Abstract:The increasing emission of nitrous oxide (N₂O) poses a serious threat to global ecological balance, to which microbes contribute a lot. Aerobic ammonia oxidation is the oxidation of ammonia to nitrite under aerobic conditions, which directly or indirectly affects the global production and release of N₂O. Ammonia-oxidizing archaea (AOA), ammonia-oxidizing bacteria (AOB), complete ammonia oxidizers (Comammox), and heterotrophic ammonia-oxidizing bacteria (HAOB) are major contributors to ammonia oxidation. It is important for the mitigation of N₂O emission to clarify how N₂O is produced by the four types of microorganisms. This paper reviewed the N₂O production pathways in AOA, AOB, Comammox and HAOB driven by the aerobic ammonia oxidation process. The roles of some key enzymes in the N₂O production pathways were elucidated through enzymatic analysis. Ammonia monooxygenase (AMO) is the first key enzyme in the autotrophic aerobic ammonia oxidation process, and the direct product of hydroxylamine oxidation is NO instead of NO₂^-, which is then converted to NO₂^- by an unknown enzyme. The aerobic ammonia oxidation process and related enzymes of HAOB are not completely clear, and the issue of N₂O emission needs to be further studied. This paper aims to lay a theoretical basis for regulating biological N₂O emission.

Abstract:Pine wilt disease caused by Bursaphelenchus xylophilus is a severe disease with strong transmission and destructive damage to the forest ecosystems in China. It is therefore urgent to prevent this disease. Compared with the physical and chemical control measures of B. xylophilus, biocontrol being environmentally friendly has a wide research prospect. Filamentous fungi and their secondary metabolites have less impact on the environment and more specific lethal effects on B. xylophilus than chemical nematicides. Therefore, it is of great significance to isolate antinematodal metabolites from filamentous fungi and determine their structures and activities. This paper introduces the structures and activities of metabolites from filamentous fungi against B. xylophilus. A total of 57 metabolites against B. xylophilus have been discovered in the past two decades, with diverse structures and varied activities. This paper systematically summarizes the structures and activities of all the metabolites to facilitate the research in this field and then summarizes and prospects the research in this field. It is hoped that this review will provide reference for the biocontrol of pine wilt disease and the further research on the metabolites of filamentous fungi against B. xylophilus.

Abstract:Human sapoviruses (HuSaV) are major pathogens causing sporadic acute gastroenteritis and related epidemics worldwide, especially in high-risk populations such as infants and immunocompromised patients. With rich antigenic and genetic diversity, human sapoviruses exhibit antigenic diversity and immunogenicity mainly in the P2 subdomain. The immunogenicity of the capsid protein is the theoretical basis for the development of vaccines against the viruses. With strong tolerance to capsid mutations without losing viral function, human sapoviruses can evolve rapidly through continuous amino acid mutations in the host, with mutations accumulating mainly in the P domain of VP1 and rarely in non-structural proteins and VP2. Sequence and structural changes enable human sapoviruses to escape the existing population immunity. Therefore, it is essential to explore the immune escape mechanisms of human sapoviruses and their antagonistic effects on host immune responses. This paper systematically reviews the studies of human sapoviruses in terms of genome characteristics, antigenic diversity characteristics, and genetic evolution mechanism, and puts forward the problems to be solved in the future research.

Abstract:Akkermansia muciniphila (AKK) promotes the secretion, maintains the dynamic balance, and regulates the barrier function of intestinal mucus, playing a key role in metabolic regulation and immune response. As a promising next-generation probiotic, AKK can mitigate diseases such as intestinal inflammation, metabolic disorders, neuroinflammation, and cancers. We introduced the therapeutic potential of AKK for diseases of the digestive system, the nervous system, metabolic disorders and cancers, aiming to provide a systematic review of the potential role and molecular mechanism of AKK in disease treatment.

Abstract:The thymus is the central immune organ responsible for the development, differentiation, and maturation of T cells. In addition to age-related factors, radiotherapy, chemotherapy, infections, and tumors are also major factors for the structure, cellularity, and function changes of the thymus. Here, we introduce the thymus changes induced by bacterial infections and the underlying mechanisms and summarize the therapies for ameliorating infection-associated thymus injuries, aiming to provide references for preventing or repairing infection-induced thymus injuries in clinical practice.

Abstract:Transporters are membrane proteins that mediate the transmembrane transport and signal exchange of chemicals inside and outside the biological membrane. Organic acid transporters play a key role in the transmembrane transport of microbial organic acid metabolism. They can be classified into uptake transporters and efflux transporters according to the different directions of organic acid transport. In microbial metabolism, some organic acids can directly participate in the metabolism as energy, and some are important intermediates in energy conversion. The overexpression of uptake transporters promotes the absorbance of energy substances and increases the yield of target products in microorganisms. The knockout of uptake transporters or the overexpression of efflux transporters contributes to the efflux of target products from chassis cells and thus promotes the biosynthesis of organic acids. Studying the structures and functions of organic acid transporters helps to decipher the mechanism of organic acid biosynthesis and utilization in microbial cells, which is of great significance for improving the utilization and biosynthesis of organic acids in industrial microorganisms. This paper introduces the classification, structures, transport modes, and transport functions of organic acid transporters in microorganisms and the application of these transporters in organic acid production. It provides references for the efficient biosynthesis and future development of organic acids in industrial microorganisms.

Abstract:Most microorganisms respond to glucose via complicated sensing and signaling mechanisms. In yeast cells, glucose represses the expression of genes involved in the tricarboxylic acid cycle, gluconeogenesis, glyoxylate cycle, and alternative carbon source metabolism mainly through the Snfl/Mig1 signaling pathway. Alternative carbon sources such as xylose, galactose, sucrose, ethanol, and organic acids reset the sugar metabolism only upon the full consumption of glucose in the environment. Derepression of glucose is of great significance for improving the production efficiency of modern microbial industry and addressing environmental and energy issues. This paper introduces the repression mechanism of Snf1/Mig1 signaling pathway and the active sites of related transcription factors. Specifically, we introduce the application of alternative carbon sources and the corresponding mechanisms of glucose repression. Finally, we summarize the solutions to relieving glucose repression on the basis of different backgrounds. This review aims to provide new ideas for expanding the applications and improving the efficiency of modern yeast production.

Abstract:Bacterial protein secretion is an important way of cell-cell interaction between species, which plays a crucial role in interbacterial competition and pathogenesis. Gram-negative bacteria could use the type VI secretion system (T6SS) to deliver effector proteins to the extracellular environment or prokaryotic and eukaryotic cells, thereby promoting their fitness during competition between microorganisms or host-microbe interactions. This paper summarizes the latest advances in the structures and components of T6SS, with focus on effector assembly and effector-immunity pairs, providing new strategies for the development of antibacterial drugs in the future.

Abstract:Tick-borne encephalitis virus is the pathogen of tick-borne encephalitis, which causes severe central nervous system symptoms. The number of tick-borne encephalitis cases reported annually in Europe, Russian Far East, Japan, and northern China reaches 10 000- 12 000, and the incidence is gradually increasing in China and several European countries. This disease is becoming a potential hazard to human. Active immunization is an effective measure to prevent infection, and vaccines with high safety have been developed in multiple countries including China. However, the vaccination is limited in the provinces where tick-borne encephalitis is popular. The design of specific antivirals may be a feasible way for the treatment of this disease. The non-structural proteins NS2B-NS3 and NS5 of tick-borne encephalitis virus play key roles in viral genome replication, capping, and host immunomodulation, thus becoming key targets for antiviral development. In this review, we outlined the three-dimensional structures and the development of inhibitors of NS2B-NS3 and NS5 of tick-borne encephalitis virus. This review provides a reference for probing into the molecular mechanism of tick-borne encephalitis virus infection and the development of antivirals.

Abstract:Parkinson's disease is a common neurodegenerative disease with the cause remaining unknown. The available therapies are characterized by high costs, limited efficacy, and strong side effects. Gastrointestinal dysfunction is common in the patients with Parkinson's disease. Studies have demonstrated the association between Parkinson's disease and intestinal flora, and the patients are expected to be treated by probiotics which can restore the intestinal flora. The engineering of probiotics makes it feasible to modify probiotics according to people's wishes and improve their stability and targeting, thus giving full play to the role of probiotics in disease treatment. We reviewed the relevant studies in recent years, elaborated on the possible treatment mechanisms, and further analyzed the feasibility of engineering probiotics in the treatment of Parkinson's disease, aiming provide new ideas for the safe treatment of this disease.

Abstract:Myocardial fibrosis is the end-stage manifestation and main pathogenic factor of a variety of cardiovascular diseases, such as coronary heart disease, myocardial infarction, and heart failure. Immune and inflammatory processes play a decisive role in the pathogenesis of myocardial fibrosis. Recent studies have demonstrated that gut microbiota plays a crucial role in the pathogenesis and development of myocardial fibrosis. The dysregulation of gut microbiota can lead to the transfer of microbial metabolites, such as short-chain fatty acids, lipopolysaccharides, and trimethylamine oxide into the blood circulation. These metabolites directly or indirectly induce immune responses to tissue damage and systemic activation of inflammatory responses which then affect myocardial fibrosis. How to mitigate myocardial fibrosis by modifying gut microbiota has become the focus of current research, and the measures include dietary interventions, antibiotics, probiotics, prebiotics, and fecal microbiota transplantation. This paper reviews the interactions of gut microbiota and its metabolites with myocardial fibrosis and introduces the research progress in alleviating myocardial fibrosis by modulating gut microbiota, providing new ideas for the treatment of myocardial fibrosis.

Abstract:Intestinal microflora homeostasis plays a crucial role in maintaining human health. As a guard of the natural intestinal microflora, probiotics improve the microecological balance in the host. Lactobacillus is a representative of intestinal probiotics. The exopolysaccharide (EPS) synthesized by Lactobacillus can optimize the intestinal microecology by promoting the growth of other probiotics in the intestine. Moreover, EPS is praised for anti-tumor, antioxidant, cholesterol-lowering, blood pressure-lowering, and immunity-enhancing activities. This article reviews the research progress in the genetics, biological activities, structure-activity relationship of Lactobacillus EPS in recent years.

Abstract:Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that can cause persistent infection in immunocompromised patients. The infection of P. aeruginosa is dependent on its virulence factors, most of which are regulated by the quorum sensing (QS) system. P. aeruginosa has four QS systems:las, rhl, pqs, and iqs. The 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal, PQS), as a signal molecule in the pqs system of P. aeruginosa, can not only regulate the expression of diverse virulence factors but also affect a variety of physiological processes of microorganisms and hosts. This review summarizes the biological functions of PQS, such as mediating the QS system, regulating biofilm formation, mediating outer-membrane vesicle biogenesis, iron acquisition regulating host immune activities and cytotoxicity and providing population protection. The purpose of this review is to highlight the functional diversity of P. aeruginosa PQS and provide guidance for studying new PQS functions and developing antimicrobial agents.

Abstract:[Objective] To reconstitute the partial biosynthetic pathway of the type II polyketide kinamycin in a cell-free system starting from CoA, acetyl-CoA and malonate.[Methods] The polyketide synthases (PKSs) and two supplemental proteins (MCAT and MatB) were purified and quantitated. The one-pot enzymatic synthesis of kinamycin intermediates was accomplished, and the products were analyzed by high performance liquid chromatography (HPLC). The system was used to explore the function and exact substrate of the stand-alone thioesterase, AlpS, in the biosynthetic pathway. The parameters including temperature, pH, and the concentrations of minimal PKSs and AlpS were optimized by the single-variable method. [Results] Eight PKSs and two supplemental proteins (MCAT and MatB) were expressed and purified. The intermediates and shunt products in kinamycin biosynthesis were produced in vitro, including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin. The yields of these products, especially prejadomycin and dehydrorabelomycin, were all increased when AlpS was added. The optimal conditions were 30℃, pH 7.0, 2.8 μmol/L minimal PKSs (AlpA, AlpB, and RavC, respectively), and 7.2 μmol/L AlpS, under which the yield of prejadomycin was increased to 302 mg/L. [Conclusion] A one-pot enzymatic synthesis system with high yields of kinamycin-related products including SEK15, UWM6, rabelomycin, prejadomycin, and dehydrorabelomycin was successfully constructed. Furthermore, the chain-release function and substrate specificity of AlpS in the synthetic pathway were investigated.

Abstract:[Objective] To identify the l-cysteine transporter of Cryptococcus neoformans and study the effect of the transporter on the fungal pathogenicity. [Methods] We constructed the candidate gene-deleted strains, and examined the growth of the mutants with l-cysteine as the sole sulfur source. We then tested the effect of the l-cysteine transporter Mup1 on the expression of virulence factors and the growth of C. neoformans under different stress conditions. Further, we used the Galleria mellonella infection model and the mouse infection model to explore the effect of MUP1 on the pathogenicity of C. neoformans. RNA-Seq and yeast one-hybrid assay were employed to investigate the regulatory relationship between the master transcription factor Cys3 and Mup1. [Results] Mup1 could transport l-cysteine, l-cystine, dl-cystathionine, and l-homocysteine. The deletion of gene MUP1 did not affect the expression of virulence factors or the cellular response to stress. Mup1 had no significant effect on the pathogenicity of C. neoformans in G. mellonella and mice. Cys3 might indirectly regulate the transcription of MUP1. [Conclusion] Mup1 is the transporter of l-cysteine, l-cystine, dl-cystathionine, and l-homocysteine in C. neoformans. It does not affect the pathogenicity of C. neoformans and may be indirectly regulated by Cys3.

Abstract:[Objective] Vicia villosa Roth. is widely grown as important forage and green manure in Southwest China, which is beneficial to improving soil nitrogen and the yield and quality of succeeding crops. Inoculation of beneficial microorganisms is one of the important strategies to promote biological nitrogen fixation and legume crop growth. Thus, a plant growth-promoting fungus Careporia lacerata HG2011 was selected to study its effects on biological nitrogen fixation and growth of V. villosa Roth. and the underlying mechanisms. [Methods] Microbial culture, plant culture, and field trials were carried out to investigate the mobilization capacity of phosphorus and iron, metabolite composition, and interaction with Rhizobium sophorae S3 of C. lacerata and its effects on nodulation, growth, yield, quality of V. villosa Roth., and soil available phosphorus and iron. [Results] There was no mutual antagonism between C. lacerata and rhizobia. Liquid chromatography-mass spectrometry (LC-MS) analysis found that C. lacerata fermentation broth contained allelochemicals such as amino acids, organic acids, and flavonoids, which enhanced the chemotaxis and biofilm formation of rhizobia. In addition, this fungus could release gibberellins, salicylic acid, siderophores, and mobilize insoluble organic and inorganic phosphorus. In the plant culture experiment, inoculation with either C. lacerata or rhizobia alone promoted the growth of V. villosa Roth., but co-inoculation had the highest plant biomass. C. lacerata was able to colonize the roots of V. villosa Roth. and significantly increased root length, surface area, and nodule number. Field trials showed that the number of root nodules, nodule mass, nitrogenase activity, soil available iron, and phosphorus content, as well as phosphatase activity, were improved after C. lacerata inoculation. Compared with the conventional fertilization treatment, C. lacerata application increased the yield of V. villosa Roth. by 12.15%, and there were no negative effects on plant quality. [Conclusion] C. lacerata can colonize in the rhizosphere and promote nodulation, nitrogen fixation, and growth of V. villosa Roth. by secreting allelochemicals, auxin, siderophores, and mobilizing soil phosphorus and iron. C. lacerata inoculant is easy to prepare and use, which is valuable for improving the yield and quality of legume crops.

Abstract:[Objective] This paper aimed to analyze the role of TvRpd3 encoding histone deacetylase in the biocontrol of Trichoderma viride Tv-1511 by constructing the engineered strain with TvRpd3 deleted (∆TvRpd3). [Methods] Fusion PCR technique and homologous recombination method were employed to delete TvRpd3 in T. viride Tv-1511. Confrontation culture, phenotype observation, immunohistochemistry detection, and metabonomics analysis were carried out to compare the histone acetylation level, secondary metabolite synthesis, antagonism to pathogens, and field biocontrol effect between the engineered and the wild-type strains. [Results] Compared with the wild type, ∆TvRpd3 exhibited enhanced inhibitory effects on pathogens. The biocontrol effects of ∆TvRpd3 fermentation broth on wheat powdery mildew, tobacco black shank, and tomato Fusarium wilt increased by 62.27%, 57.45%, and 70.71%, respectively. Meanwhile, the knockout of TvRpd3 significantly changed the species and yields of secondary metabolites produced by Trichoderma. Specifically, the yields of several vital antibiotic substances including trichomycin, sorbicillinoids, and monocillin I produced by ∆TvRpd3 increased compared with those of the wild type. [Conclusion] TvRpd3 mediates histone deacetylation to play a role in the biocontrol of T. viride.

Abstract:[Objective] Streptococcus suis is a major zoonotic pathogen. This study aims to characterize the acetyltransferase toxin of the toxin-antitoxin system in Streptococcus suis and analyze the functions of the toxin, which may contribute to exploring the role of the toxin-antitoxin system in the infection of S. suis. [Methods] We first predicted the putative type Ⅱ toxin-antitoxin system in the genome of S. suis serotype 5 strain HN105. We further determined the activity and analyzed the phylogenetic relationship of the toxin-antitoxin system. Western blotting was employed to determine the expression of the toxin in S. suis. We constructed the toxin-deficient strain, antitoxin-deficient strain, and toxin-antitoxin-deficient strain with HN105 as the parental strain to explore the effects of the system on the adhesion, biofilm formation, anti-phagocytosis, and intracellular survival of S. suis. [Results] The Gcn5-related N-acetyltransferase (GNAT) toxin of the toxin-antitoxin system encoded by DF184_RS00980-DF184_RS00985 in the genome of the strain HN105 was predicted and identified, and the system was named SsMarR-SsGNAT based on the conserved domain. SsGNAT toxin exerted toxicity in the periplasmic space of Escherichia coli, and the antitoxin could neutralize the toxicity. SsGNAT toxin showed low amino acid sequence identity and distant genetic relationship with other acetyltransferase toxins. SsGNAT toxin was cleaved in S. suis, and the reduced expression of antitoxin upregulated the expression of SsGNAT toxin. SsGNAT toxin had no effect on the biofilm formation, anti-phagocytosis or intracellular survival of S. suis, while it affected the adhesion of S. suis to human laryngeal cancer epithelial cells (Hep-2) and human brain microvascular endothelial cells (HBMEC) cells. [Conclusion] We discovered and identified the novel type Ⅱ toxin-antitoxin system SsMarR-SsGNAT in S. suis. SsGNAT toxin exerted toxicity in the periplasmic space of E. coli, and its deletion promoted bacterial adhesion to cells. The findings provide a reference for further unraveling the pathogenic mechanism of S. suis.

Abstract:[Objective] The polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) is essential for the biocontrol effect of Pseudomonas fluorescens 2P24. However, the genetic elements that modulates 2,4-DAPG production remains to be ascertained. [Methods] Previously, Tn5 transposon mutagenesis was employed to get P. fluorescens W3 with the complete loss of antagonism to Rhizoctonia solani, and the strain W3 was then used to screen out the novel gene that influencing the production of 2,4-DAPG. In this study, the effect of the mutated gene in W3 on the 2,4-DAPG production and biocontrol effect of 2P24 was explored. [Results] One of the insertion mutations, exhibiting no activity against R. solani, was mapped to opgG, which encodes the protein for the synthesis of osmoregulated periplasmic glucan (OPG). The deletion of opgGH impaired the production of 2,4-DAPG and bacterial swimming motility, while it did not affect the production of quorum sensing (QS) signals, the generation of hydrocyanic acid, or the biofilm formation. The transcriptional fusion assay indicated that the expression levels of rsmY, rsmX1, and gacA were not changed in the opgGH mutant compared with the wild type. The data suggested that opgGH regulated the production of 2,4-DAPG in a Gac/Rsm-independent manner. [Conclusion] The opgGH operon is essential for the regulation of 2,4-DAPG production, swimming mobility, and antifungal effect, serving as a key regulator for the biocontrol capacity of P. fluorescens 2P24.

Abstract:[Objective] To realize the heterologous expression of dibasic endopeptidase genes sckex2 and pkkex2 from Saccharomyces cerevisiae and Pichia kudriavzevii in Escherichia coli BL21 and then study the properties of the recombinant KEX2 enzymes and their efficiency of hydrolyzing soybean protein in collaboration with alkaline protease. [Methods] The kex2 from S. cerevisiae and P. kudriavzevii was optimized according to the codon bias of E. coli. After analysis of non-functional region, we redesigned their C-terminal and N-terminal residues by truncation. Four truncated genes, sckex2∆3, sckex2∆4, pkkex2∆3, and pkkex2∆4, were respectively cloned into the vector pGEX-6P-1, and the recombinant plasmids were transformed into E. coli BL21 competent cells. After confirmed by DNA sequencing, the four recombinant strains, E. coli BL21/pGEX-ScKEX2∆3, E. coli BL21/pGEX-ScKEX2∆4, E. coli BL21/pGEX-PkKEX2∆3, and E. coli BL21/pGEX-PkKEX2∆4, were constructed. The recombinant enzymes were purified by GST affinity chromatography and PreScission protease and then their pH stability and thermal stability were determined. With the soybean protein hydrolysis by alkaline protease alone as the control, the yield of small-molecule peptides from the hydrolysis with the recombinant KEX2 in collaboration with alkaline protease was determined. [Results] The recombinant KEX2 and the wild type showed soluble expression in E. coli BL21. SDS-PAGE showed that the purified recombinant enzymes produced single bands. Under the optimal conditions, the wild type showed no enzyme activity, while the mutants showed the maximum specific activity of 47.32 U/g, K_m of 23.61 μmol/L, k_cat of 50.18 s⁻¹, and k_cat/K_m of 2 125.06 L/(mmol·s). After incubation at pH 5.0 for 2 d, the maximum relative activity of the recombinant enzymes was more than 40%. After incubation at 35℃ for 1 h, the relative activity was still over 60%. When the recombinant KEX2 and alkaline protease were used together to hydrolyze soybean protein, more than 39% of the hydrolysates were active peptides with molecular weight less than 500 Da, and the content of active peptides with the molecular weight less than 100 Da was 50% higher than that from the hydrolysis with the alkaline protease alone. [Conclusion] Through terminal truncation, the soluble expression of truncated KEX2 mutants was realized in E. coli. The recombinant enzymes demonstrate high catalytic efficiency and can efficiently hydrolyze soybean protein to release active peptides. This work lays a solid foundation for the value addition and deep hydrolysis of protein-rich biological resources.

Abstract:[Objective] To type and identify 29 strains of Burkholderia cepacia complex (Bcc) isolated from daily chemical products in 2020–2022, re-identify 6 isolates identified as Burkholderia lata from daily chemicals products before 2020, and reveal the antibiotic resistance of Burkholderia aenigmatica sp. nov. [Methods] Bcc isolates from daily chemical products were subjected to multilocus sequence typing (MLST). Seven housekeeping genes, atpD, gltB, gyrB, recA, lepA, phaC, and trpB, were amplified by PCR. The sequencing results were compared with the data in MLST database to obtain the accession number of each housekeeping gene and the sequence type of each strain. Multilocus sequence analysis (MLSA) was employed to construct a phylogenetic tree based on the nucleotide sequences of alleles in MLST. The resistance of Bcc to common preservatives (1,3-dimethylmethylol-5,5-dimethylhydantoin, kathon, sodium benzoate, and potassium sorbate) and antibiotics (ceftazidime, kanamycin, and tetracycline) were determined by the minimum inhibitory concentration (MIC) method. [Results] The 29 Bcc isolates were identified as 5 species (B. cenocepacia, B. contaminans, B. aenigmatica, B. vietnamiensis, and B. stabilis) and typed as 15 sequence types. Seven new alleles and seven new sequence types (ST2118, ST2120, ST2122, ST2127, ST2128, ST2129, and ST2130) were identified in this study. The six B. lata strains from the samples before 2020 were re-identified as B. aenigmatica sp. nov. Only one of the 11 B. aenigmatica isolates was resistant to ceftazidime, and nine and eight B. aenigmatica isolates were resistant to kanamycin and tetracycline, respectively. Kathon and 1,3-dimethylmethylol-5,5-dimethylhydantoin (DMDMH) within the maximum permissible concentrations can effectively inhibit the growth of B. aenigmatica. Nine B. aenigmatica strains showed resistance to sodium benzoate and potassium sorbate. [Conclusion] The identification of Bcc is complex and there are unknown alleles and sequence types. B. aenigmatica has become the main Bcc species contaminating daily chemical products. Most strains of B. aenigmatica are resistant to aminoglycosides and tetracyclines. Most B. aenigmatica isolates from the daily chemical products have resistance to sodium benzoate and potassium sorbate.

Abstract:[Objective] To address the problem that the detection of drug resistance gene usually relies on specialized devices and the lack of rapid detection methods, we aimed to establish a rapid detection method for the drug resistance gene mecA of Staphylococcus aureus based on clustered regularly interspaced short palindromic repeats (CRISPR). [Methods] Firstly, we designed and screened the recombinase-aided amplification (RAA) primers and CRISPR RNA (crRNA) with high sensitivity according to the conserved region of mecA. Then, we established the detection method for mecA with the easy-readout and sensitive enhanced (ERASE) strip. Finally, we used simulated samples and clinical isolated samples to compare the established method with the traditional method. [Results] A set of efficient RAA primers and crRNAs targeting mecA were successfully screened out, and a highly sensitive CRISPR-ERASE-based nucleic acid detection method was established for mecA. This method can detect mecA at a minimum concentration of 10 copies/μL. Among 32 clinical isolates of S. aureus, 24 strains were tested positive for mecA by the established method, which was in 100% agreement with the results of antimicrobial susceptibility test and quantitative real-time PCR (qPCR). [Conclusion] A CRISPR-ERASE-based simple and sensitive method for detecting the drug resistance gene mecA was established.

Abstract:[Objective] To explore the oligotrophic culturomics for the isolation of human gut microbiota. [Methods] Oligotrophic media were obtained by diluting the enrichment medium (blood culture bottle supplemented with sheep blood and rumen fluid), yeast casitone fatty acid (YCFA) agar plate, and YCFA broth. Healthy human fecal samples were cultured with the original medium (0), 5, 10, 20, 30, and 40-fold dilutions of enrichment medium. At different time points (0, 3, 6, 9, 15, 27, and 30 d) during the enrichment, the YCFA agar plate was used to isolate colonies, and then single colonies were picked for further enrichment with YCFA broth. The strains were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and 16S rRNA gene sequencing. The effects of the above six oligotrophic conditions on the isolation of gut microbiota were compared. Three enrichment media with better isolation effects, i.e., 0-, 10-, and 30-fold dilutions of the enrichment medium, were selected and combined with YCFA agar plate and YCFA broth with the same dilution factors to form nine culture conditions for the optimization of the culture conditions of gut microbiota. [Results] Among the six oligotrophic enrichment media, the 0-, 10-, and 30-fold dilutions of the enrichment medium isolated more species than the other dilutions, and the 10-fold dilution isolated the most species. Twenty-four species were only isolated by the oligotrophic media and not by the original medium. Further optimization showed that the following three combinations isolated the most bacterial species:0-fold diluted enrichment medium+10-fold diluted YCFA plate and broth, 10-fold diluted enrichment medium+0- or 10-fold diluted YCFA plate and broth. As for the three combinations, 20 species were only isolated by the oligotrophic media, and the combination of 10-fold diluted enrichment medium+0-fold diluted YCFA plate and broth isolated the largest number of species. [Conclusion] The oligotrophic culture conditions obtained by diluting the enrichment medium, YCFA agar plate, and YCFA broth can isolate about 40% of species that cannot be isolated by conventional culture conditions, which provide an effective method for isolating more species from the human gut microbiota.

Abstract:[Objective] To establish a multi-target method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), and swine influenza virus (SIV), and the detection of the general type, H1 type and H3 type of SIV. [Methods] We designed six pairs of primers and corresponding extension probes according to the conserved sequences of six pathogens and conducted single reaction tests. The primer concentrations and the reaction conditions were optimized for the established method. After examination of the specificity, repeatability, and sensitivity, the MALDI-TOF MS method was used to detect clinical samples and pig-derived products, and the results were compared with those obtained by fluorescence quantitative PCR. [Results] The established method only generated peaks corresponding to the positions of the target viruses, and other common pathogens causing swine diseases did not appear in the mass spectra, which indicated that the system had good specificity. The in-batch positive coincidence rate of each virus at high, medium, and low concentrations was 100%, and the inter-batch coincidence rate was ≥ 96.7%, indicating that the system had good repeatability. The minimum limit of detection for each virus in the system varied within 6.73–21.25 copies/μL. We then used the MALDI-TOF MS method to detect 124 tissue, feed, and pork samples. A total of 12 positive samples were detected, including nine PRRSV positive samples and three SIV positive samples (two SIV-H1 positive samples and one SIV-H3 positive sample). We then compared the above results with the results from fluorescence quantitative PCR, and found that the total coincidence rate of the two methods for detection of each pathogen was 99.2%–100%. [Conclusion] A method for the simultaneous detection of six RNA viruses causing porcine respiratory diseases was established based on MALDI-TOF MS. This sensitive and specific high-throughput multi-target detection method facilitates the surveillance and rapid identification of related diseases in the import and export animal quarantine.

Abstract:[Objective] To clone and express three β-agarase genes agaW3418, agaW3419, and agaW3472 in Vibrio natriegens WPAGA4 isolated from the deep-sea sediment and study the enzymatic properties of the proteins. [Methods] The three agarase genes were expressed in Escherichia coli BL21(DE3) cells, and the 3,5-dinitrosalicylic acid (DNS) method was employed to measure the activities of the recombinant agarases. [Results] The three agarases, AgaW3418, AgaW3419, and AgaW3472, belonged to the GH50 family, with the highest activities at 50, 60, and 30℃ and pH 6.0, 7.0, and 7.0, respectively. AgaW3472 showed the highest activity and maintained good stability at 20℃. The highest production of AgaW3472 was detected in the super optimal broth (SOB) medium with 1% (W/V) lactose as the carbon source, 20 mmol/L MgCl₂, and 0.1 mmol/L isopropyl-β-d-thiogalactoside (IPTG) at the induction temperature of 37℃. [Conclusion] The agarase AgaW3472 has high enzyme activity, low temperature adaptability, and good stability, serving as a potential bio-tool for the development of agar degradation-related industries. The three recombinant agarases provided a foundation for understanding the agarose metabolism in V. natriegens WPAGA4 and the role of this strain in the marine carbon cycling.