Sains Malaysiana 53(1)(2024): 99-110

http://doi.org/10.17576/jsm-2024-5301-08

In-vitro Inhibitory Activities of Potential Probiotic Isolated from Pangasius nasutus against Aeromonas hydrophila and Streptococcus agalactiae

(Aktiviti Perencatan In-vitro Probiotik Berpotensi Diasingkan daripada Pangasius nasutus terhadap Aeromonas hydrophila dan Streptococcus agalactiae)

SHAFIQ JOHAR¹, PUVANESWARI PUVANASUNDRAM^1,2, CLEMENT ROY DE CRUZ^1,3, CHOU MIN CHONG^1,2, MD YASIN INA-SALWANY^1,2, KENG CHIN LIM¹, NOORDIYANA MAT NOORDIN⁴ AND MURNI KARIM^1,3,*

¹Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

²Aquatic Animal Health and Therapeutics Laboratory, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

³Laboratory of Sustainable Aquaculture, International Institute of Aquaculture and Aquatic Sciences, Universiti Putra Malaysia, 71050 Port Dickson, Negeri Sembilan, Malaysia

⁴Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia

Received: 27 October 2023/Accepted: 28 December 2023

Abstract

In aquaculture, using probiotics is crucial for strengthening the immune system and encouraging the growth and survival of many aquatic organisms, including the Pangasius species. This approach is particularly significant given the impact of bacterial diseases on Pangasius survival. This study aimed to assess the effectiveness of probiotics isolated from Pangasius nasutus as alternatives to antibiotics for combating infections caused by Aeromonas hydrophila and Streptococcus agalactiae. Potential bacteria were isolated from the intestine and stomach of healthy P. nasutus. Seventy probiotic strains were successfully isolated and further screened using A. hydrophilaand S. agalactiae as pathogens in an in vitro disc diffusion assay. Preliminary screenings indicated that five probiotic strains inhibited the growth of A. hydrophila. Stomach-derived strain S1 and intestine-derived strain L1 suppressed A. hydrophila growth with inhibition zones of 10.5±1 mm and 8.5±1 mm, respectively. Likewise, strains L2, L8, and L12 from the intestine showed inhibitory zones of 6.0±1 mm, 6.5±1 mm, and 6.0±1 mm, respectively. Of these, only L12 inhibited the growth of S. agalactiae with a clear zone of 5.0±1 mm. In the elimination of pathogenic strains, potential strains S1 and L1 did not grow on the Aeromonas isolation medium. Co-culture assays demonstrated that both potential strains significantly inhibited Aeromonas hydrophila growth at concentrations of 10⁶ and 10⁸ CFU mL^-1 over 48- and 96-hour periods, respectively. The potential bacterial strains were identified using 16s rRNA gene sequencing and were classified as follows: S1 - Lactococcus lactis, L1 - Weissella confusa, L2 - Cosenzaea myxofaciens, L8 - Lactococcus garvieae, and L12 - Plesiomonas shigelloides. Strain S1 L. lactis and strain L1 W. confusa are suggested for further evaluation and acquired additional research to fully elucidate their mechanisms and potential as probiotics.

Keywords: Antagonistic activity; in vitro screening; Pangasius species; pathogens; potential probiotics

Abstrak

Dalam akuakultur, penggunaan probiotik adalah penting untuk menguatkan sistem keimunan dan menggalakkan pertumbuhan dan kemandirian hidup pelbagai organisma akuatik, termasuk spesies Pangasius. Pendekatan ini amat penting memandangkan kesan buruk penyakit bakteria terhadap kelangsungan hidup spesies Pangasius. Kajian ini bertujuan untuk menilai keberkesanan probiotik yang dipencilkan daripada Pangasius nasutus sebagai alternatif kepada antibiotik untuk melawan jangkitan penyakit yang disebabkan oleh Aeromonas hydrophila dan Streptococcus agalactiae. Bakteria berpotensi sebagai probiotik telah diasingkan daripada organ usus dan perut P. nasutus yang sihat. Tujuh puluh strain probiotik berjaya diasingkan dan seterusnya disaring dengan menggunakan A. hydrophila dan S. agalactiae sebagai patogen dalam ujian penyebaran cakera in vitro. Pemeriksaan awal menunjukkan bahawa lima strain probiotik dapat merencat pertumbuhan A. hydrophila. Strain S1 yang dipencilkan daripada perut dan strain L1 daripada usus dapat merencat pertumbuhan A. hydrophila dengan zon perencatan masing-masing sebanyak 10.5±1 mm dan 8.5±1 mm. Begitu juga, strain L2, L8 dan L12 daripada usus menunjukkan zon perencatan ke atas A. hydrophila masing-masing sebanyak 6.0±1 mm, 6.5±1 mm dan 6.0±1 mm. Manakala, hanya L12 merencat pertumbuhan S. agalactiae dengan zon yang jelas 5.0±1 mm. Dalam pengasingan strain patogen, S1 dan L1 tidak hidup di atas medium pengasingan Aeromonas. Ujian kultur bersama menunjukkan bahawa kedua-dua SI dan LI dengan ketara merencat pertumbuhan A. hydrophila pada kepekatan 10⁶ dan 10⁸ CFU mL^-1 dalam tempoh 48 dan 96 jam. Semua strain bakteria dikenal pasti menggunakan penjujukan gen 16s rRNA dan dikelaskan seperti berikut: S1 - Lactococcus lactis, L1 - Weissella confusa, L2 - Cosenzaea myxofaciens, L8 - Lactococcus garvieae dan L12 - Plesiomonas shigelloides. Strain SI L. lactis dan L1 W. confusa dicadangkan untuk penilaian lanjut dan memerlukan penyelidikan tambahan untuk menjelaskan sepenuhnya mekanisme dan potensi sebagai probiotik.

Kata kunci: Aktiviti antagonis; kajian in vitro; patogen; probiotik berpotensi; spesies Pangasius

REFERENCES

Alonso, M., Lago, F.C., Vieites, J.M. & Espineira, M. 2012. Molecular characterization of microalgae used in aquaculture with biotechnology potential. Aquaculture International 20: 847-857.

Assefa, A. & Abunna, F. 2018. Maintenance of fish health in aquaculture: Review of epidemiological approaches for prevention and control of infectious disease of fish. Veterinary Medicine International 2018: 5432497.

Ayisi, C.L., Apraku, A. & Afriyie, G. 2017. A review of probiotics, prebiotics, and synbiotics in crab: Present research, problems, and future perspective. Journal of Shellfish Research 36(3): 799-806.

Banerjee, G. & Ray, A.K. 2017. The advancement of probiotics research and its application in fish farming industries. Research in Veterinary Science 115: 66-77.

Barman, P., Banerjee, A., Bandyopadhyay, P., Mondal, K.C. & Das Mohapatra, P.K. 2011. Isolation, identification and molecular characterization of potential probiotic bacterium, Bacillus subtilis PPP 13 from Penaeus monodon. Biotechnology, Bioinformatics, Bioengineering 1(4): 473-482.

Chapela, M.J., Ferreira, M., Varela, C., Arregui, L. & Garrido-Maestu, A. 2018. Development of a multiplex real-time PCR method for early diagnosis of three bacterial diseases in fish: A real-case study in trout aquaculture. Aquaculture 496: 255-261.

Chuah, L.O., Effarizah, M.E., Goni, A.M. & Rusul, G. 2016. Antibiotic application and emergence of multiple antibiotic resistance (MAR) in global catfish aquaculture. Current Environmental Health Reports 3: 118-127.

Chowdhury, M.A., Roy, N.C. & Chowdhury, A. 2020. Growth, yield and economic returns of striped catfish (Pangasianodon hypophthalmus) at different stocking densities under floodplain cage culture system. The Egyptian Journal of Aquatic Research 46(1): 91-95.

Devirgiliis, C., Zinno, P. & Perozzi, G. 2013. Update on antibiotic resistance in foodborne Lactobacillus and Lactococcus species. Frontiers in Microbiology 4: 301.

DOF. 2019. Annual Fisheries Statistic 2019 (Volume 1). Department of Fisheries. https://www.dof.gov.my/sumber/i-extension/laporan-tahunan/

Dong, H.T., Nguyen, V.V., Le, H.D., Sangsuriya, P., Jitrakorn, S., Saksmerprome, V., Senapin, S. & Rodkhum, C. 2015. Naturally concurrent infections of bacterial and viral pathogens in disease outbreaks in cultured Nile tilapia (Oreochromis niloticus) farms. Aquaculture 448: 427-435.

El‐Bouhy, Z.M., Reda, R.M., Mahboub, H.H. & Gomaa, F.N. 2021. Chelation of mercury intoxication and testing different protective aspects of Lactococcus lactis probiotic in African catfish. Aquaculture Research 52(8): 3815-3828.

FAO. 2022. The State of World Fisheries and Aquaculture: Towards Blue Transformation. The State of World Fisheries and Aquaculture (SOFIA) 2019. Food and Agriculture Organization.

Fitri, M.Y. & Christianus, A. 2019. Morphological approaches to elucidate two closely related pangasius catfish: Morphological approaches in pangasius catfish. International Journal of Allied Health Sciences 3(4): 967-975.

Fuller, R. & Fuller, R. 1992. History and development of probiotics. In Probiotics: The Scientific Basis. Dordrecht: Springer. pp. 1-8.

Fulbright, S.P., Chisholm, S. & Reardon, K.F. 2016. Growth inhibition of Nannochloropsis species by Bacillus pumilus. Algal Research 20: 70-76.

Grossart, H.P., Schlingloff, A., Bernhard, M., Simon, M. & Brinkhoff, T. 2004. Antagonistic activity of bacteria isolated from organic aggregates of the German Wadden Sea. FEMS Microbiology Ecology 47(3): 387-396.

Gustiano, R. 2003. Taxonomy and phylogeny of Pangasiidae catfishes from Asia (Ostariophysi, Siluriformes). Ph.D. Thesis, Katholieke Universiteit Leuven (Unpublished).

Hai, N.V. 2015. The use of probiotics in aquaculture. Journal of Applied Microbiology 119(4): 917-935.

Janda, J.M., Abbott, S.L. & McIver, C.J. 2016. Plesiomonas shigelloides revisited. Clinical Microbiology Reviews 29(2): 349-374.

Jasmin, M.Y., Wagaman, H., Yin, T.A., MY, I.S., Daud, H.M. & Karim, M. 2016. Screening and evaluation of local bacteria isolated from shellfish as potential probiotics against pathogenic Vibrios. Journal of Environmental Biology 37(4): 801-809.

Knipe, H., Temperton, B., Lange, A., Bass, D. & Tyler, C.R. 2021. Probiotics and competitive exclusion of pathogens in shrimp aquaculture. Reviews in Aquaculture 13(1): 324-352.

La Fata, G., Weber, P. & Mohajeri, M.H. 2018. Probiotics and the gut immune system: Indirect regulation. Probiotics and Antimicrobial Proteins 10: 11-21.

Lee, O.W.S., Puvanasundram, P., Lim, K.C. & Karim, M. 2022. In vitro assessment of multistrain probiotic on its safety, biofilm formation capability, and antimicrobial properties against Aeromonas hydrophila. Pertanika Journal of Tropical Agricultural Science 45(4): JTAS-2482-2022.

Li, C., Ren, Y., Jiang, S., Zhou, S., Zhao, J., Wang, R. & Li, Y. 2018. Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka. Fish & Shellfish Immunology 74: 69-75.

Lim, C., Webster, C.D. & Lee, C.S. 2015. Feeding practices and fish health. In Dietary Nutrients, Additives and Fish Health, edited by Lee, C.S., Lim, C., Gatlin III, D.M. & Webster, C.D. Wiley-Blackwell. pp. 333-346.

Martinez-Villaluenga, C., Peñas, E. & Frias, J. 2017. Bioactive peptides in fermented foods: Production and evidence for health effects. In Fermented Foods in Health and Disease Prevention, edited by Frias, J., Martinez-Villaluenga, C. & Peñas, E. Massachusetts: Academic Press. pp. 23-47.

Mahmud, N.S., Abdullah, S.Z., Jalal, K.C.A., Rimatulhana, R. & Amal, M.N. 2019. Assessment of bacteria and water quality parameters in cage cultured Pangasius hypophthalmus in Temerloh, Pahang River, Malaysia. Nature Environment & Pollution Technology 18(5): 1479-1486.

Mamun, A.A., Nasren, S., Rathore, S.S. & Mahbub Alam, M.M. 2022. Histopathological analysis of striped catfish, Pangasianodon hypophthalmus (Sauvage, 1878) spontaneously infected with Aeromonas hydrophila. Jordan Journal of Biological Sciences 15(1): 93-100.

Maqsood, S., Singh, P., Samoon, M.H. & Munir, K. 2011. Emerging role of immunostimulants in combating the disease outbreak in aquaculture. International Aquatic Research (Islamic Azad University, Tonekabon Branch) 3(3): 147-163.

Masuda, M., Ide, M., Utsumi, H., Niiro, T., Shimamura, Y. & Murata, M. 2012. Production potency of folate, vitamin B12, and thiamine by lactic acid bacteria isolated from Japanese pickles. Bioscience, Biotechnology, and Biochemistry 76(11): 2061-2067.

Meyburgh, C.M., Bragg, R.R. & Boucher, C.E. 2017. Lactococcus garvieae: An emerging bacterial pathogen of fish. Diseases of Aquatic Organisms 123(1): 67-79.

Meidong, R., Nakao, M., Sakai, K. & Tongpim, S. 2021. Lactobacillus paraplantarum L34b-2 derived from fermented food improves the growth, disease resistance and innate immunity in Pangasius bocourti. Aquaculture 531: 735878.

Nahar, S., Rahman, M.M., Ahmed, G.U. & Faruk, M.A.R. 2016. Isolation, identification, and characterization of Aeromonas hydrophila from juvenile farmed pangasius (Pangasianodon hypophthalmus). International Journal of Fisheries and Aquatic Studies 4(4): 52-60.

Newaj‐Fyzul, A. & Austin, B. 2015. Probiotics, immunostimulants, plant products and oral vaccines, and their role as feed supplements in the control of bacterial fish diseases. Journal of Fish Diseases 38(11): 937-955.

Parven, M., Alam, M.M., Khalil, S.M.I., Hamom, A., Goni, O., Rahman, M.M. & Abdullah-Al-Mamun, M. 2020. Identification of pathogenic bacteria from diseased Thai Pangas Pangasius hypophthalmus with their sensitivity to antibiotics. Microbiology Research Journal International 30(3): 7-21.

Shotts Jr., E.B. & Rimler, R. 1973. Medium for the isolation of Aeromonas hydrophila. Applied Microbiology 26(4): 550-553.

Siti-Zahrah, A., Zamri-Saad, M., Firdaus-Nawi, M., Hazreen-Nita, M.K. & Nur-Nazifah, M. 2013. Detection of channel catfish virus in cage-cultured Pangasius hypophthalmus (Sauvage, 1878) in Malaysia. Journal of Fish Diseases 37(11): 981-983.

Sharma, R., Tyagi, S., Panwar, A., Kumar, S., Tyagi, C. & Kumar, Y. 2020. On annual cycle of monogenean parasites infestation in freshwater fish Pangasius pangasius. The Scientific Temper 11(1&2).

Soni, P., Pradhan, P.K., Swaminathan, T.R. & Sood, N. 2018. Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878). Acta Tropica 182: 215-222.

Sun, Y.Z., Yang, H.L., Ma, R.L., Song, K. & Li, J.S. 2012. Effect of Lactococcus lactis and Enterococcus faecium on growth performance, digestive enzymes and immune response of grouper Epinephelus coioides. Aquaculture Nutrition 18(3): 281-289.

Trung, N.B. & Dung, T.T. 2023. Antimicrobial resistance and the prevalence of integron in Aeromonas hydrophila from hemorrhagic diseased Pangasius catfish of the Mekong Delta: Veterinary Integrative Sciences 21(2): 333-347. https://doi. org/10.12982/VIS. 2023.025 

Vargas-Chacoff, L., Martínez, D., Oyarzún, R., Nualart, D., Olavarría, V., Yáñez, A., Bertrán, C., Ruiz-Jarabo, I. & Mancera, J.M. 2014. Combined effects of high stocking density and Piscirickettsia salmonis treatment on the immune system, metabolism and osmoregulatory responses of the Sub-Antarctic Notothenioid fish Eleginops maclovinus. Fish & Shellfish Immunology 40(2): 424-434.

Vendrell, D., Balcázar, J.L., Ruiz-Zarzuela, I., De Blas, I., Gironés, O. & Múzquiz, J.L. 2006. Lactococcus garvieae in fish: A review. Comparative Immunology, Microbiology and Infectious Diseases 29(4): 177-198.

Vinderola, G., Ouwehand, A.C., Salminen, S. & von Wright, A. 2019. Lactic Acid Bacteria: Microbiological and Functional Aspects. Boca Raton: CRC Press.

Wang, A., Ran, C., Wang, Y., Zhang, Z., Ding, Q., Yang, Y., Olsen, R.E., Ringø, E., Bindelle, J. & Zhou, Z. 2019. Use of probiotics in aquaculture of China - A review of the past decade. Fish & Shellfish Immunology 86: 734-755.

Yusof, F. & Nakajima, M. 2019. Morphological tool to elucidate two closely related Pangasius catfish. International Journal of Allied Health Sciences 3(3): 765-765.

Zhou, X., Wang, Y., Yao, J. & Li, W. 2010. Inhibition ability of probiotic, Lactococcus lactis, against A. hydrophila and study of its immunostimulatory effect in tilapia (Oreochromis niloticus). International Journal of Engineering, Science and Technology 2(7): 73-80.

*Corresponding author; email: murnimarlina@upm.edu.my