Characteristic Analysis And Application Research Of A Polyvalent Broad-spectrum Escherichia Phage Tequatrovirus EP01

Salmonella and Escherichia coli are common foodborne pathogens,widely found in contaminated animal-origin foods such as meat products and dairy products and often cause foodborne illness.Phage can lyse host bacteria specifically,and they are considered to be a promising method to control food-borne contamination due to their availability,low cost,nontoxicity,and environmental protection.Electrochemical biosensor has been widely studied as a rapid,sensitive,simple and low-cost detection method.Phage-based electrochemical biosensor is a new method of pathogen detection,and it has a good application potential in the detection and monitoring of pathogenic microorganisms.In this study,a polyvalent broad-spectrum lytic phage named Tequatrovirus EP01 was isolated from sewage of a pig farm in Nanning,Guangxi.Phage EP01 could form a uniform,clear and bright plaque without halo on double-layer agar plates with diameter of 3 mm.EP01 has a wide host range and could infect 1 S.enteritidis strain and 31 E.coli isolates of different sources and serotypes simultaneously.The incubation period was 10 min,and the burst volume was 80 PFU/cell.EP01 remained relatively stable at p H 4～10and temperature at 30～60℃,and EP01 could still be detected at 80℃after incubation of 30 min.EP01 belongs to T4-like viruses genus,Myoviridae family.The genome size of EP01 was 165577 bp（Genbank OM135583.1）,and there were 12 CDSs related to receptor binding proteins in the genome.No genes related to virulence factor,lysogeny and drug resistance were found.In this study,we tested the inhibitory effect of phage EP01 on S.enteritidis,E.coli O157:H7,E.coli O114:K90（B90）,and E.coli O142:K86（B）in liquid broth medium（LB）.The results showed that EP01 could significantly reduce the number of all tested strains.We further examined the effectiveness of EP01 in controlling bacterial contamination on artificially contaminated pork surface and milk,respectively.EP01 significantly reduced the viable counts of all the tested bacteria(2.18–6.55 log₁₀CFU/sample,p<0.05).A significant reduction of 6.55 log₁₀CFU/cm²,4.3 log₁₀CFU/m L（p<0.001）in bacterial counts on the surface of pork and in milk was observed with EP01 treatment,respectively.Generally,the inhibitory effect exhibited more stable at 4℃than that at 28℃,whereas the opposite results were observed in milk.The antibacterial effects were better at MOI of 1 than that at MOI of0.001.We evaluated the efficacy of EP01 on chicks infected with E.coli O157:H7 by in vivo safety experiment and in vivo treatment experiment.The results showed that the chicks were in good health and no pathological changes were found in tissues or organs after injection of EP01,indicating that EP01was safe and had no pathogenicity to chicks.Intraperitoneal injection of EP01with high potency（1×10⁹PFU/m L）could increase the survival rate of chicks infected by 60%,and the color of liver,spleen and intestine of chicks treated with EP01 gradually returned to normal,and the symptoms of necrosis and hemorrhage were relieved.After 24 h of EP01 treatment,the number of viable bacteria in liver,spleen and intestine of chicks was significantly decreased by1.29-3.56 orders of magnitude（p<0.01）.These results showed that EP01 had a good therapeutic effect on chicks infected with E.coli O157:H7.In this study,a phage electrochemical sensor for the detection of E.coli O157:H7 GXEC-N07 was established based on the high efficiency and specificity of phage EP01 in recognizing E.coli O157:H7 GXEC-N07.Due to the large specific surface area and good electrochemical performance of carboxyl graphene oxide and conductive carbon black,CFGO was selected as the carrier of EP01 and CB as the electrochemical signal enhancer.The two nanomaterials were mixed with EP01 and dropped onto the surface of GCE and sealed with BSA.Since EP01 has a specific recognition effect on GXEC-N07,and the adhesion of GXEC-N07 to the surface of the sensor leads to the electron shielding effect of the cell membrane,which obstructs the interface electron transfer and causes the reduction of the electrochemical signal,thus achieving the quantitative analysis of GXEC-N07.This study also explored the effects of carboxyl activation time,blocking time and scanning rate on the performance of the sensor,and determined the optimal detection conditions by studying the effects of phage sensor and incubation time of GXEC-N07.Different concentrations of GXEC-N07 were detected under optimal conditions.The results showed that CFGO could immobilize phages well and CB had good electron conduction ability.The phage sensor showed good linearity in the range of 1×10²-1×10⁷CFU/m L,with a linear correlation coefficient of 0.97194 and a detection limit of 11.8 CFU/m L.The whole detection process could be completed within 30 min.Finally,we carried out real sample detection in milk samples,and the results showed that the recoveries ranged from 63.3%to 114.2%.In conclusion,polyvalent broad-spectrum phage EP01 has the potential to control foodborne E.coli and Salmonella,as well as treat animal infected with E.coli,and has a good application prospect in animal food safety and phage therapy.Bacteriophage electrochemical sensor based on carboxyl graphene oxide and conductive carbon black has the advantages of simple operation,low cost,short detection time,low detection limit,strong anti-interference and good reproducibility.Phage-based electrochemical sensor provides a new idea for the detection of foodborne pathogens,and it has a good application prospect.