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

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.