Abstract:[Background] Investigations of coral-bacterial and Symbiodiniaceae-bacterial interactions are crucial for unravelling the mechanisms of coral health maintaining. Labeling coral-associated bacteria with stable fluorescence is useful for visualization of interactions between bacteria and Symbiodiniaceae or coral host in real time. Presently, the lack of the genetic manipulation system of coral-associated bacteria hampers the research on coral-bacterial and Symbiodiniaceae-bacterial interactions. [Objective] We tried to establish a genetic manipulation system that is suitable for obligate marine bacteria, and then label a coral-associated bacterial strain with an enhanced green fluorescent protein gene (eGFP). The interaction between eGFP-labeled strain and Symbiodiniaceae can be visualized. [Methods] The recombinant plasmid was transferred to the donor strain (Escherichia coli WM3064) by electroporation. The donor strain was mixed with the recipient strain SCSIO 12696 (Porticoccaceae, isolated from the tissue of coral Pocillopora damicornis), growth of which requires seawater, at different cell amount ratios (4:1, 2:1, 1:1). The mixed cells were then cocultured on the modified LB medium at 25 °C and 30 °C. The interaction between Symbiodiniaceae and the eGFP-labeled strain was observed by using microscopy. [Results] The modified LB medium met the growth requirement of the obligate marine bacterium SCSIO 12696, and was preferably used in the conjugational transfer experiment. The efficiency of conjugational transfer was influenced by the ratio of the donor and recipient bacterial cells and the incubation temperature. The optimum incubation temperature was 30 °C, and the optimum ratio of donor to recipient bacterial cells was 1:1. Strain SCSIO 12696 was labeled with an eGFP gene by using the conjugational transfer approach established in this study. The interaction between eGFP-labeled SCSIO 12696 and Symbiodiniaceae was clearly observed by using confocal laser scanning microscopy. [Conclusion] A genetic operation system that is suitable for the obligate marine bacteria has been established. The coral-associated bacterial strains can be labeled with fluorescent proteins by using this system. Moreover, the fluorescence-labeled bacteria can be used for investigations of the Symbiodiniaceae-bacterial and coral-bacterial interactions, which is helpful to revealing the ecological function of coral-associated bacteria and its mechanism.

Abstract:[Background] Bio-desulfurization is an effective method of removing hydrogen sulfide from industrial gas, biogas, natural gas, etc. In the process of bio-desulfurization, the formation and metabolism of sulfur particles is the key steps that directly affect the efficiency of desulfurization. However, our understanding of the methods of regulating sulfur particles is still insufficient. [Objective] The effects of different surfactants on the formation and metabolism of sulfur particles by Thialkalivibrio versutus D301 were studied. [Methods] T. versutus D301 was cultured in erlenmeyer flask, and the sulfur-oxidizing bacterica and sulfur particles were characterized by X-ray diffraction (XRD), cold field emission scanning electron microscopy (SEM), energy dispersive system (EDS) and Fourier transform infrared spectroscopy (FTIR). [Results] The elemental sulfur in sulfur particle mainly exists in the form of S8. The addition of Tween-80 or SDS has a significantly effect on the morphology and production of sulfur particles. In the control experiment, the sulfur particles generated were regular spherical, smooth and intact, and biological macromolecules such as proteins cover to the surface of the sulfur particles. When 0.01 g/L Tween-80 is added, long rod-shaped and larger-sized sulfur particles are formed and the metabolic rate of elemental sulfur slows down. When 0.3 g/L SDS is added, the sulfur particles are in the form of short rods. Less sulfur particles are formed, and they are rapidly metabolized by T. versutus D301. [Conclusion] Regulating the morphology and metabolic rate of sulfur particles can be achieved by adding specific surfactants during bacterial cultivation.

Abstract:[Background] Biocathode microbial fuel cell is a promising wastewater treatment system due to its low construction cost and sustainable development of the cathode. However, the oxidation-reduction properties of cathode microorganisms limit its further development in practical applications. [Objective] In order to improve the performance of the biocathode, it is necessary to understand the microbial community that affects the redox performance of the cathode. [Methods] The 16S rRNA gene high-throughput sequencing technology was used to analyze and compare the microbial diversity and structural changes of the original inoculated sludge samples and the domesticated cathode electrode biofilm samples. [Results] The sequencing results showed that there were significant differences in the types and structures of microbial communities in the original inoculated sludge and the domesticated cathode electrode biofilm samples. The relative abundance ratios of Proteobacteria, Gammaproteobacteria and Trueperaceae in the cathodic electrode biofilm samples after domestication were higher than those of the original sludge samples, which became the dominant flora. [Conclusion] Domestication has a significant impact on the biofilm community of the cathode electrode of the system. With the output of electricity production, the dominant bacterial community is continuously enriched, and finally a new microbial community adapted to the experimental environment is formed. Discussing the structure and changes of the dominant flora, which provide more biological theoretical foundations for the research of biocathode.

Abstract:[Background] Zealenone (ZEN) is a mycotoxin with estrogen-like effect, which often contaminates grains and feed, and threatens the health of animals and humans seriously. As an ideal method to remove ZEN, the biological detoxification has been received with wide attentions. However, there are few related degradation strains, which need to be further screened. [Objective] To clarify the biological classification status of a ZEN-degrading bacterium and optimize the degradation conditions. [Methods] The phylogenetic tree was constructed by 16S rRNA gene sequences. Optimization of culture conditions with single factor analysis was carried out, and degradation kinetic of ZEN was analyzed. [Results] Based on 16S rRNA gene sequence similarities, the closest relative of WLB-29 was Stappia indica B106T (97.47%), which suggested WLB-29 was a potential novel species of the genus Stappia (GenBank accession number MT196321). Results of the optimization showed that the best conditions for strains to degrade ZEN were followed: LB culture medium, 37 °C, pH 8.0, 2% inoculum and initial concentration of 10 mg/L ZEN. Under the conditions, the degradation rate could reach 92.56% after cultured 144 hours. [Conclusion] The strain WLB-29 could degrade ZEN. It provides a research basis for further analyzing the mechanism of ZEN degradation by the strain, and utilization of the strain to ZEN detoxification in future.

Abstract:[Background] Plant endophytic bacteria not only inhibit the infection of pathogens on plants, but also promote plant growth. Screening the antagonistic and growth-promoting endophytic bacteria can provide a theoretical basis for the development of microbial fertilizers. [Objective] Screen superior antagonistic and growth-promoting bacteria resource from plant endophytic bacteria. [Methods] Four kinds of isolation media were used to isolate endophytic bacteria from Euphorbia humifusa, colony morphological characteristics were combined with 16S rRNA gene sequence analysis to identify the classification of endophytic bacteria. The antagonistic activity of endophytic bacteria on three pathogenic fungi, Rhizoctonia solani, Fusarium graminearum, Bipolaris maydis, was tested by plate confrontation method. Through nitrogen fixation, phosphate solubilization, production of indole acetic acid (IAA) and synthesize siderophore capacity to evaluate the growth-promoting potential of endophytic bacteria in Euphorbia humifusa. [Results] A total of 133 endophytic bacteria were isolated, which belonged to 4 phyla, 5 classes, 8 orders, 13 families, and 25 genera. Proteobacteria was dominant phylum (52.63%), Bacillus was dominant genus (15.79%). eight strains are found to be similar less than 98.65%, may be potential new species. The result of antagonistic activity showed that 22 endophytic bacteria had different antagonistic activity. Among them, strains DHL56, DHN17, DHP3 and DHP8 against to both of these three kinds of pathogenic fungi, they are different species of Bacillus genus. Strain DHP8 had the strongest antagonistic activity, with the inhibitory rates of F. graminearum, R. solani being 73.80% and 71.25% respectively, and the inhibitory rates of B. maydis being 61.70%. The results of growth-promoting experiments showed that 76 strains shave the ability of nitrogen-fixing; Nineteen strains showed phosphate solubilization ability; Thirty-seven strains showed IAA-producing activity, and strain DHL55 produced 105.67 mg/L of IAA; Seven strains had the ability to synthesize siderophore. Nine strains showed the ability of fix nitrogen, phosphorus, and produce IAA. Strain DHP8 has the ability to fix nitrogen, phosphorus, and synthesize iron. In addition, the strain DHP8 not only displays significant role in plant growth, but also has a remarkable antagonistic activity against pathogenic fungi we used. So that, need further study. [Conclusion] The species of endophytic bacteria are abundant, and many strains have excellent antagonistic and growth-promoting activities, which provides some new sources for the further studies.

Abstract:[Background] The development and utilization of cassava residues (CR) as feed resources can reduce environmental pollution and realize the local transformation of these resources. Microbial fermentation can reduce the crude fiber content of CR, improve their palatability, and increase feed conversion efficiency. [Objective] This study was conducted to screen microbial starter cultures with better fermentation effects on CR and their fermentation duration. [Methods] Three kinds of complex microbial starter cultures, including A (Bacillus, Lactobacillus, and Saccharomyces cerevisiae), B (Pediococcus pentosaceus, and Saccharomyces cerevisiae), and C (Lactobacillus plantarum, Saccharomyces cerevisiae, and Bacillus subtilis) were selected and mixed with fresh CR. Then, they were randomly divided into seven groups with five replicates per group. Samples were collected on days 0, 1, 3, 7, 14, 28, and 56 of the fermentation, respectively, to determine the nutritional composition. [Results] Compared with fresh CR, starter culture A and B increased (P<0.05) the crude ash content of CR; Starter culture C increased (P<0.05) the crude protein content of CR; The three kinds of starter cultures decreased (P<0.05) the neutral detergent fiber content of CR and increased (P<0.05) the crude fat content. [Conclusion] Microbial fermentation can effectively improve the nutritional value of CR, of which the use of starter culture C for short-term fermentation has the most significant increase in their nutritional value, especially crude protein and crude fat contents.

Abstract:[Background] Environmental factors are important regulatory factors for the growth and metabolism of microorganisms. Therefore, the analysis of their influence on the succession of microbial communities in the fermentation process plays an important role in the controlled fermentation of highland barley Baijiu. As important nutrient substrates and environmental factors in microbial growth and metabolism, the effects of amino acids on microbial succession are still unclear. [Objective] To revealing the role of environmental factors in driving microbial community succession and flavor metabolism during highland barley Baijiu fermentation. [Methods] Gas chromatography-mass spectrometry was used to compare the changes of flavors during fermentation of dulihuang and walan fermented grains. High throughput sequencing and multivariate statistical analysis were used to compare the microbial structure characteristics in two kinds of fermented grains. The effects of environmental factors on microorganisms were determined by Mantel test and correlation analysis. The results were verified by simulated fermentation in vitro. [Results] The content of esters, amino acids and the relative abundance of Lactobacillus in dulihuang fermented grains was significantly (P<0.05) lower than that in walan. Through bioinformatics analysis, it was found that the content of free amino acids drove the microbial succession during the fermentation process, and the relative abundance of Lactobacillus was significantly correlated with the content of free amino acids. In vitro validation confirmed that the lack of amino acids can affect the growth of Lactobacillus. [Conclusion] The study revealed the driving effect of free amino acids on the assembly of microbial community in the process of highland barley Baijiu, and further affected the flavor quality, laying a foundation for the controlled brewing of highland barley Baijiu.

Abstract:[Background] Kombucha is a traditional tea beverage fermented by a symbiotic consortium of bacteria and yeasts. A number of metabolites in kombucha are beneficial to human health such as promoting digestion, anti-inflammatory, antibacterial and anti-diabetes. The beneficial metabolites are mainly produced by complex microbial interactions dominated by acetic acid bacteria and yeasts. Therefore, kombucha is an excellent model for studying metabolic process in a symbiotic microbial consortium. [Objective] This study aims to study the effects of interactions among different microorganism on metabolites in kombucha by culturing microbial strains isolated from kombucha singly or in combination under different culture conditions. [Methods] Dominant microbial strains were isolated from kombucha consortium by agar plate dilution method. The isolated microbes were identified by morphological observation and molecular biology methods. Different culture combinations were designed to investigate the effects of single-culture and mixed-culture on the pH value, acidity, reducing sugar and total polyphenol contents in fermentation broth. [Results] The isolated microbial strains grew well in tea sugar medium singly or in combination, but with different growth rate. The microbial growth rate was closely related to the fermentation ability of the tested strain to produce reducing sugar. pH value of fermentation broth was significantly affected by Komagataeibacter sp. strain C6, which showed a strong ability to produce organic acids. The fermentation process by microbes in the broth significantly increased the contents of total polyphenols in the fermentation broth. Compared to the single-culture, the mixed-culture of three strains contributed to the stability of the microbial community, and significantly promoted the production of reducing sugar, organic acid and decreasing of pH value, and slight increase of polyphenols in kombucha. [Conclusion] Kombucha is a symbiotic consortium-fermented system with complex interactions between bacteria and yeasts. The multi-strains co-culturing contributes to formation of the stable microbial communities, and also promotes the metabolism of organic acids, reducing sugars and polyphenols in kombucha. The symbiotic fermentation with different microbes is the biochemical basis for the popular kombucha drinks with flavor taste and beneficial physiological activity.

Abstract:[Background] As a preservative added in bread and other foods, calcium propionate has a certain antimicrobial effect, but most of its current researches focus on biochemical and group levels. [Objective] To explore the mechanism of antimicrobial effect of calcium propionate on Saccharomyces cerevisiae at the molecular level. [Methods] Transcriptome sequencing and analysis were performed on logarithmic growth period of high-glucose resistant yeast BH1 in experimental group and control group, and real-time fluorescent quantitative PCR was performed for verification. [Results] Compared with the 6 h control group (no calcium propionate treatment; control group, CG), there were 1 438 differentially expressed genes (DEGs) in the 6 h experimental group (calcium propionate 2 h group, CP2G), of which 643 genes were up-regulated and 795 gene down-regulation. Compared with the 4 h experimental group (calcium propionate 0 h group, CP0G), there were 1 921 differentially expressed genes in CP2G, of which 1 438 genes were up-regulated and 483 genes were down-regulated. Among them, DEGs involved in multiple pathways, including the mitogen-activated protein kinase (MAPK) signal pathway, cell cycle pathway, and meiotic pathway. In addition, genes involved in cell wall synthesis also differentially express. [Conclusion] The molecular mechanism of calcium propionate’s antimicrobial effect on yeast was explored, which provided a theoretical basis for further revealing the mechanism of calcium propionate’s antimicrobial effect.

Abstract:[Background] With the development of the aquaculture industry and the improvement of intensive aquaculture, the aquaculture environment deteriorates seriously and the aquatic animal diseases take place frequently. Aquatic probiotics are widely used in aquaculture for their environmental friendliness and safety. [Objective] Bacillus subtilis was isolated from the bottom mud of Litopenaeus vannamei breeding pond. The in vitro biological characteristics and water purification effect were then explored to expand the germplasm resources of microecological preparations. [Methods] Firstly, the strains were isolated by the dilution spread plate method and its identification and classification were determined successively by morphological observation, physiological and biochemical assays, phenotypic test by microbial identification system, and 16S rRNA gene sequence analysis. Secondly, biological characteristics in vitro were studied by a single factor control method. Lastly, the contents of nitrite (NO2?-N), ammonia nitrogen (NH3-N), and chemical oxygen demand (COD) of the aquaculture water were separately measured by N-(1-naphthyl)-ethylenediamine spectrophotometry method, Nessler?s reagent method, and alkaline potassium permanganate method. [Results] The isolated strains were identified as Bacillus subtilis WH1 by the phenotypic identification and 16S rRNA gene sequence analysis. During a series of in vitro biological characteristics test studies, WH1, compared with the model bacteria, had better performances in resistance of high temperature, artificial gastric juice, and bile salts, as well as the inhibitory ability of Staphylococcus aureus and Vibrio parahaemolyticus. Especially, it was sensitive to 8 kinds of antibiotics that must be checked for Bacillus used as a feed additive according to the European Food Safety Agency rules. This also met the in vitro evaluation standard of aquatic probiotics used as feed additives. Additionally, the water purification studies showed that the strain WH1 could significantly reduce the contents of NO2?-N, NH3-N, and COD in water (P<0.05). [Conclusion] The screened Bacillus subtilis WH1 can be used as a water quality regulator and has the advantage of in vitro biological characteristics used as a feed additive, which can effectively expand the germplasm resources of microecological preparations.

Abstract:[Background] The unique development cycle of Chlamydia trachomatis is completed in inclusion. About 7%–10% genes encode membrane proteins in the inclusion. Therefore, the membrane proteins in the inclusion may play an important role in the development and pathogenesis of Chlamydia. However, its specific functions still need to be further studied. [Objective] In order to further understand the function of the membrane protein CT225 in the inclusion, the association molecules were screen. [Methods] The fusion protein GST-CT225 was first expressed. Then, the interaction molecules of CT225 were screened from the lysate of HeLa cell by affinity chromatography, and analyzed by mass spectrometry. Finally, the candidate proteins were selected and verified by Co-immunoprecipitation, glutathione S-transferase (GST) pull-down test and subcellular localization. [Results] Vimentin was the protein interacted with CT225 according to the score of mass spectrometry. [Conclusion] CT225 interacts with vimentin in HeLa cells, suggesting that CT225 has the relationship with the cytoskeleton integrity, membrane transport and lipid transport.

Abstract:[Background] Reduced susceptibility has been found in Listeria monocytogenes to antimicrobial agents such as several clinical commonly used antibiotics and nisin. However, mechanisms behind this are still unclear. [Objective] To study the role of VirAB transporter in resistance to antimicrobial agents and biofilm formation of L. monocytogenes. [Methods] Gene deletion mutants were constructed by homologous recombination, and resistance to antimicrobial agents was determined in the wild-type and mutant strains. The ability of biofilm formation was measured using the microplate method. Swimming motility of these strains was investigated by plate migration method. [Results] The mutant strain ΔvirAB showed increased sensitivity to cephalosporin, nisin and ethidium bromide compared to the wild-type strain; the mutant strain exhibited varying degrees of growth defects in the presence of sub-lethal concentrations of benzalkonium chloride, kanamycin and tetracyclines. Decreased ability of biofilm formation was observed in ΔvirAB. [Conclusion] VirAB plays an important role in resistance to antimicrobial agents and biofilm formation of L. monocytogenes.

Abstract:[Background] Isaria cicadae is a Chinese traditional medicine. A preliminary study showed that wild I. cicadae contains similar metabolites with cultured I. cicadae on Antheraea pernyi. Therefore, the cultured I. cicadae has the potential to be used as an alternative. [Objective] Reveal the metabolites difference between different parts of the cultured I. cicadae. The results will provide a theoretical basis for the rational use of the cultured I. cicadae. [Methods] HPLC-DAD-TOF/MS-based metabolomics was used to compare the metabolic difference between the insect body part, mycelia on the insect surface and coremia of I. cicadae. [Results] Different parts of cultured I. cicadae showed significantly different metabolomics. Despite that, the insect part is full of mycelia (sclerotia) which contains less small molecular metabolites than the other parts, and has only a relatively higher level of dihydroceramide and phytosphingosine. This result implied that it contained mainly insoluble macromolecular substances. The high level of phytosphingosine was possibly related to immune injury from the host insect. Lots of small molecular metabolites were detected from mycelia on the surface of the insect body implying that nutrients from the insects were preferentially absorbed by the mycelia. The coremia contain relatively few secondary metabolites. [Conclusion] Different parts of cultured I. cicadae on A. pernyi contain significantly different metabolites, and mycelia on the surface of the insect contain relatively higher level of secondary metabolites suggesting more potential of metabolites utilization.

Abstract:[Background] Injurious insect (Noctuidae) tend to develop high resistance to chemical pesticides, some of them can increase the effect of some entomogenous fungi. There is a lack of research on Nomuraea rileyi in this part currently. [Objective] To explore the physiological characteristics of UV- mutant Nomuraea rileyi mutants, which are highly resistant to dichlorvos, a common organophosphate insecticide, including mycelial growth, sporulation and the activity of chitin enzyme. [Methods] Based on the mutant strains Nr-UVY1 and Nr-UVY6, which were screened by UV-induced mutagenesis of Nomuraea rileyi, the mycelial growth inhibition rate, the subculture sporulation amount, and the chitinase activity were measured by the addition of different concentrations of dichlorvos growth medium. [Results] On the medium containing 1 291 mg/L dichlorvos, the inhibition rate of dichlorvos on the mycelial growth of the original strain reached 100%, and the mutant Nr-UVY1 (42.38%) and Nr-UVY6 (37.01%) were far away lower than the original strain. According to the mycelial growth inhibition rate curve, when the treatment concentration was higher than 1 291 mg/L, the curves of the two mutant strains increased steadily and were much lower than the original strain, indicating that the resistance of the mutant strains to dichlorvos was remarkable and stable. On the medium with different concentrations of dichlorvos, the mycelial inhibition rate of the mutant strain Nr-UVY6 was smaller than that of the mutant strain Nr-UVY1, manifesting Nr-UVY6 was more resistant to dichlorvos. On the same culture conditions, the initial sporulation time of the two mutant strains was slightly later than the original strain, but the difference was not significant. The sporulation of the mutants was significantly higher than that of the original strains in each generation. In particular, the sporulation of Nr-UVY6 was always more than 2 times higher than the original strain and more stable. The chitinase activity measured by the transparent circle method, the mutant strain Nr-UVY6 was higher than Nr-UVY1 and the starting strain. [Conclusion] The mutant strains subjected to ultraviolet mutagenesis have much higher resistance to dichlorvos than the original strains, and exhibit better traits in growth and reproduction, and it is possible to further explore their level of pathogenicity due to their high activity of chitinase enzyme.

Abstract:[Background] Intestinal microorganisms play a vital role in homeostasis of the host’s intestinal microenvironment. Many factors, such as antibiotics, diet and age, can interfere with this microbalance, causing changes in the microflora balance, which in turn affects the health of the body. There exist many pharmacological activities such as myocardial protection, immunity improvement, anti-oxidation, anti-diabetes, anti-stress, and anti-fatigue on Radix pseudostellariae. However, the regulation effect of endophytic metabolites of Radix pseudostellariae on intestinal microbes and their possible impacts on body health have not been studied so far. [Objective] The taxonomic status of endophytic bacterium RPB-32 from Radix pseudostellariae and the regulation of its metabolic extracts on the intestinal microbes of mice were studied to explore its impact on body health. [Methods] Strain RPB-32 was identified based on the traditional and molecular methods. 120 KM mice were randomly divided into four groups, viz. solvent control group (3% ethanol), the low, medium and high-dose groups of petroleum ether extract, ethyl acetate extract and n-butanol extract (n=12). After 14 days of intragastric treatments, the mice feces were collected aseptically, and the changes of intestinal microorganisms before and after intragastric gavage were detected by selective medium culture and metagenomic sequencing (solvent control (S.F.1), high-dose ethyl acetate extract group (S.F.2)). [Results] Strain RPB-32 was identified as Bacillus sp. according to the conventional and molecular methods. Compared with the blank control group, the amounts of lactic acid bacteria significantly increased in the tested groups of medium-dose ethyl acetate extracts, high-dose ethyl acetate extracts, low-dose n-butanol extracts, medium-dose n-butanol extracts and high-dose n-butanol extracts, while the amounts of Enterococcus and Enterobacter in the groups of middle-dose n-butanol extracts and high-dose n-butanol extracts were significantly reduced, respectively. The comparison of the microbial community structure showed that the amount of Firmicutes obviously reduced, but that of Bacteroidetes significantly increased in S.F.2 collated with S.F.1 at the phylum level; the amounts of Bacteroides, Prevotella, Parabacteroides, Akkermansia, Helicobacter, Butyricimonas, Alistipes, Ruminococcus, Muribaculum, Barnesiella, Odoribacter, Azospirillum, Mucispirillum, Lactobacillus obviously increased, while those of Rikenella, Angelakisella, Clostridium, Oscillibacter, Pseudoflavonifractor, Flavonifractor, Mailhella, Desulfovibrio, Faecalibacterium, Eubacterium, Lachnoclostridium, Hungatella, Butyrivibrio, Blautia, Dorea, Ruminiclostridium, Roseburia, Anaerotruncus, Butyricicoccus significantly decreased in S.F.2 contrasted with S.F.1 at the genus level; the amounts of Bacteroides sartorii, Bacteroides caecimuris, Bacteroides uniformis, Parabacteroides chinchillae, Parabacteroides glodsteinii, Helicobacter bilis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides thetaiotaomicron, Butyricimonas virosa strikingly increased, while those of Faecalibacterium prausnitzii, Rikenella microfusus, Flavonifractor plautii, Mailhella massiliensis, Clostridium clostridioforme significantly decreased in S.F.2 in comparison with S.F.1 at the species level. [Conclusion] It was preliminarily determined that the metabolites of endophytic Bacillus sp. RPB-32 (BM) from Radix pseudostellariae had the significant effects on the composition of intestinal microorganisms in mice, and might improve the degradation of glucose and lipid, anti-infective and anti-inflammatory activities in body.

Abstract:[Background] Since 2014, H5N6 avian influenza virus has continued to evolve in domestic poultry and live-poultry markets, becoming a major threat to human and animal health. [Objective] To analyze the molecular characteristic of hemagglutinin (HA) and neuraminidase (NA) proteins of highly pathogenic avian influenza H5N6 viruses from 19 human cases. [Methods] Whole-genome sequences of original specimen and virus isolates were obtained by next-generation sequencing technology. Pairwise sequence alignments and phylogenetic analysis were performed by Blast, Mega 6.1 and ClustalX softwares. [Results] In 2017–2019, 43 viruses were isolated from 189 H5 subtype poultry/environmental samples and 1 H5N6 patient throat swab samples, and 33 H5N6 viruses were sequenced. Based on the sequence of H5N6 virus collected on the internet, the HA gene of 93 H5N6 viruses was analyzed. Among 93 H5N6 strains, 78 belong to 2.3.4.4h, 9 belong to 2.3.4.4e, 4 belong to 2.3.4.4b, 1 belong to 2.3.4.4f, 1 belong to 2.3.4.4g. The cleavage sites of HA proteins of all 93 strains contain several basic amino acids, indicating that they are highly pathogenic avian influenza viruses. Q222 and G224 amino acids of HA protein of all 93 viruses did not mutate, which retained the binding characteristics of avian receptor α2-3 galactoside sialic acid (SAα2-3Gal); 158 sites lost glycosylation, and 124 sites showed a new potential glycosylation site. [Conclusion] From 2017 to 2019, the evolution of H5N6 virus infection in China is active, and there is obvious genetic diversity. It is necessary to strengthen the monitoring of the evolution of H5N6 viruses in China.

Abstract:[Background] In the past, there were few studies on the taxonomy of bifidobacteria plasmids, and the phylogenetic classification method of Bifidobacterium plasmid was missing. [Objective] In order to establish a method for phylogenetic classification and identification of natural plasmids in Bifidobacterium, and increase understanding and application of plasmids in the research of bifidobacteria biology. [Methods] We conducted to phylogenetic classification of all sequenced plasmids in Bifidobacterium by the analysis of phylogenetic relationship of replication initiation protein and genome colinearity. [Results] All known natural plasmids of Bifidobacterium were divided into six different types of plasmid families and three unique complex plasmids. Plasmids of type III and VI family were further divided into different subtype groups. Plasmids of the type III and VI family can be further divided into different subtype groups. Plasmids of the type III family were the main type and dominant family of natural plasmids in Bifidobacterium. Plasmid subtypes of type VI family were most abundantly classified. At the plasmid family level, the classification results of the two methods were completely identical. [Conclusion] These results comprehensively and accurately revealed the phylogenetic classification relationship between all the plasmids that were analyzed, and established the phylogenetic classification standard and method of natural plasmids in Bifidobacterium, which provided important theoretical references and classification basis for the future classification and identification of natural plasmids in Bifidobacterium.

Abstract:[Background] Salmonella is one of the most important food-borne pathogens. Its multidrug resistance has attracted more and more attention. [Objective] To analyze the antimicrobial-resistant characteristics and molecular subtyping of multidrug resistant (MDR) Salmonella from clinical sources in Hangzhou, the present study was performed. [Methods] A total of 339 Salmonella isolates from clinical sources were subjected to antimicrobial susceptibility testing and the minimum inhibitory concentration (MIC) was determined. The MDR isolates, which show reduced susceptibility to at least 3 of the 14 antimicrobial categories tested, were analyzed the drug-resistant characteristics, serotype distribution, and the molecular subtyping by pulsed field gel electrophoresis (PFGE). [Results] Among the 339 Salmonella isolates, 234 MDR isolates were found and the MDR rate reached 69.03%. No significant difference was observed in the MDR rate from 2017 to 2019 (χ2=0.117, P=0.943). Isolates resistant to 4?8 antimicrobial categories were most common, accounting for 56.93% (193/339) of the total number of Salmonella isolates. Most of the MDR isolates (199/234, 85.04%) showed resistance to 5?13 antimicrobial agents. The resistance patterns of the MDR isolates were quite diverse, and the relatively dominant patterns were AMP-AMS-NAL-STR-SUL (10, 4.27%) and AMP-STR-TET-MIN-DOX-SUL (7, 2.99%). The level of MDR in Salmonella Derby and monophasic Salmonella Typhimurium was prominent, which MDR rate was 100% (11/11) and 97.06% (66/68), respectively. The 234 MDR Salmonella isolates were subtyped into 162 molecular types by PFGE, with the similarity of 44.2%–100%. The PFGE profiles of the MDR isolates showed high polymorphism and no predominant PFGE profile was found, except for serotype Enteritidis and Typhimurium. For the isolates with identical PFGE profiles, their MDR patterns were either identical or different. For those with different PFGE profiles, the MDR patterns may also be identical. [Conclusion] High level of MDR was observed in Salmonella from clinical sources in Hangzhou, with the MDR patterns being quite diverse, and the PFGE profiles showed high polymorphism. The results showed that the infection of MDR Salmonella in Hangzhou was mainly sporadic. However, the genomic characteristics and the main food origin of MDR Salmonella in Hangzhou need to be further studied.

Abstract:[Background] Human rotavirus group A (RVA) is the main pathogen of gastroenteritis in infants and important cause of infant death in developing countries. There are no specific drugs until now and vaccines are the only feasible method to prevent infection. As outer capsid proteins VP7 and VP4 are the main targets of rotavirus vaccine, it is necessary to strengthen molecular epidemiological surveillance of local clinical circulating RVA strains against these genes. [Objective] To identify VP7 and VP4 genotypes and analyze their sequence characterization of RVA circulating strains in Jinzhou. [Methods] The fecal samples of infants suffered from RVA infection diarrhea in Jinzhou during 2018?2020 were collected and viral RNA were extracted. VP7 and VP4 gene fragments were amplified by RT-PCR and PCR products were sequenced. Seven RVA strains VP7 and VP4 genes were obtained. The sequencing results were analyzed by genotyping using online tool RotaC V2.0. Phylogenetic and amino acid sequence alignments analysis compared to clinical epidemic strains and vaccine strains were carried out with BLAST, DNAstar, Mega X, Bioedit. [Results] The genotyping results showed that 7 Jinzhou strains in this study was G9P[8]. Phylogenetic analysis confirmed that VP7 and VP4 belonged to lineages G9-Ⅵ and P[8]-3, and nucleotide sequence identity were 99.32%?100% and 99.41%?100%, respectively. There were 4 and 3 amino acid substitutions in the antigen epitope regions 7-1a, 7-1b, 7-2 when JZ strains VP7 were compared with vaccine strains Rotavac and Rotasiil VP7. The amino acid sequences of JZ strains VP4 were compared with vaccine strains Rotarix and RotaTeq VP4. Seven and four amino acid substitution sites located in the antigen epitope regions 8-1, 8-3 were found. [Conclusion] Seven G9P[8] RVA strains were detected in Jinzhou, Liaoning province from 2018 to 2020. The identity of VP7 and VP4 sequences was higher than 99%. G9P[8] was probably one of the main epidemic genotypes of infantile rotavirus diarrhea in Jinzhou, Liaoning province in 2018?2020. Compared to the same genotypic vaccines strains, amino acids variations located in VP7 and VP4 epitope regions of JZ strains are significant for understanding immune escape mechanisms of wild RVA strains.

Abstract:[Background] Short chain fatty acids (SCFA) produced by ruminal bacteria fermentation of carbohydrates can be used as the fuels and chemical precursors. Ethanol plays an important role in the production of caproic acid by carbon chain extension, but the effect of ethanol on the caproic acid production capacity of feed with different fibers fermented in rumen is rarely reported. [Objective] To reveal the difference in SCFA yield of ethanol on rumen fermentation of fiber feed in vitro, and to explore the potential bacteria producing C5 and C6 fatty acids. [Methods] In vitro continuous passage culture technology and Illumina HiSeq sequencing technology were used to compare the effects of ethanol on SCFA-producing capacity of six feeds, as well as the differences in bacterial community structure. [Results] The total SCFA yield of 6 kinds of fiber feed was ryegrass straw>triticale straw>oat straw>corncob>rice straw>stevia straw. The yield of valeric acid and caproic acid of triticale straw and ryegrass straw was significantly increased by adding ethanol. Firmicutes and Bacteroidetes were the dominant bacteria phylum, and ethanol significantly increased the relative abundance of Actinobacteria and Tenericutes phylum in oat straw and triticale straw group. At the species level, the relative abundance of dominant bacteria of stevia straw, rice straw and corncob was different from that of oat straw, triticale straw and ryegrass straw. Among the bacteria with the top 10 relative abundance, the relative abundance of Prevotella sp. DJF CP65, Clostridium butyricum and Bifidobacterium thermophilum showed significant positive correlation with the yield of valeric acid. C. butyricum relative abundance was significantly positively correlated with caproic acid yield. [Conclusion] More valeric acid and caproic acid can be produced by fermentation of fiber feed with ethanol in vitro. The results provide reference data for screening ruminal bacteria that can be cultured for in vitro fermentation to increase the yield of valeric acid and caproic acid, and to explore the functions of ruminal bacteria.

Abstract:Helicobacter pylori (H. pylori) infection is one of the factors that cause human gastric diseases. Outer membrane vesicles (OMVs) are vesicular structures formed by the spontaneous shedding of bacterial outer membrane, and have many components of outer membrane, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), lipids and other protein components. Increasing studies were focusing on the biological function of OMVs in the development of H. pylori infection. Meanwhile, many researches showed that H. pylori OMVs act as vaccine candidate with the potential application in the prevention and treatment of H. pylori infection. Therefore, our review summarized the research progress on the components of OMVs derived from H. pylori, and discussed the potential functions of OMVs in the survival and pathogenesis of H. pylori, as well as the role of OMVs in the treatment of H. pylori infection.

Abstract:Bacterial exopolysaccharides (EPS) are widely used in food, pharmaceutical, and chemical fields due to their unique physicochemical properties and physiological activities. Bacterial EPS such as xanthan gum, gellan gum, and thermal gel are widely applied and favored in food industry. Transcriptional regulators can regulate the expression of eps genes at transcription level to affect bacterial EPS biosynthesis. Moreover, the regulatory mechanism of most reported EPS transcriptional regulators has not been elucidated. This review summarizes the progress of bacterial EPS transcriptional regulators in recent years and focuses on the research methods and regulatory mechanisms, in order to provide reference for transcription regulation research of bacterial EPS.

Abstract:The strong hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) is the main factor to prevent their microbial degradation in soil and water environment. Surfactants have been extensively studied in microbial degradation of PAHs because the apparent solubility of PAHs can be improved. Up to now, there have been many reports on the PAHs biodegradation promoted by chemical surfactants or biosurfactants. However, some scholars have discovered the inhibitory effect of surfactants on microbial degradation of PAHs. This article reviewed the latest progress of surfactants on PAHs biodegradation from the aspects of surfactant types and toxicity, PAHs solubilization, mass transfer enhancement, cell surface properties and environmental factors. Moreover, these two conflicting research results have been explained. In addition, a new method to promote microbial degradation of PAHs in cloud point system was introduced. Finally, the research prospect of surfactants on microbial degradation of PAHs is prospected.

Abstract:The two-component signal transduction system SrrAB can sense the change of oxygen concentration in the external environment, affect the transcription of target genes through the change of phosphorylation level, and thus regulate a variety of biological characteristics of Staphylococcus. Studies have found that SrrAB is closely related to the virulence and biofilm formation of Staphylococcus, and the regulatory mechanisms under aerobic and anaerobic conditions are different. However, the role of SrrAB in staphylococcal growth and metabolism, and its mechanism in pathogen-host interaction remain unclear. Combined with the previous research of our laboratory, we reviewed the recent studies of SrrAB on the growth and metabolism in recent years, meanwhile, illuminated the interaction between SrrAB and innate immune cells so as to provide theoretical basis for the control of Staphylococcus infection.

Abstract:Rare sugars, referred to their limited existence of rare monosaccharides and their derivatives in nature, can be applied in many areas ranging from foodstuffs to pharmaceutics and nutrition, or as raw materials for various natural products and drug candidates. However, most of the rare sugars are quite expensive, and their synthetic chemical routes are both limited and economically unfeasible due to expensive raw materials. Meanwhile, due to the advantages of enzymatic techniques such as moderate reaction conditions, high specificity, efficiency, and sustainability. Therefore, enzymatic transformations have become a very powerful tool in this field. This article reviewed the biological functions and applications of rare sugar, including D-tagatose, D-allulose, D-sorbose, L-fructose, and D-allose. Meanwhile, this article reviews the studies on the enzymes for rare sugar production and its enzymatic production.

Abstract:Type VI secretion system (T6SS) that widely exists in Gram-negative bacteria has been extensive studied in recent years. It plays important roles in the virulence, colonization, spread and competition inheritance of bacteria. This paper reviews the latest research progresses in the structure, regulation and biological function of T6SS. Hopefully, the review could inspire the development of antibacterial drugs based on T6SS and provide new thoughts for the diagnosis and control of bacterial infections.

Abstract:Although the aerobic granular sludge (AGS) has the advantages of good settling property, high efficiencies of nitrogen and phosphorus removal, and better resistance to shock loads, which still faces great challenges in practical sewage treatment application due to the bottleneck of slow granulation process and easy particle disintegration. In recent years, acyl homoserine lactone (AHL)-based microbial quorum sensing (QS) has become a hotspot in this field, and it may be of great significance to the rapid formation of activated sludge and particle stabilization, including the use of AHLs can increase the secretion of extracellular polymeric substances (EPS) and to optimize the change of microbial community structure. This paper reviews the mechanism of AHLs on microorganisms, further discusses the effects of different environmental factors (including substrates, electron acceptors, sludge concentration, pH, and temperature) on AHLs, and outlines the method of adding exogenous AHLs or AHLs-secreting strains which can ultimately achieve the rapid formation and long-term stability of aerobic granular sludge. Finally, the review discusses the prospect of future research for the application of AHLs-based quorum sensing in aerobic granular sludge technology.

Abstract:Post translational modification (PTM) refers to the precursors or proteins undergoing a series of processing modifications to form the protein with multiple functions. PTMs occur at distinct amino acid side chains or peptide linkages, and most are mediated by enzyme activity. Enzymes composed of 5% proteome mediate more than 200 types of post-translational modifications types, among which acetylation is an important PTM pathway. Acetylation modification is widely studied in eukaryotic organisms, it participates in almost all physiological activities of cells and is highly conserved. Many recent studies have shown that acetylation modification is also widespread in bacteria, and the researches related to physiological functions have also made significant progress. This article summarizes the mechanisms, functions of acetylation and detection technology of acetylation modification in bacteria. Besides, we analyze the existing problems and prospects the potential application value of acetylation modification.

Abstract:Professional certification in engineering education is an internationally accepted quality assurance system for engineering education, and it is an important basis for international recognition of engineering education and qualification of engineers. Professional certification in engineering education requires that curriculum teaching should be designed in accordance with the outcome based education (OBE) concept, and the student-centered teaching model should be built. Finally, the closed-loop teaching mode is formed with continuous improvement mechanism. Environmental engineering microbiology is the core course of environmental engineering. Colleges and universities at home and abroad pay more attention to the curriculum reform, but there are some problems in the course setting and teaching methods. This paper focused on Environmental engineering microbiology, and discussed how to definite the contributions to graduation requirements, set curriculum objectives, develop the teaching syllabus, design experiments, track and evaluate students and monitor the quality, in order to achieve continuous improvement. It can provide theoretical reference for the curriculum teaching design under professional certification in engineering education.

Abstract:Medical Microbiology is an important basic course for undergraduate students in traditional chinese medical universities. In view of the practical problems that affect teaching effect in the traditional theoretical teaching process, the “three-link integration & bilateral interactive” blended teaching model combining online and offline is developed using the Super Star Learning APP. The three learning links pre-class, class and after-school study are integrated through activities such as autonomous learning, free discussion among students, and instructor-student interactive teaching. Teachers and students communicate and interact throughout the whole process with each other. The effect of blended learning was studied by comparative method. The preliminary exploration showed that, compared to the traditional one, this teaching mode improved students’ engagement, test score and autonomous learning enthusiasm, which is more conductive to students’ understanding and mastery of knowledge. Therefore, in the course of medical microbiology in traditional Chinese medical universities, blended teaching model is worthy of further research and promotion.

Abstract:The outbreak of COVID-19 pandemic has highlighted the significant social and economic impact of genetic engineering techniques, which are getting closer to our life. Aiming to facilitate the students to conduct online learning at home in the context of COVID-19 pandemic, we have adopted the blended online teaching-learning model, which is students learning basic knowledge independently via Small Private Online Course (SPOC) micro-videos, teachers dissecting practical cases via Tencent Instant Messenger (QQ) classroom, students expanding management skills cooperatively after class via QQ group and teachers answering the perplexed questions in real-time via QQ group. Based on the combined techniques of raising questions, dissecting practical cases and enlarging interdisciplinary knowledge, the basic principles and key methods were presented and the process of gene cloning, available methods and application of DNA techniques were grasped by the students. Additionally, by adopting the practical case of preventing and controlling COVID-19 pandemic, the students’ learning goals were made certain and the interdisciplinary learning interests enhanced. Moreover, under the guidance of the teachers, the interdisciplinary teaching-learning activities, based on the COVID-19 nucleic acid test, that includes designing the process to be adopted on the desired aims, defining the available methods and learning the courses knowledge, and then getting the application skills of different disciplinary knowledge, have strengthened the students’ ability of applying management skills in genetic engineering techniques to solve practical problems. Our practice indicates that the adopted online interdisciplinary model, including self-learning, dissecting case, mutual developing and answering questions in real-time, can make the set objectives achieved with the substantial equivalence to face-to-face version. In addition, via the explored teaching method that is composed of analyzing case for identifying the issue to be solved, followed by designing plan and dissecting the process, then determining methods to be used and matching the disciplines, and then learning knowledge and gaining the skills, finally setting project and realizing the aims, both the students’ problem-solving skills related to the intersection of gene manipulation techniques as well as management techniques and the interdisciplinary thinking and integrative capabilities were cultivated.

Abstract:[Background] Herpes viral haematopoietic necrosis (HVHN) caused by cyprinid herpesvirus 2 (CyHV2) is one of the main diseases in crucian carp breeding, causing serious economic losses. The disease has no effective therapeutic treatment, therefore early detection of the virus and efficient control is an effective way to prevent its outbreak. [Objective] A real-time recombinase polymerase ampli?cation (RPA) assay for CyHV2 orf72 gene was established, and its specificity and sensitivity were evaluated. [Methods] Specific primers and probes were designed in the conserved region by comparing the orf72 nucleotide sequences between the five CyHV2 strains. Five reaction temperatures were set to optimize the conditions of real-time RPA assay. Under the optimal conditions, the specificity of the real-time RPA assay was verified among different species. The sensitivity of real-time RPA and qPCR was compared using gradient diluted CyHV2 positive DNA as template. [Results] Real-time RPA assay can quickly and accurately detect CyHV2 virus within 20 minutes at 37.8 °C, with high specificity, no cross-reaction with other viruses, and the same sensitivity as qPCR. [Conclusion] The real-time RPA assay developed in this study can be used for rapid on-site detection of CyHV2.

Abstract:[Background] As a common host used in prokaryotic expression system, Escherichia coli owned many advantages like low cultivation cost, short growth period and strong operability while also shared some deficiencies isochronally. [Objective] To decrease the endotoxin biosynthesis and virulence of E. coli and improve its exogenous proteins soluble expression ability synchronously. [Methods] lpxM gene in lipopolysaccharide biosynthesis pathway of E. coli was primarily deleted by CRISPR-Cas technology for lipid A side chain structure modification. Then tig gene was integrated expressed in genome of E. coli BL21(DE3) ?lpxM to provide chaperon factor for exogenous proteins. Besides, the recombinant plasmid pET-28a-Rcodon was constructed to supply the rare codon relevant tRNA for protein expression. [Results] Bacterial somatic endotoxin level obtained a 90% reduction compared to the original strain after lpxM deletion. By utilizing the recombinant host strain and plasmid, expression level of the infectious bursal disease virus (IBDV) VP2 protein was observably enhanced. Clinical safety evaluation results indicated that the endotoxin virulence of E. coli BL21(DE3) ?lpxM::tig was apparently lower than the original strain and the relevant immunity group showed no clinical symptoms. [Conclusion] E. coli prokaryotic expression system was remolded by recombinant technologies for low virulence and high soluble expression of the exogenous proteins, which laid a certain foundation for the relevant subunit vaccines investigation.