Phytogenic Solutions: Using Plant Extracts as Antibiotic Alternatives to Mitigate AMR for Sustainable Aquaculture

1. Overview

Scientific rationale and importance: Antimicrobial Resistance (AMR) is one of the most urgent public health challenges facing both veterinary and human medicine. The excessive use of antibiotics in aquaculture negatively impacts animal and human health as well as the environment. Taking a One Health approach to finding efficacious and eco-friendly alternatives to antibiotics for the prevention of fish diseases and combating AMR in Aquaculture, we propose evaluating plant-based compounds with antibacterial activity to boost disease resilience in fish. There are only three antibiotics approved by the U.S. Food and Drug Administration (FDA) to treat food fish in the U.S. While antibiotics remain an important tool for managing bacterial infections in aquaculture, medicated feeds can be costly and must be used carefully to protect their long-term effectiveness. Even if there is no current bacterial resistance to these medications, repeated use or misuse in commercial aquaculture increases the selective pressure on bacteria and can lead to the development of AMR over time, posing a significant threat to the sustainability of the U.S. aquaculture industry. Furthermore, producers must also follow strict withdrawal periods after antibiotic use before marketing their fish, which can increase feed and maintenance costs if fish need to be held longer. As a result, there is an urgent, industry-recognized need to identify alternatives for mitigating bacterial infections without relying on antibiotics.

Confirmation of funding/facilities/ethics approvals: Additional funding provided by Texas A&M AgriLife research is available to support this initiative through start-up funds to Dr. Haitham Mohammed (PI), who is an early-career faculty member in the Department of Rangeland, Wildlife & Fisheries Management at Texas A&M University (TAMU). Facilities available include Dr. Mohammed's Fish Health & Disease Laboratory (FHDL) on TAMU's main campus, which is comprised of two distinct lab spaces. The first is a 1,285 ft2 BSL-2 Microbiology Lab, specifically designated for Fish Microbiology/Pathology research. The second is a 355 ft2 BSL-2 Wet Lab, 32-tank system, each with a capacity of 75 liters operating as flow-through, allowing for replicated disease challenge experiments. All necessary ethical approvals to conduct this research are valid and were obtained through TAMU’s Institutional Animal Care and Use Committee (IACUC) and Institutional Biosafety Committee (IBC).

Estimated timeline: 12 months (6 months for objective 1 & 6 months for objective 2).

Data sharing commitment: In alignment with the Open Science principles outlined in this program, we are fully committed to making all project-generated data publicly accessible. All datasets, metadata, and associated materials will be deposited in a recognized open-access repository (e.g., institutional repository) in compliance with FAIR (Findable, Accessible, Interoperable, and Reusable) data standards. To facilitate rapid dissemination of results, we will also publish preprints of manuscripts arising from this work on ResearchHub or other appropriate preprint platforms prior to formal peer-reviewed publication. In addition, we will provide quarterly progress updates to ensure transparency, promote collaboration, and accelerate the translation of our findings to the broader research and stakeholder community.

2. Introduction

Disease outbreaks pose a significant threat to aquaculture and result in extensive mortalities and catastrophic economic impacts ¹. For context, a 2023 study published in the journal Aquaculture estimated that the annual total disease-related economic losses in western Alabama alone exceed $11 million ². In intensive food production systems like aquaculture, animals are often crowded and raised in stressful or suboptimal conditions, making them more susceptible to bacterial infections ³. As a result, farmers frequently use antimicrobials both therapeutically and sometimes preventively to manage these disease outbreaks. Currently, more than half of global antibiotic consumption is attributed to the widespread use of antibiotics in animal production, and this usage is projected to increase by two-thirds by 2030 ⁴. Numerous studies have shown a direct connection between high levels of antibiotic use and the global rise in AMR observed in human medicine, agriculture, and aquaculture ^5,6,7.

The level of AMR in aquaculture, particularly concerning medically important antimicrobials, is alarming. The overuse and misuse of antibiotics in commercial fish farming are considered significant factors contributing to the development of AMR ^8,9,10. Approximately 80% of the antimicrobials used in aquaculture make their way into the aquatic environment. Thus, selecting for the survival of bacteria that develop resistance through random mutations or by acquiring mobile genetic elements that contain multiple resistance factors, which can be transmitted to other bacteria ³. As a result, the effectiveness of antibiotics is diminishing due to the emergence of AMR in aquatic bacteria, including fish pathogens ¹¹. Previous studies have reported that several bacteria, including Aeromonas spp., Escherichia coli, Salmonella spp., and Vibrio spp., are resistant to antibiotics from three or more antibiotic categories. These bacteria are therefore classified as multidrug-resistant ^12,13. This serves as a warning for aquaculture farmers and managers to take preventive measures against the spread of AMR in aquatic farming systems.

The utilization of plant-derived compounds, known as phytotherapeutics, as natural alternatives to chemicals or antibiotic treatments for disease control in aquaculture has gained significant attention in recent years ¹⁴. Plant extracts and phytochemicals from medicinal plants have demonstrated antibacterial activity against antibiotic-resistant microorganisms ¹⁵. Moreover, modification of fish feeding regimens through dietary administration of functional additives such as natural plant products in aquafeed formulations extends beyond satisfying the basic nutritional requirements of the fish species to enhancing growth and feed utilization, improving stress tolerance, and boosting health status and disease resistance. Therefore, phytotherapeutics may offer an additional layer of protection by supporting immune health and helping reduce the risk of disease outbreaks in aquaculture.

Hypotheses

H1: Selected plant extracts possess antibacterial properties and can inhibit the growth of major fish pathogens (e.g., Aeromonas spp., Edwardsiella spp., Flavobacterium spp., Streptococcus spp., Vibrio spp.).

H2: Incorporating effective plant extracts into fish diets as feed supplements will enhance fish resistance to bacterial infections, resulting in improved survival following controlled pathogen challenges.

Objectives

In vitro screening: Evaluate the antibacterial activity of purified plant extracts/compounds against major aquaculture pathogens.

In vivo feeding trials: Diet supplementation of the most effective plant extract identified under Objective 1. The selected extract will be incorporated into fish feed at varying concentrations and tested in controlled feeding trials. At the end of the feeding period, fish will be challenged with target pathogens to assess protective efficacy and potential improvements in survival, health, and performance.

Impact Statement: Healthy fish are the foundation of a sustainable aquaculture industry, yet intensive production systems create conditions that favor the spread of bacterial diseases. With few approved antibiotics available and no new drugs on the horizon, continued reliance on existing antibiotics accelerates the emergence of AMR in fish pathogens, threatening both animal health and global food security. This study addresses this urgent challenge by evaluating plant-based extracts as natural alternatives to antibiotics. Beyond their antibacterial properties, these phytotherapeutics have the potential to boost fish immune defenses and resilience to stress, providing multiple health benefits. The outcomes will contribute to reduced antibiotic dependence, slower AMR development, and a more environmentally responsible and resilient aquaculture sector that supports the growing demand for safe, sustainable seafood.

3. Methods

Participants/Sample

We will conduct an initial screening of pure plant extracts or compounds derived from at least 10 different plants to assess their antibacterial activity against common fish pathogens. Catfish (channel or hybrid) will serve as the model species for the in vivo trials. A power analysis will be performed to determine the appropriate sample sizes for various analyses. However, it is important to note that not all proposed activities can undergo a power analysis at this stage, as we do not have all the necessary factors available yet. Furthermore, we plan to take advantage of the comprehensive statistical support services offered by TAMU to enhance the robustness of our study. All in vitro and in vivo experiments will be conducted with appropriate replication, ensuring a minimum of three replicates for each test.

Materials and Procedures

Pathogen cultures: To determine the antibacterial activity of the plant extracts, a panel of ten fish pathogenic bacteria (F. columnare, F. covae, F. davisii, Edwardsiella ictaluri, Aeromonas hydrophila, A. veronii, Plesiomonas shigelloides, Vibrio spp., Streptococcus spp., and E. coli) will be used as indicators. The E. coli ATCC strain will be included as a quality control organism. To prepare fresh pathogen inoculum, bacteria will be streaked on appropriate agar media (according to their specific growth requirements) and incubated at 28°C for 48 h to confirm purity. Three to five (3 to 5) colonies will be picked and inoculated in appropriate broth media (such as cation-adjusted Mueller Hinton broth (CAMHB), diluted CAMHB, etc.). Cultures will be grown overnight (~20 h) in a rotary shaker with continuous shaking at 150 rpm. To ensure uniformity, the density of testing inocula of the various organisms will be standardized to a 0.5 McFarland standard. This is equivalent to a cell density of ~1-2 x 10⁸ CFU/mL, or to an absorbency/Optical Density (OD) of 0.1-0.15 at 600 nm (OD₆₀₀ = 0.1-0.2) on a spectrophotometer, which will be verified using standard plate counts in triplicate.

Agar well diffusion assay: The standardized inoculum of each strain will be swabbed on agar media plates using a cotton swab to create a homogeneous lawn of bacteria on the surface of the plate. Then, agar wells (5-mm diameter) will be made in each of these plates using a sterile cork borer. The test plant extract (50 µL) will be loaded into the corresponding wells. Antibiotic discs (30µg disc) or a diluted solution (50 µL of 0.5-1 μg/mL) of oxytetracycline or florfenicol will be used as positive controls, and 50 µL of sterile PBS or water will be used as negative controls on each plate. The plates will be allowed to settle for ~1 hour and then incubated at 28°C for 24- to 48-h. The diameters of growth inhibition zones will be accurately measured using a digital caliper and recorded. The inhibition zones produced by plant extracts will be compared to the zone of inhibition produced by the positive control antibiotic.

In vivo feeding and experimental challenge trials: A feeding trial with the most effective plant extract will be conducted with catfish juveniles (20-30 g) at the TAMU Aquacultural Research and Teaching Facility (ARTF) operated by the TAMU System in Burleson County, TX. Fish will be obtained from the A.E. Wood State Fish Hatchery operated by Texas Parks and Wildlife Department (TPWD) in San Marcos, TX. Upon arriving at the facility, fish will be acclimated to local water conditions for a minimum of 7 d in four, 500-L circular-fiberglass tanks operating as a recirculating aquaculture system (RAS). Before the commencement of the feeding trials, fish will be fed to apparent satiation twice daily with a basal (control) commercial diet with 32% crude protein (Rangen, Inc., Angleton, TX, USA).

Experimental diets will be prepared by adding incremental levels of plant extract to the basal diet at the expense of cellulose and will likely range from 1.0 to 5.0% of dry weight. Diets will be prepared by mixing the dry ingredients in a V-mixer (Blend master, Buflovak, NY, USA) machine for 30 minutes. Following acclimation, fish will be placed into glass aquaria at 20-25 fish per aquarium. The experimental diets will be fed to fish in 110-L glass aquaria supplemented with fresh water (0-3 ppt salinity) and connected as RAS. Each diet will be randomly assigned to at least three separate units, and the fish will be fed with their respective diets for 6-8 weeks. Fish will be fed twice daily (8:00 and 16:00 hours). Feeding will be conducted to apparent satiation (maximum amount of feed consumed within 30 min from the beginning of feeding), and the collective weight of the fish will be measured every other week. Temperature will be maintained at 27 ± 1°C, and nitrogenous waste will be removed by a biofilter. Supplemental aeration will be provided by an airstone in each aquarium to maintain dissolved oxygen levels close to saturation (90 - 120%), and other water quality parameters will be monitored routinely to keep them within acceptable levels. A 12:12 light: dark cycle will be maintained with fluorescent lights controlled by timers.

The challenge experiment will be performed as previously described ^16,17. Briefly, fish will be moved to 75 L aquaria (10 fish per tank) in our challenge facility. These aquaria are supplied with de-chlorinated city water through a precise water delivery system that provides a consistent flow rate of approximately 0.2-1.0 L/min, desirable for disease challenge experiments ¹⁸. For the challenge, the incoming water flow to the tanks will be turned off. Fish in each challenge tank will be exposed by immersion to 100 mL of pathogen culture inoculum (containing approximately 1.0-3.0 × 10⁷ cells) in 10 L of water for 1 h. After the 1 h static challenge, the water flow to the tanks will be restored. An additional group of mock-challenged fish will be treated with identical procedures except that they will be exposed to a volume (100 mL) of sterile growth medium (with no pathogen) in 10 L of water and will serve as negative controls. Post-challenge, fish will be monitored every 3 h, at which point moribund and dead fish will be removed and mortality and moribundity will be recorded. Fish will not be fed on the challenge day, but will be offered the same feed on the next day after the challenge and throughout the remainder of the study. Challenges will be terminated after 48 h without any mortality.

Planned Analyses

Data will be validated for homogeneity of variances and will be analyzed using repeated measures ANOVA followed by Tukey-Kramer HSD for post-hoc testing to determine significant (P<0.05) differences among mean values of treatment groups. Survivability after bacterial challenges will be compared by Kaplan–Meier survival analysis with Log-rank (Mantel–Cox) test using GraphPad Prism, version 9.0 (GraphPad Software Inc., San Diego, CA, USA).

Ethics and Data Management

AUP: IACUC 2023-0159 and IBC2023-006. All project data and metadata will be deposited in an open-access repository. Published data will be available in print or electronically from publishers, subject to subscription or printing charges. All data will also be stored electronically in computers on the campus of Texas A&M University. We will maintain regular data backup.

4. Pilot Data (Optional)

Initial results with some plant extracts show great promise. Notice the zone of growth inhibition produced by the plant extract against F. covae, the causal agent of columnaris disease (Figure 1).

5. Budget

Personnel: $1,500 student labor hourly wages; Materials and Supplies: $2,000 for microbiology supplies, reagents, and consumables; Fish, diet, and culture consumables: $1,500. Total amount = $5,000 USD

6. References

1. Hedberg, N., Stenson, I., Pettersson, M.N., Warshan, D., Nguyen-Kim, H., Tedengren, M. and Kautsky, N., 2018. Antibiotic use in Vietnamese fish and lobster sea cage farms; implications for coral reefs and human health. Aquaculture, 495, pp.366-375.

2. Abdelrahman, H.A., Hemstreet, W.G., Roy, L.A., Hanson, T.R., Beck, B.H. and Kelly, A.M., 2023. Epidemiology and economic impact of disease-related losses on commercial catfish farms: A seven-year case study from Alabama, USA. Aquaculture, 566, p.739206.

3. Cabello, F.C., Godfrey, H.P., Tomova, A., Ivanova, L., Dölz, H., Millanao, A. and Buschmann, A.H., 2013. Antimicrobial use in aquaculture re‐examined: its relevance to antimicrobial resistance and to animal and human health. Environmental microbiology, 15(7), pp.1917-1942.

4. Van Boeckel, T.P., Brower, C., Gilbert, M., Grenfell, B.T., Levin, S.A., Robinson, T.P., Teillant, A. and Laxminarayan, R., 2015. Global trends in antimicrobial use in food animals. Proceedings of the National Academy of Sciences, 112(18), pp.5649-5654.

5. Chelossi, E., Vezzulli, L., Milano, A., Branzoni, M., Fabiano, M., Riccardi, G. and Banat, I.M., 2003. Antibiotic resistance of benthic bacteria in fish-farm and control sediments of the Western Mediterranean. Aquaculture, 219(1-4), pp.83-97.

6. Brolund, A., Sundqvist, M., Kahlmeter, G. and Grape, M., 2010. Molecular characterisation of trimethoprim resistance in Escherichia coli and Klebsiella pneumoniae during a two year intervention on trimethoprim use. PLoS One, 5(2), p.e9233.

7. Buschmann, A.H., Tomova, A., López, A., Maldonado, M.A., Henríquez, L.A., Ivanova, L., Moy, F., Godfrey, H.P. and Cabello, F.C., 2012. Salmon aquaculture and antimicrobial resistance in the marine environment.

8. Reverter, M., Sarter, S., Caruso, D., Avarre, J.C., Combe, M., Pepey, E., Pouyaud, L., Vega-Heredía, S., De Verdal, H. and Gozlan, R.E., 2020. Aquaculture at the crossroads of global warming and antimicrobial resistance. Nature communications, 11(1), p.1870.

9. Santos, L. and Ramos, F., 2018. Antimicrobial resistance in aquaculture: Current knowledge and alternatives to tackle the problem. International journal of antimicrobial agents, 52(2), pp.135-143.

10. Watts, J.E., Schreier, H.J., Lanska, L. and Hale, M.S., 2017. The rising tide of antimicrobial resistance in aquaculture: sources, sinks and solutions. Marine drugs, 15(6), p.158.

11. Cabello, F.C., Godfrey, H.P., Buschmann, A.H. and Dölz, H.J., 2016. Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. The Lancet Infectious Diseases, 16(7), pp.e127-e133.

12. Jeamsripong, S., Thaotumpitak, V., Anuntawirun, S., Roongrojmongkhon, N., Atwill, E.R. and Hinthong, W., 2022. Molecular epidemiology of antimicrobial resistance and virulence profiles of Escherichia coli, Salmonella spp., and Vibrio spp. isolated from coastal seawater for aquaculture. Antibiotics, 11(12), p.1688.

13. Nhinh, D.T., Le, D.V., Van, K.V., Huong Giang, N.T., Dang, L.T. and Hoai, T.D., 2021. Prevalence, virulence gene distribution and alarming the multidrug resistance of Aeromonas hydrophila associated with disease outbreaks in freshwater aquaculture. Antibiotics, 10(5), p.532.

14. Gatlin III, D.M., Barrows, F.T., Brown, P., Dabrowski, K., Gaylord, T.G., Hardy, R.W., Herman, E., Hu, G., Krogdahl, Å., Nelson, R. and Overturf, K., 2007. Expanding the utilization of sustainable plant products in aquafeeds: a review. Aquaculture research, 38(6), pp.551-579.

15. Nascimento, G.G., Locatelli, J., Freitas, P.C. and Silva, G.L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Brazilian journal of microbiology, 31, pp.247-256.

16. Peatman, E., Mohammed, H., Kirby, A., Shoemaker, C.A., Yildirim-Aksoy, M. and Beck, B.H., 2018. Mechanisms of pathogen virulence and host susceptibility in virulent Aeromonas hydrophila infections of channel catfish (Ictalurus punctatus). Aquaculture, 482, pp.1-8.

17. Mohammed, H.H. and Arias, C.R., 2016. Protective efficacy of N igella sativa seeds and oil against columnaris disease in fishes. Journal of Fish Diseases, 39(6), pp.693-703.

18. Mitchell, A. and Farmer, B., 2010. Evaluation of an ultra‐low‐flow water delivery system for small experimental tanks. North American Journal of Aquaculture, 72(3), pp.195-200.

Phytogenic Solutions: Using Plant Extracts as Antibiotic Alternatives to Mitigate AMR for Sustainable Aquaculture

Phytogenic Solutions: Using Plant Extracts as Antibiotic Alternatives to Mitigate AMR for Sustainable Aquaculture

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