| National Action Plan to Contain Antimicrobial Resistance(2016-2020)is the public health consensus and the national will,which needs practical technical measures.In addition to accurate medication on the reliable basis of antibiotic susceptibility test,phage becomes a huge potential option with unique advantages of strong phagocytosis specificity,no cross resistance with antibiotics,no residue,no infectious for humans and animals,easy to filter and obtain.In this paper,E.coli isolates were obtained from clinic and fecal samples of breeder chickens and commodity chickens in Shandong province.The method of antibiotic susceptibility test was improved for early warning resistant strains;further more,the lytic phages were screened for the epidemic E.coli strains,and then analysed their biological properties and genetic structures.The treatments were implemented by injection and oral test,in order to explore the control of E.coli infections in such farms.Using Macconkey medium,E.coli isolates were obetanied from clinical and fecal samples.A total of 117 strains were identified with the specific primer phoA.There were 65 types in MLST typing,in which ST155 ST156 and ST69 were the majority,and the other two new ST types were found in this studyLinear gradient plate could accurately determine MIC,calculate the original test antibiotic concentrations,so as to better guarantee the reliability of the quality control test.When the linear gradient were setted as O-S,S-R,R-8R according to Clinical Laboratory Standards Institute or European Committee on Antimicrobial Susceptibility Testing standards,first of all the sensitivity range and bacterial community MIC value could be judged accurately,and resistant strains could be effectively isolated for early warning the antibiotic resistance that may occur after use,and guiding the choice of antimicrobial agents and clinical application.Optimistically ready-to-use gradient plates with the automated production would facilitate the clinical drug sensitive test and improve resistance screening technology.143 strains of phage were isolated and purified with semi solid double agar plate method for the E.coli isolates menthioned above.Three strains of phage of main ST strains were muscular tails T4 phage of caudata composed of icosahedron the head and tail by sequencing and electron microscope,in whichvB-EcolP-YSF1 was a new phage which only 11%of the similarity with the sequenced phage.The biological characteristics analysis of vB-EcolP-XT4,vB-EcolP-RZ? and vB-EcolP-YSF1 showed that their titer can reach 10~9 pfu/ml,and the thermal stability was higher that phage titer could still remained more than 10~7 pfu/ml at 60 ? heat preservation after 1 h.The optimum growth temperature were at about 37 ?commonly.High cracking activity could keep in pH 4-10.Phage best multiplicity of infection was commonly 0.1-1 infection,preclinical time was about 20 min after infection of the host.Toxic strains were selected by median lethal dose and virulence genes determination and multiplicity of infection of its phages was optimized by board test.Optimal concentration ratio of strain together with phage vB-EcolP-RZ?or vB-EcolP-XT4 was injected into chicken chest muscle,which showed good treatment effect.Mortality decreases with improvement of phage titer.However,the pair of high resistant E.coli and its phage were taken orally by SPF chickens,the pathogenic bacteria RZ?-3 reduces with one order of magnitude in spit of no clinical symptoms.Totally,it has clinical value in the actual production conditions and makes the foundation for later phage therapy to colon bacillus disease,so a large scale of purification and prevention and control of E.coli is expected in the next step in a breeder chicken farm. |
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