Isolation,Identification And Characteristics Analysis Of Bacteriophage Lising Multi-Drug Resistant Salmonella

Salmonellosis is a zoonotic disease that seriously endangers human health and the development of aquaculture.Antibiotics are generally used for the prevention and treatment of salmonellosis.Recently,the misuse and overuse of antibiotics lead to the emergence of multidrug-resistant bacteria,drug residue,food safety and the environmental imbalance,which are threats to the public health worldwide.Bacteriophages(phages)are viruses that could infect and kill the bacteria,fungi and other microorganism.Bacteriophages have been used to treat bacterial infection since their discovery.Compared with traditional antibiotics,bacteriophages show many advantages,including existence wildly,high specificity,proliferation fast,high safety and low cost.The bacteriophages become the focus for threating bacterial infection due to the growing multidrug-resistant bacteria threat.The purpose of this study is to screen the broad host spectrum phage with high efficiency of lysis for different serotypes of multi-drug resistant Salmonella,analyze its biological characteristics,sequence and analyze the whole genome,further clone and express the bacteriophage lysozyme to analyze the bacteriostatic activity,and explore the preliminary laboratory application of the phage,so as to lay the foundation for the bacteriophage to become a new type of antibacterial agent.1.Isolation,identification and characteristics analysis of a broad-host-range lytic Salmonella phage SHWT1In this study,68 strains of Salmonella pullorum bacteriophages were isolated from fecal water samples of in Shanghai poultry farm,and two broad-spectrum bacteriophages with cleavage of multiple drug-resistant Salmonella Pullorum,Salmonella Gallinarum,Salmonella Typhimurium,and Salmonella Enteritidis were purified,and one of the bacteriophages was named Salmonella phage SHWT1,or SHWT1.The ability of bacteriophage to adsorb bacteria in a short time was determined by phage host range,electron microscope and adsorption ability.Then the optimal MOI and one-step growth curve of phage were determined according to the bacteriophage adsorption ability.The optimal storage conditions of bacteriophages were determined by pH stability,thermal stability and room temperature storage test.Finally,through inhibition of Salmonella growth by phages in vitro,inhibition of bacterial biofilm formation and the preliminary application of bacteriophage in laboratory,the safety of bacteriophage and its bacteriostatic effect in vitro and in vivo were analyzed.The results showed that the phage SHWT1 was a member of the family Siphoviridae and the order Caudovirales,and has a specific for lytic Salmonella.Phage SHWT1 had a latent period of 5min and burst size of approximately 150 plaque-forming units PFU/cell.The phage was stable from pH 3-12 and 4-65?.Moreover,phage SHWT1 could inhibit the growth and biofilm formation of Salmonella Pullorum,Salmonella Gallinarum,Salmonella Typhimurium,and Salmonella Enteritidis at optimal multiplicity of infection(MOI)of 0.001,0.01,0.1,and 100,respectively.Bacteriophage SHWT1 has good safety,good in vivo activity and antibacterial treatment potential,which provides a reference for bacteriophage to become a new type of antibacterial agent.2.Genomic analysis of a novel lytic Salmonella phage SHWT1In order to analyze the phage genome comprehensively,phage DNA was extracted by phage genomic DNA rapid extraction kit and sequenced on Illumina NovaSeq PE150 platform.Then NCBI blast(http://blast.ncbi.nlm.nih.gov)was used to analyze the whole genome sequence of the phage,DNA Star was used to analyze the base composition ratio and GC content of the phage DNA sequence,tRNAscan-SE(http://lowel ab.ucsc.edu//tRNAs can-SE/)was used to identify the tRNA in the genome,and GeneMarkS software(Version 4.17)was used to predict the ORF of the sequenced genome.The ORF finder is provided by NCBI(National Center for Biotechnology Information)to verify the ORF prediction function.Finally,Antibiotic resistance gene database(https://card.mcmaster.ca/)and virulence factor database(http://www.mgc.ac.cn/VFs/main.htm)were used,detecting the presence of antibiotic resistance gene and phage encoded virulence gene in phage SHWT1 genome.The results showed that the genome of phage SHWT1 comprises 40629 bp of double-stranded DNA,with a GC content of 49.83%.The average gene length was 678 bp,including 56 ORF.Among the 56 ORFs,only 21 ORFs had annotated functions,while 35 ORFs were assigned as hypothetical proteins.The identified ORFs were categorized into four functional groups,including DNA replication/modification?structural components?packaging module and host lysis.There was no tRNA gene in phage SHWT1.Toxin genes,virulence genes,and antibiotic resistance genes were not detected in the genome of phage SHWT1.In addition,no putative integrase genes were identified,indicating that phage SHWT1 could not integrate into the host bacterial genome and might be lytic in nature.Thus,phage SHWT1 is potentially agent for controlling Salmonella infection.3.Preparation and Antibacterial activity Analysis of Salmonella bacteriophage lysozyme LysSHWTlIn order to analyze antibacterial activity of lysozyme,prokaryotic expression was carried out,and then SHWT1 protein was purified by His-tag magnetic beads.Finally,plate lysis test,phage host range and bacteriostatic test combined with membrane permeable agent EDTA were carried out.The results show that LysSHWTl had good lytic activity against S.Pullorum,S.Gallinarum,S.Typhimurium and S.Enteritidis.The diameter of lysis circle was 16,13,14 and 13 mm,respectively.The control group has no lytic circle.LysSHWTl could lyse 60 of 101 clinical Salmonella strains.It was further found that EDTA could significant increase the lytic activity of LysSHWTl lysozyme to S.Pullorum SP01,S.Gallinarum SG02,S.Typhimurium SAT52,and S.Enteritidis SE12.This study showed that LysSHWTl could effectively lyse Salmonella,indicating its potential application in the prevention and control of Salmonella infection.