Construction and Application of the Recombinant Bacteriophage PhiV10 Nanoluc
Bacteriophages are viruses that infect bacteria and can be lytic or temperate. Recently regulatory approvals of lytic bacteriophage in food applications by the FDA and USDA-FSIS have strongly advanced the use of phage as natural preservatives/antimicrobials for food safety. The three main objectives of this work are: (1) to construct a reporter phage for detection of Escherichia coli O157:H7; (2) to characterize the recombinant phage for detection of E. coli O157:H7 in food samples; (3) to develop a method to immobilize phages for intervention. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and only detect viable cells upon expression of the reporter genes. The first study used lambda (λ) Red mediated in vivo recombineering to construct the reporter phage ?V10nluc by replacing the phage recET with a kanR-nluc gene cassette. The Nluc encodes the luciferase derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by ΦV10nluc, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo ®). Enrichment assays using E. coli O157:H7 grown in LB broth with a ΦV10nluc concentration of 1.76×10 2 pfu ml-1 are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23×10 3 pfu ml-1 of phage in selective enrichments of ground beef. Using the FDA protocol for leafy greens, approximately 1 CFU g -1 of E. coli O157:H7 was detected in 10 hours using 3.50×102 pfu ml-1 of phage. In the second study, a process for immobilization of phages on inert surfaces using Fish Protein Hydrolysates (FPHs) was developed. Phages were immobilized on to Parafilm using FPHs as carriers and drying at 37°C. FPHs derived from papain treatment were the optimal carrier in terms of the least reduction of phage titer after drying. Interestingly, phages belonging to the Myoviridae family were more sensitive to drying than the Podoviridae family. However, both demonstrated acceptable stability after immobilization. Immobilized phages exhibited resistance to inactivation at ultra-low temperature (-80°C) for up to two weeks. In the third study, the reporter phage ΦV10 nluc was immobilized on Alfalfa seeds that were germinated in an agar system inoculated with E. coli O157:H7. The infection event between phages on seeds and bacteria in agar was confirmed by visualizing colonization of the lysogen E. coli O157:H7 ?V10nluc on young seedlings using a charge-coupled camera. It was found that the initial inoculation level in agar determined the cell load on seedlings on their early growth stage. These studies demonstrated that ΦV10nluc can be used to detect E. coli O157:H7 during selective enrichment even at low concentration and used to validate infective events in situ to aid in using phage as an intervention in food systems.
Applegate, Purdue University.
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