Progress 08/01/23 to 07/31/24

Outputs
Target Audience:Industry, academic and governmental food safety professionals: two posters were presented in the 2024 annual meeting of International Association for Food Protection (IAFP). Two posters were presented at the American Society for Microbiology North Central Branch 83rd annual meeting.A journal publication from this project has been published in Current Microbiology and is available for stakeholders interested in the findings. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. California State Polytechnic University, Pomona: An undergraduate student (McNair Scholar) has been trained through this project to advance their knowledge in isolating antibiotic-resistant bacteria from food products and performing genomic analysis of antibiotic resistance genes using various bioinformatics tools. The student showcased the discovery and findings via the university research conference and college research conference. Moreover, a graduate student from Cal Poly Pomona has been recruited and trained through the extensive bioinformatic analysis, including BV-BRC platform, identifying bacterial species and antimicrobial resistance genes via a variety of bioinformatic tools. 2. University of Wisconsin-La Crosse : two undergraduate students were trained and contributed to the project. One full-time technician worked on this project until late October 2023. One graduate student was trained on this project and assisted with irrigation water sample processing and the disk diffusion assay. How have the results been disseminated to communities of interest?A journal publication from this project has been published in Current Microbiology and is available for stakeholders interested in the topics. Additionally, two posters were presented at the 2024 Annual Meeting of the International Association for Food Protection (IAFP), where the audience primarily consisted of food safety professionals and other stakeholders. Two posters were presented at the American Society for Microbiology North Central Branch 83rd annual meeting. What do you plan to do during the next reporting period to accomplish the goals?We have successfully completed the retail sampling for Objective 1 and are now shifting our focus to field sampling without intervention, as well as the second growing season of our field trial. Key activities for this phase include: 1. Farm sample Collection and Analysis: We will continue collecting and analyzing the remaining 100 vegetable and environmental samples from a farm and packinghouse in Pomona, California. The goal is to investigate potential factors contributing to antibiotic resistance in fresh produce. 2. Field Trial at Desert Research and Extension Center: We will proceed with the second phase of the field trial at the Desert Research and Extension Center in Imperial County, California. This trial aims to assess the impact of irrigation water treatment on the occurrence of antibiotic resistance in produce and the surrounding environment. We will analyze samples of freshly harvested vegetables, irrigation water, and soil. 3. Whole Genome Sequencing and Bioinformatics: We will conduct whole genome sequencing and bioinformatics analysis on the remaining resistant isolates from farm samples to further understand resistance mechanisms. 4. Extension and Outreach: In addition to research activities, we will continue with our extension efforts, including developing factsheets, videos, and conducting seminars to disseminate findings.

Impacts
What was accomplished under these goals? Extended-spectrum ß-lactamases (ESBL)-producing Enterobacterales and carbapenem-resistant bacteria are considered a serious and an urgent threat, respectively, by the United States Centers for Disease Control and Prevention. Vegetables may serve as an important reservoir for antibiotic-resistant bacteria and antibiotic resistance genes, and may play a significant role in the dissemination of antibiotic resistance because they can carry a high number of commensal bacteria. Therefore, there is an urgent need to understand the role of fresh produce in the transmission of antibiotic resistance, especially resistance that is serious or urgent threats to public health. The goals of this proposed project are to assess the prevalence of antibiotic resistance in retail vegetables, and to determine critical factors contributing to antibiotic resistance transmission in vegetable production and supply chains.In the 2nd year of the project, we have completed these major activities. Objective 1: We have completed the primary tasks under Objective 1, which focused on assessing the prevalence of ESBL-producing Enterobacterales and carbapenem-resistant bacteria in retail vegetables across U.S. markets. Between 2022 and 2023, we conducted an extensive antibiotic resistance surveillance study of fresh vegetable products in three states: Arkansas, Wisconsin, and California. A total of 1,218 vegetable samples were collected and analyzed, divided into five categories: lettuce, spinach, carrots, sprouts/microgreens, and salads. After pre-enrichment in buffered peptone water (BPW) and selective enrichment in Enterobacteriaceae enrichment (EE) broth, we used CHROMagar ESBL and CHROMagar mSuperCARBA to isolate ESBL-producing Enterobacterales and carbapenem-resistant bacteria. Blue and pink colonies were selected for further resistance phenotype confirmation via standard disk diffusion assays, following the Clinical & Laboratory Standards Institute (CLSI) protocols. Carbapenemase production was determined using mCIM/eCIM assays. Positive isolates underwent whole genome sequencing to identify resistance genes. Key findings include: 1. Prevalence of Antibiotic Resistance: Out of 1,218 fresh vegetable samples, we identified 62 carbapenem-resistant isolates (5.1%) and 70 ESBL-producing Enterobacterales isolates (5.7%) through phenotypic and genomic analysis. 2. Vegetable Types and Resistance: The type of vegetable was significantly associated with the presence of antibiotic-resistant bacteria. Notably, 19 (7.9%) of 241 sprouts and microgreens samples were positive for carbapenem-resistant Enterobacteriaceae, with all isolates belonging to the Enterobacter genus and producing various carbapenemases. Additionally, 13 (5.3%) of these sprouts and microgreens samples tested positive for ESBL-producing Enterobacteriaceae, including 9 Enterobacter and 4 Klebsiella isolates. 3. Phylogenomic Insights: Phylogenomic analysis revealed that antibiotic-resistant isolates from sprouts and microgreens exhibited close genome similarity to clinical isolates from humans. For instance, the carbapenem-resistant E. asburiae isolate AR-CRE-C52 from mixed microgreens closely matched the genome of E. asburiae 15694 (GCA_023153555.1), a clinical isolate collected from human sputum. Similarly, the ESBL-producing K. pneumoniae isolate AR-ESBL-E225 closely resembled K. pneumoniae EuSCAPE_IT211 (GCA_900514335.1), a human clinical isolate. Objective 2: We have successfully collected and analyzed 508 farm samples, which included both vegetable and environmental samples from a farm and packinghouse in Pomona, California, to isolate ESBL-producing Enterobacterales and carbapenem-resistant bacteria. From these samples, we have identified 23 isolates that showed resistance to an extended-spectrum cephalosporin, cefotaxime (CTX) or ceftazidime (CAZ). In addition, 12 isolates were resistant to carbapenem antibiotics by disk diffusion assays. The taxonomic identification of resistant isolates and their resistance genes will be determined and confirmed through whole genome sequencing and bioinformatics analysis. Additionally, the first part of the field trial at the Desert Research and Extension Center in Imperial County, California, has been completed. This trial aimed to study the impact of various factors on the occurrence of antibiotic resistance in produce and its production environment. We analyzed 320 samples of freshly harvested vegetables, along with 181 irrigation water and 192 soil samples. The taxonomic identification of resistant isolates and their resistance genes will be determined and confirmed through whole genome sequencing and bioinformatics analysis.Phylogenomic analysis will be used to determine the genetic relatedness among resistant isolates from vegetables, water and soil. Objective 3: The extension team has completed the consumers survey in Qualtrics about their perceptions and concerns related to antibiotic resistance in food with specific emphasis on produce. A questionnaire was developed to assess (1) consumer awareness of antibiotic uses and antibiotic resistant bacteria; (2) concerns about antibiotic resistant bacteria in food and types of food of concern; and (3) specific concerns related to production practices and processes and occurrence of antibiotic resistant bacteria in vegetables. The questionnaire was developed, reviewed and edited by the project team, and the research protocol was approved by the UC Davis Institutional Review Board. We have received a total of 2,476 responses from consumers in 50 states. Key findings include: 1. When asked about their level of concern regarding antibiotic-resistant bacteria in food, 54.41% (n=1063) of respondents indicated either 'moderate' or 'high' concern. A substantial portion of respondents (64.56%) indicate that concerns about antibiotic resistance in food impact their purchasing and consumption practices at least sometimes. 2. Among the 542 respondents concerned about antibiotic-resistant bacteria in vegetables, 76.38% expressed higher concern for imported vegetables. Organic vegetables tend to decrease concern for 31.37% of respondents, though there is still a portion (29.52%) for whom organic methods increase concern. Non-organic (conventional) vegetables are more likely to increase concern, with 48.71% of respondents indicating increased concern, and only a small group (7.38%) reporting that conventional methods decrease their concern. 3. Pre-cut and packaged vegetables raise the highest level of concern, followed by raw vegetables, with more than half of respondents expressing increased concern for these types of vegetables. Cooking is perceived as the most effective method to decrease concern about antibiotic-resistant bacteria in vegetables. Fermented, canned, and frozen vegetables have more mixed responses, with no strong consensus in either direction. Key outcomes: These microbiologicalfindings highlight the significant public health implications of antibiotic resistance in fresh produce, particularly in sprouts and microgreens. The survey data provided important insights into consumer awareness and risk perceptions related to antibiotic-resistant bacteria.

Publications

• Type: Journal Articles Status: Published Year Published: 2024 Citation: Leighton, E. A., Gale, C. N., Huang, E., Yang, X., DiCaprio, E. L., & Li, X. (2024). A Multidrug-Resistant Extended-Spectrum Beta-Lactamase (ESBL)-Producing Enterobacter hormaechei Strain from Mixed Sprouts. Current Microbiology, 81(5), 131. https://doi.org/10.1007/s00284-024-03663-3
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Moon S.H., Li X., Yang X., DiCaprio E., Huang E. 2024. Enterobacter isolates harboring class A carbapenemase genes blaNMC-A or blaIMI on chromosomal integrative element and plasmid. International Association for Food Protection (IAFP) annual meeting, poster presentation, Long Beach, California.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Li X., Leighton E., Huang E., Yang X., DiCaprio E. 2024. Characterization of an ESBL-Producing Strain of a New Enterobacter Species from Malabar Spinach. International Association for Food Protection (IAFP) annual meeting, poster presentation, Long Beach, California.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Gale C., Leighton E., Li X. 2023. A multidrug-resistant extended-spectrum beta-lactamase (ESBL)-producing Enterobacter hormaechei strain from mixed sprouts. American Society for Microbiology (ASM) North Central Branch (NCB) 83rd annual meeting.
• Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Pintar M., Leighton E., Li X. 2023. Characterization of an ESBL-producing strain and a carbapenem-resistant strain from a pre-packed microgreen sample. American Society for Microbiology (ASM) North Central Branch (NCB) 83rd annual meeting.

Progress 08/01/22 to 07/31/23

Outputs
Target Audience:Industry, academic and governmental food safety professionals:A research talk was presented in the 2023 annual meeting of International Association for Food Protection (IAFP). Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A postdoctoral researcher has been trained in this project for advancing knowledge in isolating antibiotic resistance bacteriafrom food products and genomic analysis of antibiotic resistance genes using a variety of bioinformatics tools. How have the results been disseminated to communities of interest?The journalpublicationresulting from this project was published in an open access journal (Journal of Food Protection) which is available for all interested parties to read. A research talk was presented in the 2023 annual meeting of International Association for Food Protection (IAFP) and audience are mainly food safety stakeholders. What do you plan to do during the next reporting period to accomplish the goals?We will conduct whole genome sequencing and bioinformatics analysis for all resistant isolates from the vegetables from objective 1. We will also carry out microbiome and resistance analysis for vegetables from retail market. We will continue collecting and analyzing farm vegetable and environmental samples to investigate factors that could contribute to antibiotic resistance in vegetables. In addition, we will finish collecting consumer survey data and analyze the data.

Impacts
What was accomplished under these goals? Extended-spectrum ß-lactamases (ESBL)-producing Enterobacteralesand carbapenem-resistant bacteria are considered a serious and an urgent threat, respectively, by the United States Centers for Disease Control and Prevention. Vegetables may serve as an important reservoir for antibiotic-resistant bacteria and antibiotic resistance genes, and may play a significant role in the dissemination of antibiotic resistance because they can carry a high number of commensal bacteria. Therefore, there is an urgent need to understand the role of fresh produce in the transmission of antibiotic resistance, especially resistance that is serious or urgent threats to public health. The goals of this proposed project are to assess the prevalence of antibiotic resistance in retail vegetables, and to determine critical factors contributing to antibiotic resistance transmission in vegetable production and supply chains.Through providing information on consumer concerns related to antibiotic resistance in produce and best practices to mitigate this risk in produce, growers can engage in practices that can reduce the overall occurrence of antibiotic resistant bacteria in produce. In the first year of the project, we have completed these major activities. Objective 1:We have collected and analyzed a total of 1219 vegetable samples from markets (407samples in Arkansas, 404samples in Wisconsin, 408 samples in California) for assessingthe prevalence of ESBL-producingEnterobacteralesand carbapenem-resistant bacteria. After pre-enrichment in buffered peptone water (BPW) and selective enrichment in Enterobacteriaceae enrichment (EE) broth, CHROMagar ESBL and CHROMagar mSuperCARBA were used to isolate ESBL-producing Enterobacterales and carbapenem-resistant bacteria, respectively. Blue and pink colonies on CHROMagar agar were selected for resistance phenotype confirmation using standard disk diffusion assays following the Clinical& LaboratoryStandardsInstitute (CLSI)protocols. Carbapenemase production was determined by mCIM/eCIM assays. The phenotypic positive rates for ESBL-producingEnterobacteralesand carbapenem-resistant bacteria are as follows: Arkansas (9.83% and 7.37%), Wisconsin (10.89% and 3.46%), and California (2.94% and 5.39%). The taxonomic identification of each isolate and resistance geneswill be determined and confirmed by whole genome sequencing. Objective 2: We have collected and analyzed50 farm samples (including vegetable samples and environmental samples in a farm and packing house) for isolatingESBL-producingEnterobacteralesand carbapenem-resistant bacteria. Objective 3: The extension team has created the consumers survey and implemented the survey inQualtrics. The aim of this survey is to determine consumer concerns related to antibiotic resistance in food with specific emphasis on fresh produce. The survey remainsopen inQualtricsfor data collection. Key outcomes: This large-scale surveillance across three states in a 1-yeard periodprovided the largest datasetfor estimating the prevalence ofESBL-producingEnterobacteralesand carbapenem-resistant bacteria in fresh vegetablesin the United States.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2023 Citation: Huang, E., Yang, X., Leighton, E., Li, X. (2023). Carbapenem resistance in the food supply chain. Journal of Food Protection, 100108. https://doi.org/10.1016/j.jfp.2023.100108
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Moon S.H., Li X., Yang X., DiCaprio E., Huang E. 2023. NmcA carbapenemase-producing Enterobacter ludwigii C1 from carrots. International Association for Food Protection (IAFP) annual meeting, oral presentation, Toronto, Canada.