Isolation,Biological Characteristics And Genome Analysis Of Shiga Toxin-producing And Multidrug-resistant Escherichia Coli Phages

Shiga-toxin-producing Escherichia coli(STEC)is a common intestinal pathogenic bacterium.It usually causes diarrhea in piglets and is highly contagious.It has a high mortality rate during the early stages of piglets’ growth and can bring economic losses to the pig industry.Because of the relatively high cost of animal-protection products to prevent such diseases and the increasing attention paid to the problems caused by the use of antibiotic drugs,it is urgent to find new ways to solve the problem of bacterial resistance.Phages mainly invade host bacteria and kill bacteria through the action of lyase.Phages can be detected in natural environment and can kill target bacteria quickly,with little influence on other bacteria.After infection,lytic phages amplified and released their progeny with the help of nutrients in the host,and lysed the host bacteria at the same time.Because of its natural bactericidal properties,bacteriophages have the potential for the research and development of antibiotic substitutes and biological disinfectants.In this study,two strains of Shiga-toxin-producing and multi-drug resistant Escherichia coli GXEC007 and GXEC010 were isolated from a pig farm in Guangxi province as host bacteria.Phages that could lyse these two strains of Escherichia coli were isolated and purified and named vB＿EcoM＿BP7 and vB＿EcoM＿BP10,respectively.Then the biological and genomic characteristics of these two phages were analyzed by transmission electron microscopy,optimal infection complex number determination,one-step growth curve drawing,thermal sensitivity and p H stability evaluation,bactericidal test and whole genome sequencing.The results showed that the phage plaques of the two strains were clear and transparent with smooth edges.The optimal infection complex of vB＿EcoM＿BP7 and vB＿EcoM＿BP10 phages was 0.001 and 1,respectively.The one-step growth curve showed that the incubation period of vB＿EcoM＿BP7infection was 5 min,the outbreak period was 70 min,and the outbreak amount was 145 PFU/cell.The incubation period of vB＿EcoM＿BP10 infection was 5 min,the outbreak period was 60 min,and the outbreak amount was 51PFU/cell.VB＿EcoM＿BP7 and vB＿EcoM＿BP10 can tolerate the same temperature range of 30-60 ℃,vB＿EcoM＿BP7 can maintain stability in p H 6-9,and vB＿EcoM＿BP10 can maintain stability in p H 5-8.The bactericidal test results showed that the bactericidal effect of the phage vB＿EcoM＿BP10 was good when the infected complex number(MOI)was 1,while the bactericidal effect was most significant when the infected complex number(MOI)was 0.001.The whole genome sequencing results of phage vB＿EcoM＿BP10 gene showed that the total genome fragment length was 52 288 bp,GC content was44.16%,and there were 72 ORF reading frames.The GO functional annotation indicates that it can participate in biological processes such as small molecule metabolism and cell nitrogen compound metabolism,and has molecular functions such as nucleotide transferase activity,ion binding and DNA binding.The results of phylogenetic tree analysis showed that it was closely related to Escherichia phage ST32 and Enterobacteria phage phiecom-Gj1.To sum up,phages vB＿EcoM＿BP7 and vB＿EcoM＿BP10 have clear hosts,good thermal stability and acid-base stability,which can lay a certain theoretical foundation for the effective prevention and control of shiga-toxin multidrugresistant Escherichia coli infection in livestock production.