Abstract:

Abstract:[Objective] To explore the effects of compound probiotics on demyelination of the mice induced by bis-cyclohexanone-oxalyldihydrazone (cuprizone, CPZ).[Methods] Twenty-seven mice were randomly assigned into normal (NC) group, model (CPZ) group, and probiotics group. The mice in the NC group were fed with normal diet, and those in the CPZ group and probiotics group with the diet containing 0.2% CPZ. The mice in the CPZ group and probiotics group were fed with 0.2 mL normal saline and 0.2 mL compound probiotics (7.5 billion colony-forming units) per day, respectively. All the mice were sacrificed after 6 weeks. Luxol fast blue (LFB) staining was employed to observe myelin sheath in the cerebrum. The histopathological change and the expression of mucin and tight junction proteins (ZO-1 and occludin) in ileum and colon were detected by hematoxylin-eosin (HE) staining, Alcian blue staining, and immunofluorescence staining, respectively. The content of lipopolysaccharide (LPS) in brain homogenate was detected by ELISA. The expression of proteins involved in the Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB) signaling pathway was detected by Western blotting.[Results] Compared with the CPZ group, compound probiotics significantly alleviated the demyelination in corpus callosum. In addition, the mice in the probiotics group showed neat and dense ileum villi, increased colon glands, decreased infiltration of inflammatory cells, and up-regulated expression of mucin and tight junction proteins, declined LPS level, and down-regulated expression of TLR4, NF-κB, and P-IκB/IκB in the brain.[Conclusion] Compound probiotics may alleviate CPZ-induced demyelination in mice by reducing intestinal wall permeability.

Abstract:[Objective] To study the effects of Nostoc sphaeroids Kütz polysaccharide (NSKP) on blood lipid and gut microflora in high fat diet mice.[Methods] The healthy 8-week-old male mice were assigned into 5 groups, with 10 mice in each group. The normal group included the C57/6CNC mice administrated with normal saline by gavaged with standard diet, and the control group consisted of ApoE^-/- mice administrated with normal saline by gavaged with standard diet. The model group consisted of the ApoE^-/- mice administrated with normal saline by gavage and fed with high-fat and high-cholesterol diet. The ApoE^-/- mice in the low-dose and high-dose NSKP groups were fed with high-fat and high-cholesterol diet and received 0.4 g/kg and 0.8 g/kg BW NSKP, respectively. The experiment lasted for 22 weeks, and then the serum, liver, colon tissue, and cecal contents samples were collected from the mice. Serum lipid level was measured by the biochemical analyzer. The tissue structure and fat deposition were observed via HE staining and oil red O staining, respectively. The diversity and composition of gut microflora in cecal contents were analyzed by 16S rRNA gene high-throughput sequencing and bioinformatics tools.[Results] NSKP reduced the serum lipid level and fat deposition in liver tissue, and improved the diversity and relative abundance of gut microflora in high-fat diet mice.[Conclusion] NSKP can improve lipid metabolism and regulate gut microecological balance in high-fat diet mice.

Abstract:[Objective] To compare the allergic effects of Lactobacillus acidophilus La28 and L. plantoplantum LP45 against allergic dermatitis and allergic asthma in mice, and to analyze the strain-specific differences in related immune regulatory effects.[Methods] In order to study atopic dermatitis, we randomly assigned 40 mice into control group, model group, La28 group, and LP45 group. Except those in the control group, the mice in other three groups were treated with 2,4-dinitrofluorobenzene to induce ear swelling and dermatitis. The mice in La28 group and LP45 group received 5×10⁸ CFU/mL corresponding bacteria by intragastric administration every day for three weeks. We then measured the ear swelling rate and skin lesion score in each group, observed the pathological changes of skin, and determined the levels of interleukin-4 (IL-4) and interleukin-13 (IL-13) in the skin. To study allergic asthma, we treated mice in the other three groups except the control group with ovalbumin and aluminum hydroxide adjuvant to induce asthma. The mice in La28 group and LP45 group were fed with 5×10⁸ CFU/mL corresponding bacteria every day for four weeks. We then evaluated the asthma symptoms of mice in each group, examined the pathological changes in the lung tissue, and determined the levels of IL-4, IL-13, and interleukin-17 (IL-17) in the lung tissue.[Results] La28 and LP45 significantly relieved ear swelling and skin damage, significantly reduced IL-4 secretion, and slightly lowered the IL-13 level in the mice with allergic dermatitis. La28 and LP45 alleviated asthma symptoms. LP45 mitigated the pathological injury of the lung tissue, reduced inflammatory cell infiltration, and significantly down-regulated the levels of IL-4, IL-13, and IL-17 in the lung tissue. La28 significantly reduced the IL-13 secretion while insignificantly lowered the levels of IL-4 and IL-17.[Conclusion] The two Lactobacillus strains, especially LP45, can regulate the immune function and repair the damaged tissues to relieve atopic dermatitis and asthma in mice.

Abstract:Probiotics can improve the microbiota of human oral cavity, gastrointestinal tract, skin, and vagina and reach the targets via delivery systems for preventive and therapeutic purposes. Hydrogel is currently accepted as the most common carrier of delivery systems. The recent studies aiming at improving hydrogel materials mainly focus on matrix structure, bulking agents, and external coating. The novel hydrogel contributes to the adaptation of probiotics to the processing and storage environment, as well as human body microenvironment. Probiotics have varying requirements for delivery carriers in different human microenvironments. The specific characteristics of the oral microenvironment pose a new challenge to the colonization of probiotics, to which hydrogel may be a solution. The application of hydrogel will expedite the course of clinical research and pharmaceutical development of oral probiotics.

Abstract:[Objective] Fermented dairy are favored for their excellent nutrition and health functions concomitantly. Post-acidification is caused by the continuous metabolism of active lactic acid bacteria during the process of storage, transportation and sales, which seriously affects its sensory quality and the number of viable strains. This study screened the low-post-acidification Lactobacillus helveticus to resolve the post-acidification of its fermented dairy.[Methods] In this study, Lactobacillus helveticus L551 was used as the original strain. Using neomycin sulfate as the selection pressure select the weakly acidified Lactobacillus helveticus. Comparing the difference of fermentation characteristics between the weakly acidified strain and the original strain.[Results] The acid production rate of weakly acidified Lactobacillus helveticus L551-1 was slowed down. The acid production rate of 7-8 h at 42℃ was 9 °T/h, which was 53% lower than the original strain (19 °T/h); the acidity of fermentation at 42℃ for 24 h was 155 °T, which was 40% lower than that of the original strain (259 °T); the post-acidification of fermented milk was 122 °T stored at 4℃ for 21 days, which was 23% lower than the original strain (158 °T). Using gas chromatography-mass spectrometry (GC-MS) compared the composition and type of volatile flavor compounds in fermented milk of the two strains, and there was no significant difference.[Conclusion] Through neomycin sulfate, the weakly post-acidified Lactobacillus helveticus L551-1 was obtained, which can significantly improve the post-acidification problem of fermented milk, and have a great significance to the development and application of direct starter.

Abstract:The human gastrointestinal tract harbors trillions of microorganisms which affect the host health. Probiotics, as a group of live microorganisms, play a role in maintaining the gut mucosal barrier function, regulating immune function, and promoting the metabolism and absorption of nutrients. Probiotics have demonstrated good performance in the treatment of gut microbiome disorders and gastrointestinal diseases such as functional dyspepsia, gastroenteritis, diarrhea, constipation, colic, irritable bowel syndrome, inflammatory bowel disease, and Helicobacter pylori infection. This article briefly overviewed the effects of probiotics on gastrointestinal health, aiming to provide support for consumers and professionals to understand their role in gastrointestinal health and use probiotics in daily health management.

Abstract:The incidence of food allergy (FA) has been rising in the past two decades, which has seriously affected the life quality of patients. Immunological tolerance defect owing to low exposure to exogenous antigens in the early life stage is the main cause of FA. FA is closely related to multipartite interactions between host and microbiome. A healthy microbiome promotes the establishment of a mature immune system in the early life of the host and reduces the susceptibility to FA. Probiotics, prebiotics, and postbiotics have been reported to alleviate the symptoms and reduce the incidence of FA by improving the microbiome and regulating body immunity, which is of great importance in the treatment of FA. In this review, we summarized the therapeutic effects and mechanisms of probiotics, prebiotics, and postbiotics on FA and the results from different studies. Furthermore, we discussed their efficacy and safety in the prevention or treatment of FA and prospected their application.

Abstract:Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria. Bacteriocin production has been considered an important trait in the selection of probiotic strains. Verified safe, they inhibit many food spoilage bacteria and pathogens. Besides, they facilitate the colonization of probiotics and regulate gut microbiota. In this article, we described the types of bacteriocins produced by probiotics, and the synthesis under specific conditions, functions, and mechanisms of them, hoping to enhance the understanding of functions and mechanisms of probiotics and lay a theoretical basis for the development of beneficial bacteriocins and probiotic products.

Abstract:[Objective] To investigate the effect of synbiotic containing Bacillus coagulans and lactulose on clinical symptoms, intestinal morphology and microbiota in mice with dextran sodium sulfate (DSS)-induced ulcerative colitis.[Methods] A total of 24 7-week-old male C57/BL6 mice with initial body weight of (22.96±1.87) g were randomized into 4 groups (6 in each):control group (CON), DSS group (2.5% DSS in water for 5 days), SYN-P group (gastric gavage of oral synbiotic once a day for 7 days before DSS administration), and SYN-T group (gastric gavage of oral synbiotic once a day for 7 days after DSS administration). After 7-day pre-feeding period and 19-day experimental period, the mice were euthanized and samples were collected.[Results] On the 10th day in the experimental period, except CON group, mice began to lose weight, among which DSS group had the fastest weight loss and the highest disease activity index (DAI). After DSS treatment was terminated (14th day), the body weight of SYN-P and SYN-T groups gradually returned to normal level, while that of DSS group did not. Compared with CON group, DSS group showed decrease in colon length (P<0.05) and increase in liver and spleen indexes (liver or spleen weight/body weight) (P<0.05), while SYN-T group demonstrated rise of liver index (P<0.05). As for the histomorphology, the height of colon villi in CON group was larger than that in DSS and SYN-P groups (P<0.05), and colonic crypt depth in DSS group was larger than that in the other three groups (P<0.05). The villus height-to-crypt depth ratio in DSS group was significantly smaller than that in SYN-P and SYN-T groups (P<0.05). As to the caecal microflora, the diversity indexes (ACE, Chao1, Shannon, and Anova) of microflora changed (P<0.05). At the phylum level, the relative abundance of Desulfobacterota and Deferribacteres in SYN-P group decreased (P<0.05) and that of Deferribacteres in SYN-T group reduced (P<0.05) compared with that in DSS group. At the genus level, the relative abundance of 13 dominant genera such as Enterococcus changed. PICRUSt analysis showed that the KEGG pathway of carbohydrate metabolism was up-regulated in SYN-P and SYN-T groups compared with that in CON and DSS groups (P<0.05). In addition, 11 pathways at KEGG level 3, including fructose and mannose metabolism, were affected.[Conclusion] Synbiotic containing B. coagulans and lactulose can relieve clinical symptoms in mice with DSS-induced ulcerative colitis, improve the intestinal morphology, and reduce the relative abundance of Desulfobacterota and Deferribacteres that caused inflammatory response in the intestinal tract through prevention and treatment.

Abstract:The skin, the largest organ of the human body, is the first defense line of the immune system. Aging of the skin may lead to dysfunction and weakened protection for our body, causing a series of health problems. Lights, especially the ultraviolet, may accelerate skin aging and destroy our skin barrier. Researchers have demonstrated that ultraviolet can directly damage the DNA and RNA in the skin cells and induce the generation of reactive oxygen species, leading to lipid peroxidation, extra-cellular matrix degradation, and inflammation, all of which ultimately accelerate skin photoaging. Growing evidence has proved the importance of microbiome to skin health, and probiotics may modulate the skin ecology and postpone skin aging through generating anti-oxidative metabolites, inhibiting the activities of matrix metalloproteinases, and reducing inflammatory cytokines. In this review, we summarized the mechanisms of skin photoaging as well as the cutting-edge studies of probiotics against skin photoaging and discussed the potential application of probiotics, aiming to provide a reference for the application of probiotics in the alleviation of skin photoaging.

Abstract:Lactococcus lactis has a variety of probiotic effects and is often used as a host strain for genetic engineering. It is generally recognized as safe (GRAS) by the Food and Drug Administration (FDA). In the past two decades, L. lactis has been widely used as a carrier in presenting viruses and bacterial antigens, playing an important role in different fields. In this review, we introduced the nisin-controlled expression (NICE) system from the aspects of composition, functional characteristics, and the application in food, vaccines, animal husbandry, and veterinary medicine. We summarized the features and improvements and then predicted the development prospects of NICE system.

Abstract:There are a large number of transcriptional regulator families in bacterial genomes, which play a crucial role in the growth, metabolism, and external signal perception and transmission of bacteria. Deoxyribonucleoside operon repressor (DeoR) family, a category of transcriptional regulators ubiquitous in prokaryotes, is mainly involved in the regulation of multiple physiological processes, including nucleotide metabolism, sugar metabolism, pathogenesis, and secondary metabolism. The ligand-binding domain at the C-terminus of DeoR usually responds to phosphorylated intermediate of the corresponding metabolic pathway. This paper reviewed the structural features, regulatory function, and ligands of DeoR family transcriptional regulators in bacteria, aiming to help researchers to decipher the regulatory mechanisms of DeoR proteins.

Abstract:In the field of wastewater treatment, algae-bacteria/fungi symbiosis can efficiently remove nitrogen and phosphorus, reduce greenhouse gas emissions, and recycle biomass after sewage treatment. Thus, researchers have shown increasing interests in the mechanisms and applications of algae-bacteria/fungi symbiosis in sewage treatment. There are few articles summarizing the interaction mechanisms of different algae-bacteria/fungi symbiotic systems in sewage treatment. In this review, we introduced the research progress on algae-bacteria/fungi symbiosis, especially the interaction mechanisms and effects, in sewage treatment from the following three aspects:algae-bacteria, algae-fungi, and mixed algae-mixed bacteria. Nutrient exchange is the basis of algae-bacteria/fungi symbiosis in sewage treatment. The molecules involved in signal transduction, such as quorum sensing molecules, can change the behavior and growth of algae or bacteria by activating gene expression or physiological activities. Fungal-assisted bio-flocculation has an immobilization effect on algae. Finally, we proposed the future research directions from the perspectives of mechanism, large-scale application, and biomass recycle.

Abstract:Sulfur, an essential component of all living cells, is ubiquitous in the nature. Sulfur metabolic pathways in microorganisms, animals, and plants demonstrate various relationships. Based on microbial sulfur metabolism, this paper summarized four main metabolic pathways of sulfur in microorganisms, animals, and plants, and highlighted the similarities, differences, and relationships. Microorganisms, the major driving force of biological sulfur cycle, participate in all the main metabolic pathways of sulfur. The dissimilatory sulfur reduction by microorganisms reduces the volatilization of methane in the environment. The assimilatory sulfur reduction by microorganisms or plants yields abundant organic sulfur sources for animals, while plants and animals lack the function of dissimilatory or assimilatory sulfur reduction. Sulfur oxidation occurs in all the three kinds of organisms with similar pathways, in which sulfur transferase diversifies the oxidation products. Sulfur mineralization in plants is still unclear, but the mineralization in animals or microorganisms enables additional inorganic sulfur substrate for sulfur assimilation in plants. In the nature, the sulfur metabolism-based ecological relationships, such as the relationships between intestinal microorganisms and host animals and between rhizosphere microorganisms and plant roots, microbial mineralization of decayed animals and plants, and microbial oxidation and reduction of sulfur, significantly enhance the biogeochemical cycling of sulfur.

Abstract:Molecular typing has been an important technique to monitor the outbreak of bacterial infections and the transmission routes of bacterial pathogens. Improvements in the next-generation sequencing technologies are facilitating rapid and cost-effective molecular diagnosis and genotyping in identification, characterization, and source tracking of bacterial pathogens. With the advancement of microbial whole genome sequencing techniques, a large volume of bacterial genome data have been produced and deposited in public databases, which necessitate the need of a variety of bioinformatics tools to analyze and interpret these data. This review provided an overview of the whole genome sequencing-powered typing and source tracking of bacterial pathogens by various cutting-edge bioinformatics tools. We also discussed the bottleneck in the deployment of this technology in clinical practice and the future application prospects in bacterial infectious disease management.

Abstract:[Objective] The aim of this study was to screen keratinase-producing strains from marine environment, investigate fermentation conditions and enzymatic properties, so as to provide strain resources and theoretical basis for the subsequent development and utilization of marine microbes to degrade feather waste.[Methods] The sludge from a marine duck farm in Beibu Gulf, Guangxi was used for bacterial isolation, and the strain with efficiently feather degradation ability was obtained by casein plate preliminary screening and keratinase activity re-screening. The strain was identified by morphology and molecular biology, and the enzyme-producing conditions were optimized by single-factor and orthogonal experiment. Finally, amino acid composition of feather degradation products and enzymatic properties were also studied.[Results] A strain of efficiently degrading feather was selected and identified as Pseudomonas aeruginosa Gxun-7. The optimum enzyme-producing conditions:feather 25 g/L, Zn²⁺ 0.10 g/L, initial pH 8.0, fermentation temperature 32.5℃, fermentation time 48 h, and the keratinase activity was 124.03 U/mL which was 2.3 times as high as before optimization. The analysis of enzyme properties showed that the optimum temperature was 70℃ and the optimum pH was 8.0. In the chemical reagent, mercaptoethanol could enhance enzymatic activity by 6.16 times, while phenylmethylsulfonyl fluoride (PMSF) decreased its relative activity to 15.00%. And it had excellent salt resistance (the relative activity in 20% NaCl retained 74.29%). There were 16 kinds of amino acids in the feather degradation products, including 7 kinds of essential amino acids. The total free amino acids content was as high as 2 329.80 mg/L, and the highest valine content was 575.89 mg/L.[Conclusion] P. aeruginosa Gxun-7 from marine environment had the ability to degrade feather keratin efficiently. The alkaline keratinase had excellent temperature and salt resistances, which had potential application value.

Abstract:[Objective] This study aimed to construct a novel T7 phage delivery platform for the recognition and packaging of eukaryotic expression vector harboring an anchor sequence, and to evaluate its feasibility for DNA vaccine research and development.[Methods] Anchor sequences were prepared by SOE-PCR method and inserted into the non-essential region of pcDNA3.0-EGFP to construct the recombinant eukaryotic expression plasmid. The recognition and packaging efficiency of recombinant plasmids by T7 phage was determined via fluorescence quantitative PCR method. Intact T7 phage particles carrying recombinant plasmids were then used as vehicle to deliver plasmids into dendritic cells. The EGFP gene expression was detected using a confocal microscope.[Results] Four PCR amplified-anchor sequence (AS1-4) were successfully inserted into pcDNA3.0-EGFP plasmid. The recombinant plasmid pcDNA3.0-EGFP-AS2 could be recognized and packaged by T7 phage at a package efficiency of approximately 95%. T7 phage packaging effectively prevented the nuclease degradation of recombinant plasmids. Moreover, intense EGFP expression was detected by confocal microscopy suggesting the successful phage-based delivery of plasmids into dendritic cells.[Conclusion] Our results demonstrate that eukaryotic expression plasmid harboring anchor sequences can be recognized and packaged by T7 phage, and the intact phage particles can be used as a vehicle to delivery plasmids into dendritic cells for endogenous gene expression. T7 phage mediated eukaryotic expression may provide a novel technical platform for the research and development of DNA vaccine.

Abstract:[Objective] This study aimed at investigating the roles of thioredoxin dependent reduction pathway in growth and metabolism of Clostridium acetobutylicum.[Methods] We used ClosTron technology to inactivate the thioredoxin reductase genes (trxB) of Trx-dependent reduction system in C. acetobutylicum to obtain the mutant, which was further verified by Southern blotting to test the copy number of the intron. Batch fermentation was implemented in minimum medium to analyze the growth and metabolic products of the wild type and mutant. The wild type and mutant were maintained at acidogenesis and solventogenesis phase to investigate the growth and production in the phosphorus-limited continuous fermentation. Antioxidant capacity was also studied by adding different concentrations of hydrogen peroxide.[Results] The results of resistance screening and gene sequencing showed that a thioredoxin reductase inactivated mutant named C. acetobutylicum trxB::int(29) was successfully constructed. In batch fermentation, the maximum optical density (OD₆₀₀) value of mutant and wild type was similar, reaching 6.5; but the maximum OD₆₀₀ of mutant was obtained at 36 h, 12 h later than that of wild type. Nevertheless, in the acidogenic phase of continuous fermentation, the growth of wild type and mutant had little change. OD₆₀₀ was stable at 4.6 and 4.4, respectively; glucose consumption and acid production were also similar. At the solventogenenic phase, OD₆₀₀ of the mutant was 3.5 and the butanol titer decreased to 36.1 mmol/L, lower than those of the wild type (4.0 and 48 mmol/L); but the acetone titer increased to 25.5 mmol/L compared to the wild type of 18 mmol/L. Furthermore, low concentration of hydrogen peroxide had little effect on the mutant, while high concentration caused more damage to the mutant than the wild type.[Conclusion] The inactivation of thioredoxin reductase gene was not a lethal mutation. The growth and acid production ability of the mutant were similar to those of the wild type, indicating that the inactivation of thioredoxin reductase gene did not affect the metabolic process of acetogenesis. The inactivation of trxB gene mainly affected the redox balance during solventogenic phase, resulting in the increase of acetone/butanol ratio.

Abstract:[Objective] This study aims to explore the mechanism of bacterial taxa adapting to repeated drying-wetting cycles in a paddy field via culture-dependent DNA/RNA-based 16S rRNA gene analysis, and to assess whether air-dried paddy soil could be used for the study of microbial community structure.[Methods] The fresh soil representative of rice production in China was collected from Changshu city of Jiangsu province, and three consecutive cycles of air drying and wetting were conducted (drying or wetting state was maintained for seven days in each cycle). We then analyzed the changes of the abundance and community composition of soil bacteria by using 16S rRNA gene high-throughput sequencing and real-time fluorescence quantitative PCR respectively.[Results] The bacterial abundance decreased by 300-771 times at the DNA level while only by 1.95-5.60 times at the RNA level during the wetting-drying cycles. The bacterial diversity in the air-dried soil showed no significant difference from that in the wet soil at the DNA level, while was significantly higher than that in the wet soil at the RNA level. Non-metric multidimensional scaling (NMDS) and co-occurrence network analysis showed that the community structure of soil bacteria changed significantly during the drying-wetting cycles (P<0.05). The drying-wetting cycles caused significant changes in relative abundance of key taxa, and a total of 8 numerically dominant phyla was consistently observed at both the DNA and RNA levels (P<0.05). The drying-wetting cycles significantly increased the relative abundance of Chloroflexi and Actinobacteria, while significantly decreased that of Proteobacteria and Acidobacteria. A total of 7 246 microbial genera were detected during the three wetting-drying cycles. The relative abundance of 35 and 58 bacterial genera changed significantly at the DNA and RNA levels, respectively, in response to the drying-wetting cycles. Among all the bacterial genera with significant changes in relative abundance, only 4 genera showed significant changes at both DNA and RNA levels, accounting for only 1.09% of the detected genera in this study. Specifically, the relative abundance of KD4-96 significantly increased and that of bacteriap25 significantly decreased at DNA and RNA levels; the relative abundance of Nitrososphaeraceae and SC-I-84 decreased significantly at the DNA level while increased significantly at the RNA level.[Conclusion] The three wetting-drying cycles resulted in significant decrease in the absolute abundance of bacterial taxa. The 16S rRNA transcripts were two orders of magnitude lower than the 16S rRNA genes, which implied that 16S rRNA transcripts could be originated most likely from intact living cells, and soil extracellular DNA could have likely resulted in drastic variations of 16S rRNA genes. Intriguingly, the composition of bacterial taxa remained generally unchanged during the drying-wetting cycles at both DNA and RNA levels, suggesting strong capacity of bacterial taxa to recover from desiccation. Our results indicate that soil microorganisms have strong functional plasticity of being adaptive to drought, and air-dried soil can be used for microbiological research under certain circumstances.

Abstract:[Objective] To construct the ygeG deletion strain of avian pathogenic Escherichia coli (APEC), and analyze its biological characteristics and pathogenicity to explore the role of ygeG plays on the pathogenesis of APEC, so as to lay a foundation for further research on the pathogenesis of type three secretion system 2 (ETT2), which ygeG locates in.[Methods] The ygeG deletion strain (APEC81-ΔygeG) and complementary strain (APEC81-CΔygeG) were constructed by Red homologous recombination technology. Then, the growth curve, motility, biofilm formation, stress resistance, serum resistance among APEC81, APEC81-ΔygeG and APEC81-CΔygeG were compared and analyzed. The effect on host infection of ygeG was investigated by cell adhesion, invasion test and inflammatory factor expression level detection by fluorescence quantitative PCR.[Results] We successfully constructed APEC81-ΔygeG and APEC81-CΔygeG. Compared with APEC81, there was no significant changes in growth characteristics in APEC81-CΔygeG (P>0.05). However, biofilm formation ability of APEC81-ΔygeG significantly decreased (P<0.01), motility of APEC81-ΔygeG extremely significantly improved (P<0.001). Compared with APEC81, APEC81-ΔygeG showed lower tolerance to acid (P<0.001) and oxidative shock (P<0.001), and higer tolerance to alkali (P<0.01), osmotic pressur (P<0.01) and heat shock (P<0.01). Furthermore, serum survival experiments results showed that deletion of ygeG significantly decreased survival abilities of APEC81 in serum (P<0.01), and extremely significantly decreased at serum concentrations of 100% and 30% (P<0.001); The adhesion of APEC81-ΔygeG to epithelial cells of chicken trachea mucosa was extremely significantly decreased (P<0.001), and the invasion ability was significantly increased (P<0.01). Furthermore, qPCR results also showed that APEC81-ΔygeG significantly up-regulated the transcription level of of inflammatory factors in chicken tracheal epithelial cells (P<0.01).[Conclusion] These data indicated that ygeG plays roles in regulation of biofilm formation, motility, stress resistance, adhesion and invasion ability, and serum resistance of APEC, it also can inhibit cell inflammatory factor expression.

Abstract:[Objective] There are some two-component systems in the genome of Saccharopolyspora pogona, including a LytR regulatory factor and a PdtaR transcription resistance termination regulator, which were analyzed through genetic engineering technology.[Methods] In this study, we performed the fusion PCR and inter-genus conjugative transfer technology to construct S. pogona-∆lytR and S. pogona-PdtaR, and studied their effects on the biosynthesis of butenyl-spinosyn.[Results] We found that the production of butenyl-spinosyn in S. pogona-∆lytR was increased compared that in the wild type, while the butenyl-spinosyn production of S. pogona-PdtaR was dropt through high performance liquid chromatography (HPLC) experiments, indicating that lytR and pdtaR negetively regulating the biosynthesis of butenyl-spinosyn. In addition, we found that S. pogona-∆lytR and S. pogona-PdtaR also had certain impacts on the growth and sporulation.[Conclusion] This stydy laid a theoretical foundation for further elucidating the regulatory mechanism of butenyl-spinosyn biosynthesis.

Abstract:[Objective] To explore the synergistical effect and mechanism of the shikonin and ebselen against S. aureus.[Methods] We used spectrophotometry assay to determine the antibacterial effect of shikonin-ebselen on S. aureus, propidium iodide (PI) staining to detect the bactericidal effect of shikonin-ebselen, and the transmission electron microscope to observe the S. aureus morphological changes after the shikonin-ebselen treatment. We used the 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) staining to detect the effect of shikonin-ebselen on bacterial ROS production, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to detect the effect of shikonin-ebselen on the thioredoxin reductase (TrxR) activity. Reactive oxygen species (ROS) was eliminated by adding ROS scavenger DL-dithiothreitol (DTT), and we further detected the relevant indicators. We isolated the multidrug resistant Staphylococcus aureus (MDRS) strains, and the antibacterial effects of shikonin-ebselen against MDRS were determined by spectrophotometry.[Results] We found shikonin and ebselen both have inhibitory effects on S. aureus, and 5 μmol/L shikonin and 10 μmol/L ebselen could work synergistically against bacteria (P<0.000 1). Shikonin-ebselen can significantly inhibit the activity of TrxR (P<0.01). Meanwhile, shikonin-ebselen can significantly induce the production of ROS in S. aureus (P<0.01). DTT can rescue the S. aureus from the shikonin-ebselen treatment. In addition, shikonin-ebselen also has a significant antibacterial effect on MDRS (P<0.000 1).[Conclusion] Shikonin-ebselen can execute antibacterial effect on S. aureus through inducing the production of a large amount of ROS by co-targeting the TrxR.

Abstract:[Objective] This study aims to analyze the drug resistance and molecular types of Klebsiella pneumoniae isolated from giant panda (Ailuropoda melanoleuca), which is expected to clarify the drug resistance and prevalence of K. pneumoniae in the captive population of giant panda and to guide clinical drug use.[Methods] For the 178 K. pneumoniae strains of giant panda collected from 2018 to 2019, the drug resistance phenotypes were analyzed with the Kirby-Bauer (K-B) disk diffusion method, drug resistance genes and mobile genetic elements by Wafergen Smartchip high-throughput fluorescent quantitative PCR, and sequence types (STs) by multi-locus sequence typing (MLST).[Results] The 178 strains had the highest resistance rate to doxycycline (15.2%) and the resistance to cefotaxime, imipenem, and azithromycin was significantly higher in strains isolated in 2019 than in 2018 (P<0.05). A total of 106 drug resistance genes (106/227) and 11 mobile genetic elements (11/19) were detected. The resistance mechanisms were mainly efflux pumps (42.0%), antibiotic inactivation (41.8%), and altered target sites of action (16.2%). MLST classified the strains into 42 STs, and ST was correlated with drug resistance.[Conclusion] The resistance rate of K. pneumoniae strains from giant panda to β-lactam and macrolide antibiotics increased from 2018 to 2019. The drug resistance mechanisms of the strains were mainly efflux pumps and antibiotic inactivation. ST17, ST23 and ST4007 may be the dominant types. In the clinical treatment of giant pandas, antibiotics should be rationally used to prevent further enhancement of drug resistance.

Abstract:[Objective] Our aim was to analyze the effect of overexpressing the effector Pi16275 on the pathogenicity of Phytophthora infestans, and to clarify the subcellular localization of Pi16275. We screened the plant proteins that interacted with Pi16275 and explored their role in plant disease resistance, and finally revealed the role of Pi16275 in the process of pathogen infecting plants.[Methods] We used the Agrobacterium-mediated method to transiently overexpress Pi16275 in the epidermal cells of tobacco leaves, and then observed the subcellular location of Pi16275 under a fluorescence microscope. After inoculating the zoospores of P. infestans at the transient expression site, we measured the area of the diseased spot. The yeast nuclear cDNA library and yeast two-hybrid assay were used to mine and verify the target protein of Pi16275 in potato. In order to explore whether gene silencing affects the plant resistance to pathogens, we used virus-mediated gene silencing technology to silence the homologous gene of the target protein gene in tobacco and inoculated the tobacco leaves with zoospores of P. infestans.[Results] The transient expression of Pi16275 in tobacco leaves significantly promoted the infection of P. infestans, and Pi16275 was located in plant cell nucleus, cytoplasm, and cell membrane. Three target proteins of potato interacted with Pi16275, including 40S ribosomal protein S5 (StRPS5), mucin 2 (StMUC2), and V-type proton ATPase subunit E-like protein (StVAEL). Silencing the homologous gene of StRPS5 significantly weakened the resistance of tobacco to P. infestans.[Conclusion] Pi16275 plays a role in the infection of P. infestans in plants.

Abstract:[Objective] The study explored the differentially expressed genes of Halomonas campaniensis under salt stress and identified the differential genes associated with anabolism of ectoine.[Methods] Three experimental groups were set up:no-salt group (NS, 0 mol/L NaCl), medium-salt group (MS, 1.5 mol/L NaCl), and high-salt group (HS, 2.5 mol/L NaCl), and then H. campaniensis XH26 was respectively cultured. Illumina HiSeq was employed for transcriptome analysis, and differential genes associated with ectione metabolism were screened and then verified by qRT-PCR.[Results] The accumulation of ectione in XH26 was closely related to salinity and the maximum (419.2 mg/L) was achieved in MS (1.5 mol/L NaCl). According to the transcriptome sequencing (n=3) result, total mapped reads accounted for 87.24%-95.87% of the clean reads. A total of 748 operons (2 182 related genes) and 941 transcription start/stop sites were annotated and 456 new transcripts were predicted in addition to 385 up-regulated genes and 326 down-regulated genes (involving 245 KEGG pathways). For the differential genes between NS and MS, synthetic ectABC and lysC were up-regulated to promote the ectoine production, and associated genes gltB, gltD, davT, hisD, alh-9, betA, acnB, pckA, and gadA were up-regulated to modulate the upstream of ectoine synthesis pathway. As for the differential genes between MS and HS, ectA, acnB, pckA, gadA, and gdhA were down-regulated, thus decreasing ectoine production. qRT-PCR result supported the transcriptome sequencing result.[Conclusion] The biosynthesis of ectoine was closely related to the aspartic acid (or aspartic acid hemiacetal), the upstream amino acid metabolism networks (e.g., asparagine, glutamate, glutamine, and histidine), and tricarboxylic acid cycle (succinic acid, fumaric acid, and oxaloacetic acid). These results can serve as a reference for the optimization or integration design of ectoine synthesis pathway in the future.

Abstract:[Objective] This paper aims to explore the influence of host proteasome 20S subunit beta 5 (PSMB5) on the intracellular growth and reproduction of the Gram-negative obligate intracellular parasite Rickettsia rickettsii.[Methods] We transfected Vero and THP-1 cells with PSMB5-specific small interfering RNA (siRNA) (si-PSMB5 group), non-specific siRNA (negative control group), and solution (Mock group), respectively. Then, we verified the down-regulation of PSMB5 by RT-qPCR and Western blotting assay. Subsequently, we infected the transfected cells with R. rickettsii (multiplicity of infection=0.1) and detected R. rickettsii proliferation by light microscopy, indirect immunofluorescence staining, and RT-qPCR.[Results] Compared with the negative control and Mock control, si-PSMB5 significantly down-regulated the mRNA and protein levels of PSMB5 in host cells and R. rickettsii load in si-PSMB5-transfected cells was significantly reduced after R. rickettsii infection.[Conclusion] Down-regulation of host PSMB5 gene inhibits the intracellular growth of R. rickettsii.

Abstract:[Objective] To understand the composition and characteristics of the human lung microbiota, and build a respiratory tract microbial bank which provides strains for further study, we isolated lung microbiota with culturomics.[Methods] Six clinical alveolar lavage fluid samples were collected, and blood culture bottles supplemented with different additives were used to pre-enrich the samples. The strains in the blood bottles were isolated, cultured, and preserved at different time points of the pre-enrichment. Isolated strains were identified with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry or 16S rRNA gene sequencing.[Results] A total of 101 known bacterial species, 6 potential new bacterial species, and 2 fungal species were obtained. The bacteria belonged to 45 genera, 35 families, 24 orders, 14 classes of 5 phyla. More isolates were obtained from the early stage of pre-enrichment, and there were more isolates in the anaerobic environment than at aerobic conditions. The additional supplement of sheep blood and rumen fluid during pre-enrichment were beneficial to strain isolation. The lung bacteria isolated in this experiment overlapped to a large extent with those from other parts (especially the intestine) of the human body.[Conclusion] Culturomics realizes the isolation of lung microbiota, and it is essential to continue to optimize the methods for isolating more bacteria from lungs.

Abstract:[Objective] This paper aims to evaluate the effect of Cordyceps cateniannulata on the growth and antioxidant enzyme activity of Solanum lycopersicum.[Methods] With the seed soaking method, S. lycopersicum was inoculated with C. cateniannulata. The plant height, root length, fresh and dry weight of root, and fresh and dry weight of shoot of S. lycopersicum were measured 30 and 60 days post inoculation (dpi) to assess the influence of the fungus on plant growth. Endophytic colonization of different plant parts by the tested fungus was confirmed 10, 20, 30, 60 and 90 dpi via a selective medium. Through morphological identification and DNA sequence alignment, we tested whether the endophytes isolated from S. lycopersicum were the inoculated strains. The content of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) in S. lycopersicum leaves was determined 30 dpi in a bid to evaluate the effect on antioxidant enzyme activity.[Results] C. cateniannulata colonized S. lycopersicum seedlings and promoted the plant growth. At all the five sampling time points, the colonization rate was in the order of root>stem>leaf. C. cateniannulata significantly increased the activity of defense enzymes in S. lycopersicum leaves, as the activity of POD, CAT, and SOD was respectively 52.21%, 75.31%, and 158.59% higher than that of the control and MDA content was 35.15% down from the control.[Conclusion] Through seed inoculation, C. cateniannulata colonizes in roots, stems, and leaves of S. lycopersicum, promoting the seedling growth and enhancing the antioxidant enzyme activity. Thus, this fungus has good application prospects in field.

Abstract:[Objective] In order to provide guidance for industrial production, this study investigated the diversity and correlation of metabolites among Rhizopus arrhizus strains in different ecological habitats, in different geographical locations, with different sporulation capacities, and of different taxonomic varieties.[Methods] Sixty-eight representative strains of R. arrhizus were inoculated in glutinous rice media for liquid fermentation, and the yields of secondary metabolites were determined by high-performance liquid chromatography. Pearson correlation coefficients between fermentation products were calculated. The correlation between strains and fermentation characteristics was assessed by multivariate analysis of variance and principal component analysis.[Results] A total of seven fermentation products were detected, including glucose (average yield in percentage:70.96%), ethanol (17.87%), lactic acid (6.63%), maltose (1.80%), malic acid (1.46%), fumaric acid (0.92%), and glycerin (0.36%). Maltose was metabolized independently, while the other six were divided into two groups with a negative inter-group correlation. The first group contained glucose, lactic acid, and glycerol, which were positively correlated with each other. The second group included ethanol, malic acid, and fumaric acid, also showing positive correlations. Fermentation characteristics were significantly correlated with sporulation capacities and taxonomic varieties, while slightly with ecological habitats and geographical locations. The strains not producing spores in synthetic mucor liquid medium had significantly stronger fermentation ability of than the SM spore-producing strains. In particular, strain XY01957 had the potential for industrial production since it could produce glucose at a yield as much as 138.30 g/L. The varieties arrhizus and tonkinensis had lower content of fumaric acid and malic acid while higher content of lactic acid and glycerin, whereas delemar was obviously opposite to the two varieties above with respect to all these components.[Conclusion] This study demonstrated high correlations among fermentation metabolites, sporulation capacities, and taxonomic varieties of R. arrhizus, laying a sound foundation for population genetic research. Some high-quality strains were screened out, providing solid support for the fermentation industry.

Abstract:[Objective] In this study, the effect of the mutant srp40³⁹ gene on isobutanol tolerance of Saccharomyces cerevisiae was studied.[Methods] Firstly, the wild-type SRP40 gene and the mutant srp40³⁹ gene were respectively cloned from the chromosome DNAs of the wild-type strain W303-1A and the mutant EMS39 of S. cerevisiae. Then, SRP40 and srp40³⁹ were respectively ligated to YCplac22 to construct the recombinant plasmids YCplac22-SRP40 and YCplac22-srp40³⁹. The obtained recombinant plasmids and the YCplac22 empty plasmid were respectively transformed into the wild-type strain W303-1A to construct the engineering strains W303-1A-SRP40, W303-1A-srp40³⁹, and W303-1A-control. The three engineering strains were then fermented in the complete media containing 1.0% isobutanol, 1.3% isobutanol, 8.0% ethanol, and 0.5% isoamyl alcohol, respectively. The cell density (OD₆₀₀) was measured, and the specific growth rate in 2-10 h was calculated. The three engineering strains were heated at 55℃ for 4 min and then diluted for observation of cell growth on the plate. Finally, the amino acid sequences of SRP40 and srp40³⁹ were analyzed by bioinformatics tools.[Results] In the medium without isobutanol (0% isobutanol), the three strains showed no significant difference. After fermentation for 24 h in the complete media containing 1.0% isobutanol and 1.3% isobutanol, W303-1A-srp40³⁹ showed the cell density 1.12 and 1.06 times that of W303-1A-SRP40, and 1.10 and 1.10 times that of W303-1A-control, respectively; the specific growth rate of W303-1A-srp40³⁹ was 1.07 and 1.10 times as high as that of W303-1A-SRP40, and 1.10 and 1.10 times as high as that of W303-1A-control, respectively. After fermentation for 24 h in the complete media containing 8.0% ethanol and 0.5% isoamyl alcohol, W303-1A-srp40³⁹ showed the cell density 1.12 and 1.01 times that of W303-1A-SRP40, and 1.17 and 1.07 times that of W303-1A-control, respectively; W303-1A-srp40³⁹ showed the specific growth rate 1.37 and 1.07 times as high as that of W303-1A-SRP40, and 1.31 and 1.09 times as high as that of W303-1A-control, respectively. Moreover, W303-1A-srp40³⁹ still grew better than the other two strains after heat shock. The bioinformatics analysis showed that Srp40³⁹ protein had a similar structure to the pleuralin-1 of diatom.[Conclusion] We found that the srp40³⁹ mutant gene can enhance the tolerance of S. cerevisiae to isobutanol, and plays a role in enhancing the tolerance to ethanol, isoamyl alcohol, and heat. Srp40³⁹ protein has a similar structure with the pleuralin-1 of diatom, which indicates that Srp40³⁹ protein may have the function of pleuralin-1 and play a role in cell wall maintenance. These findings will provide new ideas for improving the tolerance of S. cerevisiae to ethanol, isobutanol, isoamyl alcohol, and high temperature.