Molecular characterization and safety properties of multi drug-resistant Escherichia coli O157:H7 bacteriophages

Abstract

The increase in multi drug resistance (MDR) amongst food-borne pathogens such as Escherichia coli O157:H7, coupled with the upsurge of food-borne infections caused by these pathogens is a major public health concern. Lytic phages have been employed as an alternative to antibiotics for use against food-borne pathogens. However, for effective application, phages should be selectively toxic. Therefore, the objective of this study was to characterise lytic E. coli O157:H7 phages isolated from wastewater as possible biocontrol agents and access their genomes for the absence of genes that denotes virulence, resistance, toxins, and lysogeny using whole genome sequencing. E. coli O157:H7 bacteriophages showed clear plaques ranging in size from 1.0 mm to 2.0 mm. Spot test and Efficiency of plating (EOP) analysis demonstrated that isolated phages could infect various environmental E. coli strains. Four phages; vB_EcoM_EP32a, vB_EcoP_EP32b, vB_EcoM_EP57, and vB_EcoM_EP69 demonstrated broad lytic spectra against E. coli O157:H7 strains. Transmission Electron Microscopy (TEM) showed that all phages have tails and were classified as Caudoviricetes. Growth parameters showed an average latent period of 15 ± 3.8 min, with a maximum burst size of 392 PFU/cell. The phages were stable at three distinct temperatures (4 °C, 28 °C, and 37 °C) and at pH values of 3.5, 5.0, 7.0, 9.0, and 11.0. Based on their morphological distinctiveness, three phages were included in the Whole Genome Sequencing (WGS) analysis. WGS results revealed that E. coli O157:H7 phages (vB_EcoM_EP32a, vB_EcoP_EP32b, and vB_EcoM_EP57) were composed of linear double-stranded DNA (dsDNA) with genome sizes 163,906, 156,698, and 130,723 bp and GC contents of 37.61, 37, and 39% respectively. Phages vB_EcoM_EP32a and vB_EcoP_EP32b genomes were classified under the class Caudoviricetes, Straboviridae family, and the new genus "Phapecoctavirus", while vB_EcoM_EP57 was classified under the class Caudoviricetes, Autographiviridae family. Genome analysis revealed no lysogenic (integrase), virulence, or antimicrobial resistance sequences in all three Escherichia phage genomes. The overall results provided evidence that lytic E. coli O157:H7 bacteriophages in this study, are relatively stable, can infect diverse E. coli strains, and does not contain genes responsible for virulence, resistance, toxins, and lysogeny. Thus, they can be considered as biocontrol candidates against MDR pathogenic E. coli O157:H7 strains in the food industry.

Keywords: E. coli O157:H7; Antibiotic resistance; Biocontrol; Food pathogen; Phage genome; Whole genome sequencing.

Fig. 1

Plaque morphology of representative phage

Fig. 2

Transmission electron micrographs of representative phage isolates (negatively stained with 1% ammonium molybdate). The bars indicate scale (100 nm). *A = vB_EcoM_EP57; B = vB_EcoP_EP32b; C = vB_EcoM_EP32a and D = vB_EcoM_EP69

Fig. 3

One-step growth curves for four E. coli O157:H7 phage isolates

Fig. 4

Adsorption rate of E. coli O157:H7 phages

Fig. 5

Effect of temperature on E. coli O157:H7 phages

Fig. 6

The stability and viability of E. coli O157:H7 phage at 40 ºC

Fig. 7

Effect of pH on E. coli O157:H7 phage after 24 h

Fig. 8

Effect of pH on E. coli O157:H7 phage after 48 h

Fig. 9

Subsystem function of Escherichia phage vB_EcoM_EP32a (A), vB_EcoP_EP32b (B), and vB_EcoM_EP57 (C)

Fig. 10

Genomic map of Escherichia phage vB_EcoM_EP32a

Fig. 11

Genomic map of Escherichia phage vB_EcoP_EP32b

Fig. 12

Genomic map of Escherichia phage vB_EcoM_EP57

Fig. 13

Proteomic tree of Escherichia phage vB_EcoM_EP32a, vB_EcoP_EP32b and vB_EcoM_EP57 constructed based on the complete genome sequences of closely related phages selected from the entire proteomic tree on the Viptree software. The tree is constructed by BIONJ based on genomic distance matrixes, and mid-point rooted. Branch lengths are logarithmically scaled from the root of the entire proteomic tree. The numbers at the top represent the log‐ scaled branch lengths based on the SG (normalized tBLASTx scores) values

Fig. 14

Hit Map for genomic comparison of Escherichia phage vB_EcoM_EP32a, vB_EcoP_EP32b, and vB_EcoM_EP57

Fig. 15

Easyfig homology diagram of vB_EcoM_EP32a, vB_EcoP_EP32b and vB_EcoM_EP57 (with red colour) with close phage relatives in NCBI using BLASTn. Arrows represent the locations of coding sequences and the percentage of sequence similarity is indicated by the red and grey shaded lines

Fig. 16

Phylogenetic tree showing position of the Escherichia phage vB_EcoM_EP32a and vB_EcoP_32b phage (blue star) based on the amino acid sequence of the terminase large subunit (TerL) of the closest 10 sequences identified using NCBI BLASTp. Sequences were aligned using MUSCLE in MEGA 11 software. The neighbor joining method was used to generate phylogenetic tree

Fig. 17

Phylogenetic tree showing position of the phages vB_EcoM_EP32a and vB_EcoP_EP32b and vB_EcoM_EP57 (blue star) based on the amino acid sequence of the tail protein of the closest 9 sequences identified using NCBI BLASTp. Sequences were aligned using MUSCLE in MEGA 11 software. The neighbor joining method was used to generate phylogenetic tree