Significance of the bacteriophage treatment schedule in reducing Salmonella colonization of poultry

Abstract

Salmonella remains the major cause of food-borne diseases worldwide, with chickens known to be the main reservoir for this zoonotic pathogen. Among the many approaches to reducing Salmonella colonization of broilers, bacteriophage offers several advantages. In this study, three bacteriophages (UAB_Phi20, UAB_Phi78, and UAB_Phi87) obtained from our collection that exhibited a broad host range against Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium were characterized with respect to morphology, genome size, and restriction patterns. A cocktail composed of the three bacteriophages was more effective in promoting the lysis of S. Enteritidis and S. Typhimurium cultures than any of the three bacteriophages alone. In addition, the cocktail was able to lyse the Salmonella enterica serovars Virchow, Hadar, and Infantis. The effectiveness of the bacteriophage cocktail in reducing the concentration of S. Typhimurium was tested in two animal models using different treatment schedules. In the mouse model, 50% survival was obtained when the cocktail was administered simultaneously with bacterial infection and again at 6, 24, and 30 h postinfection. Likewise, in the White Leghorn chicken specific-pathogen-free (SPF) model, the best results, defined as a reduction of Salmonella concentration in the chicken cecum, were obtained when the bacteriophage cocktail was administered 1 day before or just after bacterial infection and then again on different days postinfection. Our results show that frequent treatment of the chickens with bacteriophage, and especially prior to colonization of the intestinal tract by Salmonella, is required to achieve effective bacterial reduction over time.

Fig 1

DNA restriction patterns of the bacteriophages UAB_Phi20 (lanes 3 to 5), UAB_Phi87 (lanes 6 to 8), and UAB_Phi78 (lanes 9 to 11) with the restriction enzymes EcoRI (lanes 3, 6, and 9), EcoRV (lanes 4, 7, and 10), and HindIII (lanes 5, 8, and 11). Lanes 1 and 13 correspond to HindIII-restricted bacteriophage lambda DNA markers, and lanes 2 and 12 correspond to BstEII bacteriophage lambda and Xphi174 DNA markers.

Fig 2

Electron micrographs of bacteriophages UAB_Phi20 (A), UAB_Phi78 (B), and UAB_Phi87 (C).

Fig 3

Viable concentration of S. Typhimurium ATCC 14028 (A) and S. Enteritidis LK5 (B) infected with UAB_Phi20 (○), UAB_Phi78 (×), UAB_Phi87 (△), or a bacteriophage cocktail (□). Uninfected (♦) cultures are indicated as well. The data reflect mean values from duplicate experiments, and the error bar represents the standard deviation of these replicates.

Fig 4

Absorbance of cultures of the S. enterica serovars Virchow (A), Infantis (B), and Hadar (C) infected (■) or not (□) with the bacteriophage cocktail. The time of bacteriophage infection is also shown (↓). The data reflect mean values from triplicate experiments, and the error bar represents the standard deviation of these replicates.

Fig 5

Effect of the bacteriophage cocktail treatment (□) on the survival of mice infected with S. Typhimurium UA1872. (A) Treatment applied at 8 h preinfection. (B) Treatment applied at 4 h postinfection and on days 7 and 10. (C) Treatment administered simultaneously with bacterial inoculations and at 6, 24, and 30 h postinfection. The untreated groups (■) are also shown. Details of each trial are described in Table 1.

Fig 6

Concentration of S. Typhimurium UA1872 in the cecum of chickens left untreated (♦) or treated (□) with the bacteriophage cocktail, expressed as log₁₀ CFU/g. The titer (○) of bacteriophage in the cecum of treated chickens is also presented as log₁₀ PFU/g. Trials 1, 2, and 3 (Table 2) correspond to panels A, B, and C, respectively.