Antibacterial Bacillus strains from the Mekong Delta: Combating Vibrio parahaemolyticus in intensive white-leg shrimp aquaculture https://doi.org/10.12982/VIS.2025.077

Main Article Content

Quach Van Cao Thi
Nguyen Van Le
Nguyen Phuong Thuy
Truong Quoc Tat
Thanh Tran

Abstract

Vibrio parahaemolyticus is a major bacterial pathogen causing significant losses in the global shrimp farming industry, including in Vietnam. The overuse of antibiotics for controlling V. parahaemolyticus has led to increased bacterial resistance. This study aimed to isolate and screen indigenous Bacillus strains with inhibitory activity against V. parahaemolyticus, which causes Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Disease (AHPNS) in intensively farmed white-leg shrimp (Litopenaeus vannamei) in Bac Lieu province, Mekong Delta, Vietnam. A total of forty-two Bacillus strains were isolated from the intestines of healthy shrimp, pond mud, and pond water. The well diffusion agar method revealed that twenty out of the forty-two strains exhibited inhibitory effects against V. parahaemolyticus. Among these, four strains, identified as BLN7, STNC, BLB7, and BLN8, demonstrated the strongest antagonistic activity, with inhibition zone diameters of 24 ± 0.00, 12.03 ± 0.06, 11.97 ± 0.06, and 11.93 ± 0.12 mm, respectively. Additionally, the study found that these four strains possess extracellular enzyme activities, including amylase, cellulase, and protease. Molecular identification through 16S rRNA gene sequencing, along with morphological, physiological, and biochemical analysis, confirmed that strains BLN7 and STNC belong to Bacillus spp. These findings provide a foundation for developing biological products to prevent EMS disease in white-leg shrimp.

Article Details

How to Cite
Quach Van Cao Thi, Nguyen Van Le, Nguyen Phuong Thuy, Truong Quoc Tat, & Tran, T. (2025). Antibacterial Bacillus strains from the Mekong Delta: Combating Vibrio parahaemolyticus in intensive white-leg shrimp aquaculture : https://doi.org/10.12982/VIS.2025.077. Veterinary Integrative Sciences, 23(3), 1–17. retrieved from https://he02.tci-thaijo.org/index.php/vis/article/view/269858
Section
Research Articles

References

Amara, A.A., Shibl, A., 2015. Role of probiotics in health improvement, infection control and disease treatment and management. Saudi Pharm J. 23(2), 107-14.

Amarantini, C., Satwika, D., Budiarso, T.Y., Yunita, E.R., Laheba, E.A., 2019. Screening of antimicrobial-producing lactic acid bacteria isolated from traditional fish fermentation against pathogenic bacteria. In Journal of Physics: Conference Series. 1397(1), 012045. IOP Publishing.

Balcázar, J.L., Rojas-Luna, T., 2007. Inhibitory activity of probiotic Bacillus subtilis UTM 126 against Vibrio species confers protection against vibriosis in juvenile shrimp (Litopenaeus vannamei). Curr Microbiol. 55(5), 409-12.

Barrow, G.I., Feltham, R.K.A., 1993. Cowan and Steel’s manual for the indentification of medical bacteria (3rd edn). Cambridge Univesity Press, Cambridge. 351.

Bojarski, B., Kot, B., Witeska, M., 2020. Antibacterials in aquatic environment and their toxicity to fish. Pharmaceuticals (Basel). 13(8), 189.

Buller, N.B., 2014. Bacteria and fungi from fish and other aquatic animals: a practical identification manual. CABI Publishing. 920.

Caulier, S., Nannan, C., Gillis, A., Licciardi, F., Bragard, C., Mahillon, J., 2019. Overview of the antimicrobial compounds produced by members of the bacillus subtilis group. Front. Microbiol. 10, 302.

Chalasani, A.G., Dhanarajan, G., Nema, S., Sen, R., Roy, U., 2015. An antimicrobial metabolite from bacillus sp.: significant activity against pathogenic bacteria including multidrug-resistant clinical strains. Front. Microbiol. 6, 1335.

Chen, Y., Li, J., Xiao, P., Zhu, W., Mo, Z., 2016. The ability of marine Bacillus spp. isolated from fish gastrointestinal tract and culture pond sediment to inhibit growth of aquatic pathogenic bacteria. Iran. Fish. Sci. J. 15(2), 701-714.

Dao, N.T.B., Tran, Q.K.V., Nguyen, V.K., Nguyen, Q.L., 2015. Isolation and selection of Bacillus strains to inhibit Vibrio parahaemolyticus which is the cause of EMS on shrimp farming in Thua Thien Hue Province, Vietnam. 100(01), 15-28.

Dat, T.T.H., Tam, V.T.T., Dung, T.T.K., Bui, L.M., Anh, H.L.T., Oanh, P.T.T., 2019. Isolation and screening of cellulose and organic matter degrading bacteria from aquaculture ponds for improving water quality in aquaculture. IOP Conf. Ser.: Earth Environ. Sci. 266, 012002. IOP Publishing.

Department of Fisheries, 2024. Vietnamese seafood exports have begun to recover. Viet Nam News. (Accessed June 19, 2024).

Dhar, A.K.A., Piamsomboon, P., Aranguren Caro, L.F.L., Kanrar, S., Adami, R., Juan, Y.-S.Y., 2019. First report of acute hepatopancreatic necrosis disease (AHPND) occurring in the USA. Dis. Aquat. Organ. 132, 241-247.

Drake, S.L., DePaola, A., Jaykus, L.A., 2007. An overview of Vibrio vulnificus and Vibrio parahaemolyticus. Comprehensive reviews in food science and food safety. 6(4), 120-144.

Duc, L.H., Nam, N.T., Oanh, D.T.H., Thanh, T.T., Ha, C.H., 2022. Isolation and evaluation the effect of Bacillus subtillis BLD01 strain on the survival rates and gut microbiota of Penaeus vannamei after challenge with Vibrio parahaemolyticus. Vietnam J. Biotechnol. 20(3), 505-516.

Dung, D.T.T., Le, T.B., Hoang, T.D.D., Vo, Đ.Q., Phan, T.P.T., 2017. Selection of Bacillus spp. isolates having enzyme producing ability and antagonistic to Vibrio parahaemolyticus causing the early mortality syndrome (EMS) on shrimp. Sci. Tech. Dev. J. 1(6), 23-31.

Elias, N.A., Abu Hassan, M.S., Yusoff, N.A.H., Tosin, O.V., Harun, N.A., Rahmah, S., Hassan, M., 2023. Potential and limitation of biocontrol methods against vibriosis: a review. Aquac Int. 31(4), 2355-2398.

Elshaghabee, F.M.F., Rokana, N., Gulhane, R.D., Sharma, C., Panwar, H., 2017. Bacillus As potential probiotics: status, concerns, and future perspectives. Front. Microbiol. 8,1490.

FAO, 2022. The state of world fisheries and aquaculture 2022. towards blue transformation. Rome, FAO.

Flegel, T.W., 2012. Historic emergence, impact and current status of shrimp pathogens in Asia. J. Invertebr. Pathol. 110,166-173.

Harley, J.P., Prescott, L.M., 2001. Laboratory exercises in microbiology.

Heuer, H., Krsek, M., Baker, P., Smalla, K., Wellington, E.M., 1997. Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63, 3233-3241.

Hlordzi, V., Kuebutornye, F.K., Afriyie, G., Abarike, E.D., Lu, Y., Chi, S., Anokyewaa, M.A., 2020. The use of Bacillus species in maintenance of water quality in aquaculture: a review. Aquac. Rep. 18, 100503.

Hoa, T.T.T., Fagnon, M.S., Thy, D.T.M., Chabrillat, T., Trung, N.B., Kerros, S., 2023. Growth performance and disease resistance against Vibrio parahaemolyticus of whiteleg shrimp (Litopenaeus vannamei) fed essential oil blend (Phyto AquaBiotic). Animals. 13, 3320.

Hong, T.T.T., Yanagawa, H., Khanh, T.N., Hiep, D.M., Cuong, D.V., Khai, L.T.L., Taniguchi, T., Kubo, R., Hayashidani, H., 2020. Prevalence of Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease of shrimp in shrimp, molluscan shellfish and water samples in the Mekong Delta, Vietnam. Biology (Basel). 9(10), 312.

Jamilah, I.T., Meryandini, A., Rusmana, I., Suwanto, A., Mubarik, N.R., 2009. Activity of proteolytic and amylolytic enzymes from Bacillus spp. isolated from shrimp ponds. Microbiol. Indones. 3(2), 4.

Jlidi, M., Akremi, I., Ibrahim, A.H., Brabra, W., Ali, M.B., Ali, M.B., 2022. Probiotic properties of Bacillus strains isolated from the gastrointestinal tract against pathogenic Vibriosis. Front. Mar. Sci. 9, 884244.

Kuebutornye, F.K.A., Abarike, E.D., Lu, Y.S., 2019. A review on the application of Bacillus as probiotics in aquaculture. Fish Shellfish Immunol. 87, 820-828.

Kumar, V., Roy, S., Behera, B.K., Bossier, P., Das, B.K., 2021. Acute Hepatopancreatic Necrosis Disease (AHPND): virulence, pathogenesis and mitigation strategies in shrimp aquaculture. Toxins. 13, 524.

Loc, T., Nunan, L., Redman, R.M., Lightner, D.V., Fitzsimmons, K., 2013. EMS/AHPNS: Infectious disease caused by bacteria. Global Aquaculture Advocate. 20(7), 18-20.

Loc, T., Nunan, L., Redman, R.M., Mohney, L.L., Pantoja, C.R., Fitzsimmons, K., Lightner, D.V., 2013. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis. Aquat. Org. 105, 45-55.

Minh, N.V., Nguyen, H.T.D., Do, P.Q., Tran, T.A.N., Tran, K.D., Duong, N.L., 2019. Isolation and selection of Bacillus strains with high potential probiotic that used in catfish farming (Pangasianodon hypophthalmus). Ho Chi Minh City Open University Journal of Science, 10(1), 3-16.

Monier, M.N., Kabary, H., Elfeky, A., Saadony, S., El-Hamed, N.N.A., Eissa, M.E., Eissa, E.S.H., 2023. The effects of Bacillus species probiotics (Bacillus subtilis and B. licheniformis) on the water quality, immune responses, and resistance of whiteleg shrimp (Litopenaeus vannamei) against Fusarium solani infection. Aquac Int. 31(6), 3437-3455.

Oanh, D.T.H., Truong, Q.P., Nguyen, T.P., 2022. Antibacterial resistance of Vibrio parahaemolyticus isolated from shrimp farms located in east coastal region of the Mekong Delta, Viet Nam. Can Tho University J. Science. 14(20), 1-7.

Oanh, D.T.H., Phuong, N.T., 2012. Serious diseases in marine shrimp and freshwater prawn farming in the Mekong Delta. Can Tho University Journal of Science. 2012(22c), 106-118.

Olmos, J., Acosta, M., Mendoza, G, Pitones, V., 2020. Bacillus subtilis, an ideal probiotic bacterium to shrimp and fish aquaculture that increase feed digestibility, prevent microbial diseases, and avoid water pollution. Arch Microbiol. 202(3), 427-435.

Peng, M., Ye, Z., Zengfu, S., 2019. Isolation and characterization of a Bacillus spp. against vibrio parahaemolyticus from shrimp culture ponds. Int J Microbiol Biotechnol. 4(2), 29-37.

Pereira, W.A., Mendonça, C.M.N., Urquiza, A.V., Marteinsson, V.P., LeBlanc, J.G., Cotter, P.D., Villalobos, E.F., Romero, J., Oliveira, R.P.S., 2022. Use of probiotic bacteria and bacteriocins as an alternative to antibiotics in aquaculture. Microorganisms. 2022(10), 1705.

Phuong, V.H., Vo, T.H., Nguyen, T.H.N., Le, H.P., Nguyen, H.T., Nguyen, H.L., Le, T.B.T., 2018. Evaluation of antagonism properties of Bacillus licheniformis (B1) against Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (AHPND) on shrimp in-vitro condition. Can Tho University J. Science. 2018(2), 91-100.

Proespraiwong, P., Mavichak, R., Imaizumi, K., Hirono, I., Unajak, S., 2023. Evaluation of Bacillus spp. as potent probiotics with reduction in AHPND-related mortality and facilitating growth performance of pacific white shrimp (Litopenaeus vannamei) Farms. Microorganisms. 11, 2176.

Purivirojkul, W., Areechon, N., 2007. Application of Bacillus spp. isolated from the intestine of black tiger shrimp (Penaeus monodon Fabricius) from natural habitat for control pathogenic bacteria in aquaculture. Kasetsart J. (Nat. Sci.). 41, 125-132.

Ran, C., Carrias, A., Williams, M.A., Capps, N., Dan, B.C.T., Newton, J.C., Kloepper, E.L.O., Browdy, C.L., Terhune, J.S., Liles, M.R., 2012. Identification of Bacillus strains for biological control of catfish pathogen. PLOS ONE. 1, 79.

Restrepo, L., Bayot, B., Betancourt, I., Pinzón, A., 2016. Draft genome sequence of pathogenic bacteria Vibrio parahaemolyticus strain Ba94C2, associated with acute hepatopancreatic necrosis disease isolate from south America. Genom. Data. 9, 143-144.

Saggese, A., Baccigalupi, L., Ricca, E., 2021. Spore formers as beneficial microbes for humans and animals. Appl. Microbiol. 1, 498-509.

Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4(4), 406-425.

Santos, R.A., Oliva-Teles, A., Pousão-Ferreira, P., Jerusik, R., Saavedra, M.J., Enes, P., Serra, C.R., 2021, Isolation and characterization of fish-gut Bacillus spp. as source of natural antimicrobial compounds to fight aquaculture bacterial diseases. Mar. Biotechnol. 23, 276-293.

Shleeva, M.O., Kondratieva, D.A., Kaprelyants, A.S., 2023. Bacillus licheniformis: a producer of antimicrobial substances, including antimycobacterials, which are feasible for medical applications. Pharmaceutics. 15, 1893.

Soto-Rodriguez, S.A., Gomez-Gil, B., Lozano-Olvera, R., Betancourt-Lozano, M., Morales-Covarrubias, M.S., 2015. Field and experimental evidence of Vibrio parahaemolyticus as the causative agent of acute hepatopancreatic necrosis disease of cultured shrimp (Litopenaeus vannamei) in northwestern Mexico. Appl. Environ. Microbiol. 81, 1689-1699.

Stoica, R.M., Moscovici, M.I.Ș.U., Tomulescu, C.A.T.E.R.I.N.A., Cășărică, A.N.G.E.L.A., Băbeanu, N.A.R.C.I.S.A., Popa, O.V.I.D.I.U., Kahraman, H.D.I.C.A., 2019. Antimicrobial compounds of the genus Bacillus: a review. Rom Biotechnol Lett. 24(6), 1111-9.

Tamura, K., Stecher, G., Peterson, D., Filipski, A., Kumar, S., 2013. MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Mol. Biol. Evol. 30(12), 2725-2729.

Thi, Q.V.C., Nguyen, B.T., Tu, T.D., 2025. The antibiotic resistance of Vibrio parahaemolyticus originated from intensively farmed white shrimp (Litopenaeusvannamei) in Ben Tre and Soc Trang provinces of the Mekong Delta, Vietnam. Vet. Integr. Sci. 23(1), e2025024-1-14.

Thinh, N.Q., Masashi, M., Tran, M.P., 2020. Chemical use in intensive whiteleg shrimp aquaculture in Tra Vinh province, Vietnam. Can Tho University J. Science. 56(2), 70-77.

Thompson, J., Weaver, M.A., Lupatsch, I., Shields, R.J., Plummer, S., Coates, C.J., Rowley, A.F., 2022. Antagonistic activity of lactic acid bacteria against pathogenic vibrios and their potential use as probiotics in shrimp (Penaeus vannamei) culture. Front. mar. sci. 9, 240.

Truong, Q.P., Phan, T.C.T., Vu, H.H., Pham, T.T.N., Huynh, T.G., Vu, N.U., 2021. Isolation of potential probiotic Bacillus subtilis CM3.1 and its effects on water quality and growth performance of the whiteleg shrimp Litopenaeus vannamei in the Mekong Delta, Vietnam. AACL Bioflux. 14(6), 3347-3357.

Tuan, L.C., Nguyen, H.L., Tran, T.H., Te, M.S., Le, T.T.C., Mai, N.C., Tran, N.T., Duong, V.H., Pham, Q.A.K., Truong, V.D., 2021. Use of antibiotics in white leg shrimp (Litopenaeus vannamei Boone, 1931) farming in sandy land of Thua Thien Hue province. Hue University Journal of Science: Agriculture and Rural Development. 130(3D), 131-145.

Ut, T.M., Le, M.K., Nguyen, T.N., Ly, N.D., Tu, A.D., 2021. Pathogenicity determination of Vibrio spp. causing white feces disease on white leg shrimp (Litopenaeus vannamei) in some provinces of the Mekong Delta. Jounal of Vietnam Agricultural Science and Teachnology. 08(129), 125-132.

Wang, Y., Al Farraj, D.A., Vijayaraghavan, P., Hatamleh, A.A., Biji, G.D., Rady, A.M., 2020. Host associated mixed probiotic bacteria induced digestive enzymes in the gut of tiger shrimp Penaeus monodon. Saudi J. Biol. Sci. 27(9), 2479-2484.

Zhang, F., Zhang, J., Lin, G., Chen, X., Huang, H., Xu, C., Chi, H., 2024. Antibiotic Resistance and Genetic Profiles of Vibrio parahaemolyticus Isolated from Farmed Pacific White Shrimp (Litopenaeus vannamei) in Ningde Regions. Microorganisms. 12, 152.

Zorriehzahra, M.J., Banaederakhshan, R.J.A.A.V.S., 2015. Early mortality syndrome (EMS) as new emerging threat in shrimp industry. Adv. Anim. Vet. Sci. 3(2S), 64-72.