Isolation, Characterization, and Genomic Analysis of Three Novel E. coli Bacteriophages That Effectively Infect E. coli O18

Abstract

Escherichia coli (E. coli) is one of the most common pathogenic bacteria worldwide. Avian pathogenic E. coli (APEC) causes severe systemic disease in poultry (Colibacillosis), and accordingly, has an extreme risk to the poultry industry and public health worldwide. Due to the increased rate of multi-drug resistance among these bacteria, it is necessary to find an alternative therapy to antibiotics to treat such infections. Bacteriophages are considered one of the best solutions. This study aimed to isolate, characterize, and evaluate the potential use of isolated bacteriophages to control E. coli infections in poultry. Three novel phages against E. coli O18 were isolated from sewage water and characterized in vitro. The genome size of the three phages was estimated to be 44,776 bp, and the electron microscopic analysis showed that they belonged to the Siphoviridae family, in the order Caudovirales. Phages showed good tolerance to a broad range of pH and temperature. The complete genomes of three phages were sequenced and deposited into the GenBank database. The closely related published genomes of Escherichia phages were identified using BLASTn alignment and phylogenetic trees. The prediction of the open reading frames (ORFs) identified protein-coding genes that are responsible for functions that have been assigned such as cell lysis proteins, DNA packaging proteins, structural proteins, and DNA replication/transcription/repair proteins.

Keywords: Escherichia coli; Siphoviridae; avian pathogenic E. coli (APEC); bacteriophages.

Figure 1

Stability of ZCEC10, ZCEC11, and ZCEC12 at different pH and temperatures. The figure represents the stability of phages at different temperatures: (A) ZCEC10, (B) ZCEC11, and (C) ZCEC12 and the stability of phages at different pH values: (D) ZCEC10, (E) ZCEC11, and (F) ZCEC12.

Figure 2

Time killing assay of phages against E. coli at 37 °C. These panels show the bacterial count of E. coli as the control and bacterial survival infected with three different phages: (A) ZCEC10; (B) ZCEC11; (C) ZCEC12 at different MOIs (0.01, 0.1, 1, and 10). Optical density at 600 nm was measured every 10 min up to 2.5 h. MOI: Multiplicity of infection, OD: Optical density.

Figure 3

Phylogenetic relationships between the ZCEC10, ZCEC11, and ZCEC12 phages and BLASTN top-matched phages.

Figure 4

(a–c) Genetic maps of the three phages. Only functional genes are highlighted.

Figure 5

TEM images showing the morphology of the individual phages: (a) ZCEC10 phage, (b) ZCEC11 phage, and (c) ZCEC12 phage, scale bar represents 100 nm.