Abstract:[Background] The marine environment, as the largest organic carbon store on the earth, stores a large amount of humic substances, which may harbor abundant humic substance-transforming bacteria. [Objective] To isolate potential humic substance-transforming bacteria from marine sediments, and provide bacterial resources for the biotransformation of refractory natural organic polymers. [Methods] With humic substances as the sole carbon source, the humic substance-transforming bacteria in the sediment samples from 12 stations in the western Pacific polymetallic nodule province were enriched and purified. The taxonomic status of the purified bacteria was preliminarily identified by 16S rRNA gene sequence comparison. The potential humic substance-transforming bacteria were screened out by aniline blue-containing medium. [Results] A total of 276 strains were isolated from 12 sediment samples, belonging to 56 species, 37 genera, 14 orders of Actinobacteria, Cytophagia, Flavobacteria, Alphaproteobacteria and Gammaproteobacteria, including 1 potential new genus and 2 new species. Of the 56 species, 49 species were lignin-modifying enzyme positive. [Conclusion] The medium with humic substances as the sole carbon and energy source can be used to isolate potential humic substance-transforming bacteria with high diversity.

Abstract:[Background] The ocean is the largest reservoir of carbon and the largest habitat for life on the Earth. This vast ecosystem harbors a wide variety of microorganisms that play a key role in the global carbon cycle. Hadal trench (6 000 m below the sea level) is a special habitat containing rich biological resources owing to the high hydrostatic pressure and the accumulation of massive organic matter by surface deposition. [Objective] To obtain the bacterial pure culture capable of utilizing aromatic acids as the sole carbon and energy from the hadal trench sediment samples, and analyze its aromatic acid-degrading characteristics. [Methods] The hadal trench sediment sample was cultivated in the simulated in situ high-pressure environment, and then the culture was incubated on the plates with different aromatic acids under atmospheric ambient pressure. Finally, the bacterial pure culture was selected and identified based on the morphological characteristics and the phylogenetic tree constructed with the 16S rRNA gene. The strain was then incubated with different aromatic acids, and the degradation intermediates were identified by HPLC and LC/MS. [Results] A strain capable of utilizing benzoate and 4-hydroxybenzoate was isolated from the sediment sample from Mariana trench (6 300 m below the sea level). It belonged to the genus Halomonas and was designated Halomonas sp. strain NyZ771. The intermediate in 4-hydroxybenzoate degradation was identified as protocatechuate. [Conclusion] A benzoate- and 4-hydroxybenzoate-degrading bacterium, Halomonas sp. strain NyZ771, was isolated from the trench sediment. This study enriches the microbial resources derived from the hadal trench and provides a theoretical foundation for the future research on the aromatic acid degradation in hadal trenches and the carbon cycle driven by microorganisms in the ocean.

Abstract:[Background] Compared with viruses infecting bacteria and eukaryotes, archaeal viruses are limited in number but morphologically diverse. Therefore, it is of great significance to isolate and identify new archaeal viruses. [Objective] To gain an in-depth understanding of the diversity of archaeal viruses, we isolated a new halophilic archaeal virus from water samples of Feicui Lake in Qinghai Province and analyzed its biological characteristics and taxonomic status. [Methods] Halophilic archaea were isolated by picking single colonies and the archaeal virus was obtained by plaque assay. Virus particles were concentrated and purified by two-step precipitation with PEG 6000 and CsCl density gradient centrifugation. After the negative staining with uranyl acetate, viruses were observed under a transmission electron microscope. Viral genome was extracted and sequenced and the bioinformation was analyzed. Virus samples were concentrated by trichloroacetic acid (TCA) and applied to SDS-PAGE gels for separation of viral proteins, followed by Coomassie brilliant blue staining and Sudan Black B staining, respectively, to observe the protein and lipid bands. [Results] A halophilic archaeal virus was isolated on a double-layer plate with Halorubrum sp. K2 as sensitive bacteria. The plaques were hazy with a polymorphic enveloped virus-like shape under transmission electron microscope, with a diameter of about 60 nm. It had the double-stranded circular DNA genome of 9 333 bp in size and shared about 75% identity to the reported HRPV11, HRPV12, and HRPV10 in Betapleolipovirus, which was a new viral species of this genus. According to morphology and genome characteristics, it was named Halorubrum pleomorphic virus 13 (HRPV13) in Betapleolipovirus. The virus can still infect bacteria at high salt concentration and had high viability at below 50 °C and pH 5.0–9.0. Moreover, it failed to cause significant lysis of host after infection. [Conclusion] HRPV13 is a new polymorphic species in Betapleolipovirus, as verified by electron microscopy, biological characterization, and genome sequencing. The result lays a foundation for research on the evolutionary relationship of polymorphic viruses in different regions.

Abstract:[Background] Despite the extensive research on the application of confocal Raman spectroscopy in imaging and component identification, there are few studies focusing on the rapid detection and identification of a variety of bacteria. [Objective] A method for rapid classification and identification of single bacterial cells was established with confocal Raman spectroscopy. [Methods] We optimized the excitation wavelength of confocal Raman spectroscopy by using the single cells of Escherichia coli, and studied the influence of storage time on the Raman spectrum of E. coli. Furthermore, we performed confocal Raman spectroscopy tests for Staphylococcus albus, Escherichia coli, Staphylococcus aureus, Salmonella, and Pseudomonas aeruginosa. We then analyzed the Raman spectra of the 5 bacterial species to design a rapid identification method combining confocal Raman spectroscopy with support vector machine (SVM) model for the 5 bacterial species. [Results] Among the three common Raman detection wavelengths of 532, 633, and 785 nm, 532 nm had the best excitation efficiency and spectral signal-to-noise ratio for the identification of single bacterial cells. The SVM model showed the sensitivity and specificity above 96.00% and the overall accuracy rate of 98.25% for the identification of these bacteria. Moreover, the Raman spectra showed good repeatability and stability for the E. coli stored for different time, and the matching rate of SVM model was above 90.00%. [Conclusion] Single-cell Raman spectroscopy combined with SVM model can quickly and accurately classify the five bacterial species, and different storage time has little effect on the identification of E. coli based on Raman spectra.

Abstract:[Background] β-galactosidase has important application value in many fields such as food processing, clinical medicine, and genetic engineering. Thus, the development of β-galactosidase with high activity and strong thermal stability has become a research focus. [Objective] This paper aims to screen novel β-galactosidases from the fecal microbial metagenome of Nomascus concolor and study its enzymatic properties. [Methods] β-galactosidase gene GalNC1-8 was cloned by PCR with the fecal microbial metagenomic DNA of N. concolor as template. Then, the recombinant plasmid pEASY-E2/GalNC1-8 was constructed and transformed into Escherichia coli BL21(DE3) for expression, and the enzymatic properties of recombinase GalNC1-8 was tested. [Results] The basic β-galactosidase GalNC1-8, belonging to GH35 family, had the molecular weight of 28.18 kDa. The optimal conditions for GalNC1-8 were pH 8.0 and 50 °C. After incubation at 30, 37, 40, and 50 °C for 1 h, the relative enzyme activity was still above 80%. After 1 h treatment at pH 7.0−9.0, the enzyme kept more than 54% of the activity. The activity of GalNC1-8 was hardly influenced by the reaction system containing ethanol. β-mercaptoethanol, glycerol, methanol, Na⁺, K⁺, and Li⁺ enhanced the enzyme activity. It retained above 50% activity after being treated with 0.5−3.5 mol/L NaCl at 50 °C for 1 h. [Conclusion] a novel β-galactosidase gene GalNC1-8 was screened from the N. concolor fecal microbial metagenome, which expressed in E. coli BL21(DE3). GalNC1-8 has the lowest molecular weight among the known metagenome-derived β-galactosidases. With adaptability to a wide pH range, thermal stability, and salt tolerance, it has good application prospects.

Abstract:[Background] vcrV is an important gene located in the T3SS1 gene cluster, which is one of the two sets of type III secretion systems of Vibrio parahaemolyticus and plays a pathogenic role by secreting effector proteins to host cells. [Objective] In this study, the effect of vcrV was explored on the T3SS1 pathogenesis and the biological characteristics of V. parahaemolyticus. [Methods] The vcrV-deleted strain ΔvcrV and the complemented strain CΔvcrV were constructed by homologous recombination technology, using V. parahaemolyticus POR-1 as the reference strain. The growth performance, biofilm formation ability, cell adhesion, and cytotoxicity were compared among different strains. Western Blot was employed to detect the effector proteins secreted by POR-1, ΔvcrV, and CΔvcrV under T3SS1 induction. The translocation of the effector protein VopR (Vp1683) was detected by Western Blot after each strain with the pMMB207-vp1683-CyaA overexpression vector was used to infect HeLa cells. [Results] The deletion of vcrV did not affect the growth performance, biofilm formation ability, or cell adhesion while significantly reduced the strain toxicity to HeLa cells. The effector protein VopR secretion had no significant difference among POR-1, ΔvcrV, and CΔvcrV with the overexpression vector. The translocation of VopR decreased significantly when ΔvcrV infected HeLa cells. [Conclusion] The T3SS1-mediated cytotoxicity involving with vcrV is critical for the translocation of T3SS1 effector protein in V. parahaemolyticus, while the effector protein VopR could still be secreted by ΔvcrV with overexpression vector.

Abstract:[Background] The Pseudomonas tolaasii-caused brown blotch disease of Pleurotus ostreatus has occurred on a large scale, resulting in low yields and potential safety risks. It is of great significance to find safe and effective antibacterial agents for the development of P. ostreatus industry. [Objective] In this study, five different methods were used to obtain Allium sativum leachate samples. The effects of A. sativum leachate samples on P. tolaasii and the mycelial growth of P. ostreatus were then tested. [Methods] The inhibition zone method was used to determine the antibacterial effects of five A. sativum leachate samples on P. tolaasii. The plate diffusion method was employed to screen out the leachate sample and the appropriate concentration that can promote the growth of P. ostreatus. [Results] The five A. sativum leachate samples had strong activity against P. tolaasii. Among them, 10% A. sativum leachate prepared with apricot shell wood vinegar had the best performance, with the antibacterial effect equivalent to that of 0.33 mg/mL streptomycin. Moreover, it significantly promoted the mycelial growth of P. ostreatus, as manifested by the dense mycelia and neat edges, which was better than that of the control. [Conclusion] This research lays an experimental foundation for the prevention and treatment of brown blotch disease infecting P. ostreatus by the combination of A. sativum and apricot shell wood vinegar.

Abstract:[Background] Facing temperature stress, organisms produce a large amount of heat shock protein in a short time, which can restore the conformations of damaged proteins and enhance the tolerance of organisms to stress. [Objective] To preliminarily explore the relationship between heat shock protein Vvhsp60 and low temperature tolerance of Volvariella volvacea, and thereby lay a theoretical foundation for further genetic improvement of V. volvacea for low-temperature tolerance. [Methods] The bioinformation of Vvhsp60 was analyzed. The expression of Vvhsp60 gene in low temperature-sensitive strain V23 and low temperature-tolerant strain VH3 under low temperature stress and under heat shock-low temperature treatment was analyzed by real-time fluorescent quantitative PCR (RT-qPCR). [Results] With no signal peptide, Vvhsp60 is not a secretory protein. It exerts biological functions in mitochondria and cytoplasm and is a bidirectional transmembrane protein. Low temperature significantly increased the expression of Vvhsp60 gene in mycelia of both V23 and VH3, and the expression in VH3 was significantly higher than that in V23, which suggests that the high expression of Vvhsp60 gene may help enhance the low-temperature tolerance of VH3. After heat shock treatment, the expression of Vvhsp60 gene in the two strains was significantly higher than that in those without heat shock treatment, indicating that heat shock can induce the expression of Vvhsp60 gene. [Conclusion] Vvhsp60 gene is related to the low-temperature tolerance of V. volvacea, and heat shock can induce the expression of Vvhsp60 gene.

Abstract:[Background] Fusarium solani, one of the most destructive soil-borne pathogens worldwide, seriously affects the yield and quality of crops. Therefore, it is urgent to develop broad-spectrum sustainable biocontrol agents and the abundant secondary metabolites of plants are natural sources. [Objective] To explore the activity of carvacrol and eugenol against F. solani and the possible underlying mechanism. [Methods] With the mycelium growth rate method, crossing method, and spore germination method, the inhibitory effect of carvacrol and eugenol on mycelium growth and spore germination of F. solani was analyzed. The scanning electron microscope (SEM) was used for observing the mycelial morphology of F. solani, and propidium iodide (PI) staining was used for observing the damage to F. solani cell membrane. Moreover, the extracellular conductivity, protein content, and ergosterol biosynthesis were analyzed. [Results] Carvacrol and eugenol significantly suppressed the mycelial growth and spore germination of F. solani in a dose-dependent manner with the median effective concentration (EC₅₀) values of 92.39 μL/L and 263.00 μL/L, respectively. According to the observation under SEM, the cell wall and cell membrane of F. solani were damaged after exposed to carvacrol and eugenol. As a result, the mycelia had bends, folds, and depressions rather than the normal linear morphology. PI staining revealed that two essential oils broke F. solani cell membrane and enhanced the membrane permeability, resulting in the outflow of cytoplasmic contents and the surge in extracellular conductivity and protein content. After treatment with high concentration of carvacrol (400 μL/L) and eugenol (800 μL/L) for 48 h, the ergosterol content in F. solani decreased by 78.61% and 67.73%, respectively. [Conclusion] Carvacrol and eugenol inhibit the mycelial growth and spore germination of F. solani, damage the cell membrane, enhance membrane permeability, and disrupt the ergosterol biosynthesis on cell membrane, thereby exerting the antifungal activity. In conclusion, this study lays a theoretical basis for the screening of biocontrol agents of F. solani.

Abstract:[Background] Soil microbes play an important role in regulating plant growth, improving soil nutrient utilization rate and maintaining ecosystem stability. [Objective] To explore the structure diversity of the microbial community in cucumber rhizospheric soil under the in situ treatment of waste by earthworms and the treatment with desulfurization gypsum for soil improvement, and reveal the relationship between the number of soil microorganisms and microbial diversity indexes. [Methods] We determined the number of cultivable fungi, bacteria, and actinomycetes in the cucumber rhizospheric soils treated with three different amendments (T1–T3), and employed Biolog-ECO microplate method to study the metabolic functions of microbial communities. We then compared the number and metabolic function diversity between the in-situ treatment of waste by earthworms (T1), the treatment with desulfurization gypsum (T2), and the combination of the above two treatments (T3). [Results] The number of cultivable actinomycetes of T1 and T3 treatments was significantly higher than that of CK and T2, and that of bacterial and fungal colonies had no significant difference among treatments. The AWCD values of different treatments presented an upward trend, rising rapidly within 0–96 h and then slowly to the maximum. The AWCD values of T1 and T3 were higher than those of CK and T2. The AWCD, Shannon index, Simpson index, Pielou index, and McIntosh index all showed significant differences among treatments. Compared with CK, T1 and T2 had efficient carbon source utilization. The number of bacteria and actinomycetes had positive correlations with AWCD value, Shannon index, Simpson index, McIntosh index, and Pielou index (p<0.05 or p<0.01), while that of cultivable fungi was negatively correlated with AWCD value, Shannon index, Simpson index, McIntosh index, and Pielou index (p<0.05 or p<0.01). [Conclusion] Upon the in situ treatment of waste by earthworms, the soil can harbor more microbial communities with higher diversity and higher efficiency of carbon source utilization, which reveals the good performance of earthworms in the treatment of waste and the corresponding changes of ecological functions under the new cultivation mode. The findings of this study are expected to provide a theoretical basis for new cultivation modes.

Abstract:[Background] Salt stress affects the growth of soybean seedlings, while endophytic bacteria can improve the resistance of soybean seedlings to salt stress. [Objective] This study aims to investigate the effects of endophytic Bacillus subtilis 127 and B. proteolyticus 133 on the activities of superoxide dismutase (SOD) and peroxidase (POD) in soybean seedlings under salt stress. [Methods] Pot experiments were carried out with the soybean variety ‘Xudou 20’ in three groups: control group, salt stress group, and salt stress group with strain inoculation. Under artificial climatic conditions, the soybean seedlings were respectively treated with different concentrations (50, 100, 150, 200, 250 and 300 mmol/L) of NaCl solutions and inoculated with the bacterial suspensions at OD0.33, OD0.50 and OD0.75 (OD₆₀₀ value). [Results] After 14 days, the SOD activity was 1.04 U/g-FW in the soybean seedlings treated with 300 mmol/L or 100 mmol/L NaCl solution and inoculated with B. subtilis 127 suspension at OD0.33 or OD0.75; the POD activity was the highest (7 820 U/(g·min)) in the soybean seedlings treated with 300 mmol/L NaCl solution and inoculated with B. proteolyticus 133 suspension at OD0.50. After 28 days, the SOD activity was the highest (0.88 U/g-FW) in the soybean seedlings treated with 150 mmol/L NaCl solution and inoculated with B. subtilis 127 suspension at OD0.50; the POD activity was the highest (8 300 U/(g·min)) in the soybean seedlings treated with 100 mmol/L NaCl solution and inoculated with B. proteolyticus 133 suspension at OD0.50. [Conclusion] The optimum salt concentrations for endophytes B. subtilis 127 and B. proteolyticus 133 are different, and inoculation of endophytes can increase the SOD and POD activities in soybean seedlings exposed to salt stress. Both B. subtilis 127 and B. proteolyticus 133 can improve the salt tolerance of soybean seedlings.

Abstract:[Background] Bacteria would enter a viable but non-culturable (VBNC) state under oxidative stress, and their colony forming ability may be affected by sublethal injury that imposed on bacterial cells. Currently, the quantitative detection of VBNC bacteria is based on the difference between the viable and culturable counts. Therefore, the accurate detection of culturable counts is critical to quantitating the number of VBNC cells. In addition, a proper growth medium might avoid the missing detection of viable pathogenic bacteria. [Objective] To analyze the effect of medium composition on the detection of bacteria exposed to sublethal injury caused by oxidative stresses, and to explore the formation of VBNC state of Salmonella enterica serovar Enteritidis under oxidative stresses. [Methods] The culturable cells in Luria-Bertani (LB), beef peptone yeast (BPY), and Salmonella Shigella (SS) medium were counted and compared. The formation of VBNC S. enterica serovar Enteritidis was quantified respectively with RT-qPCR and fluorescent staining under confocal laser microscope. [Results] The sublethal injured cells under oxidative stress could be detected with non-selective LB and BPY medium, and the counts of culturable cells reduced in SS medium because of bile salt. The formation rate of VBNC S. enterica serovar Enteritidis exposed to H₂O₂at 53 °C for 1.5 h was significantly higher than that in the other two cases (P<0.05). [Conclusion] For the detection of VBNC state, a suitable medium should be selected to detect culturable cells. Oxidative stress would induce the VBNC state of food-borne pathogens, which deserves special attention as H₂O₂ is widely used as a disinfectant in food processing and medical treatment.

Abstract:[Background] Carbohydrate is closely related to the colonization and pathogenicity of Streptococcus suis in hosts, and the glycogen released from host cells may be an important carbon source of S. suis. [Objective] This paper aims to analyze the effect of exogenous glycogen on the transcriptome response of S. suis, especially the virulence genes. [Methods] The virulent S. suis type 2 SC19 strain was cultured in the glycogen and glucose medium, respectively. Through high-throughput transcriptome sequencing, the effect of glycogen on the metabolic pathways and virulence genes of S. suis were identified and analyzed, followed by verification through in vitro experiment and challenge test. [Results] S. suis grew well in the medium containing glycogen as the carbon source. Transcriptome data showed 908 (46.07% of the whole genome) differentially expressed genes under the glycogen culture condition, with 501 up-regulated and 407 down-regulated. Enrichment analysis revealed that glycogen affected a wide range of basic metabolic pathways in S. suis, but the glycolysis pathway remained stable. The expression levels of 30 virulence genes significantly changed. In particular, a series of known and important virulence factors, such as SLY, ApuA, and ArcABC, were highly up-regulated (fold change>20). Moreover, results showed that the hemolytic activity, adhesion, and invasion of S. suis cultured in glycogen significantly enhanced, and that its virulence to the tested animal model predictably increased. Thus, it was confirmed that S. suis responded to glycogen and glycogen regulated the pathogenicity of S. suis. [Conclusion] Exogenous glycogen significantly affected the genome expression profile of S. suis. Such response to the carbon source suggests the adaptability of the bacteria to the changing habitats and the close relationship between the carbohydrate and S. suis pathogenicity.

Abstract:[Background] Riemerella anatipestifer (RA) causes septicemia and infectious serositis in a variety of birds such as duck, leading to serious economic losses in the poultry industry. Protein vaccination is considered to be one of the most important strategies for preventing RA infection in ducklings. At present, there are few studies on the immunogenicity of RA recombinant proteins, and their application is also limited by the insufficient specific immune response induced by the single protein antigen. [Objective] The study aims to explore the immune response induced by the molecular chaperone DnaK, outer membrane protein A (OmpA) and OmpA-DnaK vaccines in ducklings, and to evaluate whether the recombinant fusion protein OmpA-DnaK has enhanced immunogenicity, which is conducive to the further development of RA vaccines. [Methods] We amplified the DnaK and OmpA genes and linked them to the pET-32a(+) vector, respectively. OmpA gene was ligated to the upstream region of DnaK gene through the restriction sites of Nco I and BamH I. DnaK, OmpA and OmpA-DnaK proteins were prepared by prokaryotic expression and purification. The DnaK, OmpA and OmpA-DnaK vaccines were respectively used to immunize ducklings twice via subcutaneous injection, and then the serum antibody titers, lymphocyte proliferation and cytokine (IL-2 and IL-4) levels were determined. The ducklings were then challenged by RA-GH5 strain through intramuscular injection, and the histopathological changes and immune protection rate were examined. [Results] The recombinant DnaK, OmpA and OmpA-DnaK proteins were expressed, with molecular weights of approximately 90, 60 and 130 kDa, respectively. The three recombinant protein vaccines induced the humoral and cellular immune responses of ducklings, and mitigated the histopathological damage caused by RA. Compared with DnaK or OmpA alone, OmpA-DnaK significantly increased serum antibody titer, lymphocyte proliferation index, and IL-2 and IL-4 levels after vaccination. The immune protection rate of OmpA-DnaK to ducklings was 50%, and those of both DnaK and OmpA were 30%. [Conclusion] The recombinant OmpA, DnaK and OmpA-DnaK protein vaccines all induced immune responses in ducklings. Moreover, the fusion of DnaK and OmpA caused stronger humoral and cellular immune responses.

Abstract:[Background] Streptococcus parauberis is a major aquatic pathogen which has gradually evolved new serotypes and multi-drug resistance. Therefore, it is urgent to develop a new antibacterial drug for the prevention and treatment of the disease caused by this pathogen. Studies have demonstrated that a lysin encoded by a prophage can effectively kill the host and has a good prospect of antibacterial application. [Objective] In this paper, we investigated the host spectrum of the prophage lysin from S. parauberis and optimized the conditions for the lytic activity. [Methods] We used bioinformatics tools to analyze the whole genomic sequence of the S. parauberis strain KRS02083 and discovered a lysin gene Sply828. By gene cloning, protein expression and purification, we obtained the lysin Sply828. We then evaluated the activities against different bacteria and the optimal lysis conditions of Sply828 by performing the turbidity decrease assay. [Results] Sply828 displayed the best activity against fish-derived S. parauberis and exhibited the highest activity against the bacteria in the exponential growth phase. This enzyme had the highest activity at 28 °C and pH 6.2, and its activity can be improved by Ca²⁺ and Mg²⁺ while inhibited by Zn²⁺, Cu²⁺, Fe²⁺, and Ni²⁺. [Conclusion] The prophage lysin Sply828 existing in the genomic sequence of S. parauberis possesses efficient and specific bactericidal activity, providing a preliminary basis for the prevention and control of the disease caused by S. parauberis.

Abstract:[Background] In recent years, largemouth bass (Micropterus salmoides) has been prone to white skin disease during seed stage in Guangdong, Guangxi, and other places, with the symptoms obviously different from those reported clinically, which seriously endangers the production of largemouth bass seeds. [Objective] To provide scientific reference for the research and treatment of white skin disease of largemouth bass, we identified the pathogen and analyzed its growth characteristics, virulence factors, pathogenicity, and antibiotic susceptibility as well as the fish histopathological changes caused by it. [Methods] The pathogen was isolated from the focus of largemouth bass suffering from white skin disease and identified based on the morphological characteristics, physiological and biochemical indexes, and 16S rRNA and gyrB gene sequence analysis. Further, the growth curve of the strain was established, and the effects of temperature, pH, and salinity on the growth were analyzed. The activities of virulence factors were detected by plate method, and the virulence genes were screened out by PCR method. The pathogenicity of the strain and histopathological changes caused by it were confirmed by immersion challenge experiments. The susceptibility of strain to 10 antibiotics and 6 disinfectants commonly used in aquaculture was determined with microdilution method. [Results] The dominant strain ZJS18004 was isolated from the largemouth bass with white skin disease, which was identified as Aeromonas veronii. The optimum growth conditions of ZJS18004 were 30 °C, pH 8.0, and salinity 5‰. The growth curve at 30 °C showed that 0–1 h and 1–5 h were the retardation phase and logarithmic phase, respectively. Strain ZJS18004 caused hemolysis, had protease and lipase activities, and carried six virulence genes including aer, act, fla, ahyB, exu, and lip. The results of challenge experiments showed that ZJS18004 had strong pathogenicity to healthy largemouth bass at 23 °C, 28 °C, and 33 °C. The tested fish exhibited disease signs similar to those in natural infections. The pathogenicity was closely correlated with the bacterial concentration and water temperature. The infection of ZJS18004 caused obvious pathological damage such as muscle tissue lysis and necrosis of the liver, spleen, and kidney. The antimicrobial susceptibility assay revealed that ZJS18004 was susceptible to doxycycline hyclate, neomycin sulphate, thiamphenicol, and florfenicol. Six commonly used aquatic disinfectants had certain bactericidal effects on ZJS18004 in vitro, among which trichloroisocyanuric acid ranked first, followed by benzalkonium bromide. [Conclusion] A. veronii was the major pathogen of white skin disease attacking largemouth bass. The results of the present study will build a solid foundation for further analyzing the pathogenic mechanism of A. veronii and carrying out the research on accurate prevention and control.

Abstract:[Background] It is an important goal for amylase development to improve the stability under variable industrial production conditions. [Objective] Amylase is widely used in food processing, desizing of cloth, brewing, and breeding industry. However, current amylase in industrial production features a few sources available and poor stability in solution. Thus, efforts should be made to expand the sources of amylase and enhance the stability of the enzyme so that it can adapt to the complex and changeable production environment. [Methods] A total of 20 strains with amylase activity were screened out from the surface soil of the Yellow River Wetland in Sanmenxia with a selective medium. The activity of the crude enzyme solution was detected with the cylinder plate method and 3 strains with high amylase activity were preliminarily identified. Among them, the strain with highest amylase activity was mutagenized by ultraviolet (UV) and the lethality rate was determined. The strain with the highest enzyme activity after mutagenesis was selected. Then the culture conditions were optimized, and the activity of amylase after mutation and the ranges of action conditions were determined. 3,5-dinitrosalicylic acid (DNS) method was used to determine the enzyme activity before and after mutation for a comparison. [Results] The 3 strains with high amylase activity were numbered S03, S08, and S17. The 16S rRNA gene sequence alignment, Gram staining, and physiological and biochemical characterization revealed that strains S03, S08, and S17 are members of Aeromonas, Exiguobacterium, and Bacillus, respectively. The optimal NaCl concentration for the amylase was 10%−12%, and the optimal temperature was 45 °C (S03, S17) and 25 °C (S08). Moreover, the optimal pH was 7.0−9.0, and the optimal fermentation time was 36 h (S03 and S17) and 42 h (S08). After UV mutagenesis, the strain with the highest enzyme activity was screened out and marked as S17M. S17M grew well at pH of 8.0 and 40 °C. Mn²⁺, Ti⁴⁺, and Mg²⁺ promoted the growth of S17M, while Cu²⁺, Fe²⁺, Fe³⁺, Na⁺, and organic solvents (particularly formaldehyde and glacial acetic acid) inhibited it. S17M demonstrated high amylase activity in the presence of NaCl at 8%–14%, at pH of 5.0–10.0, and at 15–55 °C, and the inhibitory effect of metal ions and organic solvents was decreased. The maximum enzyme activity was up to 195.62 U/mL, 3.19 folds that of the original strain. [Conclusion] UV mutagenesis can significantly improve enzyme activity and stability, so that the resultant enzyme can adapt to the variable industrial production environments. Thus, it has the potential to be applied to different industrial production conditions.

Abstract:[Background] High wuyiencin-producing genetically engineered strain Streptomyces albulus OoWysR showcases pronounced antifungal effect. [Objective] To identify the active ingredients of S. albulus OoWysR. [Methods] The active ingredients of S. albulus OoWysR were separated and purified by the column chromatography of macroporous resin, ion-exchange resin column chromatography, and high-performance liquid chromatography, respectively. The chemical structures of compounds were identified by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), nuclear magnetic resonance (NMR), and other spectral methods. The biological activity of compounds was determined with the growth rate method. [Results] Four compounds were isolated and identified from S. albulus OoWysR, which were respectively (1) p-hydroxybenzoic acid, (2) pyrrole-2-carboxylic acid, (3) p-hydroxyphenethyl alcohol, and (4) yunnanmycin. Compound 1 showed inhibitory effect on Curvularia lunata, Fulvia fulva, Alternaria alternata, and Bipolaris maydis. Compound 2 exerted certain inhibitory activity against Botrytis cinerea, Sclerotinia sclerotiorum, B. maydis, Valsa ceratosperma, Fusarium graminearum, Pyricularia oryzae, and A. alternata. Compound 3 had specific inhibitory activity against B. cinerea, B. maydis, Botryosphaeria dothidea, and Colletotrichum gloeosporioides. [Conclusion] The active ingredients of S. albulus OoWysR include p-hydroxybenzoic acid, pyrrole-2-carboxylic acid, p-hydroxyphenethyl alcohol, and yunnanmycin, in addition to wuyiencin. This study provides a theoretical basis for further development and utilization of this strain.

Abstract:[Background] Pseudomonas aeruginosa (PA) is a common opportunistic pathogen in clinical practice, and its heteroresistance often leads to failure of clinical treatment. [Objective] To study the heteroresistance of PA to penicillins, and to provide a basis for the treatment of related clinical infections. [Methods] We collected 50 clinical isolates of PA and investigated their heteroresistance characteristics by using disk diffusion method (Kirby-Bauer (K-B) test), population analysis profile (PAP), growth assay and passage stability test. [Results] According to the preliminary screening results obtained with K-B test, 52%, 52%, and 54% of the PA isolates had heteroresistance to piperacillin (PIP), piperacillin/tazobactam (TZP) and ticarcillin/clavulanic acid (TIM), respectively. Thirteen (26%) strains were confirmed to have heteroresistance by PAP. Eight heteroresistant strains were randomly selected, and the frequency of resistant subpopulations ranged from 7.3×10⁻⁷ to 1.2×10⁻⁵. Without antibiotic pressure, the heteroresistant strains PAS92 and PAS57 and their three subpopulations on the plates with the highest PIP concentration showed no statistical difference in growth rate (P>0.05), while the growth of the subpopulations in the inhibition zone of PAS92 was faster than that of PAS92 within 8–12 h (P=0.002 2<0.01). The subpopulations with different levels of resistance in the inhibition zone and those with high levels of resistance selected from the plates with the highest PIP concentration in PAP were subcultured without antibiotic pressure. Only one of the subpopulations was found to be unstable and returned to the sensitivity level of the original strain, while the others had good stability of resistance. [Conclusion] In this study, PA isolates show low heteroresistance ratio while high frequency of resistant subpopulations to penicillins. Besides, they have high heteroresistance to PIP, and most of the resistant subpopulations had good passage stability. Therefore, when using this type of antibiotics, attention should be paid to the occurrence of heteroresistance to prevent the emergence of strains with higher drug-resistance from causing treatment failure.

Abstract:[Background] For calcium oxalate stone, a common clinical disease with high recurrence rate, surgery is the only solution due to the hardness of the stones, from which, however, patients suffer a lot. It has been verified that intestinal flora affects the formation of calcium oxalate stones and thus reduces the incidence. [Objective] To investigate the effect of Lactobacillus plantarum (BNCC 194165) on calcium oxalate stones in mice. [Methods] For in vitro experiment, a total of 0.02 mol/L sodium oxalate was added to the MRS broth to prepare the screening medium (MRS-OX) and then 200 μL 3.48×10¹² CFU/L L. plantarum suspension was inoculated into the MRS-OX to yield the bacteria-containing medium (B+MRS-OX). MRS-OX and B+MRS-OX at equivalent volume were cultured at 37 °C for 2 days and the concentration of residual oxalic acid was measured by the oxalic acid kit. As for the in vivo experiment, 10-week-old male Kunming mice were randomized into the control (C) group, L. plantarum group (B), glyoxylic acid (GA) group, and L. plantarum+glyoxylic acid (B+GA) group, with 5 in each group. Glyoxylic acid was used to induce calcium oxalate stone in mice. A total of 200 μL 3.48×10¹² CFU/L L. plantarum suspension was given to the model mice and then the effect on calcium oxalate stones was observed. After the experiment, the variation of body weight in each group was plotted and kidney index was calculated. Then we detected the haematological indexes (creatinine, blood urea nitrogen, blood concentration of oxalic acid) and oxidative stress indexes of total superoxide dismutase (SOD) and malondialdehyde (MDA). Moreover, the pathological sections of kidney were observed and renal crystallization score was calculated. Through the above experiments, the effect of L. plantarum on calcium oxalate stone formation in mice was preliminarily explored. [Results] In the in vitro experiment, the oxalic acid-decomposing efficiency of L. plantarum in MRS-OX was 10%. In the in vivo experiment, L. plantarum significantly inhibited the weight loss of mice and reduced the levels of plasma creatinine, urea nitrogen, and oxalic acid. Pathological observation showed that the renal crystals of B+GA group were significantly reduced and the crystallization score was different from that of the GA group (P<0.01). Moreover, the oxidative stress damage of kidney caused by calcium oxalate stones was alleviated in the B+GA group. [Conclusion] L. plantarum can relieve the damage of calcium oxalate stones to kidney and reduce the generation of kidney crystals, which can be used for preventing the formation of calcium oxalate stones.

Abstract:[Background] The traditional method of bacterial culture is the gold standard for the diagnosis of lower respiratory tract infection. However, bacterial culture needs a long cycle and has low sensitivity. Loop-mediated isothermal amplification (LAMP) as a simple and rapid method can be used for rapid detection of common bacteria in clinical respiratory tract infection. [Objective] To evaluate the detection capability of LAMP for seven common bacterial species of respiratory tract infection. [Methods] Specific primers were designed for the LAMP of seven common pathogens of respiratory tract infections, including Klebsiella pneumoniae (KP), Acinetobacter baumannii (AB), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), Streptococcus pneumoniae (SP), Moraxella catarrhalis (MC), and Haemophilus influenzae (HI). The sensitivity and specificity of this LAMP method were evaluated by gradient dilution method and cross-reaction experiment, respectively. We then retrospectively analyzed the residual tract specimens (sputum and bronchoalveolar lavage fluid) from 240 patients with suspected lower respiratory tract infection in Peking University Peoples Hospital from November 2019 to March 2021. After DNA extraction by the automatic nucleic acid extraction system, we employed LAMP to detect the seven common pathogens. Further, we compared the results obtained from LAMP and bacterial culture to evaluate the sensitivity and specificity of LAMP. The cut-off value (C_t value) of different species were optimized, and the application value of LAMP in clinical practice were discussed. [Results] The LAMP method had a good linear correlation with the template concentration. The LAMP system had good specificity as there was no cross reaction with other strains. A total of 218 sputum specimens and 22 bronchoalveolar lavage fluid specimens were detected in this study. The seven pathogens were detected in 178 specimens based on the bacterial culture method and in 176 specimens based on the LAMP method. The LAMP method generally consumed 2–3 h for detection and could simultaneously detect multiple specimens. The optimal C_t values of HI, KP, AB, MC, SA, PA, and SP were 18.5, 20, 20, 15, 25, 19, and 18, respectively. The sensitivity and specificity were as follows: KP (90.7%, 94.1%), AB (84.0%, 94.2%), PA (90.8%, 89.1%), MC (75.0%, 99.2%), SA (81.8%, 97.4%), HI (75.0%, 90.3%), and SP (33.3%, 95.4%). The LAMP results for KP, AB, and PA showed good coincidence with the semi-quantitative results of sputum culture, and the R² values were 0.855 7, 0.804 4, and 0.924 3, respectively. [Conclusion] LAMP had low requirements for operating personnel, short cycle, and high specificity (89.1%–99.2%) compared with the culture method. It has high sensitivity (81.8%–90.8%) for the detection of K. pneumoniae, A. baumannii, S. aureus, and P. aeruginosa, and thus can be used for the rapid detection of these bacteria in clinical respiratory tract infection. The results of LAMP were in good agreement with the semi-quantitative results of sputum culture. The detection of H. influenzae and S. pneumoniae needs to be further evaluated due to the lack of positive culture specimens.

Abstract:[Background] Since the 1990s, H9N2 avian influenza virus has become a major threat to human and animal health. [Objective] To understand the molecular evolutionary characteristics of H9N2 avian influenza virus in a live poultry market in central Jiangsu, 2019–2020. [Methods] The specimens were detected by real-time quantitative PCR method, and SPF chicken embryos were used for the isolation of virus strains from the specimens. The whole genome of each isolate was sequenced with specific primers. Blast, ClustalX and Mega 6 were used for sequence alignments and phylogenetic analysis. [Results] A total of 231 environmental and poultry specimens were collected from a farmers’ market from 2019 to 2020, from which of 34 influenza A virus strains were detected, including 33 strains of H9N2 subtype. Twenty strains of H9N2 virus were isolated with SPF chicken embryos, and the whole genomes of 11 strains were sequenced and aligned. The HA and NA gene sequences indicated that the 11 strains belonged to G57 genotype of Y280-like lineage. According to the evolutionary characteristics of HA and NA, the 11 strains can be divided into 5 gene constellations (A, B, C, D and E), of which constellation A (n=5) was dominant. The cleavage site of HA1 and HA2 subunits of HA protein of the 11 strains was a basic amino acid R, which indicated low pathogenicity. Four mutations (including I155T, H183N, A190T/V and Q226L) were occurred in the receptor-binding region of HA protein, indicating that these viruses had enhanced ability of binding to human SAα2-6Gal receptor. [Conclusion] The H9N2 avian influenza virus in the live poultry market in central Jiangsu is characterized by active evolution and has a risk of transmission to people. Efforts should be made to strengthen the monitoring and the research on cross-species transmission of avian influenza virus in the live poultry market.

Abstract:Phage research and applications have been booming in medicine, animal husbandry, veterinary medicine, and food production. However, as an alive microorganism with protein capsid, phage may encounter loss of activity, short storage, and inconvenient transportation in liquid state when being applied in the prevention and control of bacterial pollution and infection. Therefore, to find suitable excipients for phage encapsulation is an urgent problem to be solved for phage therapy. We reviewed the effects of the types, formulations, and encapsulation technologies of excipients used in phage preparations on the resistance and storage stability of phages, and summarized the major research achievements in this field. In this review, we hope to find suitable formulations and technologies of excipients for solid-state phage preparations, which will lay a foundation for the targeted delivery and release of phages and contribute to the clinical application of phage therapy.

Abstract:Flagella are slender and curved protein filaments associated with the surface of many bacteria. As the motility ‘organ’ of bacteria, flagellum is one of structures that have been most extensively studied in microbiology. Through flagellar movement, bacteria can better adapt to their habitat or escape from unfavorable environments. In addition, flagellar plays an important role in the colonization of host surfaces by harmful or beneficial bacteria, and in their biofilm formation and other tight interaction with their hosts. Bacillus is ubiquitous in the nature containing many species and strains with great value in industrial, agricultural and medicinal applications. In this work, the research progress on the flagella and motility-related properties of Bacillus is reviewed. Firstly, we introduced the structural composition, assembly process, and expression regulation of synthetic genes of Bacillus flagella. Secondly, relationships and the underlying molecular mechanisms of Bacillus flagella motility and its motility-related characteristics of including the biofilm formation and dispersal, sporulation, competence, and production of γ-polyglutamic acid and antibiotics is discussed. This review aims at providing comprehensive knowledge and theoretical guidance for related studies in this field.

Abstract:Campylobacter jejuni, a worldwide prevalent food-borne zoonotic pathogen, is a Gram-negative microaerophilic bacterium. It is extremely sensitive to environmental conditions such as oxygen, temperature, pH, and bile salts, and suffers from adverse conditions including lethal reactive oxygen species (ROS) during the spread and colonization. Therefore, the response to ROS is an important strategy for C. jejuni. C. jejuni has evolved a variety of mechanisms to resist oxidative stress, which involve flagella and flagellum-mediated motility. Therefore, we comprehensively expounded the research progress on the mechanisms of C. jejuni in response to oxidative stress, especially that mediated by flagella, aiming to lay a foundation for further improving the regulation system of Campylobacter jejuni in response to oxidative stress and provide information for the prevention and control of Campylobacter at the source.

Abstract:The biofilm of sulfate-reducing bacteria (SRB) is regarded as the main culprit of the microbially influenced corrosion, but it is also the key to the microbial remediation of heavy metal-polluted water. The formation and regulation mechanisms of biofilm are very important for SRB control and utilization. In this study, we summarized the research progresses on SRB biofilm, including extracellular components of biofilm and biofilm control factors, and elucidated the role of regulators in the biofilm formation.

Abstract:The emerging of multidrug resistance (MDR) in Gram-negative bacteria has aroused worldwide concern. Overexpression of efflux pumps, especially the resistance-nodulation-cell division (RND) efflux pumps, has been demonstrated to be closely associated with MDR in Gram-negative pathogens. In RND family, AcrAB-TolC efflux pump, which is widely present in Gram-negative bacteria, is a decisive factor of bacteria to develop MDR. To develop effective efflux pump inhibitors, we need to clear understand the structure of AcrAB-TolC. The available studies about the structure of this pump are limited to individual components or the whole pump structures determined by X-ray crystallography or single-particle cryo-electron microscopy. The recently developed cryo-electron tomography sheds new light on the assembly and operating mechanism of this pump in the native cell membrane environment. Here, we summarize the contributions of the structural data of AcrAB-TolC efflux pump to the discovery of pump inhibitors against antibiotic resistance in bacteria.

Abstract:Bisphenols (BPs) are widely distributed in nature due to their extensive use as important components in industrial raw materials and pharmaceutical and personal care products (PPCPs). The ecological risks caused by BPs as estrogen-like hormones have aroused global concerns. Significant progress has been achieved in the microbial degradation of BPs as researchers have isolated a variety of bacterial strains or consortia via different approaches. This review summarizes relevant recent studies, focusing on key bisphenol A (BPA)-degrading genes in different pathways of degradation. The enzymes in the same pathways and their modes of action are compared in detail, and the effects of these enzymes on BPA degradation are analyzed. We review the research progress on the degradation of BPs by bacteria and discuss the future research directions on the mechanism and application of bacterial degradation of BPs.

Abstract:Biosurfactants, amphiphilic substances with both hydrophobic and hydrophilic groups, are mainly produced by microbial fermentation, and can significantly reduce surface and interfacial tensions. Compared with chemical surfactants, biosurfactants are characterized with low toxicity, high biocompatibility, and high degradability, with the application potential in a wide range of fields. However, the high production costs hinder the commercialization of biosurfactants. This review summarize the production of biosurfactants, particularly focusing on the optimization of production processes and metabolic pathways, to explore key factors affecting yield and costs, thus providing solutions for the commercial development of biosurfactants.

Abstract:Polymer microfluidic chips have a wide range of application in medicine, biological detection, chemical synthesis and other fields because of its low cost and easy fabrication. Polymer materials based on thermoplastic polymer polymethylmethacrylate (PMMA) and thermosetting polymer polydimethylsiloxane (PDMS), with good biological compatibility and optical transparency, are commonly used as the base material for the preparation of microfluidic chips in biomedicine. However, in view of the particular application scenarios, this kind of chips need to be sterilized prior to use to avoid microbial interference. The available sterilization methods for PMMA and PDMS based materials include autoclaving, ultraviolet, electron beam, ⁶⁰Co gamma radiation, supercritical carbon dioxide sterilization, ethanol, hydrogen peroxide, ethylene oxide, chlorogenic acid and detergent. This paper reviews and summarizes the related operation methods for the PMMA and PDMS based microfluidic chips from basic principle, sterilization methods, and application scenarios. Meanwhile, we analyze the applicable sterilization methods according to chip material and application, aiming to provide reference for the sterilization of microfluidic chips in biomedicine.

Abstract:The intestinal symbiotic Akkermansia muciniphila comprises 1%–3% of gut microbiota, which induces T cell-dependent immune response during intestinal homeostasis. Thus, it may be involved in immune tolerance of normal gut. Experiments on disease and model animals have proven the vital role of this bacterial species in improving metabolic functions and immune response of the hosts, which has attracted the interests of scholars. Previous studies mainly focused on the correlation between A. muciniphila and diseases, but the mechanisms have not been systematically explored. This study aims to discuss the relationships of A. muciniphila with diseases in major human systems and immunity and the mechanisms, which is expected to provide evidence and routes for effective utilization of this bacterial species.

Abstract:Anaerobic ammonium oxidation (anammox) process is the most economical bioprocess for nitrogen removal from wastewater at present, which is conducive to the energy neutral of wastewater treatment plants. Anammox bacteria play a major role in this process. Anammox granular sludge, formed with anammox bacteria, is the most promising sludge form for anammox process, owing to the high settling rate, strong retention capacity, and strong resistance to environmental stresses. Focusing on anammox granules, this paper introduced the characteristics, species, and metabolic pathways of anammox bacteria, and the formation mechanism of anammox granular sludge, and reviewed the extracellular polymeric substance (EPS) and quorum sensing, which are closely related to the aggregation of anammox granular sludge. Moreover, we summarized the future research directions of anammox granules. This review is expected to serve as a reference for the subsequent research on anammox granules and the regulation of anammox process.

Abstract:Lachancea thermotolerans, a species of non-Saccharomyces yeast, has attracted increasing attention because of its excellent enological characteristics such as enhancing aroma and taste. It is characterized by high production of lactic acid, glycerol, 2-phenylethanol, and ethyl esters while low production of ethanol and volatile acids during the fermentation of wine. The available studies have proved the distinct performance among different strains of L. thermotolerans in fermentation. We reviewed the studies about the strain diversity, effect on wine quality, and application in co-fermentation of L. thermotolerans. This review provides a basis for screening the traits and exploring the mechanism of acid and aroma production of the indigenous L. thermotolerans, which will promote the sound development of microbial germplasm resources for winemaking in China.

Abstract:The three pathogenic Yersinia species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica, and the phages can be used for the diagnosis, control, and evolutionary research of Yersinia. This review focuses on the isolation and identification of phages of the three species, compares the genomes of the phages, and summarizes the receptors of them, which is expected to lay a basis for studying and utilizing the phages of the three Yersinia species.

Abstract:Viral disease threaten human beings, animal and plant health seriously. At present, the measures and effects for preventing and treating viral disease are limited. It is necessary to develop non-toxic and eco-friendly antiviral agents to prevent and control viral disease. Probiotics Bacillus species play an antiviral role by producing metabolites or components such as ribonuclease and extracellular polymers, and enhancing immunity, which is expected to be further developed for the prevention and treatment of viral disease.

Abstract:Biological treatment technology has been widely applied in the purification of waste gases owing to its high efficiency, low cost, simple operation, and seldom secondary pollution. However, bioaerosols can be discharged into the atmosphere as secondary pollutants from waste gas treatment facilities. Bioaerosols pose potential hazards and risks to human health due to the carrying and transmission of harmful microorganisms. Bioreactors for waste gas treatment are both sinks and sources of bioaerosols. This paper reviews the emission levels, microbial community structure, and particle size distribution characteristics of bioaerosols released from bioreactors for waste gas removal. Furthermore, we discuss the formation, sources, factors, and exposure risk of bioaerosols, aiming to provide a scientific basis and reference for the identification and control of bioaerosols, especially for those from biological treatment facilities.