Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7

Abstract

Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37 degrees C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10(-6) CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10(-4) CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10(-6) CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.

FIG. 1.

Electron micrographs of phages negatively stained with 1% uranyl acetate; restriction digest of phage DNA restricted with EcoRV and phage plaque morphology. (A) Phage e4/1c and associated plaque. (B) Phage e11/2 and associated plaque. (C) Phage pp01 and associated plaque. Bar, 100 nm. Lanes: 1, 1 kb; 2, T4 DNA; 3, e4/1c DNA; 4, e11/2 DNA; 5, pp01 DNA. The DNA was run on a 0.7% agarose gel.

FIG. 2.

Comparison of the lytic ability of phage and phage cocktails to lyse E. coli O157:H7 strain P1432 at different temperatures in vitro. Challenge tests were carried out at 37°C (A), 30°C (B), and 12°C (C).

FIG. 3.

Comparison of BIM cells to the parental E. coli O157:H7 strain cells. P1432 (A) Parental E. coli O157:H7 colonies; (B) parental E. coli O157:H7 cells; (C) phage cocktail BIM colonies; (D) phage cocktail BIM cells.