EVALUATING THE EFFECT OF ENVIRONMENTAL MICROBIOTA ON BIOFILM FORMATION AND SANITIZER TOLERANCE OF LISTERIA MONOCYTOGENES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1027991
• Grant No.: 2022-67017-36556
• Cumulative Award Amt.: $605,000.00
• Proposal No.: 2021-08148
• Project Start Date: Jan 1, 2022
• Project End Date: Dec 31, 2024
• Grant Year: 2022
• Program Code: [A1332]- Food Safety and Defense

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505

Performing Department
Food Science

Non Technical Summary
Listeria monocytogenesis a bacterium that causes the deadly foodborne illness listeriosis.Listeriaand other microorganisms found in the natural environment, such as soil, can be unintentionally introduced into food processing facilities with raw foods like fruit. Once introduced in the food processing environment,Listeriaand many other environmental microorganisms can grow on surfaces into microbial layers called biofilms. Microorganisms enclosed in a biofilm produce slimy substances that protect them from the antimicrobial activity of sanitizing chemicals by slowing down their penetration into the core of a biofilm. Biofilm formation can therefore result in reduced efficacy of antimicrobial sanitizers used to inactivateListeria.This project will investigate the interactions between microorganisms found in fruit packing environments andL. monocytogenes. It will study the ability of environmental microorganisms to form robust biofilms together withL. monocytogenesand to what extent the formed biofilms protectL. monocytogenesfrom the antimicrobial activity of sanitizers. The data and information generated in this project will help optimize the cleaning and sanitizing procedures used in the fresh produce industry to improve the control ofL. monocytogenesand support the production of safe food.

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
723	5010	1100	100%

Knowledge Area
723 - Hazards to Human Health and Safety;

Subject Of Investigation
5010 - Food;

Field Of Science
1100 - Bacteriology;

Keywords

listeria monocytogenes

biofilm

produce packing

sanitizers

tolerance

Goals / Objectives
Listeria monocytogenes is a foodborne pathogen that can survive, grow, and persist at low temperatures in produce processing facilities. One of the mechanisms that can enhance the survival and persistence of L. monocytogenes in food processing environments is biofilm formation. A biofilm represents a physical barrier that reduces the effective diffusion and antimicrobial action of sanitizers and is hypothesized to increase L. monocytogenes' tolerance to sanitizers used in food processing facilities. The role of the food processing environment microbiota on Listeria monocytogenes survival within a biofilm under sanitizer pressure is poorly understood. Hence, we propose to evaluate the ability of most relevant environmental microbiota constituents found in produce processing or packing environments (i) to form a single- and multi-species biofilms with L. monocytogenes and (ii) to measure the effect of the resulting biofilms on L. monocytogenes' tolerance to sanitizers. The outputs of this project will inform targeted and enhanced cleaning and sanitation protocols to effectively control L. monocytogenes and microbiota that can facilitate its survival and persistence via biofilm formation. We propose four objectives:Obj. 1: Isolate environmental microbiota and determine their resistance to sanitizers.Obj. 2: Characterize genomes of environmental isolates using whole-genome sequencing.Obj. 3: Characterize biofilm formation ability of bacterial families and L. monocytogenes in single- and in multi-family assemblages.Obj. 4: Characterize the effect of microbial assemblages on the tolerance of L. monocytogenes to sanitizer treatment.

Project Methods
Objective 1Task 1.1: Sample collection. We will collect three samples during processing hours, using pre-hydrated sponges with neutralizing buffer. Samples will be collected from the floor under the conveyor belt of the wet processing area of each facility (N = 3, n = 9). Given that in our previous study, the relative abundances of the target families were consistent within each facility, the proposed number of samples should suffice to obtain target isolates. Samples will be homogenized with Brain Heart Infusion (BHI) broth using a stomacher. The three homogenates from each facility will be pooled and sterile glycerol will be added to cryopreserve samples until further use. Composite samples will be mixed, aliquoted, and cryopreserved at -80°C until isolation in Task 1.2.Task 1.2: Isolation of bacteria and cryo-preservation. To isolate strains from the families Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae and Flavobacteriaceae, we will thaw microbiome samples collected in Task 1.1 at room temperature within an hour and used for isolation with a massively parallel microbial cultivation instrument Prospector (GALT). Approximately 100 isolates will be randomly selected for 16S rRNA-based identification using Sanger sequencing.Task 1.3: Isolate identification. Ww will conduct a preliminary differentiation of isolates and identification of their genus using 16S rRNA gene Sanger sequencing and SNP analysis. We will confirm isolates' identity by phylogenetically comparing them with sequences of each genus' type strains that will be extracted from the Ribosomal Database Project (RDP) database. To allow for tracking of individual isolates in microbial assemblages in Obj. 3, we will additionally perform a Single Nucleotide Polymorphisms (SNP) analysis of the 16S rRNA V4 region of isolated strains. We will select one strain per genotype from each target family to include in assemblages in Obj. 3 and 4.Task 1.4: Determine isolates' resistance to benzalkonium chloride and peracetic acid. The minimum inhibitory concentration (MIC) of BAC, PAA, and Sterilex will be determined for each isolate selected in Task 1.3. A broth microdilution assay will be used, as described in the CLSI Standard. The MIC of each isolate will be determined as the lowest concentration of BAC, PAA, and Sterilex that inhibits growth of a strain, as determined by the absence of turbidity in a well.Objective 2Task 2.1: Whole-genome sequencing. The selected isolates obtained in Task 1.2 and identified in Task 1.3, will be whole genome sequenced. DNA of isolates will be extracted using DNeasy UltraClean Microbial Kit following the manufacturer protocol and libraries will be sequenced using an Illumina platform. We will perform quality control of sequences and assemble them into contigs using SPAdes.Task 2.2: Taxonomic identification and genome characterization. KmerFinder will be used for rapid preliminary identification of taxonomic species. To confirm the accuracy of taxonomic identification, an Average Nucleotide Identity (ANI) will be calculated using FastANI. To phylogenetically compare genomes of bacterial isolates that belong to the same family, we will perform core genome SNP analysis using kSNP3. To characterize the gene content of assembled genomes, we will perform functional gene annotation of the contigs obtained in Task 2.1 using the PROKKA pipeline. Roary will be used to generate a presence-absence spreadsheet of the genes identified in the assembled genomes. To compare gene content in isolates' genomes across families, we will use the OrthoFinder.Objective 3Task 3.1: Characterize biofilm formation ability of environmental microbiota and of L. monocytogenes. We will measure each family's biofilm formation ability in vitro using two complementary methods: quantification of total biomass by crystal violet staining and characterization of biofilm structure using confocal laser scanning microscopy (CLSM). In addition to environmental isolates, we will characterize biofilm formation of a cocktail of L. monocytogenes isolates collected from tree fruit packing houses in our previous project. Individual families will be tested for their ability to form biofilms in single-family cocktails in Task 3.1 and in multi-family cocktails in Task 3.2. Isolates will be individually grown overnight in R2A broth and diluted to OD 0.2. R2A medium will be used for biofilm growing and tolerance experiments because it is less nutrient-rich compared to BHI and hence more appropriately mimics the food processing environment. Inoculum cocktail will be added to 96-well plates in triplicates, followed by incubation for 2 days at 15°C. The total amount of attached biomass quantified using the crystal violate assay will be used as a proxy for biofilm formation. Since a true 3D biofilm structure is characterized by EPS, the biofilm formation will be confirmed using a CLSM on a subset of assay replicates, after LIVE/DEAD staining. To characterize the composition of biofilms formed by each bacterial family, we will use 16S rRNA amplicon sequencing.Task 3.2: Characterization of biofilm formation in multi-species cultures. We will create microbial assemblages composed of a cocktail of L. monocytogenes in combination with one, two, three or four bacterial families. Each isolate will be grown overnight in R2A broth and subsequently diluted to OD 0.2. Plates will be incubated statically for 2 days at 15°C. After incubation, the total amount of attached biomass will be quantified using the crystal violet staining procedure and biofilms will be characterized using CSLM as described in Task 3.1. To determine whether L. monocytogenes' growth is favored or suppressed in mixed-family biofilms, we will quantitatively measure viable L. monocytogenes in the biofilms using the most probable number (MPN) method adopted from the FDA BAM protocol. To further characterize the microbial composition of biofilms formed by each assemblage, we will use 16S rRNA amplicon sequencing data obtained in Task 3.1.Objective 4Task 4.1: Determine L. monocytogenes tolerance to sanitizers when present in a mixed-family planktonic culture. To determine L. monocytogenes tolerance to BAC and PAA when co-cultured with other species, we will determine the MDK99 of L. monocytogenes in each mixed-family assemblage (Table 1) in planktonic culture. Each mixed-family assemblage will be exposed to 2 x MIC of the L. monocytogenes' isolate with the highest MIC previously determined in Task 1.4. To quantify L. monocytogenes tolerance to the sanitizers we will use the MPN assay, as described in Task 3.2, at time zero and then at 2-hour intervals. MDK99 will be calculated from the kill-time curves as the time needed to reduce the concentration of L. monocytogenes by 2 logs.Task 4.2: Determine L. monocytogenes tolerance to a sanitizer when present in a mixed-family assemblages' biofilms. Each isolate will be grown overnight in R2A broth, diluted to OD 0.2. Two serial dilution of the density adjusted cultures will be prepared in sterile R2A broth. Two decimal dilutions of the density-adjusted cultures will be prepared in sterile R2A broth. Biofilms of the bacterial assemblages will be exposed to 2x MIC of the L. monocytogenes isolate with the highest MIC previously determined in Task 1.4, for a total of 8 h. In 2h intervals, the MPN assay will be used to quantify the survival of L. monocytogenes in the biofilms after antimicrobial treatment, as described in Task 4.1. MDK99 will be calculated from the kill-time curves as the time needed to reduce the concentration of L. monocytogenes by 2 logs. To quantify the effect of sanitizer to the whole microbial assemblage, aerobic plates count will be performed.

Progress 01/01/22 to 12/31/24

Outputs
Target Audience:Target audiences included: (i) Individuals participating in the project as part of research and academic training (faculty, PhD scientist, undergraduate students, a graduate student, a postdoctoral scholar); (ii) Food safety professionals, students, and academics that participated in conferences where project findings were presented; (iii) Research community, industry professionals, and the general public that accessed projects' research findings via scientific publications; (iv) Research community, industry professionals, and the general public that accessed study findings and their applied value by attending a webinar. Changes/Problems:We did not encounter any major challenges. We completed all objectives of the proposed project. Based on the data collected in year 2, we performedadditional tolerance experiments with 200 ppm benzalkonium chloride and 250 ppm peroxyacetic acid. What opportunities for training and professional development has the project provided?In year 3, the project provided an opportunity for food microbiology research training, scientific writing, and science communication to four undergraduatestudents, one graduate student, one postdoctoral scholar, and a PhD scientist. How have the results been disseminated to communities of interest?The results have been disseminated in two peer-reviewed papers published in Biofilm and Food Microbiology, oral and poster presentations at conferences (Advanced Sanitation Conference, ASM Microbe, IAFP), ina Food Safety Magazine webinar that was attended by nearly 500 attendees, and the Penn State One Health Microbiome Center seminar. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In year 3, we completed Objective 4, which included characterizing the tolerance of Listeria monocytogenesto peroxyacetic acid (PAA) and evaluating the anti-biofilm and anti-microbial efficacy of the biofilm remover. Previously, we characterized the tolerance of L. monocytogenes to 12 ppm benzalkonium chloride (BAC). Given that this concentration is below a commercially applied concentration, we conducted additional experiments in year 3, to determine the tolerance of L.monocytogenes(in biofilms) to 200 ppm benzalkonium chloride to make our findings more relevant to the food industry. Biofilms composed of microbiota previously shown to co-occur with L. monocytogenes in tree fruit packing facilities (i.e., Pseudomonadaceae, Xanthomonadaceae, Flavobacteriaceae, and Microbacteriaceae) were formed with L. monocytogenes in single- and multi-family assemblages. Multi-family biofilms were exposed to 250 or 500 ppm of PAA, or 200 ppm of BAC to determine the die-off kinetics of L. monocytogenes. Furthermore, the ability of a commercial biofilm remover to disrupt biofilms and inhibit bacteria in the formed single- and multi-family assemblage biofilms was assessed. The die-off kinetics of aerobic mesophilic bacteria and L. monocytogenes in biofilm assemblages throughout the exposure to a sanitizer weredetermined using the aerobic plate count and the most probable number methods, respectively. Biofilm assemblages that included Pseudomonadaceae resulted in an increased tolerance of L. monocytogenes to BAC and PAA compared to biofilm assemblages without Pseudomonadaceae. Further, the use of a biofilm remover at a recommended application concentration significantly disrupted biofilms and reduced the concentration of L. monocytogenes in single- and multi-family biofilms by 5 logarithmic units. Overall, we demonstrated that the tested biofilm remover effectively disrupted multi-family biofilms and significantly reducedL. monocytogenesin multi-family biofilms. Hence, itshows promise for controlling biofilms andL. monocytogenesin food processing facilities. We also demonstrated an increased tolerance of L. monocytogenes to benzalkonium chloride and peroxyacetic acid at concentrations commonly used in food processing facilities, when integrated in multi-family biofilms containing Pseudomonasspecies. Noteworthy, 500 ppm peroxyacetic acid was most effective in inactivating L. monocytogenesin multi-family biofilms. Given the common presence of Pseudomonas spp. in food processing environments and their demonstrated role in facilitating tolerance of L. monocytogenesto sanitizers when in a biofilm, it is recommended to assess sanitizer efficacy againstPseudomonas, in addition to L. monocytogenes. The tolerance findings have been published in two peer-reviewed publications, which have been submitted to PubAg. The associated datasets have been submitted to Ag Data Commons.

Publications

• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Rolon ML, Voloshchuk O, Bartlett KV, LaBorde LF, Kovac J. Multi-species biofilms of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride. Biofilm. 2024 Jan 14;7:100177. doi: 10.1016/j.bioflm.2024.100177.
• Type: Peer Reviewed Journal Articles Status: Published Year Published: 2025 Citation: Voloshchuk O, Rolon ML, Bartlett KV, Mendez-Acevedo M, LaBorde LF, Kovac J. Pseudomonadaceae increased the tolerance of Listeria monocytogenes to sanitizers in multi-species biofilms. Food Microbiology. 2025, 128:104687. doi: 10.1016/j.fm.2024.104687.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Kovac, J. (June 15, 2024). "The role of fruit processing facilities' microbiomes in the persistence and antimicrobial tolerance of Listeria monocytogenes," ASM Microbe, American Society for Microbiology, Atlanta, GA, Invited. International.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Rolon, M. L., & Kovac, J. (May 20, 2024). "Stop the spread! Control of biofilms to remove Listeria monocytogenes from food processing facilities," Advanced Sanitation Conference, Alliance for Advanced Sanitation, Online, Invited. International.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Rolon, L. M., Voloshchuk, O., Bartlett, K., LaBorde, L., & Kovac, J. (July 15, 2024). "Multi-species biofilms comprised of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride," IAFP Annual Meeting, International Association for Food Protection, Long Beach, CA, Accepted. International.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Rolon, L. M., Voloshchuk, O., Bartlett, K., LaBorde, L., & Kovac, J. (June 14, 2024). "Multi-species biofilms comprised of environmental microbiota isolated from fruit packing facilities promoted tolerance of Listeria monocytogenes to benzalkonium chloride," ASM Microbe, American Society for Microbiology, Atlanta, GA, Accepted. International.
• Type: Other Status: Other Year Published: 2024 Citation: Kovac, J. (September 27, 2024). "Microbial guardians: How microbiota shape the fate of Listeria monocytogenes in food processing environments," One Health Microbiome Center Seminar, Penn State One Health Microbiome Center, University Park, PA, Invited. Colleges.
• Type: Other Status: Other Year Published: 2024 Citation: Kovac, J. (December 9, 2024). "Microbial guardians: How microbiota shape the fate of Listeria monocytogenes in food processing environments," Department of Plant Pathology and Environmental Microbiology presentation and panel discussion, Penn State Foster Auditorium, University Park, PA, Invited. Universities.
• Type: Conference Papers and Presentations Status: Other Year Published: 2024 Citation: Kovac, J. (May 20, 2024). "Bacterial biofilms and our food," Organized session at the Advanced Sanitation Conference, Alliance for Advanced Sanitation, Online, Invited. International.

Progress 01/01/23 to 12/31/23

Outputs
Target Audience:Target audinece were undegraduate students, a graduate student, and a research technologist that were trained to conduct research on this project. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided an opportunity for training to undegraduate students, a graduate student, and a laboratory technician who conducted the experiments, analyzed data, and presented research findings. How have the results been disseminated to communities of interest?The results have been disseminated in a published paper, at an international FEMS2023 conference and in a one-day workshop titled "Controlling Listeria in Produce Packinghouses" offered on April 14th, 2023 at the Penn State Fruit Research and Extension Center in Biglerville, Pennsylvania. The workshop was attended by 48 attendees from fruit and mushroom industry, and government inspectors. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we plan on completing tolerance experiments using peracetic acid and assessing the effect of Sterilex biofilm remover on the biofilm degradation (Obj. 4).

Impacts
What was accomplished under these goals? Obj. 1 and Obj. 2 were completed in year 1 and described in the previous progress report. Obj. 3 The work under this objective was planned for project year 1 (quarter 4) through year 2 (quarter 4) and was completed by the end of year 2. We developed and tested the protocol for characterization of biofilm formation of single-family and multi-family assemblages. In addition to testing single and 1-1 assamblage biofilm formation experiments in year 1, we completed the assessment of biofilm formation for the rest of the assemblages (Listeria + 2 bacterial families, Listeria + 3 bacterial families, and Listeria + 4 bacterial families). Studied microbiota included bacterial families Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae previously shown to co-occur with L. monocytogenes in tree fruit packing facilities. The biofilm formation ability and concentration of total microorganisms and of L. monocytogenes was measured in single- and multi-family assemblages. A total of 8, 8, 6, and 3 strains of Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae, respectively, were used in the experiments. Biofilms were grown statically on pegs submerged in a R2A broth in microtiter plates for 3 days at 15°C. Biofilm formation was quantified using a crystal violet assay and confocal laser scanning microscopy, and the composition of biofilms at the experimental end point was determined using amplicon sequencing. The concentration of total microorganisms in formed biofilms was determined by spread plating. The concentration of L. monocytogenes in biofilms was quantified using the most probable number method. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all families combined had a significantly higher concentration of total microorganisms and L. monocytogenes compared to biofilms formed by just L. monocytogenes. Furthermore, L. monocytogenes was able to attach and/or grow significantly better in multi-family assemblage biofilms, compared to biofilms formed by L. monocytogenes alone. Obj. 4 The work under this objective was planned for year 2 (quarter 4) through year 3 (quarter 4). In year 2, we completed tolerance experiments using benzalkonium chloride. The effect of formed biofilms on the tolerance of L. monocytogenes to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. The concentration of L. monocytogenes in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of L. monocytogenes were quantified using a most probable number method. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all multi-family assemblages had significantly higher concentration of bacteria, as well as L. monocytogenes, compared to biofilms formed by L. monocytogenes alone. Furthermore, multi-family assemblage biofilms increased the tolerance of L. monocytogenes to benzalkonium chloride compared to L. monocytogenes mono-species biofilms and planktonic multi-family assemblages.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Rolon, M. L., & Kovac, J.. (April 14, 2023). "Stop the spread! Control of biofilms to remove Listeria monocytogenes from food processing facilities," Listeria control in produce packinghouses, Penn State Extension, Biglerville, PA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Kovac, J.. (April 14, 2023). "Listeria - a foodborne pathogen of concern in produce packing and processing operations," Listeria control in produce packinghouses, Penn State Extension, Biglerville, PA.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Kovac, J., Rolon, L. M., & Voloshchuk, O. (July 12, 2023). "The effect of environmental microbiota on biofilm formation and concentration of Listeria monocytogenes in formed biofilms," FEMS2023 Congres of European Microbiologists, Federation of European Microbiological Societies, Hamburg, Germany.
• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Rolon, M. L., Tan, X., Chung, T., Gonzalez-Escalona, N., Chen, Y., Macarisin, D., LaBorde, L. F., & Kovac, J. (2023). "The composition of environmental microbiota in three tree fruit packing facilities changed over seasons and contained taxa indicative of L. monocytogenes contamination." Microbiome 11(1), 24. DOI: 10.1186/s40168-023-01544-8.

Progress 01/01/22 to 12/31/22

Outputs
Target Audience:The target audiences reached included one graduate student, one undergraduate student, one postdoctoral scholar, and one laboratory technologist who were trained to conduct experimental work proposed in the project proposal. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In the first year, this project provided a laboratory and professional development training to one graduate student, one undegraduate student, one postdoctoral associate, and one research technologist. They developed laboratory technical skills, data analyses skills, and scientific communication skills via presentations in lab meetings and departmental food microbiology meetings. How have the results been disseminated to communities of interest?The results have not yet been disseminated in the first year of the project. What do you plan to do during the next reporting period to accomplish the goals?We plan on completing biofilm formation and characterization experiments, as oultined in Objective 3. Furthermore, we plan to start quantifying the tolerance of Listeria monocytogenes in biofilms to select sanitizers, as outlined in Objective 4.

Impacts
What was accomplished under these goals? Obj. 1: Isolate environmental microbiota and determine their resistance to sanitizers. The work under this objective was planned for year 1 (quarter 1 through quarter 3) and was completed as planned. A total of 901 bacterial isolates were isolated from the environmental microbiota of three tree fruit packing facilities, using four isolation strategies. A total of 510 isolates were randomly selected for identification using Sanger sequencing of the PCR amplified 16S rRNA gene region. Out of the 510 isolates that were identified using Sanger sequencing, 115, 94, 39, and 6 strains were identified as Pseudomonadaceae, Microbacteriaceae, Xanthomondaceae, and Flavobacteriaceae, respectively. After comparison of Sanger sequences with sequences of species type strains from the RDP database, we identified 17, 26, 9, and 5, distinct genotypes of Pseudomonadaceae, Microbacteriaceae, Xanthomondaceae, and Flavobacteriaceae, respectively, which were sent for whole genome sequencing for species-level taxonomic identification and isolate selection. A total of 8, 6, 8, and 3 isolates of Pseudomonadaceae, Microbacteriaceae, Xanthomondaceae, and Flavobacteriaceae, were selected for further experiments. All selected environmental isolates (n=25) and 7 L. monocytogenes isolates (which were previously isolated by Simonetti et. al, 2021, from the three tree fruit packing facilities) were tested using broth microdilution method. For each isolate, we determined the minimal inhibitory concentration (MIC) of benzalkonium chloride (BAC), peroxyacetic acid (PAA), and Perquat disinfectant. Obj. 2: Characterize genomes of environmental isolates using whole-genome sequencing. The work under this objective was planned for year 1 (quarter 1 through quarter 4) and was completed as planned. All selected environmental isolates' (n=25) and 7 L. monocytogenes isolates' genomes were annotated to identify genes involved in resistance to sanitizers that are commonly used in tree fruit packing facilities, and to detect genes involved in a biofilm formation. Twenty isolates possessed at least one gene that has been previously reported to provide resistance to benzalkonium chloride. All Flavobacteriaceae, Pseudomonadaceae, Xanthomonadaceae, and 5 Microbacteriaceae isolates possessed a gene associated with the production of exopolymeric substances commonly involved in biofilm formation. Obj. 3: Characterize biofilm formation ability of bacterial families and L. monocytogenes in single- and in multi-family assemblages. The work under this objective was planned for year 1 (quarter 4) through year 2 (quarter 4). One biological replicate of crystal violet experiemnts was completed. We developed and tested the protocol for characterization of biofilm formation of single-family and multi-family assemblages. All assemblages that contained Pseudomonadaceae and Xanthomonadaceae showed significantly higher biofilm formation abilities compared to other assemblages, as measured using the crystal violet assay. Obj. 4: Characterize the effect of microbial assemblages on the tolerance of L. monocytogenes to sanitizer treatment. The work under this objective has been planned for year 2 (quarter 4) through year 3 (quarter 4). Experiments will start in the second year of the project.

Publications