Team:UCSC/Implementation

• Project + Project
  • Description
  • Engineering + Engineering
    • Design + Design
      • Gene Elimination
      • Phage Infection
      • Conjugation
    • Proof of Concept
    • Detection
  • Results
  • Model
  • Contributions
  • Implementation + Implementation
    • Future Directions
  • Future Lab Directions
• Human Practices + Human Practices
  • Integrated
  • Collaborations
  • Partnership + Partnership
    • Wet Lab
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  • Outreach
• Journal + Journal
  • Notebook
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• Glossary

Implementation

• Implementation
• Future Directions

Overview

Progenie was built around the guidance and information provided by our human practices interviews. After speaking to professionals in the agricultural industry, many pointed out the need for ways to detect and eliminate STEC. Through our contacts in the agricultural industry, we identified the niches that Progenie can fill as well as learned how our project could be useful for eliminating other pathogenic bacteria besides STEC. This page highlights the feasibility of implementing our product in the real world in conjunction with farming practices, food processing in the status quo, and involvement in the health field.

Detection of STEC using a Lateral Flow device

Seeing as there are many steps from farm to retail where E. coli contamination could occur, we designed our detection to be adaptable to different mediums such as soil, manure, and lettuce leaves. Additionally, results are available within an hour of testing, allowing farmers to prevent further spread once a source is identified. The color indicator on the lateral flow device clearly distinguishes positive vs. negative results. In contrast with traditional lateral flow devices, which use antibodies as an indicator, our design uses aptamers because they are more cost effective to produce, and bind to antigens more precisely. To use the device, solid food products are mixed with a stx analyte that suspends the contents. The lateral flow device is then dipped into the mixed solution, allowing a sample pad to absorb the liquid. The “conjugate pad” contains the aptamers and conjugated labels (gold nanoparticles that bind to the aptamers). If the molecule is present, the aptamer and label will bind the molecule and move down the test. Binding reagents along a nitrocellulose membrane then bind the target molecule, creating the colored line that indicates the presence of STEC. A thicker line indicates a higher presence of STEC.

Elimination of STEC using Progenie

Our industry contacts indicated that eliminating Shiga toxin at its source would be the most effective way to decrease outbreaks. Stopping outbreaks at the source would entail applying a treatment to the gut of cattle, an unfortunately difficult place to get to. Previous studies have indicated that most bacteriophages cannot pass through the acidic gut of a cow without getting destroyed, thus never reaching the rectum [1]. Additionally, the rectum has little to no oxygen, so any phages used have to be effective under anaerobic conditions [2]. In order to deliver a phage orally to cattle would require extra steps taken in order to protect the phage and ensure its delivery to the rectum. Sabouri et al. suggest microencapsulation of phage particles in polymers as a method to increase survivability of phages traveling through the gut, however the method worked well in vitro but not in cattle.

Another possible entry route is applying our phage delivery vector up the rectum using suppositories. Again, encapsulation would be required but the distance to the source of STEC infections is a lot less than if taken orally, thus increasing the chances of delivery to the pathogenic population. However, inserting suppositories in every cow increases the amount of required labor for farmers, thereby decreasing the appeal of using Progenie.

Instead of targeting STEC at their source, it is also feasible to target them on produce and/or post-slaughter. Application of phages to vegetables or surfaces has shown a greater success than in live animals [1], opening up the possibility of creating an easy-to-use spray that contains Progenie. However, the efficacy of similar treatments relied on incubation time, temperature, and amount of phage [1], all difficult variables to control during the harvesting, packaging, and shipment of produce to consumers.

Implementation of Progenie will require additional design to choose the best way to infiltrate bacterial populations containing STEC due to the inaccessibility of the cow rectum. However, we designed Progenie such that it could be applied to a wide number of possible targets. Such adaptability allows us to change the initial delivery phage to one that is more robust and can survive in the low pH and anaerobic conditions of the gut. Additionally, given that our design is able to self-propagate, we only need one successful delivery in order to eliminate Shiga toxin genes, or any pathogenic gene, within a population.

Implementation Constraints

Despite all of the research and testing our team has done, there are still obstacles that we have to consider and work around before Progenie can be implemented. Even with the success of our in vitro proof of concept, there are safety, legislative, and ethical constraints that our team will have to sort out before we move forward into real life implementation.

Safety

There could be unintended consequences if our delivery or elimination system ended up to be not specific enough and somehow altered unintended genes in off-target bacteria. This could potentially alter the cattle microbiomes so that they experience health effects, which could harm the farmers and agricultural economies that we are hoping to improve. We would not introduce our system into in vivo experiments or real-world use until we were highly confident that our system was specific and safe enough in vitro.

Legislation

Progenie has broad applications in the agriculture sector, with possibly the most important application being in the meat industry. However, providing Progenie as a treatment to eliminate STEC in cattle would require overcoming legislation issues that present a significant challenge; The United States Department of Agriculture’s (USDA) Food Safety and Inspection Service (FSIS) only has the ability to add regulations and mandates to meat, poultry, and meat products once it has reached the processing stages (Policy). The USDA has another sector dedicated to the health of livestock, the Animal and Plant Health Inspection Service (APHIS), however this sector is concerned with the animal’s safety and has no bearing on food safety, and does not regulate farmers on food safety issues (USDA APHIS). The lack of legislation surrounding what ranchers must do for their livestock before their product arrives to be processed results in contamination on a larger scale. Leafy green farmers mentioned that they would purchase cattle vaccines for nearby ranchers to use, but that there are no policies that require ranchers to use the vaccine for STEC (Vaxxinova). Until there is further social and legislative pressure put on ranchers, there is a high possibility that our product would not be used by ranchers.

Ethics

Because our project is designed to work in meat and lettuce, any food that is treated with Progenie would be considered a genetically modified organism. Seeing as Progenie is targeted towards farmers, ranchers, and food processors, implementation would be limited to nonorganic facilities. However, by demonstrating a way to safely create GMOs that only alter the expression of STEC, Progenie could help to reduce the stigma of genetic modification when it comes to ensuring food safety.

• [1] A minireview on the in vitro and in vivo experiments with anti-Escherichia coli O157:H7 phages as potential biocontrol and phage therapy agents.
• [2] Intercrossing of phage genomes in a phage cocktail and stable coexistence with Escherichia coli O157:H7 in anaerobic continuous culture.