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Rapid Assay, Probe Technologies, and Media for Monitoring Flora in Foodstuffs

Hicks, Clair
University of Kentucky
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Objectives: 1. Continue development of phage peptides that binds with host cell receptor sites to develop probes for rapid identification of host organisms and delivery of metabolic disruptors and stimulants. 2. Development of acoustic sensors and protocols for virtual assays and cell activity detection. 3. Optimizing media systems, enzymes, and processing protocols on cheese cultures. Impact on public welfare. Many commercial, research, medical, defense, and food testing laboratories want rapid assays that are quick, accurate, and that can be used to screen large quantities of materials. Development of rapid assays would reduce human exposure to contaminated foods thus reducing food borne illnesses, quickly identify septic infections, track pathogen sources, reduce medical response times, and may impact such things as microbial antibiotic resistance.
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Non-Technical Summary: Reason for research. If rapid assays were developed that could be used in systems that contain some food solids, have high sensitivity, low preparation and assay time they would find immediate commercial application. A virtual nondestructive assay would have the greatest value in the marketplace. While there is zero tolerance for many pathogens in foods, usually enrichment methods are necessary to find these organisms using current assay procedures. Thus, if easy concentration methods were employed to collect bacteria from large samples, and an assay could measure 102 bacteria /ml or less and each assay costs less than $5.00 (including operator time), the rapid assay system would replace most other assays that require greater cost, greater preparation time, and higher skilled technicians. One method being investigated is an Acoustic Emission (AE) assay, which appears to be specific for individual bacteria, including pathogens, that it can measure pathogens at a minimum concentration within a mixture of bacteria. This assay would have an initial up front cost for the equipment but the actual assay cost would be limited to operator time, a disposable sample vessel, and millipore filter, thus the actual cost may be far lower than most assays that are currently on the market. While this test would be considered a screening test, because it does not provide actual numbers of organisms present, it may be accurate enough to suggest contamination levels. A second method being investigated is a rapid assay probe system that use bacteriophage peptide as a vector which is attached through a tether to a reporter molecule. While some commercial systems do use bacteriophage that have reporter molecules attached to them as rapid assay probes, this probe has the advantage of being much smaller, thus the number of probes that can saturate the host membrane goes up significantly, thus assay sensitivity increases with saturation. Also, once pathogen specific peptides from phage are characterized, highly specific peptides can be designed and synthesized to increase receptor binding and saturation. While this probe would be more expensive than AE assays, it may have some market application because of sensitivity. Again, this is a rapid screening technology and not a conformation technology. Currently, companies pay identification laboratories from $19.00 (negative test) per assay for a standard plate assay that requires 7 days. ELISA tests cost about $25.00 per test and require 24 to 48 hours. PCR tests cost approximately $30.00 per test, take at least 12 hours, and are positive if dead cells are present. AE fingerprint assays would replace all PCR and ELISA tests (positive for non viable cells) currently being used by testing laboratories if assay times could be cut to 30 min because only viable cells numbers would be determined. Thus, several million assays/year would be preformed for the food, regulatory, medical, and defense industries per year at a cost of greater than 120 million dollars per year (estimates derived from an average cost of $30/assay).

Approach: Bio-active peptides from virus and bacteriophage attachment proteins through hydrolyzation will be prepared using the procedures of Hicks et al., (2004). Peptide activity will be verified using the procedures of Hicks et al., (2004) and Neelakantan et al., (2009). Peptides will be used as receptor binders and to develop rapid assay probes, and delivery systems using the procedures of Neelakantan et al., (2009). Peptides specific to a class of bacteria will be used to develop fluorescent or reporter assay probes for the rapid identification of that class of organisms as demonstrated in the model system reported by Neelakantan et al., (2009). Other specific peptides will be used to develop antimicrobial probes, metabolic stimulators, or growth inhibitors where the peptide is used as a delivery system to a specific bacteria type (similar group of bacterial strains).). Molecules of immediate interest would be to synthesize reporter probes containing peptides hydrolyzed from the c2 F-protein (...Ala-Glu-Leu-Glu [N-terminal peptide] and ...Ser-Asn-Glu-Glu-Met, [calcium binding peptide]), plus the peptides from pathogens including Salmonella, Listeria, E. coli, and Staphylococcus. Probes for these bacteria would have the greatest commercial value. Also, all of these bacteria have phage that have been identified and are easy to procure. All of these phage types have attachment proteins that can be easily hydrolyzed using the procedure of Hicks et al., (2004). Peptides will be collected from the permeate of a 10,000 mwco membrane and purified through a G-15 Sephadex column and fractions collected using column fractionation. All fractions will be tested for activity using the procedures of Hicks et al., (2004). The most active fraction would be purified using dialysis and the resulting freeze dried powder would be analyzed for peptide sequence. This sequence would be replicated by a commercial peptide manufacturer and the resulting peptide would be tested again for activity and if activity was equal to the fractionated peptide the synthesized peptide would be used in the synthesis of a probe (Neelakantan et al., 2009). Other molecules of interest would be to replace the reporter portion of the molecule with tetracycline or ampicillin. Tetracycline is a broad base antibiotic, whereas ampicillin is active against all gram positive and some gram negative organisms. The synthesis would covalently bind the ampicillin through the terminal carboxyl group and tetracycline through the terminal amine group. Once these tethered antibiotics were prepared they would be tested against the host bacteria that the peptide portion of the molecule was specific for. These molecules would not be as readily absorbed through the bacteria membrane because their molecular weight would be approximately 2x greater than the native antibiotic. Thus, once the peptide delivered the antibiotic to the receptor of the bacteria hydrolyzation may need to occur before the antibiotic can cross the cell membrane. Tests will be conducted to determine the amount of antibiotic probe that is bound by the membrane and the amount that is free in solution (Neelakantan et al., 2009).

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens