Progress 09/15/23 to 09/14/24
Outputs
Target Audience:The main target audience for the period was food safety professionals responsible for environmental testing programs. These individuals are essential to ensuring food is safe to eat by the American public. Additional audiences included food safety consultants which have access to many food companies and influence their testing programs. The last target audience includes food safety regulators which are responsible for ensuring companies are providing safe food as well as investigating foodborne illness outbreaks. Changes/Problems:No problems/changes were so large that alterations in the goals, or reporting requirements were needed. Some delays resulted from difficulties in executing the given plan. This included difficulties with the ATP assay used to evaluate cell release. A new cell release assay that directly measured the growth of viable bacteria released from biofilms due to EC treatment was developed, which served as both a measure of cell release and of cell viability. It was validated against dilution plate counts of exposure supernatants. Overall, conducting the desired test plan took longer than expected resulting in our request for a 6 month no-cost extension which was approved by the USDA. This allowed GBS to have time to advance product development, allowed GBS to finish developing the test protocol with the third-party contract vendor for independent testing of the lead EC on 2 x 2 inch stainless steel coupons, and the third-party execution of that research to ensure the most accurate independent evaluation of the lead EC was possible. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The PI and a Business Development associate attended the IFT First (IFT-International Food Technologists)Annual Conferencein Chicago on July 14-17, 2024. Guild BioSciences (GBS) learned about the current market talking to many food production companies, Deputy Commissioner of Foods at the FDA Jim Jones, testing labs, regulatory consultants and researchers and other relevant potential stakeholders/end users for BioXpose. GBS produced an updated brochure containing newer data beyond the brochureused at the Food Safety Summit conference GBS attended in May 2023 and distributed it to interested parties.GBS had discussions with companies that could potentially be distributors of BioXpose in the future. GBS also contracted with a networking consultant and together we produced a survey about our proposed product, and it's use that was submitted to 30+ food safety experts from the food industry. A subset of responders were interviewed by the consultant to glean more detailed market information. While beneficial to us for product development, it also made these food industry actors aware of our work and what GBS is developing, which will increase awareness in the food industry of our developing product and the results achieved during this reporting period. Lastly, GBS started working with a consultant on pricing, distribution, and other market strategies. This is product development stuff not disseminating info to communities of interest. What do you plan to do during the next reporting period to accomplish the goals?The project is close to being finished. A 6 month no-cost extension was requested and approved by the USDA to allow time for us to get the formulation closer to a launch condition, finish developing the test protocol with the third-party contract vendor for independent testing of the lead EC on 2 x 2-inch stainless steel coupons, and the execution of that research by the vendor.
Impacts
What was accomplished under these goals?
The goal of this project is to contribute to reducing the impact of biofilms on the food industry. Biofilms are communities of organisms living on surfaces surrounded by a meshwork of extracellular polymeric substances (EPS) that acts like a cocoon protecting microbes within from extreme conditions and efforts by humans to eliminate them. What isn't well recognized is that this capability also may hide biofilms from detection. Standard surface sampling techniques for bacterial detection assays use swabs, wipes, or sponges to collect material for pathogen testing. These methods collect easily transmissible bacteria but may be ineffective at collecting bacteria living in biofilms leading to underestimating the presence of bacteria on critical surfaces and overestimating the effectiveness of sanitation efforts. This promotes food borne illnesses and costly product recalls. Guild BioSciences aim to address this problem through the development of a pre-treatment product called BioXpose that will be applied to test surfaces prior to sample collection. BioXpose is an enzyme cocktail (EC) that quickly breaks down the EPS scaffold that holds biofilms together but leaves viable bacteria and releases these microbes from surfaces so they can be more effectively collected. This will make all existing pathogen detection systems more sensitive, accurate, and reliable. Managers in the food industry working in food safety and tasked with protecting consumers from food borne illnesses will benefit from BioXpose. The second year of the project focused on Objective 2: Develop lead ECs that broadly disrupts bacterial biofilms with maximal viable bacteria release, in a product ready formulation. The first step was the selection of 3EC2 as the lead EC based on work presented in the first progress report. The work of the second year tested for further enhancement by optimizing EC quantities, testing under more product-realistic conditions, and determining a lead formulation. This was accomplished through the following research activities that were conducted: Optimized the concentration of each enzyme in the presence of the other two enzymes. Additionally, multiple nucleases including more cost attractive and food grade enzymes were compared to the original nuclease. Optimized or otherwise test the effects of non-enzyme factors including co-factors known to influence the activity of at least one of the component enzymes, stabilizers, thickeners and other chemicals. Evaluated the inclusion of detergents in the EC formulation. Eight detergents were tested for inhibitory effects on each of the enzymes. The 5 detergents with the lowest inhibitory behavior were tested with the lead EC and their effects on antibiofilm activity, cell release, and toxicity were evaluated in 3 representative bacterial species. Determine if biofilms from both susceptible and resistant strains exerted any inhibitory effects on EC performance. Evaluated whether including a fourth enzyme to the lead 3EC improved biofilm breakdown and cell release. Four individual enzymes were tested at 3 concentrations each. Evaluated if changes in pH, ionic strength, and alternative buffering agents impacted antibiofilm effectiveness. Evaluated the effects of conducting lead EC exposures at room temperature and sub 1 h exposure durations on antibiofilm effects better reflecting actual conditions BioXpose would be used under in the food industry. Evaluated if the lead EC had inhibitory effects on ATP luciferase activity using Hygiena ATP swab test. Evaluated the long-term stability of the lead EC when stored at 4oC Additionally, a formulation expert consultant was employed to assist with charting formulation aspects of the lead EC. Also, a networking consultant was employed and together we distributed a survey to evaluate market acceptance and market preferences concerning our proposed product was constructed and was submitted to 30+ food safety experts from the food industry. A subset of responders were interviewed by the consultant to glean more detailed information. Objective 3: Discussions with the third-party contract vendor have occurred and an initial protocol has been developed for executing Objective 3. Results: The year 2 research indicated that the initial lead EC was robust and needed little optimization. A detergent was selected for inclusion in the lead EC leading to improved better surface wetting, increased antibiofilm effects and increased cell release from biofilms without adding appreciable toxicity to tested bacteria. The EC+detergent was found to function effectively at room temperature and at EC exposure durations down to 5 minutes without appreciable loss in performance. As of the end of year 2 period, the stability of the lead EC stability test remains satisfactory (4 months thus far). Adding a 4th enzyme to the lead EC did not result in sufficient improvement in antibiofilm performance to merit inclusion. Thus far, bacterial inhibition by the lead EC has not been an issue at short duration exposures. Working with the networking consultant confirmed much of our market strategic plan but revealed some environmental aspects we had not considered. Overall, the results of the year 2 research have supported further EC maturation and development and progression to independent testing of the lead EC in multi-species biofilms grown on stainless steel coupons in Objective 3. The lead EC can produce effective biofilm breakdown at room temperature and using exposure durations that are in line with the expectations of the industry based on our market exploration with the networking consultant. The lead EC is also showing acceptable storage stability.
Publications
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Progress 09/15/22 to 09/14/23
Outputs
Target Audience:The target audience for the project are workers/managers in the food industry who are involved in food safety and tasked with protecting consumers from becomingsick from food borne pathogens. This could include both environmental surface sanitation/disinfection and safety/contamination testing as well as food safety regulators/inspectors/investigators and researchers interested in biofilms in the food industry. Conference Attendance The PI and a Business Development associate attended the Food Safety Summit in Chicago on May 9-11, 2023. Guild BioSciences (GBS) learned about the current market talking to many food production companies. BioXpose was discussed with several key USDA and FDA points of contact. This is an important market as we believe regulatory outbreak investigation teams will be very interested in BioXpose. GBS is working with the USDA's Laboratory Quality Assurance, Response and Coordination staff to identify a time to present BioXpose during a Steering Group meeting. GBS also identified several potential distributors if we decide to go that route. A marketing brochure was created for the conference and can be provided if desired. Contacts and Tours GBS toured the production facilities of a produce companyin July of 2023 and spoke extensively with food safety leaders within the organization. GBS learned a great deal about surfaces used, sampling methods and locations, and their processes for daily and weekly sampling. AOAC The PI and Business Development associate attended the AOAC educational seminar for their Performance Test Method (PTM) study. We have also started early conversations regarding PTM with our assigned Technical Consultants that will be performing the Validation Outline. Changes/Problems:We encountered a minor roadblock in proceeding further withinsubtask 3 of Technical Objective 1. The most common methods utilized for testing the toxicity of chemical agents on biofilms applies ultrasonic bath treatment to liberatebacterial cells from biofilms so that they can be analyzed by for example dilution plate counts (the gold standard) or live/dead fluorescence analysis. However, in Subtask 2 we tested the effect of treating biofilms for up to 30 minutes in an ultrasonic bath to release cells for collection, in both a high release and low release species. We found that cell release did not increase with increasing sonication time and testing of residual biofilms (not liberated by sonication) of no sonication and 30-minute sonication by crystal violet assay didn't indicate sonication reduced biofilm magnitude in either untreated or EC treated biofilms. Additional testing with just no sonication and 30-minute sonication treatments using an alternative method of subjecting 96-well plates to the ultrasonic bath didn't indicate sonication increased cell release in either untreated or treated biofilms, and crystal violet assay analysis indicated no reduction in cells only biofilms but did in EC treated biofilms. This caste doubts for us on the reliability of methods based on sonication to liberate cells from biofilms. As we were a bit pressed for time at this point we chose to postpone further investigation until after we achieved the preliminary milestone of Technical Objective 1, and intend to revisit this in year 2. What opportunities for training and professional development has the project provided?AOAC The PI and Business Development associate attended the AOAC educational seminar for their Performance Test Method (PTM) study. How have the results been disseminated to communities of interest?The PI and a Business Development associate attended the Food Safety Summit in Chicago on May 9-11, 2023. Guild BioSciences (GBS) learned about the current market talking to many food production companies. Summarized discussion ofresults of our Phase I and early Phase II data produced in developing BioXpose was discussed with several key USDA and FDA points of contact as well as venders and food industry top management which were overwhelmingly well received. A marketing brochure containing some of the key results was produced and distributed to interested parties. What do you plan to do during the next reporting period to accomplish the goals?The only items reported in the project change module wasthe following minor adjustment.We encounted a challenge in validating our toxicity assay using other recognized methods. ?Changes in the plan:We postponed this section to year 2 so we could maintain progress on more critical elements, and will complete this analysis during the second year.
Impacts
What was accomplished under these goals?
The goal of this project is to reduce food borne illness by addressing the impactsof biofilms on the food industry. Biofilms are microbial communities living on surfaces surrounded by a meshwork of biological fibers that acts like a cocoon protecting microbes from extreme conditions and efforts by humans to eliminate them. What isn't well recognized is that this capability also may hide biofilms from detection. Standard surface sampling techniques for bacterial detection assays useswabs, wipes, or sponges to collect material for pathogen testing. These methods collect easily transmissible bacteriabut may be ineffective at collecting bacteria living in biofilms leading to underestimating the presence of bacteria on critical surfaces and overestimating the effectiveness of sanitation efforts. This promotes food borne illnesses and costly product recalls. We aim to address this problem through the development of a pre-treatment productcalled BioXpose that will be applied to test surfaces prior to sample collection. BioXpose is an enzyme cocktail (EC) that quickly breaks down the meshwork that holds biofilms together releasing the microbes without killing them from surfaces so theycan be more completely collected. It's critical that BioXpose leave the bacteria alive as some testing systems require live bacteria. BioXpose will make all existing pathogendetection systemsmore sensitive, accurate, and reliable. Managers in the food industry working in food safety and tasked with protecting consumers from food borne illnesses will benefit from BioXpose.BioXpose will also assist with the USDA and FDA outbreak investigations by rapidly identifying any bacteria hiding in biofilms allowing for quicker responses/recalls and potentially reducing the number of illnesses and saving American lives. In the first year of the Phase II project our primary focus has been onTechnical Objective 1. For this,Task 1focused on optimizing the methods used to develop and test biofilms. InTask 2thetop 4 ECs from the Phase I project were tested against an expanded panel of food safety-relevant bacteria. ForTask 3these ECs were compared using biofilms grown on other types of materialsthat better reflect surfaces found in the food industry. The endpoint for this objectiveis selection of the lead 3-enzyme cocktail which moves on to technical objectives 2 and 3. Results: Technical Objective 1: Task 1: Methods optimization Subtask 1: Biofilm development protocol optimization. It should be noted thatbecause of the multitude of diverse directions of investigation associated with this task specifics for allthe testing completed in this subtask can't be provided. The technical report will disclose such details, and only a general summary of what resulted from the effort will be provided. Overall little adjustment to the Phase I protocol was found to be necessary, as consistent improvement across all 7 species was rarelyfound. In some cases, small adjustments to the protocol for individual species were introduced to improve biofilm characteristics. One adjustment that was enacted was to switch tousing working glycerol stock (overnight cultures combined with glycerol and aliquoted into single use volumes) rather than streaking out fresh cultures from archived glycerol stocks, followed by single colony inoculation. This was found to improve consistency. Subtask 2: Bacterial cell release assay.Many adjustments to this protocol were tested, but none proved to increase the untreated verses EC treated sample differential. Several alterations did increase overall bacterial cell release values but effected both untreated and treated samples equally providing no additional value. The assay was not altered appreciably. Subtask 3: EC toxicity assay optimization. At the time, robust testing of the luminescence-based toxicity assay we developed in Phase I was not possible. Therefore, in Phase II we explored a few aspects in the protocol that had potential to produce artifactual toxicity and found they were not an issue. We also needed to better define the benchmark separating a toxic and nontoxic effect. We used different concentrations of a representative QAS sanitizing agent Benzalkonium chloride on a susceptible strain to identify a preliminary threshold of toxic effect of a luminescence value that is 400% of the untreated control luminescence. This was validated using dilution plate count method alongside our assay. Task 2: Additional strains and species testing.The Phase I effort had only investigated single strains of the 7 species, and 3-enzyme cocktails were assessed against a multispecies biofilm rather than the full bacterial panel. Therefore, in Phase II the 4 top ECs were tested against all 7 species plus 5 additional strains of each species (total of 6 strains for each species) (species:Listeria monocytogenes, Salmonella enterica, E. coli, Lactobacillus plantarum, Acinetobacter johnsonii, Serratia proteamaculans,andPseudomonas fluorescens). In addition, 3 new pathogenic bacteria were added 1.Staphylococcus aureus, 2.Bacillus cereus, and3.Campylobacter jejuni)(total of 5 strains per species) making a total of 57 strains tested with our top 4 ECs. To date we have completed testing of the 4 ECs on all but the 5 C. jejuni strains, which are difficult to culture due to their need for a micro-oxygen environment. GBS initially utilized a scoring system to evaluate ECs performance. ECs were tested head-to-head, and the top EC received a point (ties split the point among members). The current tally is EC1: 6.33,EC2: 35.66, EC3: 4.99, EC4: 3.99, indicating EC2 is dramatically outperforming the other 3 ECs. If the % biofilm reduction scores across all the tested strains are averaged, the bacterial panel averages are: EC1: 42%,EC2: 61%,EC3: 45%, EC4: 41%. Statistical analysis (Dunn's multiple comparisons test) indicates that EC2 is significantly different from the other 3 ECs. Task 3: Testing ECs activity on biofilms grown on different materials.Most biofilm testing is conducted on 96-well polystyrene culture plates. However, this does not represent all the surfaces found in the food industry. Therefore, to test other representative materials biofilms were grown on 13 mm coupons composed of stainless steel, borosilicate glass, high density polyethylene, or natural rubber. The 7 original species strains (from PHI) were used for this. All 7 were tested for biofilm formation on the 4 test surfaces and then the 3 species that produced the most robust biofilms on each surface were used for testing the ECs. Of this analysis only the testing of 2 strains on natural rubber remains to be tested. Using qualitative scoring the tally is: EC1: 0,EC2: 8, EC3: 2, EC4: 0. If the total coupon testing results are averaged, the results are: EC1: 28%,EC2: 60%,EC3: 48%, EC4: 31.0%. Again, EC2 outperformedthe other 3 ECs and has a similar average % reduction to that seen in the 96-well plate testing. The results from Tasks 2 and 3 strongly indicate that EC2 is the 3-enzyme cocktail to move forward to Technical Objective 2 and 3. The year one work is unique because it looked at several strains of 10 species and at resistance to ECs not just biofilm formation. GBS did find biofilm variability among the strains of all species (often reported in the literature) and indicates single strain biofilm testing of a species may not represent the whole species well. Overall, the results are considered satisfactory. EC resistant bacterial strains not predicted by PHI resulted in somewhat lower average % biofilm reduction values than expected. Nevertheless, the planned addition of 1-2 additional enzymes and inclusion of non-toxic concentrations of detergent in Technical Objective 2 are expected to further improve biofilm reduction. No reportable outcomes were completed for Technical Objectives 2 and 3, though work has started on Technical Objective 2.
Publications
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