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In vitro and in vivo Studies Using Phages Isolated from Sheep to Reduce Population Levels of Escherichia coli O157:H7 in Ruminants |
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Control/Tracking Number: 03-GM-A-3160-ASM
Most outbreaks caused by Escherichia coli O157:H7 have been linked to ruminants; 28% of U.S. cattle presented for slaughter harbor O157:H7. We here evaluate the use of virulent bacteriophages as a potential management strategy to reduce the population of this pathogen in ruminant intestines. T-even phage CEV1 was isolated from a flock of sheep resistant to inoculation with E. coli O157. In a chemostat experiment under microbial and physiological conditions reflecting the ruminant gut, by 11 days CEV1 efficiently eliminated two virulent E. coli 0157:H7 strains (initially at 104 per ml and labelled with antibiotic resistance markers); they were no longer detectable even after enrichment efforts. In preparing to test CEV1’s efficacy upon re-introduction into sheep, 20/39 sheep in the flock were found to already contain phage. All 20 isolates were active against all O157 strains tested, as well as against common lab strains. Four that were further characterized were all Siphoviridae with a 120 kb genome. Preliminary in vivo experiments showed substantial reduction within 2 days in the intestinal levels of E. coli O157:H7 in sheep treated with a single oral dose of CEV1 (4x1011 pfu) three days after inoculation with 1010 cfu of strain 933. Further reduction was seen with a cocktail that also included 17 of the new phages, as well as in those sheep naturally carrying phages (where no additional phages were added). Viable phage were recovered from cecum, rectum, and fecal samples, at levels of 103 to 106 pfu/mL, from those sheep treated with the phage cocktail. These results suggest that the protective effect of bacteriophages against E coli O157:H7 may contribute significantly to reducing the incidence of human infection if used in a preventive manner. We are further exploring natural phage populations in various flocks in Washington State as well as Texas, both to establish patterns of natural protection and to isolate new phages potentially useful for this purpose. |
Bacteriophage |
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