Characterization and Application of Bacteriophages for the Biocontrol of Shiga-toxin Producing Escherichia Coli in Romaine Lettuce

Shiga-toxin-producing Escherichia coli (STEC) have been implicated with numerous outbreaks associated with contaminated fresh produce. Bacteriophages (phages) are viruses that specifically target and utilize bacterial hosts for replication. Although phage products for biocontrol are currently available, the risk of potential bacterial resistance mandates further characterization of new strains for future formulations. Previously isolated STEC phages (n = 15) were probed for their host ranges and genome composition. Phage (n = 13) phylogenetic origins were explored and annotated for the identification of potential integrase genes and virulence genes. Phages (n = 3) were then selected based on their ease of propagation and ability to target E. coli O157 for further analysis. Select phages were imaged by transmission electron microscopy and tested for their burst size, latent period, pH and temperature stability, as well as efficacy against four strains of E. coli O157 both in broth culture and on fresh Romaine lettuce at a temperature of 10 degrees C. Whole-genome nucleotide alignment revealed that the 13 phages grouped into three distinct clusters and two singletons. The lack of integrase and virulence genes demonstrated their suitability for the food industry. Phages VE04, VE05 and VE07 demonstrated increasing stability with increasing pH and remained stable at temperatures of -20 degrees C, 4 degrees C, and 22 degrees C. Phage VE04 was shown to have the shortest latent period and a burst size of 50 +/- 5. All phages were equally effective in significantly reducing E. coli O157 populations in liquid culture (P < 0.05). Likewise, phages VE04, VE05 and VE07 were effective against the tested E. coli O157 strains on Romaine lettuce. Compared to control groups, log-reduction ranged from 2.6 log colony forming units (CFU)/cm(2) to approximately 6 log CFU/cm(2) after 3 days of storage at a temperature of 10 degrees C (P < 0.05). Phages remained stable and persisted despite the absence of a STEC host in broth culture and on Romaine lettuce. The discovery of new phages that inactivate E. coli O157 supports the formulation of products with reduced potential of bacterial resistance and the development of future biocontrol methods.