Isolation,Characterization And Application Of Bacteriophages Against Shiga Toxin-Producing Escherichia Coli In Foods

The infection of foodborne pathogens is increasing year by year,which is the focus of global public health.Shiga toxin-producing Escherichia coli is one of the common foodborne pathogens.Beef cattle and dairy cows are considered to be the most important animal hosts of Shiga toxin-producing Escherichia coli,thus contaminating foods such as ground beef,fruits,vegetables and milk,increasing human exposure to the pathogen.At the same time,with the uncontrolled use of antibiotics,the production of multi-drug-resistant bacteria is even worse.In view of this crisis,more and more people call for the development of new antimicrobial agents to control foodborne pathogens.In this study,Escherichia coli O157:H7 and multi-drug resistant Shiga toxin-producing Escherichia coli E6-11-18 were used as host bacteria.Two bacteriophages were isolated from domestic wastewater,named JN01(host Escherichia coli O157:H7)and JN02(host E6-11-18),respectively.Then the biological characteristics of the two bacteriophages were identified and the whole genome was sequenced.Finally,the bacteriostatic effect of the two bacteriophages in food was evaluated.The specific results are as follows:(1)One-step growth curve of phage JN01 showed that the latent period and lysis period was 15 min and 85 min,respectively.The burst size of JN01 was about 134 PFU per infected cell.The latent period and burst size of JN02 was 15 min and 69 PFU/cell.(2)The host ranges showed that phage JN01 against 16 of 20 strains of E.coli,11 of which were more than three multi-drug resistant strains.In spot tests,18 Escherichia coli(E.coli)strains were sensitive to JN02.In efficiency of plating(EOP),4 E.coli strains were sensitive to JN02.(3)Phage JN01 was relatively stable under stressed conditions such as temperatures(30?–70?)and p H(4-13),especially could tolerate high alkaline conditions.Phage JN02had a relatively short latent period(15 min),large burst sizes(69 PFU/cell),and high physicochemical stability(temperature(30–60°C),p H(2–11),0.1%,1%pig bile salt,food additives and biocides(100%chloroform,0.1%acetic acid,1%potassium sorbate).(4)Transmission electron microscopy and whole genome sequencing showed that phage JN01 and JN02 belonged to Myoviridae phages.Importantly,no known virulence-associated,lysogenic,and antibiotic genes were identified in the genome of JN01 and JN02.(5)Phage JN01 could completely inhibit the growth of artificially inoculated 10²CFU/ml Escherichia coli O157:H7 during milk preservation at 4?for 3 days.At the same time,It could significantly reduce the concentration of 10⁴ CFU/ml Escherichia coli O157:H7 by more than 1.0 log₁₀ CFU/m L compared with the control group without phage JN01.During the 7-day preservation of beef at 4?,phage JN01 could significantly reduce the counts of Escherichia coli O157:H7 inoculated with 10⁴ CFU/cm² by more than 2.0log₁₀ CFU/cm².For phage JN02,it could completely inhibit the growth of E6-11-18inoculated with 10² CFU/ml and 10⁴ CFU/ml for 3 days,and phage JN02 could reduce the number of E6-11-18 in beef to 1.0-1.9 log₁₀ CFU/cm² during 7 days of fresh-keeping at 4?.