) assay. [Results] A PK15 cell line stably expressing POP was established. No significant differences in cell viability were observed between the stable cell line and the control cell line. The protein level of POP remained stable in the established cell line after 30 passages. The results of the viral infection assay demonstrated that the FMDV proliferation level in the PK15 cell line stably expressing POP was significantly higher than that in the control group, with this stimulatory effect being maintained across multiple passages. [Conclusion] We successfully constructed a PK15 cell line stably expressing POP. The results reveal that POP overexpression enhances FMDV replication in a passage-independent manner. These findings provide a valuable experimental model for elucidating the molecular mechanism underlying the role of POP in FMDV replication.

[Objective] To investigate the pathogenicity of Lichtheimiaramosa LYSF001 and ITS resistance to common fungicides, providing experimental evidence for the diagnosis and treatment of fungal infections in animals. [Methods] The strain was identified by morphological characterization and molecular biology techniques. PCR was employed to detect virulence genes in strain LYSF001, and an animal infection model was established to evaluate the pathogenicity of the strain. Histopathological examination, Grocott’s methenamine silver (GMS) staining, and ITS sequences amplification were employed to analyze pathological changes in the livers and kidneys of infected animals. Additionally, antimicrobial susceptibility tests were conducted to assess the sensitivity of strain LYSF001 to caspofungin, amphotericin B, and itraconazole. [Results] Strain LYSF001 was identified as L. ramosa. PCR analysis revealed that strain LYSF001 carried five virulence genes (CalA, PKP2, LaeA, Alp2, and AspF1). Animal experiments demonstrated that the strain led to the mortality of 40% and caused visceral hyperemia in mice, with the most significant pathological changes (inflammatory cell infiltration and tissue necrosis) observed in the livers and kidneys. Severe infections led to animal mortality. GMS staining revealed the presence of dark-colored hyphae in the heart and liver, and ITS sequences amplification further confirmed fungal infection. The antimicrobial susceptibility test results indicated that strain LYSF001 was resistant to caspofungin but sensitive to amphotericin B and itraconazole. [Conclusion] L. ramosa LYSF001 exhibits strong pathogenicity, capable of causing severe infections and even death in animals. Additionally, the strain showed resistance to caspofungin but sensitivity to amphotericin B and itraconazole. The findings provide important experimental evidence and technical support for the diagnosis and treatment of fungal infections in animals.

[Objective] To deeply understand the molecular evolution pattern, pathogenicity, and drug resistance mechanism and provide a scientific basis for the prevention and control of Streptococcus equi subsp. zooepidemicus (SEZ), we compared the pathogenicity, drug resistance, and genome sequences of two SEZ strains (YLCD588 and HT222) isolated from donkeys in Xinjiang. [Methods] Whole-genome sequences of the two strains were determined by next-generation sequencing and a phylogenetic tree was constructed based on multilocus sequence typing (MLST) of sequencing data and existing sequences in the database. The virulence factor database (VFDB) (https://www.mgc.ac.cn/VFs/) and the center for genomic epidemiology (CGE) (http://genomicepidemiology.org) were used for annotation of the virulence and drug resistance genes of the two strains. The growth curves, antimicrobial susceptibility, and biofilm formation of the two strains were examined and compared. Mice were challenged with these strains separately and the pathogenicity of the strains was observed and evaluated. Then, histopathological changes and bacterial loads in the lung and spleen tissues of the morbid mice were observed and determined. [Results] Sequencing data showed that the genome sizes of YLCD588 and HT222 were 2 090 225 bp (1 905 coding sequences) and 2 105 005 bp (1 995 coding sequences), respectively. HT222 and YLCD588 carried 214 and 212 virulence genes, respectively. HT222 and YLCD588 had 235 and 233 drug resistance genes, respectively. YLCD588 was assigned to a novel sequence type (ST) 545 by MLST. The MLST phylogenetic tree indicated that YLCD588 was closely related to the goat-derived SEZ strain, while HT222 was closely related to the canine SEZ strain. YLCD588 displayed resistance to six antibiotics and HT222 exhibited resistance to four. The crystal violet assay and confocal laser scanning microscopy results showed that YLCD588 exhibited stronger biofilm formation than HT222 (P<0.05), whereas HT222 caused higher mortality rate (P<0.05), higher bacterial load (P<0.01), and severer pathological damage in mice than YLCD588. [Conclusion] The two SEZ strains exhibit distinct genomic characteristics, sequence types, pathogenicity, and drug resistance. HT222 possesses more drug resistance genes and virulence genes and exhibits stronger pathogenicity than YLCD588, while YLCD588 showcases stronger biofilm formation and drug resistance than HT222. These findings broaden the understanding about the molecular epidemiology, pathogenicity, and drug resistance of different SEZ strains from donkey and provide references for the effective control of the infection and spread of SEZ and clinical treatment of SEZ infection.