Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to NRP
A SYSTEMATIC AND INTEGRATED APPROACH TO MITIGATION OF ANTIMICROBIAL RESISTANCE IN AQUACULTURE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1022268
Grant No.
2020-68015-30855
Cumulative Award Amt.
$999,948.00
Proposal No.
2019-06093
Multistate No.
(N/A)
Project Start Date
May 1, 2020
Project End Date
Apr 30, 2026
Grant Year
2020
Program Code
[A1366]- Mitigating Antimicrobial Resistance Across the Food Chain
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Food Science & Technology
Non Technical Summary
The use of antibiotics in animal agriculture is one of the drivers of antimicrobial resistance (AMR). While majority of research has been on bacteria present in land animals, very limited information is available about AMR in aquaculture. There are three antimicrobials approved by the FDA for use in aquaculture: oxytetracycline hydrochloride, sulfadimethoxine and ormetoprim, and florfenicol. These three drugs are effective for treating both warm-water fish diseases and cold-water fish infections. However, there is a critical knowledge gap that needs to be bridged for ensure the sustainable use of antibiotics, that is how water temperatures impact the formation of AMR in fish and in aquaculture environment when these antibiotics are applied at different disease break-out temperatures. To answer this question, in vivo animal treatment experiment will be conducted by UC Davis and Auburn University. The trial at UCD will use well water while the trial at Auburn will use surface water. The abundance, diversity, and changes of AMR bacteria and antimicrobial resistant genes (ARGs) present in fish and rearing water before and after antibiotic treatments will be investigated using culture-based and metagenomics approaches. An antibiotic usage guideline will be developed at the end of the experiment and will be disseminated to farmers, veterinarians, and the general public via multiple education and extension platforms.
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7120810110050%
1353719117050%
Knowledge Area
135 - Aquatic and Terrestrial Wildlife; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
0810 - Finfish; 3719 - Other cultured finfish;

Field Of Science
1100 - Bacteriology; 1170 - Epidemiology;
Goals / Objectives
The long-term goal of our team is to ensure and promote the sustainable growth and development of U.S. aquaculture by supporting the scientific and prudent use of antimicrobials without the unintended consequence of AMR development. The main goal of this application is to identify critical control points and mitigation strategies for preventing the development of antimicrobial resistance (AMR) bacteria and the horizontal transfer of antimicrobial resistance genes (ARGs) in aquaculture production systems. To achieve this goal, our experienced team will use an integrated approach to pursue the following specific objectives:Examine AMR development in catfish when aquaculture antibiotics are applied at different water temperatures using a model aquaculture system. Occurrence of the most economically important bacterial diseases in catfish is temperature dependent. Each pathogen has its preferred break-out temperature. When an outbreak occurs, a bacterial etiology is suspected, an antibiotic is prescribed provided no resistance to this antibiotic is identified from the outbreak isolate. Antibiotics used in the U.S. aquaculture are broad-spectrum, and each can be used for controlling different bacterial infections. For example, Aquaflor® and Romet® can both be used to treat enteric septicemia of catfish caused by E. ictaluri, and Terramycin® and Romet® can both be used for treating Aeromonas infections. The treatment efficacy of these drugs has been tested and proven at different water temperatures, and they have been used interchangeably for different fish infections. However, since it is also known that the half-life of antibiotics in water is influenced by water temperatures, a critical knowledge gap has become apparent regarding the use of various antibiotics in aquaculture at different temperatures and their impacts on AMR development. A key question that needs to be addressed is, as these antibiotics are efficient for controlling fish infections at a wide range of water temperatures and are used interchangeably for certain fish infections, but their half-life in water is impacted by water temperatures, are their impacts on AMR formation the same or different? The first objective of this proposal aims to answer this question and close the above knowledge gap by applying different antibiotics to a model catfish system at different water temperatures relevant to natural disease outbreaks. To ensure the results will be applicable and useful for all U.S. aquaculture farmers, two types of water sources will be used. U.S. aquaculture farmers use two main water sources for charging and recharging their ponds. While farmers in the southern U.S. have access to generous quantities of surface water, aquaculture farmers in regions like southern California rely on well water to charge their ponds. We hypothesize that the impact of antibiotics used in aquaculture on the development of AMR varies at different water temperatures and that the interaction between temperature and AMR formation may also be influenced by the water source. To test this hypothesis, channel catfish (Ictalurus punctatus), the leading catfish species farmed in the U.S., will be used as the model fish species. A treatment study in a well water system will be conducted at UC Davis, with another treatment study in a surface water system conducted at Auburn University. Three aquaculture antibiotics (Terramycin®, Romet® and Aquaflor®) will be applied to the model systems separately, and their impact on AMR formation will be examined at three different water temperatures (20°, 25°, and 30° C). Once completed, the study will yield a guideline for appropriate use of antibiotics that mitigates AMR development in aquaculture production.Determine the impact of antibiotic application at different water temperatures on ARG development and transfer in rearing water. A unique aspect of medication in aquaculture is the unavoidable release of medicated feed directly into the production water pond. How the application of medicated feed affects AMR in water system remains unknown. Answering this question is critical for minimizing the environmental impact of antimicrobials in aquaculture and also for sustaining aquaculture water systems. We hypothesize that different antibiotic-water temperature combinations will have varied impacts on the resistome present in the rearing water. To examine this hypothesis, water samples will be collected from the catfish tanks used for Objective 1. Resistomes present in the rearing water as well as the horizontal transfer of ARGs in the water will be analyzed. Educate aquaculture farmers and veterinarians about appropriate use of antibiotics by developing outreach and education programs using information and guidelines generated from this project. There are three target audiences for our extension and education programming: aquaculture farmers, field veterinarians and the next generation of veterinarians. To achieve this objective, we will enlist the support and involvement of different industrial organizations and extension programs and platforms. For farmers and field veterinarians, results and guidelines generated will be summarized into information brochures using laymen's terms and shared and distributed at face-to-face workshops and by online media, e.g. extension websites, trade association websites, and social media. To educate field veterinarians and next-generation veterinarians, the team will develop three 50-min PowerPoint presentations covering the topics of bacterial infections in fish and treatment strategies; antimicrobial resistance development and mechanisms; and mitigation based on interactions of antibiotics, disease break-out temperatures and water sources. The presentations will be given via a series of webinars and workshops, and will also be incorporated into the teaching curricula at UC Davis School of Veterinary Medicine and Auburn University College of Veterinary Medicine. Learning outcomes and knowledge gains of participating farmers and veterinarians will be evaluated via after-training/workshop surveys and/or in person interviews. The knowledge gain of the next generation veterinarians will be assessed by issuing pre-training and post-training surveys. One unique aspect about our extension and education programming is the involvement of food system institutes. Since AMR is not only about animal health but also food safety, having the Auburn University Food Systems Institute and the Western Institute for Food Safety and Security involved in our Extension programs and being part of our information communication platform, we are able to reach not only producers and veterinarians but also consumers. This will lead to an overall enhanced awareness of AMR in general public.
Project Methods
Objective 1: Examine AMR development in catfish when aquaculture antibiotics are applied at different water temperatures using a model aquaculture system.Overall design. Catfish will be acclimated to three water temperatures (20°, 25° and 30° C). After the acclimation period, fish will be fed three types of medicated feed (Terramycin®, Romet®, and Aquaflor®-added feed) at three water temperatures. Two trials will be conducted using different water sources (surface water vs. well water). The impact of drugs on the formation of AMR bacteria on fish skin and in fish gills and GI tracts will be investigated using both culture-based method and metagenomics.Objective 2: Determine the impact of antibiotic application at different water temperatures on ARG development and transfer in the rearing water.Overall design. Water samples from catfish tanks that have been treated with antibiotics will be collected before and after treatment. The impact of antibiotics on the formation of AMR commensal bacteria and the resistome in the rearing environment will be investigated using metagenomics.Objective 3: Educate aquaculture farmers and veterinarians for appropriate use of antibiotics by developing outreach and education programs using information and guidelines generated from this project.For catfish farmers, processors, and the general public (Extension and outreach). The team will develop a multi-faceted extension and outreach plan aimed at engaging and disseminating information to appropriate stakeholders. Results and guidelines will be communicated directly to catfish farmers, feed manufacturers, and catfish processors through Extension specialists working at UC Davis and Auburn University. Research results and the generated antibiotic selection guidelines will be utilized to develop a Cooperative Extension fact sheets on antibiotic resistance intended specifically for catfish producers, processors and feed manufacturers. Briefly, three fact sheets will be developed for three antibiotics that describe the target diseases of each antibiotic and the proper application practices (e.g. water temperatures and water quality at the time when disease breaks out) in order to minimize the formation of AMR. These information will also be presented in the context of food safety to let farmers and producers know the importance of controlling AMR and the impact of AMR on food safety and public health. Information about this study and results will also be reported in Fish Farming News, the newsletter of the Alabama Fish Farming Center (AFFC), which is mailed to nearly 100 catfish producers in Alabama and Mississippi. The information will also be posted on the AFFC website so that it will reach and be available to other aquaculture farmers and producers in the southern U.S. Results will be presented at the annual Catfish Update Meeting in Demopolis, Alabama; the Alabama Commodity Meeting hosted by the Alabama Farmers Federation in Montgomery, Alabama; the annual Farmers Federation Farm Expo, and the California Aquaculture Association annual meetings in Sacramento, California. Given the strong connection between AMR and food safety, results of this study will also be published on websites of the Western Institute for Food Safety and Security (WIFSS) and the Auburn University Food Systems Institute (AUFSI) website to allow the team to distribute the information to the general public and consumers, thus enhancing the awareness of AMR of the general public and consumers. By having these information on WIFSS and AUFSI websites will also ensure its long-term availability to all target audiences. PI and co-PIs on this team will also work with the food safety extension teams and regional extension agents in both states to share and disseminate project findings via various regional food safety workshops. By teaming up with food safety extension teams and regional agents, the team will be able to reach small fish processors and processing facilities that are not associated with local aquaculture organizations but handle aquaculture products.For veterinarians and the next generation veterinarians (education workshops). Antibiotic resistance is a very complex issue and requires additional knowledge beyond what is provided by the standard veterinary curricula. For this reason, team members will develop a lecture series with three 50-min presentations that will provide science-based information about antibiotic resistance mechanisms and updated research findings, and the antibiotic selection guidelines developed from this project. These lecture series will be offered twice in Years 3 and 4 via face to face workshops at UC Davis, Auburn University, and the Alabama Fish Farming Center. They will also be offered as webinars once every year. Webinars will be recorded and made available on AUFSI and WIFSS websites. In addition, since PI and co-PIs on this team are also involved in teaching the next-generation veterinarians, they will incorporate these materials into their classroom curricula so the next generation veterinarians in school are also trained in AMR and the antibiotic selection guidelines developed.Evaluation of Extension and outreach activities. To measure the success and impact of Extension and outreach programs, post-event surveys will be mailed to farmers. Farmers not responding to the survey will be visited for one-on-one completion of the survey. We expect that their knowledge on AMR and the use of the guidelines will lead to better control of AMR formation. The farmers' knowledge gain and behavior changes will be evaluated.

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:One poster presentation been given during this report period at the annual Institute of Food Technologists meeting (Chicago, July 2023) to reach to target audiences, including catfish farmers, researchers, regulatory agencies, and consumers. In addition, one manuscript summarizing the changes of microbial populations and antimicrobial resistant bacteria and antimicrobial resistant genes during the processing and storage of catfish is published in the International Journal of Food Microbiology. This paper is open access so that catfish farmers, processors, consumers, and regulatory agencies all have long term free access to it. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?A Ph.D. student (part time), one postdoc (part time), and an undergraduate student are hired during this period to work on the data analysis work. How have the results been disseminated to communities of interest?The generated results have been presented at the Institute of Food Technologists annual meeting and published in peer-reviewed journal. What do you plan to do during the next reporting period to accomplish the goals?The team is working on additional data analysis focusing on the intestine samples collected from the live catfish trials. Two manuscripts are under development right now. In the coming year, the team will focus more on the data distribution and extension work.

Impacts
What was accomplished under these goals? The main goal of this project is to evaluate the impact of temperatures at which antibiotics are applied at fish ponds on the formation of antibiotic resistant bacteria and genes. Three major aquaculture drugs are used. Two live catfish trials (involving a total of 2500 fish) have been completed, one at Davis, CA and the other one at Auburn, AL. The team spent the last year analyzing the data. Results have shown that temperatures are impacting the formation of antibiotic resistant genes. Different drugs reacted to the temperatures differently. Among all three drugs, Romet showed least impact on the formation of antibiotic resistant genes in the aquaculture system. Based on these findings, extension materials that summarize this information is now being prepared.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Li, X., Wang, H., Abdelraham, H., Kelly, A., Roy, L., and Wang, L. 2023. Factors shaping microbial communities present on Fish and Fish Products. Institute of Food Technologists annual meeting, Chicago, Jly 2023.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Li, X., Wang, H., Abdelrahman, H., Kelly, A., Roy, L., and Wang, L. 2024. Profiling and source tracking of the microbial populations and resistome present in fish products. 413, 110591.
  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Wang, H., Sheng, L., Li, X., Liu, Z., Canakapalli, S., Zhou, Y., Liao, C., Martinez, E. S., Kelly, A., Roy, L., and Wang, L. The impact of florfenicol treatment on the microbial populations and antibiotic-resistant bacteria associated with live catfish. Microbiome. Under review.


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:One poster presentation and three oral presentations have been given during this report period to reach to target audiences, including catfish farmers, researchers, regulatory agencies, and consumers. Changes/Problems:The aquatic experimental station at UC Davis had a major water issue in summer 2022. Although this accident was not directly related to this project. The facility is now under renovation since Summer 2022 and delayed the experimental progress of this project a little bit. What opportunities for training and professional development has the project provided?During this reporting period, one postdoc was hired by the Auburn University to be in charge of the catfish trial. She continued working on this project during this reporting period. How have the results been disseminated to communities of interest?The generated results have been presented at the International Association for Food Protection annual meeting in Summer 2022, at the annual Aquaculture America meeting in Feburary 2023 and the Pacific Fisheries Technologist annual meeting in March 2023. What do you plan to do during the next reporting period to accomplish the goals?A catfish trial was conducted in Alabama in May-July 2022. Upon completion of the Alabama trial, data has been collected. The impact of different aquacultural drugs on the formation of antimicrobial resistant bacteria and the formation and horizontal transfer of antimicrobial resistant genes will be analyzed. The data generated by the Alabama trial will be compared with the data generated by the California trial and analyze how the water properties impact the antimicrobial resistance in aquaculture.

Impacts
What was accomplished under these goals? As planned, another catfish trial was conducted at Auburn University in May 2023. A total of 1,200 catfish was involved in that trial. All fish and water samples have been collected and are currently being sequenced and analyzed.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Wang, H., Sheng, L., Li, X., Liu, Z., Canakapalli, S., Zhou, Y., Liao, C., Soto, E., and Wang, L. 2022. The impact of florfenicol treatment on the microbial populations associated with live catfish. Poster presentation given at the 2022 International Association for Food Protection Annual Meeting.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Wang, L., Soto, E., Kelly, A., Roy, L., Wang, H., Sheng, L., and Li, X. 2023. The impact of florfenicol treatment on the microbial populations associated with live fish. Aqaculture America annual meeting, New Orleans, LA.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Wang, L., Kelly, A., Roy, L., Li, X., Wang, H., and Abdelrahman, H. 2023. Factors shaping the microbial populations present on fish and fish products. Aquaculture America annual meeting, New Orleans, LA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Wang, H., Sheng, L., Li, X., Liu, Z., Canakapalli, S., Zhou, Y., Liao, C., Soto, E., and Wang, L. 2023. The impact of florfenicol treatment and rearing water temperatures on the microbial populations associated with catfish. Pacific Fisheries Technologists annual meeting. Oregon.


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:Two extension files were published in 2021 to introduce the "antibiotic feeds for catfish farmers" and "Using medicated feeds in catfish aquaculture" to catfish farmers located in Southern US, the major domestic catfish production area. The target audiences are catfish farmers as well as feed mill managers and veterinarians. Changes/Problems:The major challenge during this period was the Covid-19 global pandemic. Laboratory work density was reduced to decrease transmission risk and traveling within counties was restricted. A strategy of staggering work shifts implemented in PD's and co-PDs' Labs allowed work shifts both early and late hours thus reducing lab density. In addition, the postdoctoral researcher hired by Auburn University could not start in time due to travel restrictions. The genome center at UC Davis handles the city's COVID testing. Thus, the sequencing of our DNA samples took longer than planned. What opportunities for training and professional development has the project provided?A postdoc was hired for this project in January 2021. She continued working on this project during the reporting period of May 2021-April 2022. In addition, one Ph.D. student joined the lab and got trained with antimicrobial resistant gene analysis. How have the results been disseminated to communities of interest?As mentioned earlier, two extension files covering the topics of "antibiotic feeds for catfish farmers" and "Using medicated feeds in catfish aquaculture" have been published. The target audiences are catfish farmers as well as feed mill managers and veterinarians. In addition, the team submitted an abstract in January 2022 entitled "the impact of florfenicol treatment on the microbial populations associated with live catfish" to the 2022 International Association for Food Protection Annual Meeting. The team will present the poster in August 2022. What do you plan to do during the next reporting period to accomplish the goals?A catfish trial will be conducted in Alabama in May 2022. Upon completion of the Alabama trial, data will be combined and the analysis of the impact of temperatures and different aquacultural drugs on the formation of AMR bacteria and the formation and horizontal transfer of ARGs will be analyzed.

Impacts
What was accomplished under these goals? As planned, one catfish trial was conducted at UC Davis in 2020. A total of 1,200 fish were used in this trial. A total of 846 DNA samples were extracted from catfish gill, skin, and intestine. The team conducted library prep for these samples and sent for sequencing. The sequencing results came back in January 2022. The team is in the process of analyzing sequencing results.

Publications

  • Type: Other Status: Published Year Published: 2021 Citation: Roy, L., and Kelly, A. M. 2021. Antibiotic feeds for catfish farmers. Available at: https://www.aces.edu/blog/topics/fish-water/antibiotic-feeds-for-catfish-farmers/
  • Type: Other Status: Published Year Published: 2021 Citation: Kelly, A., and Roy, L. 2021. Using medicated feeds in catfish aquaculture. Available at: https://www.aces.edu/wp-content/uploads/2021/05/ANR-2758-Using-Medicated-Feeds-in-CatfishAquaculture_050621L-G.pdf
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Wang, H., Sheng, L., Li, X., Liu, Z., Canakapalli, S., Zhou, Y., Liao, C., Soto, E., and Wang, L. 2022. The impact of florfenicol treatment on the microbial populations associated with live catfish. Abstract submitted for 2022 International Association for Food Protection Annual Meeting.


Progress 05/01/20 to 04/30/21

Outputs
Target Audience: Nothing Reported Changes/Problems:The major challenge during this period was the Covid-19 global pandemic. Laboratory work density was reduced to decrease transmission risk and traveling within counties was restricted. A strategy of staggering work shifts implemented in PD's and co-PDs' Labs allowed work shifts both early and late hours thus reducing lab density. In addition, the postdoctoral researcher hired by Auburn University could not start in time due to travel restrictions. What opportunities for training and professional development has the project provided?A postdoc and a lab technician are hired for this project and are trained during this period of time. 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?A catfish trial will be conducted in Alabama in Fall 2021. Upon completion of the Alabama trial, data will be combined and the analysis of the impact of temperatures and different aquacultural drugs on the formation of AMR bacteria and the formation and horizontal transfer of ARGs will be analyzed.

Impacts
What was accomplished under these goals? As discussed earlier, One catfish trial was conducted at UC Davis during this period of time. A total of 1,200 fish were used in this trial. Three medicated feed, including Oxytetracycline hydrochloride-supplemented feed, Sulfadimethoxine and ormetoprim-supplemented feed, and florfenicol-supplemented feed, were used in this trial. No drug added feed was used for the control group. Three water temperatures were tested, including 20, 25, and 30 C. Four tanks for every temperature and feed combination were used with 25 fish in each tank. A total of 48 tanks were used for this study. Fish samples (gill, skin and intestine) and water samples were collected and DNA was extracted from these samples. The team is in the process of sequencing these DNA samples.

Publications