A cobA-based reporter bacteriophage for the detection of Escherichia coli O157:H7

Patricia L Romero, Purdue University

Abstract

Escherichia coli O157:H7 is a major cause of outbreaks of foodborne illness each year in the United States, and various rapid methods have been developed to detect this pathogen in food samples. However, these methods usually require skilled personnel, and laborious and expensive multi-step protocols. Bacteriophage-based systems take advantage of the phage host-specificity and its necessity to use the host machinery to produce new progeny, to use it for detection of viable bacterial pathogens. The objective of this research was to develop a reporter bacteriophage that can be used for the capture and subsequent detection of E. coli O157:H7 cells. Bacteriophage &phis;V10 is a lysogenic, non-virulent phage capable of infecting a wide range of E. coli O157:H7 strains. The phage was genetically modified by replacing a putative non-essential gene (recET) of its genome with a cassette comprised of a kanamycin resistance marker and its promoter, fused to reporter gene cobA, resulting in constitutive cobA expression. The gene cobA is found in Propionibacterium freudenreichii and its expression results in the formation of the fluorophore trimethylpyrrocorphin, that when exposed to UV light at 302 nm, emits a strong red fluorescence detectable with the naked eye. Modification of &phis;V10 was performed utilizing a lysogen carrying the inducible λ Red recombination genes on a temperature sensitive plasmid (pKD46). Recombinant &phis;V10 phage were able to generate new progeny and transduce kanamycin resistance to new E. coli O157:H7 cells, demonstrating that a prophage can be genetically modified to carry a reporter gene, and still remain biologically active. However, changes in the phenotype of the recombinant phage were observed, resulting in the formation of small and difficult-to-see plaques. Additionally, a rapid protocol for purification of recombinant phage using gradient column chromatography was developed, allowing for removal of proteins and DNA impurities in a single step, and yielding approximately 50% of the total initial phage concentration. The developed protocol is also scalable for large purification of phage, which is crucial for the commercialization of this technology. Although optimization of the reporter expression is necessary, this bacteriophage-based fluorescent bioreporter can become a valuable tool for the detection of E. coli O157:H7, being economical, easy, and most importantly rapid since long enrichment and incubation periods are unnecessary.

Degree

M.S.

Advisors

Applegate, Purdue University.

Subject Area

Agronomy|Microbiology

COinS