Isolation Of Bacteriophage PK88-4That Lyse Enterotoxigenic Escherichia Coli And Its Evaluation Of Antibacterial Efficacy And Safety

Enterotoxigenic Escherichia coli (ETEC) are a cause of porcine neonatal and post-weaning diarrhea, that may occur sporadically or as outbreaks leading to substantial economic losses. There are concerns that increasing antimicrobial resistance of normal flora and ETEC in pigs may compromise the therapeutic use of antimicrobials in pigs and threaten human health. Recently, more researchers support the biological control of ETEC K88with virulent bacteriophages. The goal of this research was therefore to isolate virulent bacteriophage that was active against ETEC K88, and investigate the in vitro and in vivo antibacterial feasibility of isolated bacteriophage and evaluate the safety of administration of it. This research included four trials:Trial1:To isolate, identify and characterize microbiologically the virulent bacteriophage that might target enterotoxigenic E. coli K88(ETEC K88). The bacteriophage was isolated from sewage samples of pig farms by double-layer agar plate method with ETEC K88as host. The isolated bacteriophage was investigated by electron microscopy and restriction analysis to identify morphology and type of nucleic acid. Meanwhile, the optimal input multiplicity of infection (MOI), one-step growth curve, thermo-and pH stability, lytic dynamic in vitro and antibacterial activity in vitro of the isolated bacteriophage were investigated.One virulent bacteriophage that lysed ETEC K88was isolated successfully and named PK88-4. The bacteriophage produced distinct, clear and no turbid or bull-eyed plaques. The isolated bacteriophage belongs to the Myoviridae family with a contractile tail and icosahedral head. The estimated genome size was about60kb and the restriction analysis suggested that it was dsDNA. Its optimal MOIinput was0.01.The latent period, rise period and burst size of PK88-4were about10min,40min and40,respectively.The PK88-4was considerably stable after exposure to high temperature (60℃) and pH between5and10, and could lyse efficiently K88in logarithmic growth phase within5hours.Trial2:To transform the plasmid with ampr gene to the K88strain used in challenge and to establish the mice model of lethal enterotoxigenic E. coli infection to evaluate subsequently the antibacterial effect of bacteriophage in vivo. The competent ETEC K88cells were prepared by CaCl2and the transformation of pUC19was by heatshock. Forty-two mice were equally assigned into six tested groups.Varying numbers of ETEC K88cells with ampr gene suspended in200μl saline were injected i.p. into the peritoneal cavities of mice from five of the six tested groups. As controls, equal volumes of saline alone were injected intraperitoneally into the remain mice. The clinical symptoms of mice were monitored, the time of death induced by challenge was recorded and the survival rates of mice from six tested groups were analyzed. The liver, kidney, small intestine and spleen from the stated mice were observed by macroscopic test and pathological section, and the colonization of anti-Amp ETEC K88strains in mice was investigated. The optimal dose of challenge was determined by above-mentioned study.The ETEC K88strains with the plasmid of pUC19were isolated and named K88plus. The obvious clinical symptoms of mice challenged with K88plus were observed. The absolute lethal dose of K88plus against mice was2.0×108CFU/200μL. The dose of≥2.0×108CFU/200μL K88plus was fatal in100%of mice within36h after challenge and the same fatalities within24h after challenge with2.6×108CFU/200μL. The celiac major organs of mice challenged with K88plus showed lesions signs, and concentrations of K88plus in them were so high.Trial3:To analyse the antibacterial feasibility in vivo of bacteriophage PK88-4against ETEC K88plus by evaluating effect on prophylaxes and therapies of lethal infection due to challenge with K88plus. The bacteriophage PK88-4of varying doses and timing of administration was injected i.p. into mouse challenged with K88plus of2.6×108CFU/200μL. As controls, equal volumes of LB alone were injected intraperitoneally on all test occasions. The survival rates of mice from different test were analyzed. In addition, the concentrations of PK88-4and K88plus in mice were measured and the dynamics of them were investigated.5min after challenge with K88plus, the administration of PK88-4at the dose of>10PFU/500μL was therapeutically effective in100%of mice.30min after challenge, the administration of PK88-4at a dose of1010PFU/500μL was therapeutically effective in50% of mice and in87.5%at a dose of1011PFU/500μL.60min after challenge, the administration of PK88-4at a dose of1010PFU/500μL was therapeutically effective in12.5%of mice and in75%at a dose of1011PFU/500μL.And120min or60min before challenge, the administration of PK88-4at the dose of1010PFU/500μL was prophylactically effective in100%of mice. Bacteriophage PK88-4showed the high antibacterial activity in vivo by observation of dynamics of PK88-4and K88plus.Trial4:To evaluate the non-clinical safety of bacteriophage PK88-4, and to investigate the intestinal microflora of mice administrated intragastrically with PK88-4. The bacteriophage PK88-4was used to perform the acute toxicity test on mice. The clinical symptoms of mice injected i.p. with high-dose of PK88-4were monitored and the survival rates of them were analyzed. The bacteriophage PK88-4was also used to perform the repeated doses toxicity test on mice. The possible pathological changes in mice injected i.p. with repeated-dose of PK88-4were observed by macroscopic test and pathological section.And the blood parameters and biochemical parameters in serum of blood samples from them were analyzed. In addition, diversities of bacterial community in feces sample of mice administrated intragastrically with PK88-4were monitored by PCR-DGGE analysis.The clinical abnormalities and lethality in mice tested by acute toxicity were not observed. The pathological examination, the blood parameters and biochemical parameters in serum in mice tested by repeated doses toxicity were not significantly abnormal. Continuous administration with PK88-4would not cause the dysbacteriosis of microflora in fecal specimens from mice.In conclusion, the isolated virulent bacteriophage that could lyse ETEC K88was novel and showed the high antibacterial activity in vitro. The ETEC K88plus strain was high comparatively virulent in mice, and the challenge of2.6×108CFU/200μL was considered to be optimal for establishing model of infection because of absolute lethality to mice. The bacteriophage PK88-4had the antibacterial feasibility, and could be applied to prophylaxes and therapies of piglet diarrhea due to enterotoxigenic Escherichia coli. According to our trial data, the safety of administration of bacteriophage PK88-4in mice was acceptable. And administration of PK88-4would not result in a severe damage of murine intestinal microflora.