Abstract:Traditional Chinese Baijiu products can be classified into multiple flavor types such as strong-flavor, light-flavor, soy sauce-flavor, and complex-flavor types. Due to differences in fermentation raw materials, starters, fermentation vessels, fermentation processes, and geographical environments, Baijiu products of different flavor types have unique and complex pit-mud microorganisms. As one of the core microbial groups in the complex microbial community of Baijiu, lactic acid bacteria, whose metabolites lactic acid and its derivatives are important flavor substances in strong-flavor Baijiu, play an important role in the flavor and taste of Baijiu. At present, the research on lactic acid bacteria in the brewing of strong-flavor Baijiu is scattered and not systematic enough. On the basis of our work, we review the research on the role of lactic acid bacteria in the brewing of strong-flavor Baijiu. This review encompasses the isolation and screening of lactic acid bacteria in the brewing of strong-flavor Baijiu by conventional microbiological methods and the in-depth understanding of the role of lactic acid bacteria in the brewing of strong-flavor Baijiu by modern microbiological research methods. Combining traditional with modern microbiological research methods to conduct systematic and in-depth research on lactic acid bacteria in strong-flavor Baijiu based on actual strains can provide references for deeply understanding the role of lactic acid bacteria in the brewing process and then strengthening or optimizing the brewing process.

Abstract:Streptococcus suis, a major pathogen of pigs, can cause meningitis, septicemia, and arthritis, leading to serious economic losses in the pig industry. In addition, it can also infect humans and result in diseases or death, being a zoonotic pathogen. Therefore, accurate, rapid, sensitive, and simple detection methods are critical for the prevention and control of swine streptococcosis. At present, a variety of detection methods for S. suis have been developed, including traditional detection methods of microbiology, molecular biology, and immunology and emerging technologies such as nanomaterial-based immunosensors, CRISPR-Cas12a system, and matrix-assisted laser desorption ionization-time of flight mass spectrometry. This review summarizes the principles, applications, advantages, and disadvantages of the above methods and introduces the future directions of S. suis detection methods, aiming to provide reference for the prevention and control of swine streptococcosis.

Abstract:Honeybees (Apis mellifera) are important pollinators worldwide and models for the research on development and behaviors, showcasing great economic, environmental, and scientific benefits. As the symbiont of honeybees, the gut microbiota is transmitted through social behavioral interactions and plays a crucial role in the development and health of honeybees. It not only helps honeybees digest and absorb nutrients but also helps resist pathogen invasion and enhance immunity. Recently, honeybees have emerged as the models for studying gut microbiota. Researchers not only analyzed the composition and function of the gut microbiota in honeybees but also explored the diversity and functions of strains. This paper reviews the temporospatial dynamics of the gut microbiota in honeybees, the factors affecting the gut microbiota, the influences of the gut microbiota on the biological characteristics and health of honeybees, and the functional applications of the gut microbiota, providing references for the research and application of the gut microbiota in honeybees.

Abstract:Shikimic acid (SA) is an important natural compound with many biological activities, including antiviral, antithrombotic, analgesic, antimicrobial, and anti-cancer properties. Due to its diverse applications in medicine, cosmetics, food, and agriculture, SA is considered a highly promising biomolecule. As a precursor of aromatic compounds, SA plays a crucial role in various metabolic pathways within organisms. Traditional methods for producing SA mainly rely on plant extraction (such as star anise) or chemical synthesis. However, these approaches face challenges such a high costs, low efficiency, and environmental concerns. With the ongoing advancements in synthetic biology and metabolic engineering, the production of SA through metabolic engineering has emerged as a focal point of research, offering a more sustainable and cost-effective alternative. This paper reviews the applications and production methods of SA, with a particular emphasis on recent progress and optimization strategies in its biosynthesis.

Abstract:The SaeRS system has been extensively and intensively studied for its involvement in the regulation of virulence factor expression and biofilm formation in the Gram-positive pathogen Staphylococcus aureus, mediating severe pathogenicity. The activation of the system depends on the recognition of external signals by the sensor histidine kinase SaeS and the phosphorylation status of the response regulator SaeR. With the help of auxiliary proteins SaeP and SaeQ, S. aureus is prompted to express a variety of virulence factors, coordinate its biofilm formation, and even evade the host immune response. In addition, other regulatory systems and modulators of S. aureus can synergize with the SaeRS system to participate in the regulation of virulence factor expression, enhancing bacterial pathogenicity. This paper reviews the SaeRS system and its interactions with other regulatory systems and factors to regulate the expression of virulence factors and biofilm formation. It summarizes targeted drugs against the SaeRS system and analyzes specific examples of anti-SaeRS system drugs to provide clues for the screening and design of new targeted drugs. This review is expected to provide a theoretical basis for the research on the specific regulatory mechanisms of the SaeRS system and the treatment of S. aureus-associated infections.

Abstract:CsgD, a core regulatory protein for the biofilm formation of Salmonella, exerts its function by regulating the expression of key components (curli and cellulose) of the biofilm. In recent years, significant progress has been achieved in analyzing the regulatory network of Salmonella CsgD protein and its complex influencing factors. Focusing on the regulatory function of CsgD in the biofilm formation of Salmonella, this paper systematically introduces how environmental factors affect the CsgD expression and comprehensively analyzes the multi-level regulatory effects of various regulatory factors on CsgD, aiming to enrich our understanding about the mechanism and regulatory network of the biofilm formation of Salmonella and provide possible research directions.

Abstract:In the special environment of plateau, adaptive responses and pathological changes occur in the skin of organisms, which include skin barrier dysfunction, skin vasoconstriction disorder, abnormal thickening of the stratum corneum, and fibrous pigment deposition, leading to a high incidence of skin diseases in plateau areas. In the plateau environment, the composition and diversity of skin microbiota change, which may be related to the occurrence and development of skin diseases. This paper reviews the effects of plateau environments on the skin, common skin diseases in plateau environments, and the effects of skin microbiota on skin diseases and discusses the mechanisms by which skin microbiota influences skin diseases in plateau environments.

Abstract:Polycyclic aromatic hydrocarbons (PAHs) are widely distributed in the environment and have ecological and environmental toxicological effects. Therefore, the treatment and remediation of PAH-contaminated sites have attracted much attention. Microbial degradation, as one of the remediation methods for PAH pollution, has the advantages of low cost, high efficiency, and environmental friendliness. The co-cultured microbial system usually has better degradation effect, stronger adaptability, and greater resistance to stress than single strains. This paper reviews the microbial species and mechanisms of co-cultured microorganisms for degrading PAHs and points out the strategies for constructing co-culture systems and different microbial combinations, hoping to provide reference for the application of co-cultured microbial flora in the remediation of organically polluted environments.

Abstract:Cotton Verticillium wilt is the most serious disease affecting cotton cultivation, which could cause a significant decrease in cotton yield or even complete crop failure. Cotton Verticillium wilt is caused by the filamentous fungus Verticillium dahliae. The traditional chemical control affects public health and brings about environmental pollution, and the continuous usage has induced the drug resistance of Verticillium dahliae. Therefore, it is urgent to develop environmental friendly and sustainable development control strategies against cotton Verticillium wilt. Biological control has become a good choice to prevent cotton Verticillium wilt. Based on the analysis of the recent research progress, this review discussed the screening, mechanism of action and field application of biocontrol microbial strains against cotton Verticillium wilt, and summarized the research progress of biocontrol microorganisms inhibiting the growth of pathogen through various mechanisms such as competition, antibiotic action, and inducing plant defense response. Although the application prospects of biocontrol microorganisms are expected, they still face challenges such as environmental adaptability, stability, and usage costs of these biocontrol microorganisms. To further improve the practicality of biocontrol microbial strains in agricultural production, future research should focus on genetic improvement of biocontrol microorganisms, development, and application of the microbial agents and so on.

Abstract:The toxin-antitoxin (TA) system is ubiquitous in bacteria and archaea. A typical TA system generally consists of a toxin that inhibits bacterial growth and an antitoxin that neutralizes toxin toxicity. At present, TA systems are classified into types I-VIII, of which type II system is the most extensively studied. In addition, researchers have found that there are atypical TA systems such as monocistronic TA systems and three-component TA systems. After the discovery of the first TA system (CcdB/CcdA) in the 1980s, TA systems have been shown to play a key role in the physiological processes of microorganisms. In this paper, we review the research results about the roles of TA systems in resisting bacteriophage infections in recent years and summarize the neutralization mechanisms. In particular, we brief how TA systems specifically sense the invading bacteriophages and the underlying molecular mechanisms, aiming to provide reference for the research on the roles and regulation mechanisms of unknown TA systems in the future.

Abstract:In the post-antibiotic era, phage therapy becomes a candidate for treating drug-resistant bacteria. Phages are diverse, and jumbo phages are a class of phages with genomes longer than 200 kb. Their large genomes carry abundant functional genes with scattered distribution. Jumbo phages have a variety of unique biological characteristics, such as oversized phage particles, unique replication cycles, and unique structures such as the nucleus-like compartment, the “inner body”, and the long-wavy curly tail fiber. This paper reviews the research progress in jumbo phages, focusing on their biological characteristics, genomes, evolution, and special replication mechanisms and structures. Furthermore, this review explores the application potential of jumbo phages in the treatment of drug-resistant bacterial infection, environmental management, aquaculture, and biocontrol, aiming to provide reference and implications for the research and application of jumbo phages.

Abstract:Heavy metal pollution has attracted increasing attention because of its toxicity, environmental persistence, bioaccumulation, and potential contribution to antibiotic resistance. The accurate, rapid, efficient, and sensitive detection of heavy metals in the environment is of great significance for environmental protection and human health. A whole-cell microbial biosensor (WCMB), integrating a biorecognition module and a signal processing module, provides a new strategy for heavy metal monitoring. Great progress has been achieved in the transcription factor-based WCMBs for the monitoring of heavy metals in recent years. This paper introduced the basic composition and design principles of WCMBs, summarized the construction and application of WCMBs for heavy metal detection that were developed in recent years, analyzed the optimization strategies of WCMBs by synthetic biology, and finally prospected the future research directions of WCMBs. This paper is expected to be an important reference for the effective prevention and control of heavy metal pollution in the environment.

Abstract:Objective To elucidate the phylogenetic position and mine the gene resources for synthesis of secondary metabolites from Streptomyces sp. YH02, a Gram-positive actinomycete strain isolated from the soil sediment of Yuncheng Salt Lake in Shanxi.Methods Illumina and PacBio platforms were used for whole genome sequencing of YH02, which was followed by gene prediction, functional annotation, prediction of secondary metabolite synthetic gene clusters (BGCs), comparative genomic analysis, and morphological, physiological, and biochemical characterization.Results The YH02 genome was a linear chromosome spanning 8 285 116 bp, with the G+C content of 71.77% and 7 237 open reading frames. Gene annotations in the GO, COG, KEGG, and CAZy identified 2 829, 5 478, 4 805, and 279 genes, respectively. The subcellular localization analysis predicted various secretion system-related proteins and 1 030 transporters. Additionally, 32 secondary metabolite BGCs were predicted in strain YH02, involving the synthesis of various natural products such as terpenoids, non-ribosomal peptides, polyketides, and ribosomally synthesized and post-translationally modified peptides. The comparative genomic analysis revealed 15 739 pan-genome orthologous gene clusters and 4 267 core genome orthologous gene clusters. The phylogenetic analysis based on the 16S rRNA gene sequence revealed a proximate phylogenetic affiliation between strain YH02 and Streptomyces venezuelae ATCC 10712 as well as Streptomyces zaomyceticus NBC 00278. However, the average nucleotide identity (ANI) value was below the threshold of 95.00%, and the digital DNA-DNA hybridization (dDDH) value was less than 70.00%. YH02 exhibited light pink aerial mycelia on the ISP 2 medium. It showed significant differences in tolerance to pH, sodium chloride, and growth temperature compared with its closely related strains. Additionally, this strain demonstrated weak starch hydrolysis activity, positive gelatin liquefaction, positive nitrate reduction, and slow milk coagulation.Conclusion Based on the findings from genomic, physiological, and biochemical analyses, strain YH02 is confirmed as a potential new species of Streptomyces. This study not only enriches the microbial resource pool but also provides a theoretical basis and potential genetic resources for mining the natural products with unique mechanisms of action.

Abstract:Objective To identify mazEF family type II toxin-antitoxin systems of Leuconostoc pseudomesenteroides L64 and to elucidate the molecular roles of the mazEF systems in the host exposed to environmental acid stress.Methods Putative MazF toxins were induced alone or co-expressed with their cognate antitoxins in Escherichia coli. The toxic effect of MazF on bacterial growth and the antitoxic effects of cognate antitoxins were examined. The lacZ reporter system and electrophoretic mobility shift assay (EMSA) were used to decipher the auto-regulation mechanism of the mazEF system in vivo and in vitro. The putative target genes regulated by MazE were predicted and validated through in vivo and in vitro experiments.Results Among the three putative mazEF systems in L. pseudomesenteroides L64, mazEF1-Leup (OYT_01690-OYT_01685) encoded a functional type II toxin-antitoxin system. MazE1-Leup (OYT_01685) inhibited mazEF1-Leup transcription by binding to the palindromic sequence (TAACAaaatgTGTTA) in the promoter. In addition, MazE1-Leup inhibited transcription of the dlt-acpS-alr operon by binding to the similar palindromic sequence (TAACAtattgaaatatatgTGTTA) in the promoter of dlt-acpS-alr.Conclusion mazEF1-Leup (OYT_01690-OYT_01685) encodes a functional mazEF family type II toxin-antitoxin system. Beyond regulating its own operon, MazE1-Leup regulates the transcription of dlt-acpS-alr and finally assists L. pseudomesenteroides L64 in response to low acid stress.

Abstract:Objective The biological regulatory mechanisms of soil microorganisms in response to single heavy metals or organic pollutants have been extensively studied, while the biological mechanisms of microbial responses to combined pollution remain unclear. This study aims to reveal the biological mechanisms of Enterococcus faecalis HHT-1 in response to single and combined stress of cadmium (Cd) and aniline (AN) by transcriptomics.Methods Transcriptomics was employed to explore the transcriptional regulation of HHT-1 under single (Cd: 150 mg/L; AN: 2 g/L) and combined stress (150 mg/L Cd and 2 g/L AN) at half inhibitory concentrations (IC₅₀).Results Under single Cd stress, HHT-1 upregulated the expression of genes encoding metal-binding proteins and transporters to promote the sequestration or efflux of Cd²⁺. High concentrations of Cd induced oxidative stress in cells, and HHT-1 upregulated the expression of ribosome-related genes and nucleotide-related genes to repair the protein damage and DNA damage caused by oxidative stress, and cleared intracellular reactive oxygen species (ROS) by producing catalase. Moreover, Cd stress upregulated the expression of genes related to virulence and antibiotic resistance in HHT-1. Under the single stress of AN, HHT-1 activated the expression of AN-degrading enzyme genes to reduce its toxicity and upregulated the expression of genes encoding efflux pumps to excrete AN from the cell. AN also caused intracellular oxidative stress, and HHT-1 cleared the ROS by upregulating the expression of glutathione synthase genes. Under the combined stress of Cd and AN, HHT-1 exhibited a complex array of adaptive mechanisms. Initially, HHT-1 upregulated the expression of genes encoding metal transporters and efflux pumps to transport Cd and AN out of the cell, thereby mitigating their toxic effects on the cell. At the same time, HHT-1 cleared intracellular ROS via the dual pathway of catalase and glutathione. In addition, HHT-1 upregulates the expression of virulence and antibiotic resistance genes under combined stress, which may enhance its pathogenicity and resistance. It is worth noting that HHT-1 did not significantly upregulate the expression of AN-degrading enzyme-related genes under the combined stress.Conclusion The transcriptional regulation mechanism of E. faecalis HHT-1 in response to the combined stress of Cd and AN is a combination of the transcriptional regulation mechanisms observed under single Cd stress and single AN stress, and it is more similar to the mechanism observed under single Cd stress. HHT-1 mainly responds to the combined stress of Cd and AN by enhancing cell wall synthesis, Cd²⁺ efflux, DNA repair, and ROS scavenging. Genes associated with efflux pumps and glutathione under AN stress continue to be expressed under the combined stress. Both single Cd stress and combined stress of Cd and AN may increase the potential pathogenicity and drug resistance of HHT-1.

Abstract:Objective To study the effect of Alistipes finegoldii (AF) on inflammatory bowel disease (IBD) and the underlying mechanism.Methods Six-week-old male C57BL/6J mice were administrated with streptomycin for three days and then randomly assigned into the control, phosphate buffered saline (PBS), and AF groups. Mice were administrated with AF suspension (1×10⁹ CFU, 200 μL per mouse) or PBS by gavage for two weeks, followed by drinking of the water containing 2.5% dextran sulfate sodium (DSS) for one week for the modeling of colitis. The weight loss fraction percentage, fecal characteristics, blood fecesstools, and colon length were determined. The colon tissue was stained with hematoxylin-eosin for the scoring of histopathological changes, and feces samples were collected at the beginning and end of the experiment for sequencing of 16S rRNA gene amplicons at the beginning and end of the experiment. The mRNA levels of colon tissue-associated intestinal barrier proteins and inflammatory mediators were determined by qPCR.Results The mice in the AF group had severer disease conditions than those in the PBS group regarding the weight loss percentage, disease activity index, colon shortening, and histopathological score. Compared with the PBS group, the AF group showed down-regulated mRNA levels of occludin and claudin 5 and up-regulated mRNA level of interleukin (IL)-17A. The AF group had lower alpha diversity of intestinal flora than the PBS group, and the beta diversity showed significant differences between AF and PBS groups. The linear discriminant analysis effect size (LEfSe) results revealed that the significantly differential bacteria between AF and PBS groups were Bacilli, Erysipelotrichales, Erysipelotichaceae, Odoribacter, Marinifilaceae, Dubosiella, and Dubosiella newyorkensis.Conclusion AF promotes the secretion of inflammatory mediators, impairs the permeability of the intestinal mucosa, and alters the structure and diversity of the intestinal flora, thereby promoting the development of IBD.

Abstract:Objective To identify the active components in Glycyrrhiza uralensis Fisch. that inhibit methicillin-resistant Staphylococcus epidermidis (MRSE) infections and explore their potential antibacterial mechanisms.Methods The half-dilution method was employed to assess the inhibitory activities of pharmacological components from G. uralensis against MRSE. The anti-MRSE phenotype of this medicinal herb was evaluated by microbial adhesion to hydrocarbons, crystal violet staining, scanning electron microscopy, and integrated cell imaging. Additionally, metabolomic analysis was conducted via gas chromatography-mass spectrometry (GC-MS), and the activity of intracellular oxidative dehydrogenase was measured by a commercially available reagent kit. The propidium iodide and laurdan dyes were utilized to assess the membrane damage and fluidity of cells. The challenge test was conducted with the larvae of Galleria mellonella to determine the antibacterial activities of tested pharmacological components in vivo.Results Licochalcone A, licochalcone C, and glabridin from G. uralensis demonstrated significantly inhibitory activities against MRSE. Among these compounds, licochalcone A exhibited the strongest inhibitory effect on MRSE, with a minimum inhibitory concentration (MIC) of 6.0 μg/mL and a minimum bactericidal concentration (MBC) of 12.0 μg/mL. The metabolomic analysis indicated that licochalcone A primarily influenced the metabolic pathways, secondary metabolite biosynthesis, and ATP-binding cassette (ABC) transport systems of MRSE. This compound impeded the biosynthesis of ornithine, lysine, and niacin, leading to the accumulation of 1,3-dipalmitin in the cells. Phenotypic experiments corroborated that licochalcone A downregulated the tricarboxylic acid (TCA) cycle flux and reduced the intracellular ATP level in MRSE. Furthermore, it inhibited the biofilm formation and intracellular protein expression, thereby preventing MRSE from adhering to HaCaT cells. Additionally, licochalcone A disrupted the structural integrity of the MRSE cell membrane, resulting in cell collapse, deformation, and even rupture and increased the survival rate of G. mellonella larvae following MRSE infection.Conclusion Exposure to licochalcone A alters the metabolism of sugars, lipids, and amino acids in MRSE cells, thereby influencing the biofilm formation, biosynthesis of secondary metabolites such as proteins, and the remodeling of cell membranes. Consequently, this alteration results in an antimicrobial phenotype characterized by decreased ATP production, impaired transporter function, and reduced adhesion and infection of MRSE.

Abstract:Objective To develop a safe, effective, and identifiable new vaccine.Methods The gene deletion strain S2Δbp26 was constructed by homologous recombination with S2 as the parental strain. The stability, safety, and efficacy of S2Δbp26 were then evaluated.Results The phenotype and genotype of S2Δbp26 did not change after 20 successive passages in vitro. The experiments in mice and guinea pigs showed that there was no difference in biosafety between S2Δbp26 and S2. The bacterial load was less than 2×10⁵ CFU per gram of spleen in guinea pigs inoculated with S2Δbp26 at different passages and S2, which suggested that the virulence of the mutant strain was attenuated. The mice inoculated with S2ΔBP26 had a shorter 50% recovery time than those inoculated with S2. Meanwhile, the mice immunized with S2Δbp26 could successfully resist the challenge with M28 wild-type strain at a dose of 2×10⁵ CFU/mouse.Conclusion S2Δbp26 was successfully constructed, with excellent safety and immunoprotective capacity, which provided technical reserves for the development of identifiable vaccines for brucellosis.

Abstract:Objective To explore the potential mechanism by which Prevotella copri promotes atherosclerosis (AS) from the perspective of host-gut microbiota-metabolism.Methods ApoE^-/- mice were randomized into four groups (n=8): control group (Chow group, fed with a normal diet), model group (AS group, fed with a high-fat diet), low-concentration P. copri group (P. copri-low group, administrated with P. copri at 10⁹ CFU/mL by oral gavage daily from the first day of feeding with the high-fat diet), and high-concentration P. copri group (P. copri-high group, administrated with P. copri at 10¹¹ CFU/mL by oral gavage daily from the first day of feeding with the high-fat diet). The body weight was measured and recorded weekly to evaluate the weight gain trend. After 5 weeks, oil red O staining was employed to evaluate the aortic plaque area, and enzyme-linked immunosorbent assay (ELISA) was employed to measure lipid levels, on the basis of which the impact of P. copri on AS progression was assessed. Additionally, qPCR was used to detect the abundance of P. copri in the gut, and untargeted metabolomics was employed to analyze the metabolite changes in the feces of mice.Results Compared with the Chow group, the AS group showed increases in the body weight, aortic plaque area, and plasma levels of low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC), and triglycerides (TG) and a decline in the high-density lipoprotein cholesterol (HDL-C) level. The abundance of P. copri in the gut showed no significant difference between the P. copri-low group and the P. copri-high group, indicating that P. copri successfully colonized the gut in both groups. Based on this, the P. copri-low group was selected as the standard concentration group (P. copri group) for further analysis. Compared with the AS group, P. copri colonization in the gut significantly increased the body weight and aortic plaque area and exacerbated dyslipidemia. Metabolomic analysis revealed that P. copri transplantation led to significant increases in the content of several metabolites, including Cer(d18:1/18:1(9Z)), N-palmitoylsphingosine, genistein, adenine, and linoleic acid. KEGG pathway enrichment analysis further indicated that P. copri might contribute to the development and progression of AS through key pathways such as the regulation of ABC transporters, bile acid metabolism, and neuroactive ligand-receptor interactions.Conclusion P. copri may exacerbate inflammation and lipid metabolism imbalance by regulating sphingolipid signaling, purine metabolism, and linoleic acid metabolism, thereby promoting the progression of AS.

Abstract:Objective To investigate the effects of Weizmannia coagulans BC-G44 on the intestinal microbiota structure, barrier function, and inflammatory response in the colon of the rat model of antibiotic-associated diarrhea (AAD).Methods A total of 30 five-week-old Sprague-Dawley (SD) rats with similar body weights were randomized into five groups (n=6): control (Con), model (Mod), low-dose W. coagulans BC-G44 (LBC-G44), medium-dose W. coagulans BC-G44 (MBC-G44), and high-dose W. coagulans BC-G44 (HBC-G44). The experiment encompassed a modeling period (7 days) and a recovery period (12 days). During the modeling period, rats in the Con group were administrated with normal saline at 2 mL/d by gavage, while those in the Mod, LBC-G44, MBC-G44, and HBC-G44 groups were administrated with a mixture containing clindamycin, ampicillin, and streptomycin (2 mL/d) by gavage to induce AAD. During the recovery period, the Con and Mod groups continued to receive normal saline, while the LBC-G44, MBC-G44, and HBC-G44 groups received W. coagulans BC-G44 suspensions at 10⁷, 10⁸, and 10⁹ CFU/d, respectively. On day 19, colonic tissue samples were collected for histological examination, and the concentrations of cytokines, and the expression levels of barrier proteins and inflammation-related genes in the colonic mucosa were measured. Furthermore, the microbiota composition and metabolites in the colonic chyme were analyzed.Results On day 7 of modeling, rats in the Mod, LBC-G44, MBC-G44, and HBC-G44 groups exhibited diarrhea, weight losses, and reduced food intake compared with those in the Con group (P<0.05). On day 19, compared with the Mod group, the MBC-G44 and HBC-G44 groups showed increases in the colonic mucosa thickness and goblet cell number (P<0.05). The HBC-G44 group showed increased relative abundance of Bacteroides and concentrations of lactate in the colonic chyme (P<0.05), elevated levels of d-lactic acid (d-LA) and diamine oxidase (DAO) in the colonic mucosa, and up-regulated relative mRNA levels of Claudin-1, Occludin, and MUC2 in the colonic mucosa (P<0.05). Meanwhile, compared with the Mod group, the MBC-G44 and HBC-G44 groups showed down-regulated relative mRNA levels of Toll-like receptor 4 gene (TLR4) and nuclear factor-kappa B gene (NF-κB) (P<0.05), and reduced concentrations of tumor necrosis factor (TNF)-α and interleukin (IL)-1β (P<0.05) in the colonic mucosa.Conclusion W. coagulans BC-G44 can ameliorate antibiotic-induced colonic injury, regulate the colonic microbiota composition and metabolites, enhance intestinal barrier function, and reduce intestinal inflammatory responses in rats, thus alleviating the symptoms of AAD.

Abstract:Objective To develop a DNA vaccine by loading avian pathogenic Escherichia coli ghosts with the fimbrium gene flfA of Gallibacterium anatis and evaluate the immune effect of this vaccine in chickens.Methods Taking flfA of G. anatis as the target gene, we constructed the eukaryotic expression plasmid pCAGGS-flfA from the plasmid pCAGGS-HA carrying the chicken β-actin promoter, which enhanced the efficacy of vaccine. The temperature-sensitive plasmid PBV-E-SN was then constructed and transformed into the ampicillin-sensitive avian pathogenic E. coli isolate to prepare a bacterial ghost. Finally, pCAGGS-flfA was loaded into the bacterial ghost to prepare a bacterial ghost vaccine. Seven-day-old chickens were assigned into five groups: bacterial ghost loading pCAGGS-flfA, pCAGGS-flfA, empty bacterial ghost, PBS, and normal control. The primary vaccination was carried out for 7-day-old chickens, and the booster immunization was performed two weeks after the primary immunization. The chickens were challenged with G. anatis four weeks after the primary immunization, and the immune effect of the vaccine was evaluated.Results The eukaryotic expression plasmid pCAGGS-flfA was successfully expressed in cells in vitro. The lysis rate of the constructed avian pathogenic E. coli ghosts heated at 42 °C for 210 min reached 99.94%. The specific IgG antibody titer measured by ELISA, the number of shedding bacteria in chicken cloacal swabs and throat swabs, and the bacterial loads in tissue and organs of challenged chickens showed that the immune effects in the bacterial ghost loading pCAGGS-flfA group and the pCAGGS-flfA group were much higher than those in other groups. Moreover, the bacterial ghost loading pCAGGS-flfA showed stronger immune effect than pCAGGS-flfA.Conclusion Bacterial ghosts as the carriers of pCAGGS-flfA significantly enhanced the immune effect of the DNA vaccine.

Abstract:Objective To study the probiotic properties and safety of Limosilactobacillus reuteri CHF7-2 isolated from Chahua chicken, providing a theoretical basis for the development of this strain as a microecological feed additive.Methods In vitro assays were conducted to evaluate the adhension, enzyme production, antibacterial activity, and antioxidant activity of strain CHF7-2. PacBio Sequel II and Illumina NovaSeq 6000 were used for whole genome sequencing of strain CHF7-2. Bioinformatics tools and databases were then used for genome annotation to explore the probiotic mechanisms and safety of this strain at the molecular level.Results Strain CHF7-2 possessed significant probiotic properties and safety. It exhibited high surface hydrophobicity, self-coagulation, and antioxidant activity. The strain inhibited the growth of Escherichia coli K88, Staphylococcus aureus ATCC 49521, Salmonella gallinarum CICC 21510, and Salmonella choleraesuis CVCC 3383. Strain CHF7-2 produced protease and lipase, and it was safe for use in feed since it did not exhibit hemolytic activity. Whole genome sequencing revealed that the genome size of CHF7-2 was 2 116 761 bp, with the G+C content of 38.8%, encoding 2 067 genes. Additionally, the genome carried the biosynthetic gene cluster of the class III bacteriocin EnlA and multiple genes involved in the acid tolerance, bile salt tolerance, heat stress tolerance, cold stress tolerance, adhesion, antioxidation, and organic acid synthesis, with no virulence or antibiotic resistance genes detected.Conclusion L. reuteri CHF7-2 is a potential probiotic strain and a promising candidate for use as a microecological feed additive.

Abstract:Objective To evaluate the colonization ability and biocontrol effects of Streptomyces sp. ZH-356 with antagonistic effects on plant pathogenic fungi and reveal the biocontrol mechanism of Streptomyces sp. ZH-356 by omics analysis.Methods The colonization of Streptomyces sp. ZH-356 in plants was detected by the GFP fluorescent labeling method. The biocontrol effects and potential of Streptomyces sp. ZH-356 on plant fungal diseases were evaluated based on the biocontrol effects of the inoculant in different dosage forms (seed coating agent, wettable powder, gum inoculant, and bone glue inoculant). The whole genome information of Streptomyces sp. ZH-356 was analyzed by third-generation sequencing, and its gene functions were annotated. The comparative transcriptome analysis was performed to screen the differentially expressed genes during the antagonizing process of Streptomyces sp. ZH-356 against plant pathogenic fungi, and thus the genes involved in the synthesis of antagonistic substances were predicted.Results Streptomyces sp. ZH-356 stably colonized the roots and stems of tomato and wheat plants. Different dosage forms of inoculants prepared based on Streptomyces sp. ZH-356 demonstrated strong control effects on tomato early blight and apple valsa canker. Among them, the seed coating agent prepared with Streptomyces sp. ZH-356 did not affect the germination rate of tomato seeds after treatment while protecting tomato seedlings from the infection of Alternaria solani. The wettable powder prepared with Streptomyces sp. ZH-356 showed both prevention and treatment effects on tomato early blight, with the prevention effect stronger than the treatment effect. The liquid inoculants prepared from Streptomyces sp. ZH-356 had control effects on apple Valsa canker, regardless of whether the diseased bark was scraped or not, while the control effect was better when the diseased bark was scraped and better than that of thiophanate-methyl. The whole genome sequencing results showed that Streptomyces sp. ZH-356 contained only one linear chromosome with a size of 9 435 898 bp and the average G+C content of 70.82%. A total of 8 432 coding genes, 69 tRNA genes, and 18 rRNA genes were predicted. Species annotation results showed that Streptomyces sp. ZH-356 did not belong to any Streptomyces species whose genome has been sequenced. Genome-wide analysis showed that there were 32 biosynthetic gene clusters (BGCs) for secondary metabolites in ZH-356. Transcriptomic analysis showed that the expression of the NRPS/T1PKS gene cluster ZH_356_GM000343-ZH_356_GM000422 was significantly up-regulated in the process of antagonizing plant pathogenic fungi, suggesting that it may be a BGC mediating the biosynthesis of active substances against plant pathogenic fungi in Streptomyces sp. ZH-356. Moreover, ZH_356_GM000409 may be the core biosynthetic gene of the active substances.Conclusion Streptomyces sp. ZH-356 can colonize plants, and the biocontrol agents prepared based on this strain demonstrate good control effects on plant fungal diseases. The active substances for the antagonistic effects may be synthesized by the gene cluster ZH_356_GM000343-ZH_356_GM000422. The above work lays a foundation for the industrial application of strain ZH-356 and the research on the mechanism of antagonizing plant pathogenic fungi.

Abstract:Objective Biofilm formation and adhesion are important indicators for evaluating the beneficial effects of probiotics. However, the relationship of specific genes with the biofilm formation and adhesion of Lactiplantibacillus remains unclear. The rbk gene encodes ribokinase, which is involved in ribose metabolism and may be related to biofilm formation and adhesion. This study aims to analyze the effects of rbk overexpression on the biofilm formation and adhesion of Lactiplantibacillus paraplantrum LR-1, explore the role of this gene in the regulation of quorum sensing (QS) and expression of related genes, and reveal the influencing mechanism of rbk overexpression in bacteria from a metabolic profile perspective.Methods L. paraplantarum LR-1 was selected as the target strain, and the shuttle vector pMG76e was used to construct the recombinant strain rbk-pMG76e-LR-1. The overexpression of rbk was confirmed by qRT-PCR and the enzyme activity assay. Crystal violet staining, cell adhesion assay, and qRT-PCR were employed to evaluate the effects of rbk overexpression on biofilm formation, adhesion, and expression of tuf, luxS, and rpoN. Furthermore, untargeted metabolomics analysis was conducted to assess the effect of rbk overexpression on the metabolic profile. Finally, the effect on the biofilm formation and adhesion of LR-1 was verified by exogenous addition of metabolites.Results The overexpression of rbk increased the biofilm formation of LR-1 and the adhesion to HT-29 cells by 1.55-2.34 folds and 3.58 folds, respectively. Moreover, the overexpression of rbk up-regulated the expression levels of tuf, luxS, and rpoN by 70.30, 96.94, and 45.61 folds, respectively. The untargeted metabolomics analysis revealed that rbk overexpression led to changes in the abundance of 145 metabolites. Finally, the exogenous addition of l-proline, rhamnose, and nicotinamide adenine dinucleotide (NADH) increased the biofilm formation of LR-1 by 1.27, 1.39, and 1.25 folds and the adhesion by 1.40, 1.41, and 1.52 folds, respectively.Conclusion This study demonstrates that rbk can serve as a key target for enhancing the biofilm formation and adhesion of Lactiplantibacillus.

Abstract:Zearalenone (ZEN) is a secondary metabolite produced by the filamentous fungi of Fusarium, causing serious harm to human and animal health.Objective To identify a ZEN-degrading strain and investigate its growth and degradation characteristics under different conditions.Methods A bacterial strain that can efficiently degrade ZEN was screened out with ZEN as the only carbon source from soil samples of a field with continuous maize cropping in Anhui Province, China. The strain was identified by morphological observation, biochemical tests, and phylogenetic analysis based on 16S rRNA gene sequences. The effects of temperature, pH, and incubation time on the growth rate and ZEN degradation efficiency of the strain were studied. Furthermore, the efficiency of different active components of the strain on ZEN degradation was measured, and the locations of the active components were determined.Results A total of 21 ZEN-degrading strains were isolated from the soil samples, among which strain DC-R2 showed the strongest degradation effect and it was identified as Gordonia sp. BHI medium was the optimal medium for the growth of this strain. The optimal culture conditions of the strain for ZEN degradation were 37 ℃ and pH 8.0, under which 100% ZEN (5 μg/mL) was degraded within 6 h. Intracellular enzymes were the main contributors in DC-R2 to ZEN degradation.Conclusion We isolated a strain Gordonia sp. DC-R2 capable of efficiently degrading ZEN. The intracellular enzyme present in this strain is the key to the degradation of ZEN. This provides a foundation for the purification of the key degrading enzyme in subsequent work and the potential application.

Abstract:Objective Mumps virus (MuV) is the causative agent of mumps. Nowadays, genotype F is widely prevalent in China, while genotype G appears in localized areas and is exhibiting a trend of gradual expansion. To understand the genetic characteristics of genotype G strains in China, we selected two genotype G MuV strains that were isolated from Dalian, Liaoning for analysis.Methods The whole genomes of the two strains were sequenced, and the genotypes were determined according to the WHO reference strains. Furthermore, we compared the molecular characteristics among different genotypes and within genotype G. By comparison with the sequences of genotype G strains in other areas of China, we analyzed the features of genotype G MuVs in China, as well as the genetic distance and variations of key antigenic sites between the wild-type genotype G strains and the existing vaccine strains.Results The strains isolated in this study both belonged to genotype G, and they showed the nucleotide differences ranging from 4.2% to 6.9% from other 12 genotypes, with the greatest divergence from genotype A. Among the protein coding genes, the SH coding gene exhibited the largest variation, while the NP, M, and L coding genes were conserved. The P, F and HN coding genes demonstrated significant differences among different genotypes. The genotype G strains isolated in this study were closely related to the strain isolated in Jiangsu Province (Jiangsu.CHN/22.13/2), while they were distinct from the strains previously isolated in Liaoning Province. The genotype G strains isolated in this study lacked a N-glycosylation site (aa 12-14) but gained a N-glycosylation site (aa 464-466) in the key epitope of HN protein. In addition, the genotype G strains showed considerable differences in terms of neutralizing epitopes from the genotype A vaccine strain. These differences suggested that the mutations of these sites may potentially reduce the cross-protection effects of vaccine strains against wild-type MuV strains. Although there were some mutations in F protein, the functional region was conserved.Conclusion This study details the genotypic characteristics of genotype G MuVs in Dalian, highlighting their high similarities to the genotype G strains in China and the WHO reference strains, while underscoring significant differences from the genotype A vaccine strain used worldwide. These findings suggest the necessity of continuous surveillance of MuV strains in China and further studies of their epidemiology and virology, which could provide references for tracing MuVs, cutting the transmission route, and developing immunization strategies in China.

Abstract:Objective To explore the effects of freeze-thaw on oat silage during the aerobic exposure stage.Methods Oat silage was stored at a constant temperature (20 ℃, group 20, control) and freeze-thaw conditions (20 ℃ and -5 ℃ alternating every 12 h, group S) for 60 days and then subjected to aerobic exposure. Samples were collected on day 60 of ensiling and after aerobic exposure for 1, 3, and 5 days, respectively, for the determination of fermentation quality and nutritional quality as well as for 16S rRNA gene and ITS sequencing.Results The pH rose as the aerobic exposure was prolonged. The samples subjected to aerobic exposure at room temperature showed more rapid increases in ammonia nitrogen content and pH and more rapid decreases in lactic acid and acetic acid content than those subjected to aerobic exposure at freeze-thaw temperatures. Propionic acid and butyric acid were produced more rapidly in the S group, especially in the case of aerobic exposure at room temperature. Enterobacteria and yeast increased while lactic acid bacteria decreased as the aerobic exposure was prolonged (P<0.05). Shannon and Simpson indices of bacteria increased during aerobic exposure, and the relative abundance of lactic acid bacteria kept decreasing. The bacterial community structure presented a significant difference on days 3 of aerobic exposure, and microbial succession was accelerated by aerobic exposure at room temperature compared with that at the freeze-thaw temperature, with more species and higher counts of yeast and molds indicative of spoilage in the S group.Conclusion Freeze-thaw accelerated aerobic deterioration, especially aerobic exposure at room temperature. This study provides theoretical guidance for high-quality modulation and storage of oat silage in alpine areas.

Abstract:Objective To establish a dual detection method for contaminations by six foodborne pathogens (Cronobacter, Escherichia coli O157:H7, Bacillus cereus, Staphylococcus aureus, Salmonella, and Listeria monocytogenes) in formula milk powder in a rapid manner.Methods Enzymatic recombinase amplification (ERA) is a novel isothermal amplification technology that exponentially amplifies trace amounts of DNA or RNA in 10-30 min at 25-42 ℃. The primers and probe of ERA for the detection of Cronobacter were designed. Meanwhile, the ERA primers and probes suitable for the detection of E. coli O157:H7, B. cereus, S. aureus, Salmonella, and L. monocytogenes were screened. Further, through pairwise combination and cross-reactivity analysis, as well as method optimization, the dual ERA detection system was established. The limit of detection and accuracy of the method were determined by application of this method in the detection of simulated contaminations and actual samples.Results Three groups of dual ERA systems were established, achieving the detection of six pathogens in 16 min 10 s. The established method showed the sensitivity of 1 ng/μL in the DNA detection of the combinations of Cronobacter with E. coli O157:H7, B. cereus, and S. aureus, while it showed the sensitivity of 10^-1 ng/μL in the DNA detection of Salmonella and L. monocytogenes. The results of the simulation contaminations showed that the limit of detection of the method was 1 CFU/mL. The dual ERA method established in this study was then adopted to detect 37 commercially available formula milk powder samples near the expiration date. The detection rates of B. cereus and L. monocytogenes were 37.84% and 21.62%, respectively. The results were consistent with those of the real-time PCR (industry standard method), confirming the accuracy of the dual ERA method established in this study.Conclusion The dual ERA method established in this study exhibits high specificity and high sensitivity. Moreover, it takes merely approximately 25 min from DNA extraction to obtaining the detection results, and it is capable of simultaneously detecting six pathogens, demonstrating high efficiency. This method is of importance for the rapid screening of foodborne pathogens.