Application of a Broad Range Lytic Phage LPST94 for Biological Control of Salmonella in Foods

Abstract

Salmonella, one of the most common food-borne pathogens, is a significant public health and economic burden worldwide. Lytic phages are viable alternatives to conventional technologies for pathogen biocontrol in food products. In this study, 40 Salmonella phages were isolated from environmentally sourced water samples. We characterized the lytic range against Salmonella and among all isolates, phage LPST94 showed the broadest lytic spectrum and the highest lytic activity. Electron microscopy and genome sequencing indicated that LPST94 belongs to the Ackermannviridae family. Further studies showed this phage is robust, tolerating a wide range of pH (4-12) and temperature (30-60 °C) over 60 min. The efficacy of phage LPST94 as a biological control agent was evaluated in various food products (milk, apple juice, chicken breast, and lettuce) inoculated with non-typhoidal Salmonella species at different temperatures. Interestingly, the anti-Salmonella efficacy of phage LPST94 was greater at 4 °C than 25 °C, although the efficacy varied between different food models. Adding phage LPST94 to Salmonella inoculated milk decreased the Salmonella count by 3 log₁₀ CFU/mL at 4 °C and 0.84 to 2.56 log₁₀ CFU/mL at 25 °C using an MOI of 1000 and 10000, respectively. In apple juice, chicken breast, and lettuce, the Salmonella count was decreased by 3 log₁₀ CFU/mL at both 4 °C and 25 °C after applying phage LPST94 at an MOI of 1000 and 10,000, within a timescale of 48 h. The findings demonstrated that phage LPST94 is a promising candidate for biological control agents against pathogenic Salmonella and has the potential to be applied across different food matrices.

Keywords: LPST94; Salmonella; biological control; characterization; phage.

Figure 1

(A) Comparison of the lytic ability of selected phages using S. enterica serovar Typhimurium (UK-1) as a host at multiplicity of infection (MOI) of 1 in tryptone soy broth; Lytic ability of phage LPST94 to lyse S. Typhimurium and S. Enteritidis in tryptone soy broth medium at MOIs of 100, 10, 1, and 0.1 at 37 °C in vitro. (B) S. Typhimurium UK-1, (C) S. Typhimurium ATCC 14028, (D) S. Enteritidis ATCC 13076, and (E) S. Enteritidis SGSC 4901. Values represent means with the standard deviation of three replicates of each time point.

Figure 2

Morphological and genomic characteristics of phage LPST94. (A) TEM image of phage LPST94; Bar 100 nm, and (B) Genome map of phage LPST94. Patterns were divided into four circles: the full length of the genome was indicated in the first circle; the open reading frame was indicated in the second circle, and the clockwise arrow and the counterclockwise arrow represented the forward reading frame and the reverse reading frame, respectively; GC content was indicated in the third circle; while on the fourth circle, GC skew of G-C/G+C was indicated as green and purple, and green meant the values of GC skew greater than 0 and purple meant the values less than 0. The open reading frames marked with the color of each gene refers to the functional category: phage structure (blue), replication/recombination/repair (yellow), nucleotide metabolism (maroon), transcription (orange), translation (green) additional function (purple) and hypothetical proteins (grey). The genome map generated by the BRIG.jar software.

Figure 3

Characteristics of phage LPST94. (A) One-step growth curves of phage LPST94 with Salmonella enterica serovar Typhimurium UK-1 host infected at 37 °C, (B) pH tolerance of phage LPST94 (pH 2 to 13), (C) Temperature tolerance of phage LPST94 (30 °C to 80 °C), and (D) Stability of phage LPST94 in four food samples (milk, apple juice, chicken breast, and lettuce) at 25 °C. Values represent mean with standard deviation of three determinations of each point.

Figure 4

Effectiveness of phage LPST94 in reducing the S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 in milk. (A) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14,028 in milk at 4 °C, (B) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 in milk at 25 °C, (C) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) in milk at 4 °C, and (D) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) in milk at 25 °C. Values represent mean with standard deviation of three determinations. ** Significance of p < 0.01; * Significance of p < 0.05.

Figure 5

Effectiveness of phage LPST94 in reducing the S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 in apple juice. (A) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 in apple juice at 4 °C, (B) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 in apple juice at 25 °C, (C) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) in apple juice at 4 °C, and (D) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) in apple juice at 25 °C. Values represent mean with standard deviation of three determinations. ** Significance of p < 0.01; * Significance of p < 0.05.

Figure 6

Effectiveness of phage LPST94 in reducing the S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 in chicken breast. (A) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 on chicken breast at 4 °C, (B) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 on chicken breast at 25 °C, (C) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) on chicken breast at 4 °C, and (D) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) on chicken breast at 25 °C. Values represent mean with standard deviation of three determinations. ** Significance of p < 0.01; * Significance of p < 0.05.

Figure 7

Effectiveness of phage LPST94 in reducing the S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076 in lettuce. (A) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 on lettuce at 4 °C, (B) Effect of phage LPST94 on growth of S. Typhimurium ATCC 14028 on lettuce at 25 °C, (C) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) on lettuce at 4 °C, and (D) Effect of phage LPST94 on growth of Salmonella mixture (S. Typhimurium ATCC 14028 and S. Enteritidis ATCC 13076) on lettuce at 25 °C. Values represent mean with standard deviation of three determinations. ** Significance of p < 0.01; * Significance of p < 0.05.