Genetically Engineered Virulent Phage Banks for the Detection and Control of Bacterial Biosecurity Threats

Abstract

Natural outbreaks of multidrug-resistant microorganisms can cause widespread devastation, and several can be utilized or engineered as agents of bio-terrorism (emergent strains). From a bio-security standpoint, the capacity to detect and then efficiently control, within hours, the spread and the potential pathological effects of an emergent outbreak, without recourse to either antibiotics or vaccines, become key challenges that must be met. We turned to phage engineering as a potentially highly flexible and effective means to both detect and eradicate threats originating from emergent (uncharacterized) bacterial strains. To this effect, we developed technologies allowing us to 1) concurrently modify multiple regions within the coding sequence of a gene while conserving intact the remainder of the gene, 2) reversibly interrupt the lytic cycle of an obligate virulent phage (T4) within its host, 3) carry out efficient insertion, by homologous recombination, of any number of engineered genes into the deactivated genomes of a T4 wild-type phage population and 4) reactivate the lytic cycle, leading to the production of engineered infective virulent recombinant progeny. This allows the production of very large, genetically engineered lytic phage banks containing, in an E. coli host, a very wide spectrum of variants for any chosen phage-associated function, including host-range. Rapid screening of such a bank allowed the isolation of recombinant T4 particles capable of detecting (diagnosis), infecting and destroying hosts belonging to gram-negative bacterial species evolutionarily close (Yersinia ruckeri) as well as far removed (Pseudomonas aeruginosa) from the original E.coli host (DK8).