P-059. Bacteriophage-Based Biosorbent for Specific Capture, Concentration and Detection of Bacteria

M. H. Tolba1,2, L. Brovko1,2, M. W. Griffiths1,2;
1Univ. of Guelph, Guelph, ON, CANADA, 2Canadian Res. Inst. for Food Safety, Guelph, ON, CANADA.

Bacteriophages were proven to be good alternative to antibodies as a capturing element of a biosorbent. It was shown previously that chemically biotinylated bacteriophages can be immobilized on the surface of streptavidin magnetic beads, and the obtained biosorbent efficiently captured 20-40% of target cells in the sample within 10-15 minutes. However, for efficient capturing of low number of target cells, which is characteristic to food and environmental samples, this is not sufficient. Chemical insertion of legends on the phage resulted in modification of attachment sites of bacteriophage tail, thus lost of phage infectivity. Therefore, our objective is to construct a phage based biosorbent that can efficiently detect low numbers of bacteria. As a model system, T4 bacteriophages were engineered that display Biotin Carboxyl Carrier Protein (BCCP) and Cellulose Binding Module (CBM) on the phage head. Recombinant bacteriophages were immobilized on streptavidin coated magnetic beads and cellulose-based substrates (microcrystalline cellulose and filter paper), respectively. Results showed that immobilized phages retained their ability to capture and lyse host bacteria. It was shown that 75-99% of E. coli cells in the range of 100 - 105 cfu/ml were captured by the constructed biosorbent within 15 min. The subsequent lysis was accompanied by the release of intracellular ATP and resulted in production of multiple progeny phages. ATP bioluminescent assay was applied for monitoring lysis and thus for detecting the presence of E. coli with the detection limit of <103 CFU/ml. Combining phage-mediated magnetic separation and optimized real-time PCR targeted to progeny phage particles 100 CFU/ml of E. coli was detected within 1 h. The proposed approach may be expanded for rapid detection of multiple target bacteria using bacteriophages with the specific host ranges.