Evaluation of anhydrous processing and storage methods of the temperate bacteriophage ¿V10 for integration into foodborne pathogen detection methodologies

Authors: KANACH, ANDREW - Purdue University; BOTTORFF, THERESA - Purdue University; ZHAO, MIN - Purdue University; WANG, JUN - Purdue University; CHIU, GEORGE - Purdue University; APPLEGATE, BRUCE - Purdue University

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/18/2021
Publication Date: 6/6/2021
Citation: Kanach, A., Bottorff, T., Zhao, M., Wang, J., Chiu, G.T., Applegate, B. 2021. Evaluation of anhydrous processing and storage methods of the temperate bacteriophage V10 for integration into foodborne pathogen detection methodologies. PLoS ONE. 16(4):e0249473. https://doi.org/10.1371/journal.pone.0249473.
DOI: https://doi.org/10.1371/journal.pone.0249473

Interpretive Summary: Bacteriophages (viruses which only infect bacteria) offer a unique opportunity to expand detection technologies targeting foodborne pathogens. While several research groups have expanded the uses for various bacteriophages, reliable means of translating these proof-of-principle systems to technologies which can be impactful at a society level are lacking. The research in this manuscript serves as a pioneering step in evaluating processing methods by which temperate bacteriophages can be manufactured to easily adapt them for scaled production. Specifically, this manuscript shows that temperate bacteriophages can be processed in such a way to allow for simple, room temperature storage – a highly desirable trait for developing a shelf-stable technology. With a focus on simplicity of use by the end-user and inexpensive processing techniques, bacteriophage-based technologies have the potential to integrate into standardized food testing protocols. This eventual integration can help reduce the time it takes to evaluate the safety of various food products, improving the throughput of testing, reducing financial losses to food companies, and most importantly protecting consumers.

Technical Abstract: Due to the nascency of bacteriophage-based pathogen detection technologies, several practical hurdles stand in the way between providing promising proof-of-concept data and development of robust detection platforms. One such hurdle, and the focus of this work, is the development of methods for transitioning laboratory stocks of bacteriophage into functional, consistent, and shelf-stable delivery methods in commercial detection kits. Research described here was undertaken to evaluate two methods for their ability to store the bacteriophage 'V10 at ambient temperature without aqueous storage solutions while limiting loss of viability. 'V10 is a temperate bacteriophage which solely infects the zero-tolerance food adulterant Escherichia coli O157:H7 and has been genetically modified to generate a detectable phenotype in host cells. In order to integrate this reporter bacteriophage into food-borne pathogen detection methodologies, two methods of processing phage suspensions for long-term, ambient storage were evaluated: printing solutions onto pieces of dissolvable paper and lyophilizing suspensions with sucrose. Applying phage to dissolvable paper yielded key attributes to consider when addressing phage viability, however, optimized methodology still resulted in an approximate five-log reduction in titer of viable phage. Lyophilization of 'V10 with various concentrations of the cryoprotectant molecule, sucrose, yielded losses of approximately 0.3-log after 120 days of storage at 23 . Liquid storage buffer samples with and without sucrose saw a reduction of viable phage of at least 3.9-log in the same period. Additionally, the ability for 'V10 to form lysogens in an E. coli O157:H7 host was not negatively affected by lyophilization. Drying 'V10 at ambient temperature drastically reduces the viability of the phage. However, lyophilizing 'V10 in the presence of sucrose is an effective method for dehydration and storage of the phage in ambient environmental conditions for an extended time lending to commercial application and integration into foodborne pathogen detection methodologies.