P-066. A cobA Based Bacteriophage Reporter for Detection of Escherichia coli O157:H7

P. Romero, L. Perry, M. Morgan, B. Applegate;
Purdue Univ., West Lafayette, IN.

Escherichia coli O157:H7 is a major cause of foodborne outbreaks each year, and various rapid methods have been developed to detect it in food samples. However, these methods usually require skilled personnel and laborious and expensive multi-step protocols. Bacteriophage-based systems are also being investigated for use as an effective means for detection of viable bacterial pathogens due to their host specificity. Our objective was to develop a rapid and simple method for the detection of E. coli O157:H7 using the lysogenic bacteriophage ΦV10. The phage was genetically modified to be used as a reporter for detection of these bacteria. A non-essential gene (recET) was replaced with a kanamycin resistance gene fused to cobA resulting in constitutive cobA expression. The reporter gene cobA is found in Propionibacterium freudenreichii and codes for uroporphyrinogen III methyltransferase, which catalyzes the conversion of the endogenous substrate uroporphyrinogen III into the fluorophore trimethylpyrrocorphin. When excited with UV light at 302nm, trimethylpyrrocorphin emits a strong red fluorescence detectable at the single-cell level. Modification of ΦV10 was performed utilizing a lysogen carrying the inducible λ Red recombination genes on a temperature sensitive plasmid (pKD46). Recombinant phage were harvested from a culture of modified ΦV10, propagated on E. coli O157:H7, and its infectious efficiency determined by plaque assay. This bacteriophage-based fluorescent bioreporter can become a valuable tool for the rapid detection of E. coli O157:H7 without the need for enrichment and long incubation periods, and trained personnel.