Isolation And Identification Of Pathogenic Escherichia Coli Phage From Swine And Evaluation Of Their Effect On Antibiotic-Resistant Bacteria In The Environment

Pathogenic Escherichia coli(Pathogenic E.coli)is an important pathogen that causes intestinal disease,urinary tract infections,septicaemia and meningitis in humans and animals,and is usually formed by combining virulence genes of common E.coli with other bacteria.Infants and young animals(birds)with underdeveloped immune systems are more susceptible to infection than adults.Pathogenic E.coli is one of the main pathogens causing bacterial diarrhoea in newborn piglets in China.Infected piglets mainly show symptoms such as diarrhoea,dehydration,and loose stools of greyish yellow or greyish white colour.The bacterium not only seriously affects the growth and development of piglets,but also causes their death,causing great economic losses to the pig industry in China.In addition,with the long-term overuse and even abuse of antibiotics,drug-resistant strains of pathogenic E.coli have emerged frequently and in large numbers in recent years.The medical and farming industries are in urgent need of a new "antibiotic" that can tackle drug-resistant bacteria.Phages are naturally occurring viruses that use bacteria as hosts and are more specific and safer than antibiotics,and are abundant and inexpensive in nature.Therefore,this study aims to initially investigate the prevalence and antibiotic susceptibility spectrum of pathogenic E.coli in several large-scale pig farms,and provide a reference basis for the formulation of measures such as early warning and prevention and control of pathogenic E.coli epidemics in the region and the province.At the same time,multi-drug resistant pathogenic E.coli were used as host bacteria,and phages were screened according to their strong lysis ability and wide lysis spectrum.The biological characteristics of the screened phages were analysed,and their potential and effectiveness in purifying multi-drug resistant bacteria in the environment were assessed,with the aim of providing new methods for the prevention and treatment of multi-drug resistant pathogenic E.coli in clinical settings.Details of the study are as follows:1.Isolation and identification of pathogenic E.coli strains and antibiotic susceptibility profilesIn this study,a total of 82 rectal swabs from piglets with diarrhoea in three largescale pig farms in Hubei and Guangxi were isolated and identified,and then tested for antibiotic susceptibility profiles of pathogenic E.coli isolates.The study showed that74 E.coli strains were isolated in this experiment,of which 31 strains were pathogenic E.coli;enterotoxin-producing E.coli and enteroaggregative E.coli were mainly prevalent in these pig farms;the 31 pathogenic E.coli strains showed high resistance rates to penicillin G,amoxicillin and ampicillin,with 100%(31/31),67.7%(21/31),67.7%(21/31),and low resistance to cefoxitin,ceftazidime and polymyxin B at 6.5%(2/31),6.5%(2/31)and 0%(0/31),respectively;E.coli ET2,E.coli ET8,E.coli ET17,E.coli EA14 and E.coli EA43 were multi-drug resistant strains,suitable as host bacteria for phage screening.2.Isolation and Lytic Spectrum Determination of Pathogenic E.coli phagesBased on the screened host bacteria,22 healthy pig faeces and 10 pig farm wastewater samples collected from 2 large-scale pig farms in Hubei and Anhui regions were subjected to phage isolation.The results showed that a total of 40 phage strains were obtained against pathogenic E.coli.The phage EA43P9 was able to lyse 26 pathogenic E.coli strains with a lysis rate of 83.9%(26/31),one E.coli engineered strain DH5α,and six strains of Salmonella of porcine origin.Phage ET2P2 was able to lyse 23 pathogenic E.coli strains with a lysis rate of 74.2%(23/31)and could not lyse Salmonella.3.Biological Characterization of Pathogenic E.coli phages EA43P9 and ET2P2Phage EA43P9 forms circular,transparent,and well-defined plaques on doublelayered agar plates,with a halo.Under transmission electron microscopy,the head exhibits a symmetrical icosahedral structure with a diameter of 87 ± 2 nm,and the tail measures 175 ± 2 nm in length,belonging to the family of long-tailed Myoviridae.Temperature and p H tolerance tests show that EA43P9 maintains good activity between4°C and 60°C,and p H 3.0 to 10.0.The optimal multiplicity of infection(MOI)for EA43P9 is determined as 0.01.Adsorption time determination results indicate that 74.6%of EA43P9 phages have adsorbed to host bacteria within 5 minutes,and 91.8% within10 minutes.One-step growth curve analysis reveals that the latent period of EA43P9 is approximately 30 minutes,with a burst period of about 60 minutes,and a burst size of approximately 31.4 plaque-forming units(PFU)per cell.Phage ET2P2 forms circular,transparent,and well-defined plaques on doublelayered agar plates,with a halo.Under transmission electron microscopy,the head exhibits a symmetrical icosahedral structure with a diameter of 62 ± 15 nm,and the tail measures 125 ± 2 nm in length,belonging to the family of long-tailed Myoviridae.Temperature and p H tolerance tests show that ET2P2 maintains good activity between4°C and 60°C,and p H 4.0 to 12.0.The optimal MOI for ET2P2 is determined as 0.1.Adsorption time determination results indicate that 83.2% of ET2P2 phages have adsorbed to host bacteria within 5 minutes,and 95.5% within 10 minutes.One-step growth curve analysis reveals that the latent period of ET2P2 is approximately 20 minutes,with a burst period of about 70 minutes,and a burst size of approximately 55.3PFU per cell.4.Evaluation of the killing effect of phage on multidrug-resistant bacteria in the environmentResults from in vitro bactericidal tests indicate that phage EA43P9 and ET2P2 exhibit very good lytic activity and that pathogenic E.coli fluids with phages are clear and transparent.The results of simulated direct sunlight and UV irradiation tests showed that the bactericidal activity of phage EA43P9 and ET2P2 decreased depending on the duration of sunlight and UV exposure.The phage was applied to the simulated bacterial contaminated environment by spray.The results showed that the killing efficiency of multi-drug resistant pathogenic Escherichia coli in the environment could reach more than 98.5 % within 30 min,and the killing effect could reach 99 % after 1-2 h.Studies have shown that phage EA43P9 and ET2P2 can quickly and effectively kill host bacteria in the environment,and have the potential to be developed as a convenient and efficient spray fungicide.