Characterization of a lytic Escherichia coli phage CE1 and its potential use in therapy against avian pathogenic Escherichia coli infections

Abstract

The high incidence of Avian pathogenic Escherichia coli (APEC) in poultry has resulted in significant economic losses. It has become necessary to find alternatives to antibiotics due to the alarming rise in antibiotic resistance. Phage therapy has shown promising results in numerous studies. In the current study, a lytic phage vB_EcoM_CE1 (short for CE1) against Escherichia coli (E. coli) was isolated from broiler feces, showing a relatively wide host range and lysing 56.9% (33/58) of high pathogenic strains of APEC. According to morphological observations and phylogenetic analysis, phage CE1 belongs to the Tequatrovirus genus, Straboviridae family, containing an icosahedral capsid (80 ~ 100 nm in diameter) and a retractable tail (120 nm in length). This phage was stable below 60°C for 1 h over the pH range of 4 to 10. Whole-genome sequencing revealed that phage CE1 contained a linear double-stranded DNA genome spanning 167,955 bp with a GC content of 35.4%. A total of 271 ORFs and 8 tRNAs were identified. There was no evidence of virulence genes, drug-resistance genes, or lysogeny genes in the genome. The in vitro test showed high bactericidal activity of phage CE1 against E. coli at a wide range of MOIs, and good air and water disinfectant properties. Phage CE1 showed perfect protection against broilers challenged with APEC strain in vivo. This study provides some basic information for further research into treating colibacillosis, or killing E. coli in breeding environments.

Keywords: avian pathogenic Escherichia coli; bactericidal activity; broiler; genome sequencing; phage therapy; phage vB_EcoM_CE1.

Figure 1

Morphological characteristics of phage CE1. Clear plaques produced by phage CE1 on an agar plate (A); TEM image of phage CE1 consisting of an icosahedral head (80 ~ 100 nm in dimeter) and a retractable tail (120 nm in length) (B).

Figure 2

Biological properties of phage CE1. One-step growth curve of phage CE1. The data were expressed as means ± SD (n = 3) (A); Sensitivity of CE1 to temperature. There was little effect on the activity of CE1 when the temperature varied from 40°C to 60°C for 1 h, while CE1 was quickly inactivated when the temperature rose to 70°C for 40 min or 80°C for 20 min. (B); Sensitivity of CE1 to pH. Phage CE1 was relatively stable within the pH range of 4 to 10 when incubated at different pH values (2 to 13) for 3 h (C).

Figure 3

In vitro bactericidal activity of phage CE1. Detection of OD₆₀₀ from phage CE1 against APEC strain SD-C888 under different MOIs (1, 0.1, 0.01, 0.001) (A); Detection of CFUs from phage CE1 against APEC strain SD-C888 under different MOIs (1, 0.1, 0.01, 0.001) (B); Detection of bactericidal activity in the spray disinfection (C); Detection of bactericidal activity in water disinfection (D). All data were expressed as means ± SD (n = 3).

Figure 4

Dynamic change of cytokines in the animal experiment. Levels of TNF-α in different group. **P < 0.01, *P < 0.05 (A); Levels of IFN-α in different group. **p < 0.01, *p < 0.05 (B); Levels of IL-1β in different group. **p < 0.01, ***P < 0.001 (C); Levels of IL-8 in different group (D).