Naturally resident and exogenously applied T4-like and T5-like bacteriophages can reduce Escherichia coli O157:H7 levels in sheep guts

Abstract

In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome∼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ∼2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (∼10(11) total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (∼99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (∼99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants.

Figure 1

Electron micrograph of bacteriophage CEV2.

Figure 2

Pulsed-field gel electrophoresis (PFGE) of undigested and restriction enzyme digested bacteriophage CEV2 DNA. (A) Lane A SmaI:CEV2; Lane B EcorI:CEV2; Lane C XhoI:CEV2; Lane D TESC Lab Phage PFGE Ladder consisting of PEV3 [290 kb], T4 [175 kb], T5 [121.9 kb] and PEV2 [72.7 kb] and 816a [42.7 kb]; Lane E NEB HindIII:λ ladder [23.1, 9.4, 6.6, 4.4 kb]; Lane F undigested CEV2; Lane G EcorV:CEV2; Lane H, PvuII:CEV2; Lane I HindIII:CEV2. (B) Lane A HhaI:CEV2; Lane B HaeIII:CEV2; Lane C McrBC:CEV2; Lane D BamHI:CEV2; Lane E TESC Lab Phage PFGE Ladder consisting of PEV3 [290 kb], T4 [175 kb], T5 [120 kb] and PEV2 [70 kb] and 816a [39 kb]; Lane F NEB HindIII:λ ladder [23.1, 9.4, 6.6, 4.4 kb]; Lane G undigested CEV2.

Figure 3

High MOI CEV2 infections of E. coli O157:H7 NCTC 12900 growing either aerobically (A, MOI∼5.3) or anaerobically (B, MOI∼8.3) in TSB. Infection graphs shown are representative of at least three replicates. Bacterial survivors CFU mL⁻¹ (▵), OD_{600 nm} (○) and phage PFU mL⁻¹ after the addition of chloroform (□).

Figure 4

High MOI (∼3.9) co-infection of E. coli O157:H7 NCTC 12900 growing anaerobically in TSB with phages CEV1 (∼2.06) and CEV2 (∼1.88). Bacterial survivors (▵), OD_{600 nm} (○) and phage titers, CEV1 (solid line) and CEV2 (dashed line) after the addition of chloroform (□).

Figure 5

The use of O157:H7-infecting phage CEV1 or a cocktail of CEV1+CEV2 as a pre-slaughter treatment to remove resident E. coli O157:H7 EDL 933 from the intestines of ruminants (sheep). Group 1 (white): Control Group, O157:H7-infecting phage free and receiving no phage treatment. Group 2 (gray): Cocktail (CEV1+CEV2). Treatment Group, O157:H7-infecting phage free and treated with CEV1 and CEV2. Group 3 (black): Naturally Resident CEV2 Phage Group: Sheep in which CEV2 was naturally present. Error bars indicate standard deviations.