Source: KANSAS STATE UNIV submitted to NRP
NOVEL SANITATION APPROACHES TO CONTROL LISTERIA BIOFILMS IN THE ORGANIC PRODUCE INDUSTRY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
OTHER GRANTS
Reporting Frequency
Annual
Accession No.
1028972
Grant No.
2022-51300-37885
Cumulative Award Amt.
$1,499,844.00
Proposal No.
2022-04066
Multistate No.
(N/A)
Project Start Date
Sep 1, 2022
Project End Date
Aug 31, 2026
Grant Year
2022
Program Code
[113.A]- Organic Agriculture Research & Extension Initiative
Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506
Performing Department
Animal Sci & Industry - AES
Non Technical Summary
Organic farming is an important sector of the United States of America (USA) agricultural economy and represents an alternative to conventional production systems.Consumers prefer organic products because of perceived health benefits, environmental consciousness, and animal welfare concerns. Since 2011, the fresh produce industry, including the organic market, has been linked to outbreaks and recalls associated with L. monocytogenes contamination: from the deadly episode connected to cantaloupe grown at the Jensen Farms in Colorado to the most recent voluntary recall for salads processed in Yuma, Arizona. Despite the recognized presence of L. monocytogenes in farm operations, no strict requirements are in place to prevent contamination and/or cross-contamination in facilities under the FDA Food Safety Modernization Act Produce Safety Rule. The fast-paced harvesting, handling, and processing operations, the design of equipment, the creation of debris and little time between shifts provide ideal niches for Listeria cell adhesion and biofilm formation. Furthermore, fresh produce distribution does not include a pathogen inactivation step and both pre-harvest and post-harvest equipment are often not designed for optimum mechanical and chemical disruption of pathogens and biofilms, e.g., cleaning and sanitation. The reality for organic growers is even more limited regarding sanitation alternatives, since the National Organic Program (NOP) Standards need to be followed to maintain "organic-certified status": few options are available to control foodborne pathogens, therefore placing the organic produce industry at a competitive disadvantage. In this project we will : (1) Assess the growth of Listeria monocytogenes sessile and biofilm forms on different surfaces representative of organic produce harvesting, handling and processing facilities; (2) Characterize microbial dynamics and composition by imaging, spectroscopy and genomic approaches; (3) Optimize the simultaneous use of lactic acid and UV-C in lab bench settings; (4) Develop an integrated sanitation system from lab model to industrial prototyping; (5) Validate the system on commercial harvesting equipment and in packing facilities representative of different commodities and different production capabilities; (6) Offer workshops and demonstrations on cleaning and sanitation practices for organic growers around the nation, while evaluating the economic costs for the developed prototype.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71214991100100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
1499 - Vegetables, general/other;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The overall goal of this project is to develop and validate novel sanitation strategies to control Listeria biofilms in the organic produce industry on food-contact surfaces and equipment.and biofilm forms on different surfaces representative of organic produce handling and processing facilities; (ii) Characterize microbial dynamics and composition by imaging, spectroscopy and genomic approaches; (iii) Develop and optimize the simultaneous use of UV-C and lactic acid from lab bench settings to industrial prototyping; (iv) Validate the system in commercial harvesting and packing facilities representative of different commodities and different production capabilities; (iv) offer workshop and demonstration on cleaning and sanitation practices targeting organic growers around the nation, and evaluate the economic costs and benefits as well as the risks and implications for the developed prototype.
Project Methods
1.Two bacteria forms will be investigated in this project: sessile and biofilm. The sessile form is the first state of confluent growth of bacteria on agar plates, while a biofilm is a mature and more complex form of a bacterial community living in a complex matrix of EPS and nucleic acids, in addition to the microorganisms. Both conditions are relevant and practical to understand sanitizers' effectiveness and microbial susceptibility. We will measure the ability of bacteria to frowth on the selected food contact and we will asessrecovery yield, adhesion and repeatability of results2. Laser Scanning Confocal Microscopy and Spetroscopy and WHole GEnome Sequencing will be perform on the biofilms to understandchemical and physical changes in bacterial structure and attachment to surfaces coupled with the investigation of gene responses to the different growth conditions and treatments will allow us to obtain a comprehensive overview of the factors that affect Listeria survival and growth.3. Different antimicrobial interventions will be tested and lactic acid simultaneously with UV-C will be applied against bacteria4. Field testing will be performed in different location to validate the efficacy of lab bench experiments

Progress 09/01/23 to 08/31/24

Outputs
Target Audience:For the current reporting period our audience has been mainly the scientific community, since we worked on Objective 1(Assess the growth of Listeria monocytogenes sessile and biofilm forms on different surfaces representative of organic produce harvesting, handling and processing facilities) and we start working on Objective 2 (Characterize microbial dynamics and composition by imagining, spectroscopy and genomic approaches) and Objective 3 (Optimize simultaneous use of sanitizer and UV in lab setting). Changes/Problems:Transportation of the UV tunnel system continues to be a challenge. We are investigating ways to make sue we have support when we will conduct the field trials. From an analytical point of view determining biofilm thickness was challenging sue to the limintation posed by the surfaces and growing conditions. We used several techniques such as profilometry, spectral reflectance spectroscopy and fluorescent microscopy in order to understand and identified biofilm thichness. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Yes, we had several meetings with chemical companies updating on the enhancement of sanitizing action when UV was applied simultaneously and also about the difficulty of inactivating Listeria on certain surafaces (e.g. PVC and Teflon). With these obseravations and meetings, we would like to provide scientific information to the organic produce industry regarding also the cleanability of certain materials that are commonly used in facilities and maybe encourage them to find alterantives if from our experiments those surfaces resulted very difficult to clean. What do you plan to do during the next reporting period to accomplish the goals?In the next 6 months or so , we are planning to test the UV- tunnel system that was built and optimize the treatment choosing one or two sanitizers in combination with UV-C. We will then contact organic producers in Florida, Georgia and Arizona to set up dates to visit and perform the field trials. We will continue to studymicrobial dynamics and composition by imaging, spectroscopy and genomic approaches. During the field trials we will set up demonstrations for producers and start organizing workshops.

Impacts
What was accomplished under these goals? Santizer application of lactic acid, Silver Dihydrogen Citrate , Peracetic acid alone or in combination with UV-C light at both contact times resulted in a >5 log reduction of L. monocytogenes in all types of surfaces for sessile cells. The log reductions observed for Listeria biofilms were different. THe best log reduction was obstained with peracetic acid (~ 4 log reduction). The simultaneous application of UV-C light and sanitizers did not significantly enhance the effectiveness of sanitizers for both sessile and biofilms cells (p>0.05). UV-C light only applied for 5 min was the least effective treatment, and efficacy was influenced by surface material. Teflon and PVC were the materials were inactivation activity against Listeria was difficult to observed. Longer application improved the effectiveness of sanitizers for biofilm control(p<0.05), but no difference was instead observed for sessile cells. These results help validate sanitation strategies including LA (4%), SDC (4.85% citric acid), and PAA (120 ppm) that can be utilized by the organic fresh produce industry to control L. monocytogenes on FCS in combination with UV to enhance the control of Listeria. The influence of various environmental factors, such as surface properties, shear forces, and nutrient availability influence biofilm formation and architecture. In a series of experiments (within objective 2) Listeria biofilms were grown under static and dynamic conditions on five different surfaces common to food processing (i.e., Teflon, plastic, nylon, steel, wood). The biofilm architecture was characterized using various chemical and biophysical techniques. Determining biofilm thickness is challenging due to limitations posed by the substrate's surface topology and varying biofilm thicknesses. We employed multiple techniques for measuring film thickness, including stylus profilometry, spectral reflectance spectroscopy, and fluorescence microscopy with staining, each presenting unique challenges. We identified film thicknesses ranging from a few micrometers (μm) up to 60 μm, depending on the growth conditions. The spatial composition of extracellular polymeric substances (EPS) and distribution of microorganisms were analyzed using IR microscopy, which allowed for the identification of the polymeric network entrapping the microorganisms. This polymeric network determined surface hydrophobicity, assessed using an optical drop tensiometer. Additionally, small molecules responsible for quorum sensing were analyzed using GC-MS coupled with solid-phase microextraction (SPME). Overall, our integrated approach combining chemical, biophysical, and microbial information provided a systematic understanding of the formation and nature of Listeria biofilms under various environmental conditions, affecting their attachment to surfaces and resistance to sanitation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Appolon, Llacsahuaga, Widmer, Mmaduabuchi, Bhullar, Yucel, Trinetta,Dunn and Critzer, 2024. Synergistic efficacy of lactic acid and UV-C in the inactivation of Listeria monocytogenes on soiled food contact surface materials. Technical presentation at IAFP (International Association of Food Protection) Annual Meeting, California, JUly 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Deniz and Trinetta, 2024. Formation and Control of Listeria monocytogenes biofilms on various food processing surfaces. Technical Oral presentation at IAFP (International Association of Food Protection) Annual Meeting, California, July 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Llacsahuaga, Trinetta, Widmer, Bhullar, Yucel, and Critzer, 2024. Effectiveness of novel sanitizer and Ultraviolet (UV-C) light to control for Listeria monocytogenes in the organic fresh produce industry. Technical presentation at European IAFP (International Association of Food Protection) Annual Meeting, Switzerland, May 2024.


Progress 09/01/22 to 08/31/23

Outputs
Target Audience:For the current reporting period our audience has been mainly the scientific community (excahnge of scientific output and ideas) and the sanitation industy that was instrumental to help us building the sanitation system (Objective 4) Changes/Problems:no changes but the major challange we encounter was to develop an integarated sanitation system that is easy to transport in all the field visit that we are planning to do. What opportunities for training and professional development has the project provided? Nothing Reported 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?We are planning to: - finalize the treatment- time concentration on both Listeria sessile and biofilm form on all the different surfaces - optimize the integrate sanitation system - characterize the microoganisms by image and spectroscopy

Impacts
What was accomplished under these goals? - Initial experiments to assess the growth of Listeria monocytogenes sessile and biofilms were performed. A statistifical difference between microbial growth stage (sessile vs biofilm) was identified, with sessile form being generally more sensitive to treatment as compared to biofilms. A signigicant difference among materials was also identified with regard of efficacy of sanitizer treatment. Bacteria growth on smooth surfaces are easier to control. - Optimization of times and concentration of treatments with UV has been also accomplished and different concentration-treatment times were identified for the sessile and biofilm forms: biofilms generally require more contact time with sanitizers in order to observed a significant log reduction as compared to the sessile form - the integrated sanitation system was built

Publications