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, 7 (8), 4836-53

Bio-Control of Salmonella Enteritidis in Foods Using Bacteriophages

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Bio-Control of Salmonella Enteritidis in Foods Using Bacteriophages

Hongduo Bao et al. Viruses.

Abstract

Two lytic phages, vB_SenM-PA13076 (PA13076) and vB_SenM-PC2184 (PC2184), were isolated from chicken sewage and characterized with host strains Salmonella Enteritidis (SE) ATCC13076 and CVCC2184, respectively. Transmission electron microscopy revealed that they belonged to the family Myoviridae. The lytic abilities of these two phages in liquid culture showed 104 multiplicity of infection (MOI) was the best in inhibiting bacteria, with PC2184 exhibiting more activity than PA13076. The two phages exhibited broad host range within the genus Salmonella. Phage PA13076 and PC2184 had a lytic effect on 222 (71.4%) and 298 (95.8%) of the 311 epidemic Salmonella isolates, respectively. We tested the effectiveness of phage PA13076 and PC2184 as well as a cocktail combination of both in three different foods (chicken breast, pasteurized whole milk and Chinese cabbage) contaminated with SE. Samples were spiked with 1 × 10(4) CFU individual SE or a mixture of strains (ATCC13076 and CVCC2184), then treated with 1 × 10(8) PFU individual phage or a two phage cocktail, and incubated at 4 °C or 25 °C for 5 h. In general, the inhibitory effect of phage and phage cocktail was better at 4 °C than that at 25 °C, whereas the opposite result was observed in Chinese cabbage, and phage cocktail was better than either single phage. A significant reduction in bacterial numbers (1.5-4 log CFU/sample, p < 0.05) was observed in all tested foods. The two phages on the three food samples were relatively stable, especially at 4 ºC, with the phages exhibiting the greatest stability in milk. Our research shows that our phages have potential effectiveness as a bio-control agent of Salmonella in foods.

Keywords: Salmonella Enteritidis (SE); bio-control; food; phage cocktail; phages.

Figures

Figure 1
Figure 1
Transmission electron micrographs (TEM) of phage PA13076 (A) and PC2184 (B).
Figure 2
Figure 2
The thermal stability of phages PA13076 (A) and PC2184 (B); and the pH stability of phages PA13076 and PC2184 (C).
Figure 3
Figure 3
Lytic effects of phage PA13076 and PC2184 against the specified hosts of liquid cultures in vitro: (A) PA13076 and (B) PC2184.
Figure 4
Figure 4
Effects of individual phages on growth of SE ATCC13076 and CVCC2184 on the surface of chicken breast at 4 °C and 25 °C. Each chicken breast sample was inoculated with either SE ATCC13076 (A,B) or SE CVCC2184 (C,D) (1 × 104 CFU), and phage PA13076 or PC2184 was applied (1 × 108 PFU) later (CK, inoculated control without phage; T, treated with phage). The titers of phage were also detected each sampling time (indicated in a dotted line). Date represent the mean ± S.D. (n = 3), * represents p < 0.05 (Duncan’s test).
Figure 5
Figure 5
Effects of individual phages on growth of SE ATCC13076 and CVCC2184 in milk samples at 4 °C and 25 °C. Each milk sample was inoculated with either SE ATCC13076 (A,B) or SE CVCC2184 (C,D) (1 × 104 CFU), and phage PA13076 or PC2184 was applied (1 × 108 PFU) later (CK, inoculated control without phage; T, treated with phage). The titers of phage were also detected each sampling time (indicated in a dotted line). Date represent the mean ± S.D. (n = 3), * represents p < 0.05 (Duncan’s test).
Figure 6
Figure 6
Effects of individual phages on growth of SE ATCC13076 and CVCC2184 on the surface of Chinese cabbage samples at 4 °C and 25 °C. Each sample was inoculated with either SE ATCC13076 (A,B) or SE CVCC2184 (C,D) (1 × 104 CFU), and phage PA13076 or PC2184 was applied (1 × 108 PFU) later (CK, inoculated control without phage; T, treated with phage). The titers of phage were also detected each sampling time (indicated in a dotted line). Date represent the mean ± S.D. (n = 3), * represents p < 0.05 (Duncan’s test).
Figure 6
Figure 6
Effects of individual phages on growth of SE ATCC13076 and CVCC2184 on the surface of Chinese cabbage samples at 4 °C and 25 °C. Each sample was inoculated with either SE ATCC13076 (A,B) or SE CVCC2184 (C,D) (1 × 104 CFU), and phage PA13076 or PC2184 was applied (1 × 108 PFU) later (CK, inoculated control without phage; T, treated with phage). The titers of phage were also detected each sampling time (indicated in a dotted line). Date represent the mean ± S.D. (n = 3), * represents p < 0.05 (Duncan’s test).
Figure 7
Figure 7
Efficacy of phage cocktail on reducing SE mixture treated food samples at 4 °C and 25 °C: (A,B) chicken breast; (C,D) pasteurized whole milk; and (E,F) Chinese cabbage. Each sample was inoculated with 1 × 104 CFU of SE mix (CK), or 1 × 104 CFU of SE mix and 1 × 108 PFU of phage cocktail (T). Date represent the mean ± S.D. (n = 3), * represents p < 0.05 (Duncan’s test).
Figure 8
Figure 8
Stability of phage PA13076 and PC2184 over 72 h of incubation on the three kinds of food at 4 °C and 25 °C (the data for the milk sample at 48 h was lacking). Each sample was inoculated with 4 × 108 PFU of phage. Date represent the mean ± S.D. (n = 3), * represents p < 0.05 compared to the original phage numbers (Duncan’s test).

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