R&D Excellence: Development of Bio-active Probes Derived from Bacteriophage

Objectives: 1. Develop a rapid assay probe specific for a bacterial class. 2. Develop a antibiotic probe specific for a bacteria class that would kill the bacteria and reduce antibiotic resistence of other non-pathogenic bacteria. A model system using non-pathogenic bacteria (Lacotcoccus lactis ssp. lactis) will be used to determine if bacteriophage peptides can be used to deliver a marker for rapid identification and an antibiotic for killing select cells. Phage peptides derived from the hydrolysis (.02% Ficin at 26°C for 6 h) of c2phage (109 concentration) that are partially purified (3,000 mwco ultrafiltration) will be combined with a lucifer compound or luminol to produce a rapid assay probe. The c2 phage peptide causes L. lactis ssp. lactis C2 host to clump (agglutinate) which makes the cells easier to separate from a food mass. Phage peptides linked to a luminescing compound such as a lucifer compound or luminal would bind to the receptor sites on the C2 host and when illuminated using a flourescent wave length would cause the luminescing compound to emit a photon at an alternate wave length. The intensity of light being emitted would be proportional to the concentration or number of bacteria in the sample. Light intensity should be sufficient to detect bacteria at concentrations of 102 bacteria/ml with a 30 min preparation time. Current rapid assays for bacteria are limited to 104 and 105 bacteria/ml with 4 h of preparation time. Attachment of the c2 phage peptide to ampicillin would produce a bio-active probe that would selectively attach to the receptors on L. lactis ssp. lactis C2 and close relatives to greatly enhance the concentration of ampicillin in the proximity of the cell. Ampicillin would be released by esterases produced by the cell and become available for transport across the cell membrane. Thus cell kill rates might be achieved by ampicillin at concentrations that would be 1000 times lower than if ampicillin was added to the growth medium alone.