Evaluating the Phenotypic and Genomic Characterization of Some Egyptian Phages Infecting Shiga Toxin-Producing Escherichia coli O157:H7 for the Prospective Application in Food Bio-Preservation

Abstract

Shiga toxin-producing E. coli (STEC) is considered a worldwide public health and food safety problem. Despite the implementation of various different approaches to control food safety, outbreaks persist. The aim of study is to evaluate the applicability of phages, isolated against STEC O157:H7, as prospective food bio-preservatives. Considering the relatively wide host range and greatest protein diversity, two phages (STEC P2 and P4) from four were furtherly characterized. Complete genome analysis confirmed the absence of toxins and virulence factors-encoding genes. The results confirmed the close relation of STEC P2 to phages of Myoviridae, and STEC P4 to the Podoviridae family. The phages retained higher lytic competence of 90.4 and 92.68% for STEC P2 and P4, respectively with the HTST pasteurization. The strong acidic (pH 1) and alkaline (pH 13) conditions had influential effect on the surviving counts of the two phages. The lowest survivability of 63.37 and 86.36% in STEC P2 and P4 lysate, respectively appeared in 2% bile salt solution after 3 h. The results confirmed the strong effect of simulated gastric fluid (SGF) on the survivability of the two phages comparing with simulated intestinal fluid (SIF). Therefore, the two phages could be applied as a natural alternative for food preservation.

Keywords: Shiga toxin-producing Escherichia coli; bacteriophage; biological characterization; food bio-preservation; genome sequence.

Figure 1

Transmission electron microscopy (TEM) micrographs of E. coli phage particles (STEC P1–STEC P4) stained negatively by 2% (w/v) phosphotungstate at pH 7.2. STEC P1: (A) direct magnification: 60,000×; STEC P1: (B) direct magnification: 120,000×; STEC P2: (C) direct magnification: 80,000×; STEC P2: (D) direct magnification: 60,000×; STEC P3: (E) direct magnification: 10,000×; STEC P3: (F) direct magnification: 80,000×; STEC P4: (G) direct magnification: 80,000×.

Figure 1

Transmission electron microscopy (TEM) micrographs of E. coli phage particles (STEC P1–STEC P4) stained negatively by 2% (w/v) phosphotungstate at pH 7.2. STEC P1: (A) direct magnification: 60,000×; STEC P1: (B) direct magnification: 120,000×; STEC P2: (C) direct magnification: 80,000×; STEC P2: (D) direct magnification: 60,000×; STEC P3: (E) direct magnification: 10,000×; STEC P3: (F) direct magnification: 80,000×; STEC P4: (G) direct magnification: 80,000×.

Figure 2

SDS-PAGE analysis of phage structural proteins. Lane M, protein marker. Lanes 1–4, structure proteins from STEC P1–STEC P2.

Figure 3

Lytic activity of STEC P2 after infection of STEC O157:H7 wild type strain 93,111 at MOI of 1, 0.1, 0.01 and 0.001 for 7 h. LSD value at 0.05: 0.7.

Figure 4

Lytic activity of STEC P4 after infection of STEC O157:H7 wild type strain 93,111 at MOI of 1, 0.1, 0.01 and 0.001 for 7 h. LSD value at 0.05: 0.523.

Figure 5

STEC P2 stability in 1 and 2% bile salt. LSD value at 0.05:0.296.

Figure 6

STEC P4 stability in 1 and 2% bile salt. LSD value at 0.05:0.296.

Figure 7

Effect of simulated gastric fluid on the counts of STEC P2 and STEC P4. LSD value at 0.05:0.286.

Figure 8

Effect of simulated intestinal fluid on the counts of STEC P2 and STEC P4. LSD value at 0.05:0.449.

Figure 9

Map of the genome organization of Escherichia phage ST2 and ST4. Different structural proteins are indicated with different colors.

Figure 10

(A) phylogenetic tree of Escherichia phage ST2 based on the sequence of terL gene, (B) shows the phylogenetic GBDP tree and (C) genome comparisons with the closest phages obtained from the NCBI GenBank using BRIG software version 0.95.

Figure 11

(A) phylogenetic tree of Escherichia phage ST4 based on the sequence of terL gene, (B) shows the phylogenetic GBDP tree and (C) genome comparisons with the closest phages obtained from the NCBI GenBank using BRIG software version 0.95.

Figure 12

Comparison of ORFs between Escherichia phage ST2 (above) and the most closed phages Escherichia phage vB EcoM PhAPEC2 (middle) and Shigella phage Shf125875 (below). Analysis was performed using EasyFig software version 2.2.5.

Figure 13

Comparison of ORFs between Escherichia phage ST4 (middle) and the most closed phages Escherichia phage vB EcoP PhAPEC7 (above) and Escherichia phage PGN829.1 (below). Analysis was performed using EasyFig software version 2.2.5.