EAEC噬菌体PNJ1809-11和PNJ1809-13作为环境消毒剂的杀菌效果评估

Evaluation of sterilization efficiency of EAEC phage PNJ1809-11 and PNJ1809-13 as environmental disinfectants

摘要:

[目的] 分析2株肠聚集性大肠杆菌（EAEC） CVCC232噬菌体PNJ1809-11、PNJ1809-13的生物学特性，并对其作为环境消毒剂的杀菌效果进行评估。[方法] 透射电镜下观察PNJ1809-11、PNJ1809-13的形态；通过宿主谱、最佳感染复数（MOI）、一步生长曲线、对pH和温度耐受性的测定分析PNJ1809-11、PNJ1809-13的生物学特性；比较2株噬菌体的体外杀菌效果和喷雾、雾化处理后的杀菌效果；检测噬菌体在模拟养殖环境下的耐受性，及喷雾处理的噬菌体制剂在模拟养殖环境下的杀菌效果和对宿主菌生物被膜的清除效果；分析宿主菌的抗性突变率。[结果] 电镜下观察PNJ1809-11、PNJ1809-13均为肌尾病毒科噬菌体；PNJ1809-11可裂解155株大肠杆菌，PNJ1809-13可裂解46株大肠杆菌；噬菌体PNJ1809-11、PNJ1809-13的最佳感染复数（MOI）均为10，最适pH均为7；与PNJ1809-13相比，PNJ1809-11具有较好的热稳定性；在模拟的封闭装置中，将两株噬菌体分别经喷雾、雾化处理后，二者对宿主菌的杀灭效果均可达99%以上；对人工污染的粪便表面细菌的杀灭效果均达到99%以上。噬菌体PNJ1809-11、PNJ1809-13及两者的混合制剂（噬菌体鸡尾酒）对宿主菌CVCC232形成的生物被膜的裂解效率分别为78%、30%和83%。噬菌体PNJ1809-11在养殖温度、粪便pH以及阳光照射下，其耐受性均强于PNJ1809-13。宿主菌对PNJ1809-11、PNJ1809-13的抗性突变率分别为2.5×10^–3和1.0×10^–3。[结论] 综上，噬菌体PNJ1809-11的环境耐受力更强，噬菌体鸡尾酒对生物被膜的裂解效果更好，提示噬菌体PNJ1809-13或噬菌体鸡尾酒经喷雾处理后具有作为环境杀菌剂的潜力。

Abstract:

[Objective] Analyzing the biological characteristics of two enteroaggregating Escherichia coli (EAEC) CVCC232 phages PNJ1809-11 and PNJ1809-13, and evaluating their bactericidal effects as environmental disinfectants. [Methods] We observed the morphology of PNJ1809-11 and PNJ1809-13 under a transmission electron microscope, analysed their biological characteristics, including the host spectrum, optimal multiplicity of infection, one-step growth curve, and tolerance to pH and temperature, also compared the sterilization efficiency of atomized and sprayed phages. We next determined the bactericidal effect of the sprayed phage in a simulated livestock farm environment as well as the effect of the phages on the biofilm disruption of the host bacteria. At last we tested the tolerance of the two phages to the environment of simulated livestock farm and the mutation frequency of phage-resistant strains. [Results] The results of electron microscope observation showed that phages PNJ1809-11 and PNJ1809-13 both belong to Myoviridae phages. PNJ1809-11 and PNJ1809-13 could lyse 155 and 46 Escherichia coli strains, respectively. Biological assays showed that the MOI of the two phages are both 10; the optimal pH of them are both 7; compared with PNJ1809-13, PNJ1809-11 showed better thermal stability. In the simulated closed device, the sterilization efficiency of spryed and atomized PNJ1809-11 and PNJ1809-13 on the host bacteria was as high as 99%; the sterilization efficiency on the surface of artificially contaminated feces was also higher than 99%. The lysis efficiency of PNJ1809-11, PNJ1809-13 and their mixture (phage cocktail) against biofilm of CVCC232 were 78%, 30% and 83% respectively. Phage PNJ1809-11 had better tolerance than PNJ1809-13 to the temperature, fecal pH and sunlight of simulated livestock farm. The mutation frequency of phage-resistant strains was 2.5×10^-3 and 1.0×10^-3 respectively. [Conclusion] In summary, phage PNJ1809-11 was more tolerant to the environment, and the phage cocktail showed higher lysis effect on biofilm, which suggested that sprayed phage PNJ1809-11 or the cocktail of PNJ1809-11 and PNJ1809-13 had the potential as an environmental disinfectant.