Application of a novel lytic phage vB_EcoM_SQ17 for the biocontrol of Enterohemorrhagic Escherichia coli O157:H7 and Enterotoxigenic E. coli in food matrices

Abstract

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and Enterotoxigenic E. coli (ETEC) are important foodborne pathogens, causing serious food poisoning outbreaks worldwide. Bacteriophages, as novel antibacterial agents, have been increasingly exploited to control foodborne pathogens. In this study, a novel broad-host range lytic phage vB_EcoM_SQ17 (SQ17), was isolated, characterized, and evaluated for its potential to control bacterial counts in vitro and in three different food matrices (milk, raw beef, and fresh lettuce). Phage SQ17 was capable of infecting EHEC O157:H7, ETEC, and other E. coli strains. Morphology, one-step growth, and stability assay showed that phage SQ17 belongs to the Caudovirales order, Myoviridae family, and Mosigvirus genus. It has a short latent period of 10 min, a burst size of 71 PFU/infected cell, high stability between pH 4 to 12 as well as thermostability between 30°C and 60°C for 60 min. Genome sequencing analysis revealed that the genome of SQ17 does not contain any genes associated with antibiotic resistance, toxins, lysogeny, or virulence factors, indicating the potential safe application of phage SQ17 in the food industry. In Luria-Bertani (LB) medium, phage SQ17 significantly decreased the viable counts of EHEC O157:H7 by more than 2.40 log CFU/ml (p < 0.05) after 6 h of incubation at 37°C. Phage SQ17 showed great potential to be applied for biocontrol of EHEC O157:H7 in milk and raw beef. In fresh lettuce, treatment with SQ17 also resulted in significant reduction of viable cell counts of EHEC O157:H7 and ETEC at both 4°C and 25°C. Our results demonstrate that SQ17 is a good candidate for application as an EHEC O157:H7 and ETEC biocontrol agent in the processing stages of food production and food preservation.

Keywords: EHEC O157:H7; ETEC; biocontrol; foodborne pathogen; foods; phage.

Figure 1

Morphology of EHEC O157:H7 phage SQ17. (A) The plaques formed by phage SQ17 on the lawns of EHEC O157:H7 EO157-1. TEM analysis of phage SQ17 with its tail in the non-contracted (B) and contracted (C) states negatively stained with uranyl acetate (possibly with an empty head). Scale bar, 100 nm.

Figure 2

One-step growth curve of phage SQ17 following infection of EHEC O157:H7 EO157-1 at an MOI of 0.1 in LB medium at 37°C. The latent period is 10 min. The values represent the means and standard deviations (SD; n = 3).

Figure 3

Biological characteristics of phage SQ17 at different pH values and temperatures. Tolerance of phage SQ17 at different pH values (A) and temperatures (B). The values represent the means and standard deviations (SD; n = 3).

Figure 4

Effect of phage SQ17 on EHEC O157:H7. (A) Phage inhibition of viable EHEC O157:H7 EO157-1 cell counts at 0, 3, and 6 h in LB medium at an MOI of 10, 1, 0.1, or 0.01, respectively. (B) Lytic activity of phage SQ17 against EHEC O157:H7 EO157-1 in LB medium at an MOI of 10, 1, 0.1, or 0.01. Absorbance measurements at 600 nm were made every 30 min. All cultures were incubated at 37°C. The control contained EHEC O157:H7 EO157-1 only. The values represent the means and standard deviations (SD; n = 3).

Figure 5

Genome map of phage SQ17. The genome was generated using CGview server beta and is annotated and colored based on predicted molecular functions of identified genes. The center of the genome map provides % GC content (black) and the GCskew+ and skew-are shown in light green and purple, respectively.

Figure 6

Comparative genomic analysis of phage SQ17 with phages SF, vB_EcoM-ZQ3 and vB_EcoM_JS09 using Mauve. Nucleotide sequence similarity is indicated by the height of the colored bars, while regions that are dissimilar are in white.

Figure 7

Phylogenetic tree of phages with similar short tail fiber protein sequences to phage SQ17. Host bacteria of phages are in blue.

Figure 8

Effect of phage SQ17 against EHEC O157:H7 EO157-1 in LB medium at 4°C (A) and 25°C (B). The error bars show the standard error of the mean.

Figure 9

Effect of phage SQ17 against EHEC O157:H7 EO157-1 in milk. The viability of EO157-1 cell in UHT skim milk (A) and UHT whole milk (B) incubated at 4°C, and in UHT skim milk (C) and UHT whole milk (D) incubated at 25°C. The error bars show the standard error of the mean.

Figure 10

Effect of phage SQ17 against EHEC O157:H7 EO157-1 and ETEC EK99-F41 in solid food matrices. The viability of EO157-1 cell on raw beef incubated at (A) 4°C and (B) 25°C and on fresh lettuce incubated at (C) 4°C and (D) 25°C. The viability of EK99-F41 cell on fresh lettuce incubated at (E) 4°C and (F) 25°C. The error bars show the standard error of the mean.