Source: GUILD ASSOCIATES, INC. submitted to
DEVELOPMENT OF A NOVEL REAGENT FOR RELEASE OF BIOFILM-EMBEDDED MICROBES FROM SURFACES FOR DETECTION BY MONITORING TECHNIQUES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SMALL BUSINESS GRANT
Reporting Frequency
Annual
Accession No.
1025776
Grant No.
2021-33530-34362
Cumulative Award Amt.
$99,426.00
Proposal No.
2021-00843
Multistate No.
(N/A)
Project Start Date
Jul 1, 2021
Project End Date
Feb 28, 2023
Grant Year
2021
Program Code
[8.5]- Food Science & Nutrition
Recipient Organization
GUILD ASSOCIATES, INC.
5750 SHIER-RINGS ROAD
DUBLIN,OH 43016
Performing Department
(N/A)
Non Technical Summary
Food borne infectionsfrom pathogens originating fromfood processing continue to be a problem for the food industry resulting in sickness, death, and substantial financial losses. Current contamination monitoring tests used on food industry surfaces utilizeswabbing/wiping methods to collectmaterial to test for the presence of pathogenic microorganisms on these surfaces. This type of collection captureseasily transferable pathogens,but fails to collectmicrobesentrenched in hardened biological surface communities called biofilms that are protected fromcleaning and disinfection processes but can still contaminate food. The failureto collect these tightly bound surface bacteria reduces the sensitivity of contamination monitoring testsand canmask the true degree of contamination (as most biofilm occupants remain on the surface not on the swab/wipe.and never reach the test to be detected).The proposed project will develop, BioXpose, an innovative enzyme-based product concept that will be a liquid containing several different enzymes that collectively disintegratethe web-like support structure of biofilms releasinglivebacteria from the sampledsurfacesso they can be efficiently collected and detected by currentand future contamination monitoring tests. Using BioXposewill makethesetests more sensitive and accurate which will provide safer food industry facilities and will reduce food borne illnesses.To achieve this, the project will develop a test system ( antibiofilm assay) specifically ulitizing pathogenic bacteria and non-pathogenic residentbacteria commonly collected fromfood industry surfaces (as opposed to using generic pathogenic bacteria). Once an antibiofilm assay that works withall 8 bacteria inthe projectis developed,aset of 13 enzymes well recognized for their ability to break down biofilms will be combined in pairsand tested in antibiofilm assays with biofilms made byeach of the bacteria and the top 6 selected for further work. Single species antibiofilm assays are the norm in biofilm research, but are simple test systems that don't closely represent natural biofilms, which usually have a number of different bacteria present. To provide a more challenging and realistic test system, a secondresearch thread will developantibiofilm assays composed of 4 differentbacterial species (a multi-species antibiofilm assay) grown on either plastic or stainless steel surfaces (two very common surface types in the food industry) will bemore robust and will be moreresistancetotypical agents used for cleaning/disinfecting food industrysurfaces. The multi-species antibiofilm assays developedwill be used to evaluate the 6 top performing2-enzyme combinations identified in the single species antibiofilm assays, and theneven more effective 3 enzyme candidate cocktails will be evaluated with thesemore realistic assays. Finally, to further approach real life test conditions,a commercial ATP assay that is widelyused in the food industry to test for surface contamination and biofouling will be used to evaluate how much improvement in performance thedeveloped enzyme cocktails provideover the standard applicationof the commercial system.We expect that this project will provide solid evidence of the feasibility of thestrategy of using enzyme cocktailsto liberate viable bacteria from biofilms so they can be collected and anayzed by current surface contamination test systems. It is then expected that more rigorous and comprehensive testing and formulation development effortswill be accomplished ina larger SBIR Phase II project. Thiseffort will result in a prototype enzyme formulation that will be developed into acommercial product,BioXpose, which will be used by food safety managers to improve thesensitivity, accuracy, and value of current and future surface contamination tests increasing food industry safety and reducing food borne infections in the US and beyond.
Animal Health Component
35%
Research Effort Categories
Basic
35%
Applied
35%
Developmental
30%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71253401100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
5340 - Nonfarm structures and related facilities;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Goal: Theproject will develop, BioXpose, an innovative enzyme-based product concept whose purpose istoincreasethe effectiveness in the extraction of viable microbes out ofbiofilms on surfaces through biofilm disintegration. Use of BioXpose withcurrent surface contamination monitoring tests will make themmore sensitive and accurate.. The project will consist of three objectives.Objective 1, Develop a preliminary enzyme cocktail that dissolves mono-species biofilms. Aset of thirteen enzymes in binary pairs will be tested against in vitro biofilms of eight bacteria representing major food borne pathogens and model resident species of food industry surfaces. The top 6 2-enzyme cocktails will move on to Objective 3Objective 2: Develop a more realistic multi-species bioflm model with increased tolerance to cleaning and disinfection. Multi-species (MS) in vitro biofilms assays that employeither plastic and stainless steel surfaces will be developed as more realistic representations of actual contaminated food processing surfaces. The measure for assessing the quality of different species combinations will be increased resistance to disinfectant treatment, ensuring the final MS biofilms will be more robust than single-species biofilms and a stronger more realistic challengefor the candidate enzyme cocktails.Objective 3: Develop an advanced 3 enzyme enzyme cocktail that dissolves multi-species biofilms.The top binary enzyme pairs from Objective 1 will be tested against the two MS biofilm models developed in Objective 2, and then a set of 25 3-enzyme combinations will be tested using these two systems. The top final enzyme cocktailswill be tested using a widely used commercial contamination detection assay to gauge how the use verses no use of the enzyme cocktails improves theperformance of this real world product.
Project Methods
To achieve Objective 1, we will first develop an in vitro antibiofilm assay that can be commonly utilized with all the pathogens that are part of the project basedon methods from the literatureand our previous experience with in vitro assay in general as well as specifically biofilm assay experience. Development will include finding robust biofilm developmentconditions (media, environmental, duration,etc) acceptable to all 8 modelorganisms andevaluating potentialantibiofilm assay endpoints using DNase I as a model antibiofilm enzyme. Next a set of 12 enzymes recognized inthe literature to have well documented antibiofilm activity will be tested in different binary combinations for antibiofiilm activity in monoculture antibiofilm assays of the 8 selected model bacteria representing both food borne pathogens and common "resident" bacteria known to form biofilms on food industry surfaces which are capable of harboring pathogens that don't form biofilms. All pairing will include DNase I which has a large body of evidence supporting its antibiofilm activity to reducethe number of binarytests needed. Forevaluation a scoring matrix will be developed that applies criteria that representthe preferred characteristics of the final product (e.g. biofilm disruption efficacy across species tested, degree of bacterial release from the biofilm, released bacteria viability, time to completion, degradative effects on other enzymes etc). It is notable that these criteria while similar are distinct from those that would be applied for solely a cleaning application. The top 6 enzyme combinations will move to Objective 3 with the minimum requirement being at least one combo must display an average of 75% biofilm reduction across all 8 species tested.In Objective 2, will diverge out of the early part of Objective 1 (afterthe basic biofilm assay is developed). From this, a multi-species antibiofilm assay will be developed as a more robust and realistic approximation of in vivo biofilms on food industry surfaces. Most natural biofilms are thought to be multi-species which is believe to increase their resistance to cleaning and disinfection. First theObjective will evaluate different combinations of the 8 model species in a 96-well plate assay format with the goal ofproducing4 species biofilms that shows 2 fold higher resistance to a model disinfectant than any monospecies biofilm. Once achieved a second assay that utilizes stainless steel coupons (rather than the plastic plate bottom)will be developed representing a second common surface in the food industry and will have the same performance criteria as the first.In Objective 3, the two research threads will come together. First the top six binary enzyme combinations from Objective 1will be tested on the two more robust multispeices biofilms produced in Objective 2 to establish a baseline performance. Then 24 3-enzyme cocktailswill be evaluated on these two assays with the same criteria scoring as applied in Objective 1. Finally, the top 2 3-enzyme cocktails will be tested by a commercially available ATP assay which is commonly used in the food industry to assess surface cleaniness and safety. A larger surface area assay using eitherplastic and stainless steel surfaces will be tested and the limit of detectionof the ATP assay compared between enzyme treated and non-treated conditions. The final target milestone is that enzyme treatment should increase the limit of detection of the commercial assay at least 10 fold over no enzyme treatment.

Progress 07/01/21 to 08/01/22

Outputs
Target Audience:The audience that will have interest in the product that results from this project would primarily be food industry executives, safety managers and related staff tasked with keeping the food products associated with their facility free of contamination by food born pathogens. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?As the results of this project have substantial potential for development into a commercial product, the results are not currently planned to be released to communities of interest. At some future point, some of the fundamental testing methods developed, once further optimized might be released as a journal publication. A US provisional patent has been filed. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Current food industry contamination testing can collect loosely associated microbes on surfaces, but are ineffective at recovering microbes growing in biofilms,which can result in underestimating the microbial burden and can increasesafety risks. The envisioned product, BioXpose, an innovative enzyme-based product concept, willincreasethe effectiveness of collectingviable microbes from biofilmson surfaces bybreaking down the biofilm's structural support fibers releasing formerly protectedmicrobes, so they are susceptible to collection and then detection.The applicationof BioXpose is planned to be compatible with allcurrent surface contamination monitoring tests and will make themmore sensitive and accurate. This improvementwill allow food industry safety managers to more effectively produce safe production facilities which will reduce product recalls and food-borne illness outbreaks caused byfood industry-sourced contamination. The demonstration of feasibility was achieved through three technical objectives and the accomplishments achieved for each is given below. Objective 1: The initial work was to develop an antibiofilm assay that was compatible with the different model biofilm forming bacteria chosen for the study. A protocol was developed that used 24 hour biofilmsand exposed to test binary enzyme cocktails (ECs) for 1 hour. The commonly utilized Crystal Violet assay (CVA)was used to determine the amount of biofilm that remained after EC treatmentcompared to untreated controls was used as the initial endpoint. In total 12enzymes (that have been demonstrated in the literature to have antibiofilm activity)were tested in binary combinations against 7 model biofilm-forming bacteria representing either food pathogens or nonpathogenic food industry resident bacteriawhose strong biofilms can harbor and protect pathogens fromantibiofilm activity. Binary ECswere also evaluated using a bacterialrelease assay that quantified the amount of bacteriareleased from biofilms due toEC treatment as opposed to the amount of residual biofilm remaining from EC treatment as the CVA measured.This was considered a more practical assessment of the potential of ECs since it better mimickedhowthe future product would be used(releasing bacteria from surfaces for collection and detection testing). Post-EC exposure the solutioncovering eachbiofilmwas removed, centrifuged and the cell pellets resuspended inPBS before an ATP luminescence assay (BacTiterGlo, Microbial cell viability assay, Promega) was used to quantify thebacteriareleased from the biofilms. The luminescencevalues from EC treatments were compared to untreated controls to quantifythe increase inbacterial release. In addition, ECs were evaluated for toxicological effects on the model species, since viable microbes are needed for some types of microbial detection assays. The ECs shouldn't kill the bacteria. As planned,6 binary enzyme cocktails (ECs) were identified that had superior antibiofilmactivity against themodel bacterial species with average % biofilm reductions across all 7 model bacteria ranging from 31% to 68%.The bacterial release foldincrease in these 6 ECs ranged from 1.9 to 4.4 fold over controls and only 1 EC had an elevated toxicity against one bacterial species. These 6 binary ECs were used in Objective 3. Objective 2: Although single-species (ss) biofilmtesting isubiquitous in biofilm research, natural biofilms are thought to be largely multi-species (MS) biofilm communities, and there are many reportsthat MS biofilms are more resistant to antibiotic/antibiofilm/sanitizing agents than ssbiofilms. Therefore,Objective 2investigated if in vitroMSbiofilms could be developed that weremore resistant to a model sanitizing agent than ss biofilms which would make fora more realistic test for top performing ECs. The 2-species MS biofilm testing was based on the protocol and CVA assay endpoint developedin Objective 1but usedequal quantities of each test bacteria in biofilm formation andusedss biofilmsof the two MS membersfor comparison. As planned, a model quaternary ammonium substance (QAS) was used as the test agentfor evaluating biofilm resistance.Althoughthere are ample reports that MS biofilms are more resistance than mono-species biofilms, we did not see substantialevidence of this with the selected model biofilm species tested. In fact, we found that often mono-species biofilms were completely or almost completely resistant to the QAS treatment (when used at a concentration and treatment duration reported to be effective against biofilms in the literature).In no casedid we see 2 member MSbiofilms that were substantially more resistant to QASthan both memberspecies in ss biofilms. It is probablethat the rather simplistic method of developing MS biofilms was too artificial to effectively mimicnatural biofilms, but many reports in the literature have used very similar methods and showed improved resistance in MS biofilms over ss biofilms. A number of model bacteria of this study are very strong biofilm producers, so it is possible that these species combinations producedmore robust biofilms than those in other studies. Nevertheless, two MS biofilms did showmarginal improvement in QAS resistance over eithermember in ss biofilms, so 3-species biofilms were then investigated based on these MS biofilmsby similar methods. A single 3-species biofilm proved superior and was selected for use in Objective 3. Additionally, one ssbiofilm model (Serratia proteamaculans) was strongly resistant to all but one binary ECs in Objective 1, so this ss biofilmwas also used to evaluate 3-ECs in Objective 3. Objective 3: In Objective 3, 3-EC combinations developed fromthe top 6 binary ECsthat moved forward from Objective 1, using either the selected MS biofilm model of Objective 2 orthe single strongly resistant ssbiofilm model from Objective 1. Both biofilm reduction via the CVA assay, and the bacterial release assay wereused for 3-EC evaluation. It was found that tested 3ECs performed very efficiently against the MS biofilm model with most 3ECs achieving over 75% biofilm reduction and the top 5 3-ECs reaching 92 to 95% biofilm reduction.However, against S. proteamaculans biofilms most 3ECs were not effective and only 3-ECs containing the single enzyme found effective against this species in the binaryEC testinghad over 50% biofilm reductionactivity . The most surprising aspect of the project was that 3-ECs often did not perform substantially better than the 2ECs against either biofilm model,which was unexpected and will require further study to understand. Cell release testing results were also surprisingly low against S. proteamaculansbiofilms across the board, whereas against the MS biofilms 3-ECs displayed good bacterial release reaching up to 7 fold higherrelease overcontrols in a few cases. Our data suggestsa QAS sanitizer may notbe a good predictor of resistance to antibiofilm ECs probably due to differentmechanisms (killing vs scaffold disintegration).Nevertheless, several 3EC did achieve effectivebiofilm reduction in both models, (details in the technical report) and three-3ECs made the top 5 list on both biofilms. The top 3ECs plus an alternate will move on to be further tested, optimized, and converted into a product ready for market introduction.

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