Abstract:[Background] The Taklimakan Desert is a special geographical location with less human interference and extreme living environment, and it has uniquely bacteria resources. [Objective] To assess the community structure, diversity and factors influencing of bacteria in the eastern margin of the Taklimakan Desert. [Methods] Analysis the physicochemical properties of sand samples collected from the eastern margin of the Taklimakan Desert, and high-throughput sequencing of environmental 16S rRNA genes based on Illumina MiSeq was used to analyze the diversity and factors influencing of bacteria from the eastern margin of the Taklimakan Desert. [Results] The bacteria of eastern margin of the Taklimakan Desert belonged to definite taxonomic bacteria of 21 phylum 42 classes 304 genera, the dominant bacterial phyla were Bacteroidetes (31.26%), Proteobacteria (29.47%), Actinobacteria (15.71%) and Firmicutes (15.69%). At genus level of taxonomic, the dominant genus was Salinimicrobium (10.06%), followed by Halomonas (7.39%), Bacillus (3.25%), Pontibacter (3.14%), Aliifodinibius (2.76%) and Kocuria (1.76%). TP, SO₄^2- and HCO₃^- were significant effects on the microbial community structure of the eastern margin of the Taklimakan Desert. [Conclusion] This study revealed the community structure and the species diversity of bacteria in the eastern margin of the Taklimakan Desert. This study provides significant theoretical references for the investigation of microbial resource in the Taklimakan Desert.

Abstract:[Background] Microorganisms in reservoir sediments are an important part of the aquatic ecosystem and play an important role in the circulation of sediment materials. [Objective] To reveal the characteristics of bacterial community structure and the influencing factors in the surface sediments of the reservoir under the influence of antimony-containing wastewater. [Methods] Based on the Illumina high-throughput sequencing technology, the bacterial community structure of the surface sediments in Lengshuigou Reservoir was studied and its correlation with the physical and chemical properties of these sediments was analyzed too; the bacterial function was predicted and analyzed based on FAPROTAX. [Results] The high-throughput sequencing results showed that the bacterial community in Lengshuigou Reservoir is relatively rich, and could be divided into 81 phyla. The dominant phyla included Proteobacteria (40.32%-20.19%), Bacteroidetes (25.89%-4.44%) and Desulfobacter (9.43%-2.02%). Pearson correlation analysis indicated that different speciation of antimony are significantly correlated with a number of bacterial communities under different taxonomy levels. FAPROTAX was used to predict the function of bacteria. The results showed that aerobic heterotrophic bacteria dominated (14.59%-23.58% of the total bacteria), including chemoheterotrophic and aerobic heterotrophic bacteria. In addition, the relative abundance of functional microorganisms related to carbon, nitrogen and sulfur cycling and human pathogenic microorganisms (accounting for 12.42%-32.89% of the total bacteria) were also at a high level, which was significantly related to the geographical conditions of the reservoir and the surrounding environment and the type of pollutants. The heavy metal pollution load index method was used to evaluate the heavy metal pollution in the reservoir. [Conclusion] This study revealed the bacterial community structure characteristics and influencing factors in the sediments of the reservoir (built in 2015) affected by antimony mine wastewater, and analyzed and predicted the related functions of bacteria to study the biogeochemistry of antimony in the reservoir.

Abstract:[Background] Yersinia enterocolitica, an important food-borne zoonotic pathogen, is commonly exposed to cell envelope stresses due to its living environment and epidemic properties. The envelope stress responses of Y. enterocolitica play a very important role in maintaining its environmental tolerance and virulence. [Objective] Explore the role of Y. enterocolitica in regulation of cell envelope stress. [Methods] Polymyxin B was used to disrupt the cell membrane stability of Y. enterocolitica, and we explore the response of regulator of capsule synthesis (Rcs) system to the envelop stress by polymyxin B from the changes in growth, motility ability, biofilm formation ability and related gene expression of Y. enterocolitica. [Results] The cell membrane pressure caused by polymyxin B inhibited the motility and biofilm formation of Y. enterocolitica, which were restored after blocking the Rcs signaling pathway. The expression of genes regulated by Rcs system, such as flhC, hmsS and hmsT, were analyzed. The results further confirmed the regulation of Rcs system in response to the membrane pressure induced by polymyxin B. After sensing the envelop stress signals, Rcs system could actively regulate gene expression and cause pleiotropic changes to improve the resistance of Y. enterocolitica to polymyxin B. [Conclusion] This study clarified the specific regulatory role of the Rcs system in response to membrane stress caused by polymyxin B, and will deepen the understanding of the environmental response mechanisms of Y. enterocolitica.

Abstract:[Background] Clostridium cellulolyticum is a mesophilic model strain for ethanol production from lignocellulose. Constructing the gene targeting technology with controllable induction is an important means to study the genetic regulation of C. cellulolyticum. [Objective] To construct an anhydrotetracycline-inducible ClosTron gene targeting system in C. cellulolyticum.[Methods] Firstly, we determined the sensitivity of C. cellulolyticum to anhydrotetracycline and selected the appropriate inducer concentration, and then tested the inducible effect using β-glucosidase gene as reporter gene. Secondly, we constructed an anhydrotetracycline-induced ClosTron plasmid by combining the anhydrotetracycline-inducible operon with group II intron elements. Finally, the mspI, ldh and ack were selected as targets to determine the targeting efficiency in C. cellulolyticum.[Results] The anhydrotetracycline-inducible system could induce the ClosTron expression in C. cellulolyticum, and the targeting efficiency at mspI, ldh and ack sites were 29.48%±15.51%, 23.61%±7.08% and 28.09%±6.97%, respectively. [Conclusion] The anhydrotetracycline-inducible ClosTron system was successfully constructed in C. cellulolyticum, which laid a foundation for genetic engineering of Clostridium.

Abstract:[Background] Sea island cotton is more susceptible to fusarium wilt than upland cotton. Once it occurs, it is difficult to cure, making fusarium wilt gradually become the main disease threatening the development of sea island cotton industry in Xinjiang, but the pathogenesis is still unclear. [Objective] In order to reveal the genetic variation and pathogenic mechanism of Fusarium oxysporum, the green fluorescent protein (GFP) labeled transformants of F. oxysporum were obtained and used to observe its infection pathway to sea island cotton. [Methods] Agrobacterium tumefaciens-mediated transformation, Agrobacterium tumefaciens-mediated transformation (ATMT) mediated genetic transformation of cotton fusarium wilt race st89 is carried out and the transformation conditions were optimized. [Results] The optimum conditions for ATMT transformation of F. oxysporum:150 mg/L hygromycin could completely inhibit the growth of F. oxysporum, 200 mg/L cefotaxime sodium completely inhibited the growth of Agrobacterium LBA4404, the initial concentration of Agrobacterium is OD₆₀₀ 0.2, the pre-culture time of Agrobacterium is 8 h, The concentration of conidia is 10⁵ spores/mL, the volume ratio of spore suspension of F. oxysporum to Agrobacterium LBA4404 is 1:1. The concentration of acetosyringone in co-culture is 200 μmol/mL, co-culture time is 4 d, culture temperature after transformation 25℃. Using the optimized transformation system, GFP gene is successfully transferred into F. oxysporum. Maximum transformation efficiency can be achieved 252±7.37 transformants/10⁵ spores. PCR amplification and fluorescence observation showed that GFP gene could be expressed normally. [Conclusion] The acquisition of GFP transgenic F. oxysporum laid a foundation for further study on the invasion mechanism of cotton Fusarium wilt.

Abstract:[Background] Aureobasidium pullulans PA-2 is a fungal strain isolated from the infected poplar leaves in Ping'an district, Haidong city of Qinghai province, China. Previous studies demonstrated that this strain showed herbicidal and antimicrobial activities, suggesting that it may have potential in biological pesticide. [Objective] To understand the information of genome sequence of A. pullulans strain PA-2 and mine gene clusters related to its biocontrol function. These results provide genetic basic information in the future to analyze its biocontrol mechanism and functional modification. [Methods] Based on Illumina HiSeq high-throughput sequencing platform, A. pullulans strain PA-2 was sequenced. Bioinformatics methods were used to genome assembly, gene prediction, functional annotation, prediction of carbohydrate active enzymes and secondary metabolite synthetic gene clusters. Hydrolase activity was measured by Congo red staining and other methods. [Results] The size of the genome is 28 932 793 bp with an average of GC content at 50%, which might code the potential 10 839 genes. At the same time, 4 known secondary metabolite synthetic gene clusters were also predicted, which might code the melanin, burnettramic acid A, ACR-toxin I and abscisic acid. The strain can hydrolyze cellulose and pectin. [Conclusion] This study provides useful information to analyze the internal causes of the biocontrol mechanism of strain PA-2 at the genomic level, and offers reference for further understanding of secondary metabolite synthetic pathways of A. pullulans. It is of great significance for further studies involved in strain PA-2.

Abstract:[Background] Proteus mirabilis (PM) is a zoonotic pathogen, which is widely distributed in environment. In recent years, diseases caused by PM and the drug tolerance to PM has gradually increased, raising the importance on the prevention and control of PM. [Objective] To isolate and identify PM from chicken, and to analyze its biological characteristics, such as drug resistance, pathogenicity, and biofilm formation ability. [Methods] Polymerase chain reaction (PCR) was used to identify 52 clinical PM strains which were isolated from sick chicken between 2019 and 2020. The drug resistance, virulence genes and biofilm formation of the clinical strains on PM were investigated by the drug-resistance phenotype, PCR, crystal violet staining method respectively. [Results] The results of 16S rRNA gene sequencing on 14 selected representative strains indicated that all the detected strains were PM. All isolates were resistant to clindamycin (CLDM), azithromycin (AZI), erythromycin (EM), tetracycline (TET) and rifampin (RFP), and the resistance rates of drugs other than chloramphenicol and ceftriaxone was higher than 50%. The results of detection of 13 virulence genes showed that all PM strains contained hpmA, hpmB, rpoA, mrpA, fliL, zapA, ureC, atfC, atfA and pmfA, while the detectable rates of ucaA, rsbA and hlyA were 19.23% (10/52), 48.08% (25/52) and 0.0% (0/52), respectively. The results of biofilm formation ability showed that all strains could form biofilm. 19.23% (10/52) of the isolates had strong ability of biofilm formation and the 25℃ temperature condition had stronger ability of biofilm formation than at 37℃. [Conclusion] PM from avian harbored various virulence genes, and had strong biofilm formation ability. The drug resistance to PM has increased significantly and resistance patterns were complex. It suggested that the tighter monitor on pathogenicity and drug resistance of PM should be carried out to reduce the risk of PM infection.

Abstract:[Background] Lactobacillus paracasei, as one of the important strains in lactic acid bacteria, is often considered as a potential resource for the development of probiotics. [Objective] L. paracasei PC-01 and L. paracasei Zhang were taken as examples to analyze the genomic differences and genetic backgrounds of different L. paracasei strains, in order to laying a foundation for the identification and development of strains. [Methods] the whole genome sequencing of L. paracasei PC-01 was sequenced by PacBio SMRT third-generation sequencing technology, combining with 2 L. paracasei model strains and the whole genome data of 36 strains which had published. through the comparative genomics methods to revealed the differences of 39 L. paracasei strains. [Results] The genome of L. paracasei PC-01 has one chromosome, with a size of 2 829 251 bp and it's GC content was 46.64%; L. paracasei Zhang contained a plasmid and the genome size was 2 898 456 bp and it's GC content was 46.51%. There were some differences of L. paracasei strains about genome size, the number of plasmid and GC content. L. paracasei is an open genome with a high genome diversity. Phylogenetic tree constructed by core genes has the best effect on species differentiation of L. paracasei. What's more, L. paracasei PC-01 and L. paracasei Zhang were in different evolutionary branches. L. paracasei PC-01 and Zhang had a fragment matching rate is 91% of their genomes, and L. paracasei PC-01 has 10 specific genes related to transcriptional regulation and rhamnoglycan metabolism, such as aguA and clpB. L. paracasei Zhang contains mshA, ltrA specific genes were related to glycosyl transfer and other functions. [Conclusion] Through the comparative genomics revealed the genetic information of L. paracasei PC-01 and Zhang and found the differences of L. paracasei strains, which laid the genetic foundation for the identification and application of L. paracasei in the future.

Abstract:[Background] Streptomyces has always been the main producer of bioactive compounds. However, as the abusing of antibiotics, environmental pollution and drug resistance are becoming increasingly serious problem. The discovery of efficient bio-control factor and novel antibiotics becomes the main methods to solve these problems. [Objective] Obtain the whole genome sequence of the Streptomyces sp. SAT1 and the information about its secondary metabolite gene clusters; analyze the particularity and generality with other streptomycetes by the technology of comparative genomics and pan genomics. Form this, we could provide theoretical basis for illuminating the mechanism of bacteriostasis and growth-promoting in SAT1, and reliable data to reveal the ecological function of Streptomyces. [Methods] The sequence of SAT1 was completed by the third generation sequencing platform PacBio Sequel, then annotated and classified by bio-information technology; the software RAxML and PGAP was used to construct phylogenetic tree and analyze pan-genome, respectively. The prediction and analyze of the secondary metabolite gene clusters was achieved by antiSMASH. [Results] From the complete genome map of SAT1, the length of linear chromosome is 7.47 Mb, with 73% GC content, and four plasmids exist in the strain. Additionally, there are 7 550 genes which encoded proteins and 37 secondary metabolite gene clusters which classified by 29 types in SAT1. And the moenomycin gene cluster was highly homologous to Streptomyces ghanaesis ATCC14672 moenomycin gene cluster. In the 42 streptomycetes, it exists about 20-55 secondary metabolites gene clusters in each strain which classified into PKS, Terpene, Nrps and Heterozygous gene clusters. The dispensable genome was huge in these research objectives. [Conclusion] Streptomyces sp. SAT1 has many common points in the trait of genome and secondary gene clusters with other streptomycetes. We speculate the moenomycin and hygromycin_B gene clusters play an important role in the antibacterial activity of SAT1. In the 42 research objectives, the number of gene clusters and the size of genome has a positive correlation. In addition, the existence of abundant heterozygous gene clusters and large number of dispensable genome illustrate Streptomyces has high levels of horizon gene transfer over long periods of evolution, which possesses important environmental functions.

Abstract:[Background] Abnormal lipid metabolism is an important metabolic event in the development of liver cancer. Role of lactic acid bacteria in regulating glucose and lipid metabolism has been widely reported. [Objective] To investigate whether Lactobacillus plantarum CGMCC8198 could regulate the HMGCR/SMYD3 lipid metabolism pathway, thereby affecting the occurrence and development of liver cancer cells. [Methods] HepG2 cells were treated with different concentrations (5, 10, 15 μg/mL) of Lactobacillus plantarum CGMCC8198 crushed supernatant (LpS) for different times. Western Blot, oil red staining and real-time quantitative PCR (RT-qPCR) were used to detect the effects of LpS on fatty degeneration of liver cancer cells and the key pathway of HMGCR/SMYD3 lipid metabolism; MTT, cell scratch test and flow cytometry were used to detect the effect of LpS on the proliferation, migration and apoptosis of HepG2 cells in the process of lipid metabolism disorders. [Results] The expression of key lipid metabolism genes such as HMGCR, SMYD3, SREBP-2 could be inhibited by LpS in lipid metabolism disorders liver cancer cells, while the proliferation and migration could be inhibited and the cell apoptosis could be promoted of liver cells in the process of lipid metabolism disorders. [Conclusion] LpS could inhibit the lipid metabolism of liver cancer cells by inhibiting the expression of key transcriptional regulators of lipid metabolism SREBP-2 and HMGCR, thereby promoting endogenous apoptosis of liver cancer cells.

Abstract:Colorectal cancer is one of the most common cancers in the western world. Lots of studies suggest a link between gut microbiota dysbiosis and colorectal cancer. And scientists suppose that there is a delicate regulatory network between gut microbiota and the human body. Recently, microRNA has been showed as a crucial mediator of this network. A growing number of studies indicate that gut microbiota can influence the expression and function of human cells' microRNA. In turn, human cells derived microRNA can regulate the gut microbiota ecosystem. Therefore, this review summarizes the specific mechanism of the interaction between gut microbiota and host microRNA, especially focus on the studies related to the occurrence, development, and treatment of colorectal cancer. It can provide the newest insight in the relationship between gut microbiota and colorectal cancer.

Abstract:Microorganisms exist widely in nature, and the interactions between microorganisms have an important impact on community structure and function. At present, great attention has been paid to the mechanism of microbial interactions, and the important genes affecting the growth of an individual microorganism in the interactions have been identified by high-throughput genome sequencing and statistical analysis. In order to further study the genetic mechanism of microbial interactions, the role of genes related to motility, drug resistance, nutrient absorption and metabolic regulation in Escherichia coli under the interaction conditions was reviewed in this paper. The genetic mechanism of interactions among Escherichia coli and other microorganisms was discussed from these aspects. In general, these genes are very important for the survival of E. coli. These studies enhance our understanding of the mechanism of bacterial interactions, and lay a theoretical foundation for the future study of more complex genetic mechanisms of microbial community interactions.

Abstract:The discovery and use of antibiotics provide us a powerful weapon against bacterial infection. However, the long-term use of antibiotics leads to bacterial resistance and limits their clinical applications. Fortunately, the rapid development of nanotechnology provides new ways to solve the problem. In this review, nano-enzymes were classified into two types:one is "the composites of enzyme and nanomaterial", another is "the nanomaterials possessing enzyme-mimic activities-named as nanozymes". To the best of our knowledge, Ag nanoparticles have been widely studied as nano-antibacterial agents and their antibacterial mechanisms are diverse. Therefore, the antibacterial mechanism and the latest progress of Ag nanoparticles as nano antibacterial agents are discussed. Compared to natural enzymes, the nanozyme-based antibacterial agent can integrate more than one strategy to yield synergistic effects and thus increase their bacterial killing efficacies. Therefore, in this review, we focus on recent progress in the design and mechanism of nanozyme-based antibacterial agents including the Ag nanoparticles, composites of enzymes and nanomaterials and nanoparticles possess enzyme mimic activities (nanozymes). Finally, the current challenges and an outlook for the development of more effective and safer antibacterial nano-enzymes are also included.

Abstract:Bacillus has good protein expression and secretion ability, and is widely used in the production of industrial enzymes. There are still many bottlenecks to achieve protein secretory expression with a generally high efficiency, although it is an ideal industrial host strain. In this review, the secretion pathway of protein in Bacillus is briefly described, and we summarize the strategies of host strain optimization for recombinant protein production from five different aspects, including promoter, signal peptide, secretion pathway, host strain and culture conditions, which has certain reference value for the efficient production of industrial enzymes in Bacillus. The research direction of optimizing secretory expression of Bacillus in the future is prospected. The development of various new biotechnology will certainly promote the further application of Bacillus in secretory expression.

Abstract:噬菌体是专一感染细菌等微生物的病毒，是地球上多样性最高和最丰富的生物体，是生物学研究中重要的模式生物，同时是抗生素耐药菌的天然抗菌剂。噬菌体研究的相关成果极大地推动了生物学各个领域的发展。

Abstract:[Background] Antimicrobial resistance is a serious threat to global public health, and the animal breeding is an important source of pathogen spreading. Further, our country has created a set of strategies to combat rising resistance, which include of optimizing the use (and preventing the overuse) of antibiotics. Since, novel, safe and efficient alternatives to antibiotic are highly in demand. Bacteriophages (phages) are being considered as promising potential antibacterial agents. However, phages, as living microorganisms, have some drawbacks such as poor stability and low utilization during storage and use. [Objective] Phage powder was prepared to improve the stress resistance and stability of phage, and to provide technical support for the application of phage in the breeding industry. [Methods] In this study, we produce a phage powder of E. coli phage BpEP4 with block polyether F-68 and the defatted rice bran excipents. The double agar plate method was used to study its stability against high temperature, pH stability and storage stability at room temperature. [Results] The phage powder can maintain the activity of phage at 100℃ and could survive in a pH range of 2.0-12.0. There is no significant reduction in phage titer after 3 months at room temperature. [Conclusion] The phage powder can significantly improve the stress resistance and stability of the phage, and has good application value and promotion prospects.

Abstract:[Background] Phage lysin has good safety and antibacterial properties, and is expected to replace antibiotics as a feed additive to improve the intestinal flora of livestock and poultry and prevent animal diseases. [Objective] White feather broilers were fed with complex phage lyase and antibiotic preparations to evaluate the effect of phage lysin on the intestinal flora of livestock and poultry and its application potential as an alternative feed additive. [Methods] Three-day-old healthy white feather broilers were fed, and the basic diet was supplemented with (200 mg/kg) complex phage lysin prepared from Thermus bacteriophage lysin (TSPphg) and Subthermus bacteriophage lysin (MMPphg) the lysin preparation and the 50 μg/g chlortetracycline preparation were respectively compared for feeding management. The high-throughput sequencing on the intestinal flora of white feather broilers at 28 days using the third-generation high-throughput sequencing technology, With the help of bioinformatics analysis of intestinal flora diversity and the relative abundance of dominant flora (top ten relative abundance), reveal the differences between and within each group, and further compare the liver oxidase activities. [Results] Complex phage lysin and chlortetracycline preparation had a certain effect on the intestinal flora of white feather broilers, both of which decreased the abundance and diversity of intestinal flora, and the Shannon index was significantly different compared with the blank group (P<0.05). In the dominant intestinal bacteria, Bacteroidetes significantly increased and Firmicutes significantly decreased in the complex phage lysin group compared with chlortetracycline group and blank group (P<0.05). At the same time, at the genus level, the relative abundance of Faecalibacterium increased significantly, while the relative abundance of Helicobacter decreased significantly (P<0.05), this trend was similar to that of most alternative antibiotic products after feeding poultry intestinal flora, which was more conducive to the health of poultry intestines. In addition, compared with chlortetracycline group and blank group, the activities of catalase (CAT) and superoxide dismutase (SOD) in complex phage lysin group were significantly increased go up (P<0.05). [Conclusion] Phage lysin can effectively improve the intestinal flora of livestock and poultry, promote the micro-ecological health of the intestinal livestock and poultry and improve the immunity of the body. It has a good prospect to replace antibiotics with phage lysin in livestock and poultry breeding and is worth further study.

Abstract:[Background] With the reduction of sequencing costs, more and more scientists have used high-throughput sequencing (HTS) technology to study the genome sequence of phages. Some termini analysis theory or methods were developed by some scientists to determine genomic terminal sequences of dsDNA phages. However, these methods are based on the commands under the Linux system. There is no software under the Windows operating system. [Objective] A free software PhageGT, which can be used in the Windows platform, was developed and can detect details of the genome termini of dsDNA phages genome using HTS Reads data and the complete sequence of phage genome. [Methods] A dialog-based Microsoft foundation classes (MFC) application was developed by Visual Studio 2019 and C++ language. Corresponding algorithms were designed for reading HTS Reads data and calculation. [Results] The frequency of the Reads can be calculated and ranked in descending order. The ratio of the highest frequency of the extracted sequence to the average frequency of the sequence (R) can be calculated. [Conclusion] This software PhageGT is very practical. The software PhageGT and all the test data used in this article can be downloaded for free from the website, https://zenodo.org/record/4543997#.YEhtG50zYhQ.

Abstract:[Background] Bacteriophage depolymerase is a kind of antibacterial protein produced by bacteriophage in the process of bacterial lysis. There are few reports on the capsule typing and common types of Acinetobacter baumannii bacteriophage depolymerases. [Objective] The KL2 type A. baumannii was selected as the research object. The depolymerase was cloned from the phage IME-AB2, and soluble expression was carried out in Escherichia coli, and its activity in vitro was studied. [Methods] A. baumannii capsular typing was identified through next generation sequencing and bioinformatics analysis. The IME-AB2 whole genome was analyzed. The putative tail fiber gene of ORF76 was cloned. The recombinant expression vector pEASY-Blunt-E1-gp76 was constructed and induced to express in E. coli BL21(DE3). The depolymerase was purified by Ni-NTA affinity chromatography and the gp76 antibacterial activity was studied in vitro. [Results] The recombinant plasmid of pEASY-Blunt-E1-gp76 was successfully constructed and expressed in E. coli. In vitro activity analysis was showed that the recombinant protein had good antibacterial activity against all KL2 type A. baumannii in vitro, and the depolymerase combined with human or dog serum had good bactericidal activity. [Conclusion] It is of great significance to identify depolymerase and improve its antibacterial spectrum, and it is also one of the important problems to be solved urgently in the field of bacteriophage and depolymerase used in the treatment of drug-resistant bacteria.

Abstract:[Background] With the increasingly serious drug resistance of Enterococcus faecium, it is urgent to screen and obtain new lytic bacteriophages with high lysis efficiency and clear genetic background, to enrich phage resources and provide appropriate strains for phage therapy. [Objective] To evaluate the biological characteristics and genome analysis of an Enterococcus faecium bacteriophage isolated from the fecal sample of Nanjing Xigang Dairy Farm. [Methods] The bacteriophage was purified by double-layer agar plate. The purified bacteriophage was negatively stained with phosphotungstic acid and observed by the transmission electron microscope (TEM). Then, one-step growth curve, pH tolerance, temperature tolerance, and optimal multiplicity of infection were detected. Also, its genomics characteristics and evolutionary history were analyzed. [Results] The results are as follows:A lytic Enterococcus faecium bacteriophage, named as vB_EfaS_29, was isolated and purified. Its plaques were round, bright, and clear. TEM analysis revealed that bacteriophage vB_EfaS_29 had a twenty-side head and a long tail. The head diameter was about 52.4 nm, and the tail length was about 157.1 nm. It belongs to the Siphoviridae family. The optimal multiplicity of infection of bacteriophage vB_EfaS_29 was 0.1; the maximum tolerable temperature was about 70℃; during pH 6.0-9.0, it was stable. The complete genome sequences of bacteriophage vB_EfaS_29 comprised of 41 014 bp with a GC content of 34.99% and contained 63 putative open reading frame (ORF). Only 36 of the ORF were well predicted for functions. Undesirable genes such as lysogenic, antibiotic resistance, or virulence were not detected in the genome. The phylogenetic analysis of vB_EfaS_29 based on amino acid of the endolysin, demonstrated that vB_EfaS_29 had more than 99% of sequence homology to FelixO1-like phages. However, using the amino acid of capsid as the targeted protein, vB_EfaS_29 had a long genetic distance with other Enterococcuss bacteriophages. [Conclusion] The above results provided theoretical data and resources for bacteriophage research and bacteriophage therapy.

Abstract:[Background] Shigella is a class of pathogenic bacteria that can cause diarrhea in humans and animals. As the problem of drug resistance is becoming more and more serious due to the abuse of antibiotics, alternative antimicrobial agents and treatment methods have become an urgent need. [Objective] To detect the pathogenicity of Shigella to broiler chickens, isolate and purify a bacteriophage (phage) that can lyse the strongly pathogenic Shigella, and study its biological characteristics. [Methods] Shigella was isolated from intestinal mucosa of diseased chickens. Healthy broiler chickens were used as animal models to test the drug resistance of highly pathogenic strains. The phage was isolated by using the host bacterium, and concentrated phage was purified by polyethylene glycol (PEG) precipitation. The morphological characteristics of the phage were observed by transmission electron microscopy (TEM). The double-layer plate method was used to determine the host spectrum, multiplicity of infection, one-step growth curve, pH, and thermal stability of the phage. [Results] This study has isolated 26 Shigella species, designated BDS1-BDS26. Among, BDS8 was the most pathogenic with multiple drug resistance and was identified as Shigella flexneri. Severe diarrhea and bloody stools were observed in the broiler chickens after feeding BDS8. Anatomical symptoms were mainly manifested as cardiac hypertrophy and obvious mesenteric haemorrhage, etc. Furthermore, the phage ΦDS8 was isolated from BDS8. TEM results showed that the head of ΦDS8 was icosahedral, with a diameter of 61±2 nm and a tail length of 165±2 nm. ΦDS8 belongs to the family of Siphoviridae. ΦDS8 could maintain high activity in the range of pH 4.0-10.0 and below 50℃. The infection cycle of ΦDS8 was about 120 min, including an incubation period of 75 min and an outbreak period of 45 min. The outbreak volume was 52 PFU/cell. [Conclusion] The phage ΦDS8 has host specificity and a better tolerance to general environments. This study provides a theoretical basis for phage therapy of shigellosis flexneri.

Abstract:[Background] The development of a bacteriophage-based product for the control of Campylobacter jejuni is a promising biocontrol strategy. However, bacteriophage isolation is a complicated challenge. [Objective] Response surface methodology was used to optimize the enrichment conditions of Campylobacter jejuni phages. [Methods] The effects of culture medium, culture temperature, shaking and ion additive on phage enrichment were analyzed through the single factor test. Based on response surface methodology (RSM), the recovery efficiency of phage was selected to evaluate enrichment culture conditions. [Results] The optimal culture conditions for enrichment were 37℃ and 0 r/min, recovery efficiency was 354.12%. The main procedures of bacteriophage isolation include sampling and sample preparation, incubation of sample filtrated with indicator strains, bacteriophage isolation and identification. Compared to the single spot method without enrichment, this enrichment method could increase the phage isolation rate by 269.23% from chicken fecal samples. [Conclusion] The optimized enrichment conditions of Campylobacter jejuni phages could improve the isolation efficiency of phages. It provides a new idea for phage isolation.

Abstract:[Background] Klebsiella pneumoniae (K. pneumoniae) is an important pathogen that can infect both humans and bovine. The wide spread of Klebsiella pneumoniae and its high drug resistance have caused great difficulties in the treatment of the disease. [Objective] To isolate a lytic Klebsiella pneumoniae bacteriophage. Its biological properties were studied, and the entire genome analysis of the bacteriophage was performed. [Methods] Klebsiella pneumoniae bacteriophage was isolated by the double-layer agar culture method from a dairy farm trough in Sichuan province. We used the same method to conduct host range, thermostability, pH, optimal multiplicity of infection (MOI), and one-step growth experiment. Phage morphology was observed by transmission electron microscopy (TEM). The whole genome of the bacteriophage was sequenced. [Results] A lytic Klebsiella pneumoniae bacteriophage vB_Kpn_B01 was isolated which had strong specificity. The morphology of plaque was round, transparent plaques with clear boundaries. Phage found in this study was double-stranded DNA bacterial viruses belonging to the Siphoviridae family, and the size of it was 113 227 bp. Comparative genome analysis revealed that vB_Kpn_B01 genome possesses the highest similarity to the bacteriophages in the genus of Sugarlandvirus. The genome of vB_Kpn_B01 comprises 149 coding sequences (CDS) and 25 tRNAs, but does not contain any known antibiotic resistant genes or virulent genes. [Conclusion] Phage vB_Kpn_B01 has a strong lysis ability and the necessary genes to lyse host bacteria. It has the potential to be used in the prevention and treatment of multi-drug resistant bacteria in animal husbandry.

Abstract:[Background] Previous studies have shown that phage cocktails (multi phage combinations) specifically infecting Ralstonia solanacearum significantly reduce bacterial wilt occurrence. Though bio-organic fertilizer (BOF) can suppress disease incidence of bacterial wilt, its inhibitive effects are not stable due to complex environment in the fields. [Objective] In order to improve biocontrol efficiency of BOF and targeted suppress bacterial wilt, we explored the effect of phage cocktail combined with bio-organic fertilizer fermented by Bacillus amyloliquefaciens on the biocontrol of tomato bacterial wilt. Meanwhile, impacts of this method on the population of R. solanacearum and the bacterial community in the rhizosphere was also investigated. [Methods] BOF obtained by the secondary fermentation of B. amyloliquefaciens T-5 was applied as a base fertilizer in spring in the greenhouse. Phage cocktail which contained four lytic phages of R. soalanacearum were soil-drenched during the flowering period of tomato. The incidences of bacterial wilt and the abundance of R. solanacearum in tomato rhizosphere were measured. Changes of bacterial communities were analyzed via high-throughput sequencing. [Results] Compared with conventional fertilization, the combination of bio-organic fertilizer and phage cocktail (BOF+P) significantly reduce diseased the incidence of tomato bacterial wilt. Beta-diversity was significantly changed after treated with BOF+P, among which relative abundance of Bacteroidetes increased but Gemmatimonadetes reduced. [Conclusion] Phage cocktail combined can improve bio-control efficiency of BOF significantly, which indicates an application potential.

Abstract:[Background] Pseudomonas aeruginosa is one of the main opportunistic pathogen that has an important role in nosocomial, acute, and chronic infections. The multidrug resistant (MDR) P. aeruginosa remains a common cause of severe nosocomial infection. Phages are viruses that infect and can kill bacteria, and phage therapy is an alternative treatment to eradicate MDR P. aeruginosa infection. [Objective] To isolate virulent phages that infect carbapenem-resistant P. aeruginosa and characterize its biological and genomic properties of phages for future development as phage therapy. [Methods] Environmental water samples were collected, and phages were isolated using double agar overlay plaque assay. The morphology, one-step growth curve, optimal multiplicity of infection and other biological characteristics of phages were studied. Phage genome was sequenced using the Illumina MiSeq sequencing platform. Newbler 3.0, GeneMarkS, BlASTp, and Mauve 2.4.0 were used for genome annotation, comparative genomics and evolutionary analyses. [Results] A new phage, PHW2, which is a member of Myoviridae, was isolated from pond water. It is able to lyse seven carbapenem-resistant P. aeruginosa clinical isolates. The optimal multiplicity of infection (MOI) is 0.1. In addition, one step growth curve showed that the latent period and rise period of PHW2 was 100 min and 360 min, respectively. The burst size was about 25 PFU/cell. The phage PHW2 was stable in the temperature of 25-50℃ and pH 6.0-8.0. The activity of PHW2 decreased significantly after UV irradiation for 7 min. Moreover, it was shown that the activity of PHW2 is unaffected after treatment with 5% chloroform for 100 min. The genome of PHW2 is 65 984 bp, with GC content of 55.69% and comprises of 92 ORFs. tRNAscan-SE analysis indicated that phage PHW2 does not possess tRNAs. The genome of PHW2 had high similarity with other PB1-like phages. Phage PHW2 inhibited the biofilm formation of P. aeruginosa PA001 within 24 h. [Conclusion] We isolated PHW2, a new PB1-like phage that shows potent lytic effect against several carbapenem-resistant P. aeruginosa clinical isolates. Biological characteristics and the in vitro biofilm test indicated that the phage has the potential to be used as a biological agent to control nosocomial multidrug resistant P. aeruginosa infections.

Abstract:[Background] Escherichia coli (E. coli) is one of the most common Gram-negative bacteria causing neonatal meningitis and poultry meningitis. The strain E. coli capsular K1 is an important pathogen. Current methods for detecting E. coli capsular K1 have some disadvantages.[Objective] A rapid detection method for E. coli capsular K1 was established by using the host specific phage PNJ1809-36. [Methods] Phage PNJ1809-36 was labeled with fluorescent dye SYBR gold, and 33 tested strains were infected by this fluorescent phage. The specificity was determined by observing the 33 test strains using the fluorescence microscopy. The sensitivity was determined by observing the host bacteria DE058 with gradient tenfold dilution after being infected with fluorescence labeled phage. Eight simulated feces were detected by fluorescent labeled phages. Moreover, the titer change and detection efficiency of fluorescent labeled phages stored at 4℃ in darkness were investigated. [Results] Among the 33 strains tested, 8 strains of 9 E. coli can be observed with fluorescence around the bacteria, but one could not be detected. No fluorescence was observed in 20 non-E. coli capsular K1 strains and 4 non-E. coli strains. The detection sensitivity was 100 CFU/mL. The detection results of 8 simulated fecal samples showed that fluorescence could be observed in 3 samples containing E. coli capsular K1, and no fluorescence could be detected in 5 samples without E. coli capsular K1. After stored at 4℃ for 4 months, the titer of fluorescence labeled phages showed no obvious decrease, and the detection efficiency did not change significantly. [Conclusion] The fluorescence labeled phage PNJ1809-36 could be used to detect E. coli capsular K1 rapidly and intuitively within 15 min.

Abstract:DNA manipulation tools derived from bacteriophage, such as site-specific recombinases, cosmid libraries, and homologous recombinases, play an important role in genetic engineering. The homologous recombinases Redα/Redβ from Lambda phage or RecE/RecT from Rac prophage can efficiently mediate the recombination between homologous arms as short as 35-50 bp. Recombineering based on phage homologous recombinases Redα/Redβ and RecE/RecT can be used to precisely and efficiently modify target DNA molecules without limitation of endonuclease recognition sites and the size of DNA molecule, which has been developed into a new genetic engineering technology. This article reviews the phage homologous recombinases and their functional mechanism, their application and development in Escherichia coli and other bacteria. The broad use of recombineering has been demonstrated in the mining of microbial secondary metabolites, animal and plant transgenes, and viral genome cloning and modifications. In situ activation of a silent gene cluster accomplished by promoter engineering requires host specific recombineering system. The initial step of heterologous expression of secondary metabolites is direct cloning of large DNA fragment, which is based on RecET. Construction of large transgenic vector for both animal and plant mainly rely on Red recombineering. Generation and modification of infectious clone in E. coli mediated by RecET direct cloning and Red recombineering is the most efficient method for functional research of viral genome and vector vaccine development.

Abstract:Bacteriophage is the virus that infects bacteria. To resist bacteriophage infections, bacteria have evolved various antiviral mechanisms, which significantly promote the development of gene editing and lay down foundations for bacteriophage treatment. This review briefly concludes the molecular resistance mechanisms of host bacteria against bacteriophage infection. Next, the effects of such defense systems on bacteria physiology and ecology are discussed. Then, we summarized the limitations and future directions of bacteriophage resistance research.

Abstract:Quorum sensing is the ability of single-celled bacteria to regulate and synchronize target gene expression among a local population in response to cell density, so that a coordinate response is produced by whole population. Bacteria release signaling molecules called autoinducers that accumulate and increase in concentration according to cell density. The sensing of autoinducers by a bacterial population at a threshold stimulatory concentration leads to change in gene expression by switching the genes on that controlled by quorum sensing. Since Fuqua proposed the concept of quorum sensing in 1994, quorum sensing has become a research hotspot in the field of microbiology. In recent years, quorum sensing has been increasingly discovered in fungi and bacteriophages. Especially since 2017, Professor Erez has found quorum sensing phenomenon in a variety of phages including Bacillus subtilis phages, which regulates the lysogeny-lysis pathway conversion. In addition, a nunmber of other studies have found quorum sensing in other phages. This paper attempts to summarize the recent development in phage quorum sensing systems and their molecular mechanisms.

Abstract:Klebsiella pneumoniae is a member of the Enterobacteriaceae family and exists widely in a variety of environments. It can cause human and various animal diseases including mastitis, pneumonia, urinary tract infection, wound infection and lead to bacteremia and suppurative abscess, et al. Nowadays, the bacterial tolerance to antibiotics is becoming more and more serious, and the emergence of highly virulent strains has brought considerable challenges to the prevention and control of the bacteria. Bacteriophages are a group of viruses that can infect and kill host bacteria, have attracted much attention because of their potential to treat drug-resistant bacterial infections. Cases of successfully using bacteriophages to treat drug-resistant bacterial infections have been reported all over the world. Based on the research data of K. pneumoniae and its bacteriophage at home and abroad, this paper summarizes the epidemiological investigation of K. pneumoniae and the application of bacteriophage in the treatment of this bacterial infection, in order to provide references for authors in the antibacterial research and clinical application based on bacteriophage of K. pneumoniae.

Abstract:Klebsiella pneumoniae is one of the common pathogenic bacteria that causes a variety of clinical infections. The emergence of multi-drug resistant Klebsiella pneumoniae strains has brought numerous obstacles in prevention and control of bacterial infections. The depolymerase encoded by Klebsiella pneumoniae phage is a biological enzyme with high stability and strong specificity. It has many functions such as decomposing bacterial extracellular polysaccharides and restricting the bacterial growth, etc. The depolymerase provides new ideas to prevent and control Klebsiella pneumoniae infection and has broad prospects in antibacterial applications. This article reviews the advance of depolymerase employed by phage of Klebsiella pneumoniae.

Abstract:As a virus that infects bacteria, bacteriophage can specifically recognize its host bacterium. Extended overuse of antibiotics has resulted in a rapid rise of antibacterial resistance in bacterial population. Bacteriophages have emerged as a viable alternative to combat bacterial infections. In the long-term coevolution of bacteria and bacteriophage, both have evolved a series of defense strategies. The mechanisms of bacteria against bacteriophages include inhibiting bacteriophage adsorption, preventing bacteriophage DNA from entering, digesting the bacterio phage genome, abortion infection, and quorum sensing regulation of bacteriophages. The counteracted strategies adopted by bacteriophage are also reviewed. In addition, the related detection methods are exemplified. The study of the mechanisms provides a theoretical basis for the application of bacteriophages in bacterial control.

Abstract:Bacteria possess a variety of resistant mechanisms against phage infection in the long-term coevolution with phages. Among them, quorum sensing has become a hot topic in recent years as a means for bacteria to resist phage infection. Bacterial quorum sensing mainly depends on bacterial density and regulates the expression of target genes, as well as the infection of phage. Therefore, bacterial quorum sensing is closely related to phage. However, the interactions between quorum sensing and phage are complex and diverse, quorum sensing has been proved to be unfavorable or beneficial to phage infection, and regulates the switch of phage lysis-lysogeny. Therefore, this paper will discuss the role of quorum sensing in phage infection, the decisive role of quorum sensing in phage lysis-lysogeny switch, and the other interactions between quorum sensing and phage, which provides theoretical basis for phage therapy in bacterial diseases.

Abstract:The gut is the largest reservoir of human flora. In the last decade, with the increasing investigations on the relationships between gut microbiota and human health diseases, gut bacteriophage has also drawn more attention. However, the research of gut bacteriophage is still in its infancy, hampered by delayed progress in the experimental and bioinformatical technology for gut bacteriophage and virome. Thus, this review first summarizes the research methodology of gut bacteriophage in computation and experiments, including sequening analysis and phage isolation, etc. Then, phage taxonomy, phage-host interactions, and the clinical applications of gut bacteriophage are also discussed. Finally, this review looks forward to the challenges and opportunities for gut bacteriophage research in data and physical resources, bioinformatical and experimental technology, interactions with intestinal bacteria, interventions on human diseases, etc.

Abstract:More recently, the frequency of multi-drug-resistant strains has sped up with the misuse of antibiotics. Since the number of deaths caused by bacterial infection is increasing year by year and human health is facing a substantial challenge, it is urgent to develop novel antibacterial drugs. Bacteriophage lysin is a type of protein which synthesized by double-stranded DNA bacteriophage at the late stage of genome replication. It can hydrolyze the peptidoglycan in the bacterial cell wall to release the progeny bacteriophage and kill the bacteria. Bacteriophage lysin is derived from bacteriophages and has a unique advantage of evolutionary selection. It is not only efficient and fast quickly kill multi-drug resistant bacteria, and it is not easy to cause bacteria to develop new drug resistance. Because of its highly efficient bactericidal capacity and high host specialization, bacteriophage lysin has become one candidate for a new generation of antibacterial agents. A review on the structure and mode of action of lysins and their application were presented.

Abstract:Biological wastewater treatment is a process conventionally used to achieve wastewater purification and resource recovery through the degradation of pollutants by microbial flora. Bacteriophages, which are known to infect bacteria, are widely distributed viruses found in biological wastewater treatment systems. They can specifically control the microbial flora in wastewater, thereby regulating sludge characteristics and affecting wastewater treatment performance. Therefore, it is vital to study the distribution of bacteriophages and explore the significance of their activity in biological wastewater treatment systems. This review summarized the distribution of bacteriophages in different biological wastewater treatment processes and preliminarily analyzed methods of isolation, cultivation, and identification of bacteriophages, along with the advantages and disadvantages of these methods. This review also explored the following functions of bacteriophages:(1) regulating microbial community structure and affecting wastewater treatment, (2) being used as an indicator organism for environmental monitoring, (3) controlling pathogenic bacteria, sludge bulking, sludge foaming, and membrane fouling, and (4) reducing sludge production. Additionally, this review expounded the associated influencing factors, including temperature, pH, multiplicity of infection, and charged particles. Furthermore, this review discussed potential problems that may be encountered during the practical biological wastewater treatment process, along with corresponding solutions. Finally, this review proposed the prospective development of the use of bacteriophages in the biological wastewater treatment process and aimed to provide a reference for the development and application of biological wastewater treatment systems, in order to promote the healthy development of wastewater treatment.

Abstract:The battle for survival between microbes such as bacteria and archaea and viruses (bacteriophages) is an arms race. Bacteria and archaea have evolved innate and adaptive immune systems to protect themselves from viruses. Viruses use different counter-defense strategies to evade these phage defense mechanisms. The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) system is an adaptive immune system that is widely encoded by bacteria and archaea to resist foreign genetic elements such as viruses. At the same time, viruses also evolved specific anti-CRISPR to resist the immunity of the CRISPR-Cas system. In this paper, the discovery process, classification and mechanism of anti-CRISPR have been systematically reviewed, and their potential applications have prospected.

Abstract:In recent years, phages have shown promising applications in the control and detection of harmful microorganisms in food processing and preservation due to their specificity in infecting bacteria. For example, phages can be sprayed on food surfaces or combined with food packaging materials to control food-borne pathogenic bacteria and spoilage bacteria, and genetic engineering can be used to construct reporter phages for rapid detection of foodborne pathogenic bacteria. However, phage is also one of the most important detrimental factors in food fermentation. Phage contamination often leads to the failure of the whole fermentation process, causing huge economic loss. At present, phage contamination is mainly prevented by the methods of disinfection and inactivation, fermentation bacterial strain change, etc. This paper reviews the current research status of phage application and hazards in food industry to provide theoretical basis for broadening the application of phage in food industry and developing new technologies for phage contamination prevention.

Abstract:Phage, the most abundant organisms on the earth, play a vital role in the shaping of natural ecosystems and the driving of bacterial evolution. In the struggle with the host, phage can choose the following two ways to determine fate of itself and the host:(1) Lysis:lysing the host cell, and finally releasing a large amount of phage particles; (2) Lysogen:integrating its chromosome into the host cell, then establishing a potential coexistence relationship with the host. For some temperate phages, this tendency is further regulated by the diversity of infections, where single infections are mainly lytic, while multiple infections are mostly lysogenic. Lysogenic phage can not only activate the lysis-lysogen switch based on the physical and chemical factors of the external environment, but also use the bacteria's own quorum sensing system to initiate the lysis-lysogen switch, and then determine the fate of its host bacteria. Meanwhile, the host bacteria have evolved means to target phage during the long-term struggle. In general, phage profoundly affects bacterial community dynamics, genome evolution, and ecosystems, etc., and all of this depends on the mode of struggle between phages and host (lysis/lysogenic infection). This paper discusses the influencing factors that cause temperate phage to lyse the host bacteria-lysogenic fate, and systematically summarizes the latest research on coping strategies of bacteria facing phage infection, hoping to provide help and suggestions for the follow-up study on the interaction between phage and host.