Isolation, Characterization, and In Vitro Evaluation of Bacteriophages for Controlling the Fish Pathogen Aeromonas hydrophila

Nasri Julaini; Dinamella Wahjuningrum; Widanarni; Sukenda

Issue

2026: JIPK VOLUME 18 ISSUE 1 YEAR 2026 (FEBRUARY 2026, ISSUE IN PROGRESS)

Issue Published : June 23, 2025

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Submitted

October 1, 2025

Accepted

October 29, 2025

Published

November 7, 2025

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Aquaculture Aquatic Resource Management Fisheries Biotechnology Molecular Genetics Fish Diseases Fish Health Management

Isolation, Characterization, and In Vitro Evaluation of Bacteriophages for Controlling the Fish Pathogen Aeromonas hydrophila

Nasri Julaini

Student of Aquaculture Science Study Program, IPB Graduate School, Bogor, 16680. Indonesia

https://orcid.org/0009-0008-8016-4166

Dinamella Wahjuningrum

Lecturer of the Department of Aquaculture, Faculty of Fisheries and Marine Sciences, IPB University, Bogor. Indonesia

https://orcid.org/0000-0003-0883-7852

Widanarni

Lecturer of the Department of Aquaculture, Faculty of Fisheries and Marine Sciences, IPB University, Bogor. Indonesia

https://orcid.org/0000-0002-0821-3225

Sukenda

Lecturer of the Department of Aquaculture, Faculty of Fisheries and Marine Sciences, IPB University, Bogor. Indonesia

https://orcid.org/0000-0001-9465-8329

Corresponding Author(s) : Dinamella Wahjuningrum

dinamellawa@apps.ipb.ac.id

Jurnal Ilmiah Perikanan dan Kelautan, 2026: JIPK VOLUME 18 ISSUE 1 YEAR 2026 (FEBRUARY 2026, ISSUE IN PROGRESS)
Article Published : November 7, 2025

Abstract

Graphical Abstract

Highlight Research

1. A lytic bacteriophage was successfully isolated from catfish pond water in Dramaga, Bogor, with a high density of 5.68 × 10⁹ PFU/mL.

2. Characterization revealed round to irregular plaque morphology with high clarity and diameters reaching 0.35 cm.

3. The bacteriophage exhibited high host specificity, being effective only against the A. hydrophila AH03 isolate from the AAHL collection.

4. Treatment at MOI 10 resulted in more effective inhibition of A. hydrophila growth compared to other treatments.

Abstract

Aquaculture plays a vital role in global protein supply, yet its increasing production faces disease-related challenges, particularly A. hydrophila infections. This pathogen causes Motile Aeromonas Septicemia (MAS), leading to mass mortality in catfish and significant economic losses. While antibiotics have been the primary solution, their use is now restricted due to antimicrobial resistance, necessitating safe and sustainable alternatives. Phage have emerged as environmentally friendly, specific biocontrol agents to combat such infections. This study aimed to evaluate the effectiveness of lytic phage in controlling the fish pathogen A. hydrophila through an in vitro approach. The research stages included phage isolation (density assay), characterization (plaque morphology and host range testing), and evaluation of phage against A. hydrophila (bacteriolytic efficacy assay). A completely randomized design was employed, with six treatments and three replicates, media control (K-), A. hydrophila control (K+), antibiotic control (enrofloxacin 0.1 mg/mL, KA), and phage treatments at multiplicities of infection (MOI) of 0.1, 1, and 10 (P0.1, P1, P10) for the bacteriolytic efficacy assay. Phage isolated from catfish ponds in Dramaga, Bogor, exhibited a high titer (5.68 × 10⁹ PFU/mL) and formed clear, round plaques (0.17-0.35 cm diameter). They demonstrated a narrow host range, lysing only 1 of 10 A. hydrophila isolates. In efficacy assays, phage treatment at an MOI of 10 significantly reduced bacterial density (p < 0.05) at 24 hours post infection, decreasing the OD 600 nm by 38.7% compared to the positive control. These results indicate the potential of these phage as an antibiotic alternative for controlling A. hydrophila in aquaculture.

Keywords

Aeromonas hydrophilaAquacultureBacteriophage TherapyBiocontrol AgentIn Vitro AssayMotile Aeromonas Septicemia

Nasri Julaini, Wahjuningrum, D., Widanarni, & Sukenda. (2025). Isolation, Characterization, and In Vitro Evaluation of Bacteriophages for Controlling the Fish Pathogen Aeromonas hydrophila. Jurnal Ilmiah Perikanan Dan Kelautan. Retrieved from https://e-journal.unair.ac.id/JIPK/article/view/79707

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References

Abedon, S. T. (2017). Active bacteriophage biocontrol and therapy on sub-millimeter scales towards removal of unwanted bacteria from foods and microbiomes. AIMS Microbiology, 3(3):649-688.
Ács, N., Gambino, M., & Brøndsted, L. (2020). Bacteriophage enumeration and detection methods. Frontiers in Microbiology, 11(1):1-7.
Akmal, M., Rahimi-Midani, A., Hafeez-ur-Rehman, M., Hussain, A., & Choi, T. J. (2020). Isolation, characterization, and application of a bacteriophage infecting the fish pathogen Aeromonas hydrophila. Pathogens, 9(3):1-13.
Altinok, I., Capkin, E., & Kayis, S. (2008). Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens. Veterinary Microbiology, 131(3-4):332-338.
Brye, K. (2023). Aquaculture: A sustainable solution for fish cultivation and global food security. Journal of Agriculture, 6(4):91-93.
Choliq, F. A., Martosudiro, M., Istiqomah, I., & Nijami, M. F. (2020). Isolation and testing of bacteriophage capabilities as control agents for bacterial wilt disease (Ralstonia solanacearum) in tomato plants. Viable, Jurnal Ilmiah Ilmu-Ilmu Pertanian, 14(1):8-20.
Clokie, M. R. J., & Kropinski, A. M. (Eds.). (2009). Bacteriophages (Vol. 501). Humana Press.
D’accolti, M., Soffritti, I., Mazzacane, S., & Caselli, E. (2021). Bacteriophages as a potential 360-degree pathogen control strategy. Microorganisms, 9(2):1-14.
Dien, L. T., Ky, L. B., Huy, B. T., Mursalim, M. F., Kayansamruaj, P., Senapin, S., Rodkhum, C., & Dong, H. T. (2022). Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug‐resistant Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus). Transboundary and Emerging Diseases, 69(4):435-450.
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Glonti, T., & Pirnay, J. P. (2022). In vitro techniques and measurements of phage characteristics that are important for phage therapy success. Viruses, 14(7):1-23.
He, P., Cao, F., Qu, Q., Geng, H., Yang, X., Xu, T., Wang, R., Jia, X., Lu, M., Zeng, P., & Luan, G. (2024). Host range expansion of Acinetobacter phage vB_Ab4_Hep4 driven by a spontaneous tail tubular mutation. Frontiers in Cellular and Infection Microbiology, 14(1):1-12.
Irshath, A. A., Rajan, A. P., Vimal, S., Prabhakaran, V. S., & Ganesan, R. (2023). Bacterial pathogenesis in various fish diseases: Recent advances and specific challenges in vaccine development. Vaccines, 11(2):1-14.
Jo, S. J., Lee, Y. M., Cho, K., Park, S. Y., Kwon, H., Giri, S. S., Lee, S. B., Jung, W. J., Park, J. H., Hwang, M. H., Park, D. S., Park, E. J., Kim, S. W., Jun, J. W., Kim, S. G., Kim, J. H., & Park, S. C. (2025). Standardization of the agar plate method for bacteriophage production. Antibiotics, 14(1):1-14.
Jun, J. W. (2024). A concise overview of studies on successful real-world applications of bacteriophages in aquaculture. Viruses, 16(12):1-13.
Laguna-Castro, M., & Lázaro, E. (2022). Propagation of an RNA bacteriophage at low host density leads to a more efficient virus entry. Frontiers in Virology, 2(1):1-12.
Le, T. S., Nguyen, T. H., Vo, H. P., Doan, V. C., Nguyen, H. L., Tran, M. T., Tran, T. T., Southgate, P. C., & Kurtböke, D. İ. (2018). Protective effects of bacteriophages against Aeromonas hydrophila causing Motile Aeromonas Septicemia (MAS) in striped catfish. Antibiotics, 7(1):1-11.
Liang, S., Liang, R., Raza, S. H. A., Huang, Q., Li, T., Bai, H., Feng, C., Xin, L., Guo, H., Wang, S., Yu, J., Cao, Y., Liu, N., AlMalki, F., Zhang, D., Sun, W., & Zhang, L. (2025). Biological analysis of phage vB_AhaP_PT2 and treatment rescued crucian carp infected with Aeromonas hydrophila. Aquaculture, 595(3):1-14.
Liu, J., Gao, S., Dong, Y., Lu, C., & Liu, Y. (2020). Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila. BMC Microbiology, 20(1):1-13.
Lyu, S., Xiong, F., Qi, T., Shen, W., Guo, Q., Han, M., Liu, L., Bu, W., Yuan, J., & Lou, B. (2024). Isolation and characterization of a novel temperate bacteriophage infecting Aeromonas hydrophila isolated from a Macrobrachium rosenbergii larvae pond. Virus Research, 339(1):1-10.
Maimaiti, Z., Li, Z., Xu, C., Chen, J., & Chai. W. (2023) Global trends and hotspots of phage therapy for bacterial infection: A bibliometric visualized analysis from 2001 to 2021. Frontiers in Microbiology. 13(1):1-17.
Makky, S., Abdelsattar, A. S., Habashy, M., Dawoud, A., Nofal, R., Hassan, A., Connerton, I. F., & El-Shibiny, A. (2023). Phage ZCSS1 from isolation to application against Staphylococcus sciuri and biofilm: A prospect of utilizing temperate phage and its products. Gene Reports, 32(3):1-13.
Nhinh, D. T., Le, D. V., Van, K. V., Giang, N. T. H., Dang, L. T., & 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):1-17.
Pepi, M., & Focardi, S. (2021). Antibiotic-resistant bacteria in aquaculture and climate change: A challenge for health in the Mediterranean Area. International Journal of Environmental Research and Public Health, 18(11):1-31.
Richards, G. P. (2014). Bacteriophage remediation of bacterial pathogens in aquaculture: A review of the technology. Bacteriophage, 4(4):1-12.
Sankappa, N. M., Kallappa, G. S., Boregowda, K. K., Ramakrishna, N. M., Suresh, P. K., Balakrishna, D. S., Ballamoole, K. K., Thangavel, S., Sahoo, L., Lange, M. D., Deshotel, M. B., & Abernathy, J. W. (2024). Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila. Scientific Reports, 14(1):1-14.
Semwal, A., Kumar, A., & Kumar, N. (2023). A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon, 9(3):1-23.
Shaniyah, A., Kasin, R., Lembayung, S., Sunarti, R. N. (2023). Isolation of bacteriophages Escherichia coli and Salmonella sp. from drinking water refill and livestock waste. Prosiding Seminar Nasional Biologi, 41-48.
The State of World Fisheries and Aquaculture 2024. (2024). The state of world fisheries and aquaculture 2024.
Tomás, J. M. (2012). The main Aeromonas pathogenic factors. International Scholarly Research Notices, 2012(1):1-22.
Ture, M., Cebeci, A., Altinok, I., Aygur, E., & Caliskan, N. (2022). Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages. Aquaculture, 558(14):1-9.
Valencia-Toxqui, G., & Ramsey, J. (2024). How to introduce a new bacteriophage on the block: A short guide to phage classification. Journal of Virology, 98(10):1-10.
Youssef, O., Agún, S., Fernández, L., Khalil, S. A., Rodríguez, A., & García, P. (2023). Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODIΔAmi and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production. International Journal of Food Microbiology, 399(14):1-10.
Zaheen, Z., War, A. F., Ali, S., Yatoo, A. M., Ali, M. N., Ahmad, S. B., Rehman, M. U., & Paray, B. A. (2022). Common bacterial infections affecting freshwater fish fauna and impact of pollution and water quality characteristics on bacterial pathogenicity. Bacterial Fish Diseases, 133-154.

References

Abedon, S. T. (2017). Active bacteriophage biocontrol and therapy on sub-millimeter scales towards removal of unwanted bacteria from foods and microbiomes. AIMS Microbiology, 3(3):649-688.

Ács, N., Gambino, M., & Brøndsted, L. (2020). Bacteriophage enumeration and detection methods. Frontiers in Microbiology, 11(1):1-7.

Akmal, M., Rahimi-Midani, A., Hafeez-ur-Rehman, M., Hussain, A., & Choi, T. J. (2020). Isolation, characterization, and application of a bacteriophage infecting the fish pathogen Aeromonas hydrophila. Pathogens, 9(3):1-13.

Altinok, I., Capkin, E., & Kayis, S. (2008). Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens. Veterinary Microbiology, 131(3-4):332-338.

Brye, K. (2023). Aquaculture: A sustainable solution for fish cultivation and global food security. Journal of Agriculture, 6(4):91-93.

Choliq, F. A., Martosudiro, M., Istiqomah, I., & Nijami, M. F. (2020). Isolation and testing of bacteriophage capabilities as control agents for bacterial wilt disease (Ralstonia solanacearum) in tomato plants. Viable, Jurnal Ilmiah Ilmu-Ilmu Pertanian, 14(1):8-20.

Clokie, M. R. J., & Kropinski, A. M. (Eds.). (2009). Bacteriophages (Vol. 501). Humana Press.

D’accolti, M., Soffritti, I., Mazzacane, S., & Caselli, E. (2021). Bacteriophages as a potential 360-degree pathogen control strategy. Microorganisms, 9(2):1-14.

Dien, L. T., Ky, L. B., Huy, B. T., Mursalim, M. F., Kayansamruaj, P., Senapin, S., Rodkhum, C., & Dong, H. T. (2022). Characterization and protective effects of lytic bacteriophage pAh6.2TG against a pathogenic multidrug‐resistant Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus). Transboundary and Emerging Diseases, 69(4):435-450.

FAO. (2022). The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. Rome, FAO.

Feng, C., Jia, K., Chi, T., Chen, S., Yu, H., Zhang, L., Raza, S. H. A., Alshammari, A. M., Liang, S., Zhu, Z., Li, T., Qi, Y., Shan, X., Qian, A., Zhang, D., Zhang, L., & Sun, W. (2022). Lytic bacteriophage pzl-ah152 as biocontrol measures against lethal Aeromonas hydrophila without distorting gut microbiota. Frontiers in Microbiology, 13(1):1-14.

Glonti, T., & Pirnay, J. P. (2022). In vitro techniques and measurements of phage characteristics that are important for phage therapy success. Viruses, 14(7):1-23.

He, P., Cao, F., Qu, Q., Geng, H., Yang, X., Xu, T., Wang, R., Jia, X., Lu, M., Zeng, P., & Luan, G. (2024). Host range expansion of Acinetobacter phage vB_Ab4_Hep4 driven by a spontaneous tail tubular mutation. Frontiers in Cellular and Infection Microbiology, 14(1):1-12.

Irshath, A. A., Rajan, A. P., Vimal, S., Prabhakaran, V. S., & Ganesan, R. (2023). Bacterial pathogenesis in various fish diseases: Recent advances and specific challenges in vaccine development. Vaccines, 11(2):1-14.

Jo, S. J., Lee, Y. M., Cho, K., Park, S. Y., Kwon, H., Giri, S. S., Lee, S. B., Jung, W. J., Park, J. H., Hwang, M. H., Park, D. S., Park, E. J., Kim, S. W., Jun, J. W., Kim, S. G., Kim, J. H., & Park, S. C. (2025). Standardization of the agar plate method for bacteriophage production. Antibiotics, 14(1):1-14.

Jun, J. W. (2024). A concise overview of studies on successful real-world applications of bacteriophages in aquaculture. Viruses, 16(12):1-13.

Laguna-Castro, M., & Lázaro, E. (2022). Propagation of an RNA bacteriophage at low host density leads to a more efficient virus entry. Frontiers in Virology, 2(1):1-12.

Le, T. S., Nguyen, T. H., Vo, H. P., Doan, V. C., Nguyen, H. L., Tran, M. T., Tran, T. T., Southgate, P. C., & Kurtböke, D. İ. (2018). Protective effects of bacteriophages against Aeromonas hydrophila causing Motile Aeromonas Septicemia (MAS) in striped catfish. Antibiotics, 7(1):1-11.

Liang, S., Liang, R., Raza, S. H. A., Huang, Q., Li, T., Bai, H., Feng, C., Xin, L., Guo, H., Wang, S., Yu, J., Cao, Y., Liu, N., AlMalki, F., Zhang, D., Sun, W., & Zhang, L. (2025). Biological analysis of phage vB_AhaP_PT2 and treatment rescued crucian carp infected with Aeromonas hydrophila. Aquaculture, 595(3):1-14.

Liu, J., Gao, S., Dong, Y., Lu, C., & Liu, Y. (2020). Isolation and characterization of bacteriophages against virulent Aeromonas hydrophila. BMC Microbiology, 20(1):1-13.

Lyu, S., Xiong, F., Qi, T., Shen, W., Guo, Q., Han, M., Liu, L., Bu, W., Yuan, J., & Lou, B. (2024). Isolation and characterization of a novel temperate bacteriophage infecting Aeromonas hydrophila isolated from a Macrobrachium rosenbergii larvae pond. Virus Research, 339(1):1-10.

Maimaiti, Z., Li, Z., Xu, C., Chen, J., & Chai. W. (2023) Global trends and hotspots of phage therapy for bacterial infection: A bibliometric visualized analysis from 2001 to 2021. Frontiers in Microbiology. 13(1):1-17.

Makky, S., Abdelsattar, A. S., Habashy, M., Dawoud, A., Nofal, R., Hassan, A., Connerton, I. F., & El-Shibiny, A. (2023). Phage ZCSS1 from isolation to application against Staphylococcus sciuri and biofilm: A prospect of utilizing temperate phage and its products. Gene Reports, 32(3):1-13.

Nhinh, D. T., Le, D. V., Van, K. V., Giang, N. T. H., Dang, L. T., & 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):1-17.

Pepi, M., & Focardi, S. (2021). Antibiotic-resistant bacteria in aquaculture and climate change: A challenge for health in the Mediterranean Area. International Journal of Environmental Research and Public Health, 18(11):1-31.

Richards, G. P. (2014). Bacteriophage remediation of bacterial pathogens in aquaculture: A review of the technology. Bacteriophage, 4(4):1-12.

Sankappa, N. M., Kallappa, G. S., Boregowda, K. K., Ramakrishna, N. M., Suresh, P. K., Balakrishna, D. S., Ballamoole, K. K., Thangavel, S., Sahoo, L., Lange, M. D., Deshotel, M. B., & Abernathy, J. W. (2024). Novel lytic bacteriophage AhFM11 as an effective therapy against hypervirulent Aeromonas hydrophila. Scientific Reports, 14(1):1-14.

Semwal, A., Kumar, A., & Kumar, N. (2023). A review on pathogenicity of Aeromonas hydrophila and their mitigation through medicinal herbs in aquaculture. Heliyon, 9(3):1-23.

Shaniyah, A., Kasin, R., Lembayung, S., Sunarti, R. N. (2023). Isolation of bacteriophages Escherichia coli and Salmonella sp. from drinking water refill and livestock waste. Prosiding Seminar Nasional Biologi, 41-48.

The State of World Fisheries and Aquaculture 2024. (2024). The state of world fisheries and aquaculture 2024.

Tomás, J. M. (2012). The main Aeromonas pathogenic factors. International Scholarly Research Notices, 2012(1):1-22.

Ture, M., Cebeci, A., Altinok, I., Aygur, E., & Caliskan, N. (2022). Isolation and characterization of Aeromonas hydrophila-specific lytic bacteriophages. Aquaculture, 558(14):1-9.

Valencia-Toxqui, G., & Ramsey, J. (2024). How to introduce a new bacteriophage on the block: A short guide to phage classification. Journal of Virology, 98(10):1-10.

Youssef, O., Agún, S., Fernández, L., Khalil, S. A., Rodríguez, A., & García, P. (2023). Impact of the calcium concentration on the efficacy of phage phiIPLA-RODI, LysRODIΔAmi and nisin for the elimination of Staphylococcus aureus during lab-scale cheese production. International Journal of Food Microbiology, 399(14):1-10.

Zaheen, Z., War, A. F., Ali, S., Yatoo, A. M., Ali, M. N., Ahmad, S. B., Rehman, M. U., & Paray, B. A. (2022). Common bacterial infections affecting freshwater fish fauna and impact of pollution and water quality characteristics on bacterial pathogenicity. Bacterial Fish Diseases, 133-154.