In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7

Abstract

This study investigated the effect of bacteriophages (phages) e11/2 and e4/1c against Escherichia coli O157:H7 in an ex vivo rumen model and in cattle in vivo. In the ex vivo rumen model, samples were inoculated with either 10³ or 10⁶ CFU/ml inoculum of E. coli O157:H7 and challenged separately with each bacteriophage. In the presence of phage e11/2, the numbers of E. coli O157:H7 bacteria were significantly (P < 0.05) reduced to below the limit of detection within 1 h. Phage e4/1c significantly (P < 0.05) reduced E. coli O157:H7 numbers within 2 h of incubation, but the number of surviving E. coli O157:H7 bacteria then remained unchanged over a further 22-h incubation period. The ability of a phage cocktail of e11/2 and e4/1c to reduce the fecal shedding of E. coli O157:H7 in experimentally inoculated cattle was then investigated in two cattle trials. Cattle (yearlings, n = 20 for trial one; adult fistulated cattle, n = 2 for trial two) were orally inoculated with 10(10) CFU of E. coli O157:H7. Animals (n = 10 for trial one; n = 1 for trial two) were dosed daily with a bacteriophage cocktail of 10(11) PFU for 3 days postinoculation. E. coli O157:H7 and phage numbers in fecal and/or rumen samples were determined over 7 days postinoculation. E. coli O157:H7 numbers rapidly declined in all animals within 24 to 48 h; however, there was no significant difference (P > 0.05) between the numbers of E. coli O157:H7 bacteria shed by the phage-treated or control animals. Phages were recovered from the rumen but not from the feces of the adult fistulated animal in trial two but were recovered from the feces of the yearling animals in trial one. While the results from the rumen model suggest that phages are effective in the rumen, further research is required to improve the antimicrobial effectiveness of phages for the elimination of E. coli O157:H7 in vivo.

FIG. 1.

E. coli O157:H7 (J21-nas) numbers following exposure to bacteriophages e4/1c and e11/2 in a model rumen system. The assay used E. coli O157:H7 inocula of 10³ (A) and 10⁶ (B) CFU/ml. Phages e11/2 and e4/1c were added at MOIs of 100 and 1,000, respectively (A), and at MOIs of 1 and 100, respectively (B). A control underwent the same assay with the E. coli O157:H7 inoculum but without the addition of phage (▪). E. coli O157:H7 counts obtained following exposure to e4/1c (⋄) and e11/2 (□) over 24 h at 39°C were determined. All assays were replicated at least three times. The dotted line represents the limit of detection for direct plating.

FIG. 2.

Mean log₁₀ counts of E. coli O157:H7 shed in the feces of cattle with (□) and without (▪) bacteriophage treatment and the mean plaque counts of bacteriophages (⋄) shed in the feces of animals dosed with bacteriophages. Twenty animals were dosed with 10¹⁰ CFU of E. coli O157:H7 cocktail (NCTC 12900-nas, DAF454-nas, and 13C1T3-nas) on day 0. Ten animals were dosed with a 10¹¹ PFU cocktail of e11/2 and e4/1c phages on days 1, 2, and 3 (indicated by the arrows).

FIG. 3.

Mean number of E. coli O157:H7 bacteria in the rumen (A) and feces (B) of cattle inoculated with (⋄) and without (▪) phage treatment and mean plaque counts of bacteriophages (*) recovered in the rumens of animals dosed with bacteriophages. Two fistulated animals were dosed with 10¹⁰ CFU of E. coli O157:H7 cocktail (NCTC 12900-nas, DAF454-nas, and 13C1T3-nas) on day 0. One animal was dosed with a 10¹¹ PFU cocktail of e11/2 and e4/1c phages on days 1, 2, and 3 (indicated by the arrows). Plaques were not found in fecal samples (B) directly or following enrichment and are not shown in the figure. This experiment was performed once with two animals.