DEVELOPMENT OF PHAGE-BASED ANTIBACTERIALS TO IMPROVE ANIMAL HEALTH AND FOOD SAFETY

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010661
• Project No.: IND010930
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021

Project Director
Ebner, PA, D.

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Animal Sciences

Non Technical Summary
Over 9 million people suffer some form of foodborne illness each year in the US alone. These illnesses result in over 55,00 hospitalizations and 1,351 deaths (Scallan et al., 2011). At same time, the US FDA has in very recent past reduced the number and type of antibiotics available for use in livestock and poultry production (USFDA 2013, 2014). The purpose of this project is duo fold. We aim harness the antibacterial properties of bacteriophages, natural predators of bacteria, to develop technologies that: 1) reduce foodborne pathogen contamination in agriculture and food processing environments; and 2) prevent bacterial infections in live animals as an alternative to antibiotics. Taken together, these technologies have great potential to practically and effectively improve animal health while assuring that the US food supply remains among the safest in the world.

Animal Health Component

50%

Research Effort Categories

Basic

(N/A)

Applied

100%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3299	1100	10%
311	3299	1101	10%
311	3599	1100	10%
311	3599	1101	10%
712	3299	1100	10%
712	3299	1101	10%
712	3599	1100	10%
712	3599	1101	10%
712	5399	1100	10%
712	5399	1101	10%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3299 - Poultry, general/other; 5399 - Structures, facilities, and equipment, general/other; 3599 - Swine, general/other;

Field Of Science
1101 - Virology; 1100 - Bacteriology;

Keywords

bacteriophage

food safety

e. coli

salmonella

swine

poultry

efficacy

phage therapy

Goals / Objectives
Our primary goal is to develop phage-based therapeutics to limit bacterial pathogens in livestock and food processing environments. Specifically, we aim to:Determine if phage-based therapies can prevent diarrheal infections with bacterial etiologies. Such infections are often predicted to increase with the reduction in antibiotic use in food animal production;Measure the efficacy of phage-based treatments in reducing or eliminating foodborne bacterial contamination in food processing environments; andComprehensively examine the safety of phage-based therapies in terms of impact on the host (e.g., gut microbiome, deleterious immune responses, etc.), development of phage resistance, and impact of long-term use of bacteriophages as biocontrol measures.

Project Methods
The following are methods used in various permutations to: 1) identify and characterize phages; and 2) measure their efficacy in live animals, food matrices, and food processing facilities. 1) Phage Characterization. Most phages used in our laboratory are originally isolated from various environments including wastewater treatment facilities, livestock manure systems, or processed animals. Phages are isolated for specific bacteria and we have a growing library of over 100 different phages for different purposes. Once isolated, there is often a lengthy characterization to define phage spectrum, lytic capacities, and survivability. When possible, the phage genomes are sequenced and annotated to better determine if phages may contain genes associated lysogeny, virulence, or toxin production. 2) Live Animal Trials. Previously, we have designed preliminary experiments to determine "proof of concept" or "prototype development". To determine the potential of the phage treatment in preventing bacterial infections, young animals are divided into various treatment groups (e.g., phage treatment, antibiotic treatment, positive and negative controls, etc.). In most cases, animals are then challenged with the targeted bacterial pathogen. Following challenge, fecal sample are collected daily over a set time to measure both challenge bacteria and phage concentrations. Animals are always monitored for various signs of infection and at set times post-challenge, animals are euthanized. Ileal, cecal, and fecal samples are then collected to measure challenge bacteria and phage. Gut health is monitored in all pigs by examining ileal and jejunal tissues for villus height, crypt depth, villus height:crypt depth as well as intestinal lesions. Impact on the host is measured in various manners, namely innate immune responses, global changes in microbial communities in the gut, and phage resistance development.3) Food Processing Facilities/Food Matrices. We currently have an on-going project focused on the use of phages in food processing systems. Participating retail food processors or producers are identified and undergo baseline testing to determine the level of contamination with specific pathogens. Once pathogen contamination levels and frequencies are established, retail outlets are randomly divided into two groups: 1) phage; and 2) control. The phage intervention is applied at established intervals. Samples are collected usually over a six-month period. Efficacy of the treatment is based on its ability to reduce both pathogen prevalence (qualitative) and concentrations (quantitative) in treated stores compared to both previously established baseline measurements and prevalence and concentrations in untreated stores.To test phage efficacy in food matrices, food samples are inoculated with challenge bacteria, and then inoculated with different phage treatments. Samples are incubated under various conditions and temperatures. The efficacy of the treatment is based largely on the disappearance of challenge bacteria in phage-treated samples vs. non-treated samples. 4) Assessments and Benchmarks. We regularly conference with industry partners regarding updates and progress. These meeting inform new directions that are synergistic with industry research and industry goals.

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:Greater scientific community, US and international poultry producers, government officials Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Currently, four graduate students are involved in the project (3 MS, 1 PhD). We have previously hired laboratory coordinators who have taken leadership roles in some aspects of the research, including authorship of peer-reviewed manuscripts. How have the results been disseminated to communities of interest?In the past year, we have published four peer-reviewed research articles on results from this project. We have also (prior to COVID restrictions), shared goals and results of the project with industry leaders to gather feedback and insight. However, because of the commercialization focus of the project, we have not shared some results widely to allow pursuit of IP protection both in the US and abroad. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? As described in a previous report, we recievedmulti-national grant to pursue development and commercialization of our technologies with universities from a patnering country. All research groups have made progress towards our goals and we have published four peer-reviewed articles detailing our results thus far. We have created phage-based prototypes to target (prevention or treatment) bacterial infections and have begun measuring the efficacy of those prototypes in live-animal trials.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Mosimann S, Desiree K, Ebner P. 2021. Efficacy of phage therapy in poultry: A systematic review and meta-analysis. Poultry Science. https://doi.org/10.1016/j.psj.2021.101472
• Type: Journal Articles Status: Published Year Published: 2021 Citation: Desiree K, Myoda S, Ebner P. 2021. Efficacy of phage therapy in swine: Sytematic review and meta-analysis. Journal of Animal Science. 99(7). doi.org/10.1093/jas/skab157
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq A, Ebner P, Abbas Q, and Ubaid ur Rehman, H. 2020. Employing list-shield bacteriophage as a bio-control intervention for Listeria monocytogenes from raw beef surface and maintain meat quality during refrigeration storage. LWT, 32.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ishaq A, Syed QA, Ebner PD, and Ubaid ur Rahman, H. 2021. Multiple hurdle technology to improve microbial safety, quality and oxidative stability of refrigerated raw beef. LWT, 138.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:Greater scientific community, international poultry producers, government officials Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two graduate students and one technician are employed through the project. An additional PhD student is devoting 25% to the project. How have the results been disseminated to communities of interest?Because of COVID and the commercialization focus, our dissemination of results has been limited. What do you plan to do during the next reporting period to accomplish the goals?Keep progreessing towards goals and milestones stated in our current project.

Impacts
What was accomplished under these goals? We have received a multi-national grant to pursue development and commercialization of our technologies with universities from a patnering country. All research groups have made progress towards our goals.

Publications

• Type: Journal Articles Status: Submitted Year Published: 2021 Citation: Lytic capacity survey of commercial Listeria phage against Listeria spp. with varied genotypic and phenotypic characteristics

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:Poultry producers (international) Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided opportunities to provide training for international partners. Two new graduate students are slated to beging in January of 2020. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We are now focused on prevention of pathogen transmission in poultry and are examining the efficacy of phages in combination with other therapeutis.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Industry groups, other research groups. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?We have six undergraduates working on these projects for either undergraduate research credit or pay. One MS student graduate from the lab in August of 2018. How have the results been disseminated to communities of interest?The latest results were shared at the International Association of Food Protection Annual Meeting, which is a highly appropriate forum for this research. What do you plan to do during the next reporting period to accomplish the goals?We have established partnerships with different labs and are currently working with economists to more quantifiably identify barriers to adoption of the technology, from regulatory aspects to adoption by clinicians.

Impacts
What was accomplished under these goals? Our most recent research indicates that phage-based treatments may be effective in limiting Listeria monocytogenes contamination of surfaces common in food processing facilities. We have also branch into other areas where this same technology may be appropriate, but those results are still pending and not published.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jia Liu, Haley Oliver, MaryKate Harrod, Rachel Makowski, Danielle Marks, Kristen Sequiera, Brooke Siefert, Aishwarya Chitnis and Paul Ebner1 Efficacy of Bacteriophages Alone or as a Co-Treatment in Reducing Listeria monocytogenes Contamination of Non-food Contact Surfaces. IAFP Annual Meeting.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Liu, J. 2018. Efficacy of Listeria Phage in Reducing Listeria monocytogenes under Both Experimental and Food Processing Conditions. MS Thesis. Purdue University

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Animal health industries Human health industries Livestock producers Meat processors Changes/Problems:No major changes. What opportunities for training and professional development has the project provided?One post-doctoral student contributed to this project (in partnership with another lab). One MS student (100%) will graduate in August 2018 and another graduate (partner lab) in August of 2017. Six undergraduates participated in the project for course credit or as employees. How have the results been disseminated to communities of interest?Research articles have been published in peer-reviewed journals. Results have also been disseminated via a book chapter (in press). We have also presented the research at numerous research conferences, both in the US and abroad (Pakistan). What do you plan to do during the next reporting period to accomplish the goals?We are currently focusing on employing phage therapy to decontaminate non-food contact surfaces. This research comprehensively measures efficacy under various real-life conditions.

Impacts
What was accomplished under these goals? Our research indicates that phage therapy, unlike traditional antibiotic therapy, may have minimal impact on the gut microbiome. LIkewise, we have shown that phage treatment does not result in strong acute immune responses in pigs. Together, these two finding help to answer outstanding questions regarding phage therapy safety. Other research from our lab, however, has shown that bacteria may rapidly become resistant to phages under some conditions. Surprisingly, resistance development may be influenced by the surround microbial environment and may beless likely to occurin more complex microbial environments (e.g., ground meat) and more likely to occur in environments with fewer competing bactria (e.g., egg contents [not shell]).We have published two key manuscripts describing the impact of phage treatment on the gut microbiome in pigs as well the role phage resistance may play in treatment efficacy. These results were also detailed in a book chapter we authored focused on alternatives to antibiotics in swine production.

Publications

• Type: Book Chapters Status: Accepted Year Published: 2017 Citation: Ebner PD and Hong Y. 2017. Dealing with the challenge of antibiotic resistance in pig production. In: Achieving Sustainable Production of Pig Meat. (accepted; in press).
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Hong Y, Schmidt K, Marks D, Hatter S, Marshall A, Albino L, Ebner PD*. 2016. Treatment of Salmonella contaminated eggs and pork with a broad-spectrum, single bacteriophage: Assessment of efficacy and resistance development. Foodborne Pathogens and Diseases. 13:679-688.