Colorimetric and Bacteriophage-Based Detection of Foodborne Pathogens

Reason: There are at least 2 patent filings in production currently based on data within this dissertation, so we would like a chance to formalize this intellectual property protection before the data become publicly disclosed.

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Colorimetric and Bacteriophage-Based Detection of Foodborne Pathogens

thesis
posted on 27.04.2021, 18:41 by Andrew T KanachAndrew T Kanach

The significant toll of foodborne pathogens on human health and the economy has been well-established. Thus, the primary goal for many researchers and the agencies charged with monitoring the food supply is to improve upon detection systems for these pathogens via reductions in cost, complexity, limits of detection, and time to detection. One such avenue which has been explored in this work is the development of foodborne pathogen detection systems which result in easily visible color changes to media in the presence of the target bacterium. To this end, a scientific marriage between two naturally occurring phenomena, chromoproteins from coral and integrative temperate bacteriophage ɸV10, was explored as a means of specifically targeting, stably infecting, and ultimately detecting Escherichia coli O157:H7. A gene originally from the coral Acropora millepora, amilCP, was optimized for expression in E. coli O157:H7 and homologously recombined into the ɸV10 bacteriophage genome. The expression of amilCP in infected E. coli O157:H7 results in the accumulation of the chromoprotein, causing the cells to appear purple in color. Infection kinetics and limits of detection with this system were explored, as well as ways to optimize the resulting color production through culturing variables.

Additionally, the process of translating recombinant ɸV10 detection strategies into formulations suited for integration into commercial applications was pursued in this work. Methods for storage of recombinant ɸV10 without the needs for refrigeration and aqueous buffers were investigated to facilitate their eventual transition to commercial detection platforms. ɸV10 stocks were printed onto dissolvable paper, lyophilized using various formulations, and attempted to be covalently linked to superparamagnetic particles. The conclusions from these experiments provided key insights into how ɸV10 reporter phages respond to various processing conditions and may point to a successful anhydrous packaging strategy to yield extended shelf-life at ambient temperatures.

Finally, a semi-quantitative, differential culturing method was developed targeting Listeria species in fluid milk. This process involves combining two established protocols: the standardized initial enrichment medium used by the European Union for Listeria, and a field-ready most probable number enumeration technology. The data presented here give legitimacy to the expansion of bacterial targets of this technology to include Listeria, a deleterious foodborne pathogen in its own right.

Funding

USDA ARS Project No. 8072-42000-077

History

Degree Type

Doctor of Philosophy

Department

Biological Sciences

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Bruce Applegate

Additional Committee Member 2

Laszlo Csonka

Additional Committee Member 3

Arun Bhunia

Additional Committee Member 4

George Paoli

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