Characterization and use of the temperate bacteriophage ΦV10 for the detection of Escherichia coli O157:H7

Udit Minocha, Purdue University

Abstract

Bacteriophages are obligate intracellular parasites that infect prokaryotes. Host specificity of phages varies as some phages can infect an entire genus of bacteria while some are species specific and others are strain specific. Phage host specificity has been exploited as a tool for the detection of pathogens using both whole phage in a reporter format or the use of their host specific binding proteins. This research focuses on the characterization and potential use of the temperate bacteriophage &PHgr;V10 for the detection of Escherichia coli O157:H7. The complete genome of &PHgr;V10, was sequenced and determined to be 39104bp containing 55 predicted genes. The predicted gene ORF 26 was tentatively identified to belong to the acyltransferase family associated with seroconversion. Primers were designed and ORF 26 was amplified from the phage genome and cloned into the expression plasmid pBAD-TOPO. Expression of ORF26 in O157:H7 was determined to confer &PHgr;V10 immunity using plaque assays. LPS was also isolated from the strain expressing ORF 26 and was found to inactivate &PHgr;V10. Further analysis of the LPS using nuclear magnetic resonance spectroscopy showed the acetylation of the O-antigen suggesting it is the receptor for &PHgr;V10. However the strains with the modified O-antigen are still detectable by commercial antibody-based O157 test kits. Reporter phage &PHgr;V10KanCobA was constructed by replacing the non-essential recET with a cobA-kanamycin gene cassette. E. coli O157:H7 infected with &PHgr;V10KanCobA and plated on plates containing kanamycin grow as colonies due to the integration of &PHgr;V10KanCobA into the chromosome. A two dimensional matrix was used with decreasing concentrations of &PHgr;V10KanCobA (107-104 pfu) and O157:H7 (106-10 2 cfu) and incubated for 1 h at room temperature. A 100 μl aliquot of each phage-bacteria mixture was spread on LBkm50 agar plates and incubated at 3°C overnight. The data showed a linear correlation with an R 2 value of 0.9866. The matrix analysis showed that as few as 10 4 pfu could detect approximately 105 cfu. However 10 7 pfu were required to detect as few as 102 cfu. The lysogen assay using &PHgr;V10KanCobA for the detection of E. coli O157:H7 is convenient, reproducible and inexpensive and when coupled with a selective enrichment has low limits of detection.

Degree

Ph.D.

Advisors

Applegate, Purdue University.

Subject Area

Food Science|Microbiology

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