Isolation, Characterization, and Identification of Bacillus spp. Strains from the Digestive Tract of Mad Carp (Leptobarbus hoevenii) and Their Potential Probiotic Properties
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Abstract
The present study aimed to collect isolates of Bacillus spp. from the digestive tracts of mad carps (Leptobarbus hoevenii) and to screen these for probiotic properties. In this study, 85 bacterial isolates were obtained from the digestive tracts of mad carps. Of these, 73 isolates were Gram-positive, rod shaped, and catalase positive, while only 48 bacterial isolates were endospore forming. Evaluation of antagonistic effects of Bacillus spp. against pathogenic bacteria causing motile Aeromonas septicemia (MAS) in fish indicated that a total of 18 out of the 73 isolates exhibited antimicrobial activity, and especially the isolate MCSU14 expressed a significantly enlarged zone of inhibition (p<0.05). Study on hemolytic activity of these 18 antimicrobial isolates revealed that 6 isolates were γ-hemolytic. Furthermore, the 6 γ-hemolytic isolates survived acidic and bile salt exposures, and produced extracellular digestive enzymes. Among these six Bacillus spp., the isolate MCSU14 exhibited the most potent probiotic properties. Based on its biochemical characteristics and molecular analyses, the Bacillus sp. isolate MCSU14 is closely related to Bacillus velezensis. Our findings indicate that Bacillus spp. isolates from the digestive tracts of mad carp can be screened to find potent probiotics against MAS in aquaculture.
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References
Beveridge, M. C. M.; Thilsted, S. H.; Phillips, M. J.; Metian, M.; Troell, M.; Hall, S. J. Meeting the food and nutrition needs of the poor: The role of fish and the opportunities and challenges emerging from the rise of aquaculture. J. Fish Biol. 2013, 83(4), 1067-1084. https://doi.org/10.1111/jfb.12187
Zhang, W.; Belton, B.; Edwards, P.; Henriksson, P. J. G.; Little, D. C.; Newton, R.; Troell, M. Aquaculture will continue to depend more on land than sea. Nature 2022, 603, E2-E4. https://doi.org/10.1038/s41586-021-04331-3
Hanson, L. A.; Hemsteet, W. G.; Hawke, J. P. Motile Aeromonas Septicemia (MAS) in Fish; Southern Regional Aquaculture Center: USA, 2019; SRAC Publication No. 0478.
Nhinh, D. T.; Le, D. V.; Van, K. V.; Huong Giang, N. T.; Dang, L. T.; Hoai, T. D. Prevalence, virulence gene distribution, and alarming multidrug resistance of Aeromonas hydrophila associated with disease outbreaks in freshwater aquaculture. Antibiotics 2021, 10(5), 532. https://doi.org/10.3390/antibiotics10050532
Zhao, X. L.; Jin, Z. H.; Di, G. L.; Li, L.; Kong, X. H. Molecular characteristics, pathogenicity, and medication regimen of Aeromonas hydrophila isolated from common carp (Cyprinus carpio L.). J. Vet. Med. Sci. 2019, 81(12), 1769-1775. https://doi.org/10.1292/jvms.19-0025
Ran, C.; Qin, C.; Xie, M.; Zhang, J.; Li, J.; Xie, Y.; Wang, Y.; Li, S.; Liu, L.; Fu, X.; Lin, Q.; Li, N.; Liles, M. R.; Zhou, Z. Aeromonas veronii and aerolysin are important for the pathogenesis of motile Aeromonad septicemia in cyprinid fish. Environ. Microbiol. 2018, 20(9), 3442-3456. https://doi.org/10.1111/1462-2920.14390
Chen, F.; Sun, J.; Han, Z.; Yang, X.; Xian, J. A.; Lv, A.; Hu, X.; Shi, H. Isolation, identification, and characteristics of Aeromonas veronii from diseased crucian carp (Carassius auratus gibelio). Front. Microbiol. 2019, 10, 2742. https://doi.org/10.3389/fmicb.2019.02742
Hoai, T. D.; Trang, T. T.; Van Tuyen, N.; Giang, N. T. H.; Van Van, K. Aeromonas veronii caused disease and mortality in channel catfish in Vietnam. Aquaculture 2019, 513, 734425. https://doi.org/10.1016/j.aquaculture.2019.734425
Dos Santos, S. B.; Alarcon, M. F.; Ballaben, A. S.; Harakava, R.; Galetti, R.; Guimarães, M. C.; Natori, M. M.; Takahashi, L. S.; Ildefonso, R.; Rozas-Serri, M. First report of Aeromonas veronii as an emerging bacterial pathogen of farmed Nile tilapia (Oreochromis niloticus) in Brazil. Pathogens 2023, 12(8), 1020. https://doi.org/10.3390/pathogens12081020
Kumar, K.; Prasad, K.; Tripathi, G.; Raman, R.; Kumar, S.; Tembhurne, M.; Purushothaman, C. Isolation, identification, and pathogenicity of a virulent Aeromonas jandaei associated with mortality of farmed Pangasianodon hypophthalmus, in India. Isr. J. Aquacult. Bamidgeh 2014, 67. https://doi.org/10.46989/001c.20727
Assane, I. M.; de Sousa, E. L.; Valladão, G. M. R.; Tamashiro, G. D.; Criscoulo-Urbinati, E.; Hashimoto, D. T.; Pilarski, F. Phenotypic and genotypic characterization of Aeromonas jandaei involved in mass mortalities of cultured Nile tilapia, Oreochromis niloticus (L.) in Brazil. Aquaculture 2021, 541, 736848. https://doi.org/10.1016/j.aquaculture.2021.736848
Carriero, M. M.; Mendes Maia, A. A.; Moro Sousa, R. L.; Henrique-Silva, F. Characterization of a new strain of Aeromonas dhakensis isolated from diseased pacu fish (Piaractus mesopotamicus) in Brazil. J. Fish Dis. 2016, 39(11), 1285-1295. https://doi.org/10.1111/jfd.12457
Bartie, K. L.; Desbois, A. P. Aeromonas dhakensis: A zoonotic bacterium of increasing importance in aquaculture. Pathogens 2024, 13(6), 465. https://doi.org/10.3390/pathogens13060465
Bondad-Reantaso, M. G.; MacKinnon, B.; Karunasagar, I.; Fridman, S.; Alday-Sanz, V.; Brun, E.; Le Groumellec, M.; Li, A.; Surachetpong, W.; Karunasagar, I.; Hao, B.; Dall’Occo, A.; Urbani, R.; Caputo, A. Review of alternatives to antibiotic use in aquaculture. Rev. Aquacult. 2023, 15(4), 1421-1451. https://doi.org/10.1111/raq.12786
Hoseinifar, S. H.; Sun, Y. Z.; Wang, A.; Zhou, Z. Probiotics as means of disease control in aquaculture, a review of current knowledge and future perspectives. Front. Microbiol. 2018, 9, 2429. https://doi.org/10.3389/fmicb.2018.02429
Martínez Cruz, P.; Ibáñez, A. L.; Monroy Hermosillo, O. A.; Ramírez Saad, H. C. Use of probiotics in aquaculture. ISRN Microbiol. 2012, 2012, 916845. https://doi.org/10.5402/2012/916845
Talukder Shefat, S. H. Probiotic strains used in aquaculture. Int. Res. J. Microbiol. 2018, 7(2), 43-55. https://doi.org/10.14303/irjm.2018.023
Lin, S.; Mao, S.; Guan, Y.; Luo, L.; Luo, L.; Pan, Y. Effects of dietary chitosan oligosaccharides and Bacillus coagulans on the growth, innate immunity, and resistance of koi (Cyprinus carpio koi). Aquaculture 2012, 342-343, 36-41. https://doi.org/10.1016/j.aquaculture.2012.02.009
Liu, C. H.; Chiu, C. H.; Wang, S. W.; Cheng, W. Dietary administration of the probiotic, Bacillus subtilis E20, enhances the growth, innate immune responses, and disease resistance of the grouper, Epinephelus coioides. Fish Shellfish Immunol. 2012, 33(4), 699-706. https://doi.org/10.1016/j.fsi.2012.06.012
Bernardeau, M.; Lehtinen, M. J.; Forssten, S. D.; Nurminen, P. Importance of the gastrointestinal life cycle of Bacillus for probiotic functionality. J. Food Sci. Technol. 2017, 54(8), 2570-2584. https://doi.org/10.1007/s13197-017-2688-3
Cheng, A. C.; Lin, H. L.; Shiu, Y. L.; Tyan, Y. C.; Liu, C. H. Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-Fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture. Fish Shellfish Immunol. 2017, 67, 270-279. https://doi.org/10.1016/j.fsi.2017.06.006
Soto-Marfileño, K. A.; Molina Garza, Z. J.; Flores, R. G.; Molina-Garza, V. M.; Ibarra-Gámez, J. C.; Gil, B. G.; Galaviz-Silva, L. Genomic characterization of Bacillus pumilus Sonora, a strain with inhibitory activity against Vibrio parahaemolyticus-AHPND and probiotic candidate for shrimp aquaculture. Microorganisms 2024, 12(8), 1623. https://doi.org/10.3390/microorganisms12081623
Rahayu, S.; Amoah, K.; Huang, Y.; Cai, J.; Wang, B.; Shija, V. M.; Jin, X.; Anokyewaa, M. A.; Jiang, M. Probiotics application in aquaculture: Its potential effects, current status in China, and future prospects. Front. Mar. Sci. 2024, 11, 1455905. https://doi.org/10.3389/fmars.2024.1455905
Van Doan, H.; Hoseinifar, S. H.; Ringø, E.; Ángeles Esteban, M.; Dadar, M.; Dawood, M. A. O.; Faggio, C. Host-associated probiotics: A key factor in sustainable aquaculture. Rev. Fish. Sci. Aquacult. 2020, 28(1), 16-42. https://doi.org/10.1080/23308249.2019.1643288
Nakharuthai, C.; Boonanuntanasarn, S.; Kaewda, J.; Manassila, P. Isolation of potential probiotic Bacillus spp. from the intestine of Nile tilapia to construct recombinant probiotic expressing CC chemokine and its effectiveness on innate immune responses in Nile tilapia. Animals 2023, 13(6), 986. https://doi.org/10.3390/ani13060986
Kang, M.; Su, X.; Yun, L.; Shen, Y.; Feng, J.; Yang, G.; Meng, X.; Zhang, J.; Chang, X. Evaluation of probiotic characteristics and whole genome analysis of Bacillus velezensis R-71003 isolated from the intestine of common carp (Cyprinus carpio L.) for its use as a probiotic in aquaculture. Aquac. Rep. 2022, 25, 101254. https://doi.org/10.1016/j.aqrep.2022.101254
Yousuf, S.; Jamal, M. T.; Al-Farawati, R. K.; Al-Mur, B. A.; Singh, R. Evaluation of Bacillus paramycoides strains isolated from Channa fish sp. on growth performance of Labeo rohita fingerlings challenged by fish pathogen Aeromonas hydrophila MTCC 12301. Microorganisms 2023, 11(4), 842. https://doi.org/10.3390/microorganisms11040842
Srithongthum, S.; Au, H.-L.; Amornsakun, T.; Musikarun, P.; Mok, W. J.; Halid, N. F. A.; Kawamura, G.; Lim, L. S. Reproductive characteristics of the pond-farmed sultan fish (Leptobarbus hoevenii). J. Ilmiah Perikanan Kelautan 2021, 13(2), 171-180. https://doi.org/10.20473/jipk.v13i2.27264
Sunarto; Sukenda; Widanarni. Screening of probiotic bacteria from intestine and culture environment of Hoeven’s slender carp Leptobarbus hoeveni Blkr to control pathogenic bacteria. J. Akuakult. Indones. 2010, 9(2), 127-135. https://doi.org/10.19027/jai.9.127-135
Hellany, H.; Assaf, J. C.; Barada, S.; el-Badan, D.; Hajj, R. E.; Abou Najem, S.; Abou Fayad, A. G.; Khalil, M. I. Isolation and characterization of Bacillus subtilis BSP1 from soil: Antimicrobial activity and optimization of fermentation conditions. Processes 2024, 12(8), 1621. https://doi.org/10.3390/pr12081621
Lertcanawanichakul, M.; Sawangnop, S. A comparison of two methods used for measuring the antagonistic activity of Bacillus species. Walailak J. Sci. Technol. (WJST) 2008, 5(2), 161-171.
Baharudin, M. M. A.; Ngalimat, M. S.; Mohd Shariff, F.; Balia Yusof, Z. N.; Karim, M.; Baharum, S. N.; Sabri, S. Antimicrobial activities of Bacillus velezensis strains isolated from stingless bee products against methicillin-resistant Staphylococcus aureus. PLOS One 2021, 16(5), e0251514. https://doi.org/10.1371/journal.pone.0251514
Ritter, A. C.; Paula, A.; Correa, F.; Veras, F. F.; Brandelli, A. Characterization of Bacillus subtilis available as probiotics. J. Microbiol. Res. 2018, 8(2), 23-32.
Dabiré, Y.; Somda, N. S.; Somda, M. K.; Compaoré, C. B.; Mogmenga, I.; Ezeogu, L. I.; Traoré, A. S.; Ugwuanyi, J. O.; Dicko, M. H. Assessment of probiotic and technological properties of Bacillus spp. isolated from Burkinabe Soumbala. BMC Microbiol. 2022, 22(1), 228. https://doi.org/10.1186/s12866-022-02642-7
Santong, K.; Chunglok, W.; Lertcanwanichakul, M.; Bangrak, P. Screening and isolation of Bacillus sp. producing thermotolerant protease from raw milk. Walailak J. Sci. Technol. 2008, 5(2), 151-160.
Proca, I. G.; Diguta, C. F.; Jurcoane, S.; Matei, F. Screening of halotolerant bacteria producing hydrolytic enzymes with biotechnology applications. Sci. Bull. Ser. F Biotechnol. 2020, XXIV, 197-202.
Latorre, J. D.; Hernandez-Velasco, X.; Wolfenden, R. E.; Vicente, J. L.; Wolfenden, A. D.; Menconi, A.; Bielke, L. R.; Hargis, B. M.; Tellez, G. Evaluation and selection of Bacillus species based on enzyme production, antimicrobial activity, and biofilm synthesis as direct-fed microbial candidates for poultry. Front. Vet. Sci. 2016, 3, 95. https://doi.org/10.3389/fvets.2016.00095
Zalma, S. A.; El-Sharoud, W. M. Diverse thermophilic Bacillus species with multiple biotechnological activities are associated within the Egyptian soil and compost samples. Sci. Prog. 2021, 104(4), 368504211055277. https://doi.org/10.1177/00368504211055277
Tamura, K.; Stecher, G.; Kumar, S. MEGA11: Molecular evolutionary genetics analysis version 11. Mol. Biol. Evol. 2021, 38 (7), 3022-3027. https://doi.org/10.1093/molbev/msab120
Wu, Z.; Qi, X.; Qu, S.; Ling, F.; Wang, G. Dietary supplementation of Bacillus velezensis B8 enhances immune response and resistance against Aeromonas veronii in grass carp. Fish Shellfish Immunol. 2021, 115, 14-21. https://doi.org/10.1016/j.fsi.2021.05.012
Zhang, D. X.; Kang, Y. H.; Zhan, S.; Zhao, Z. L.; Jin, S. N.; Chen, C.; Zhang, L.; Shen, J.-Y.; Wang, C. F.; Wang, G. Q.; Shan, X. F.; Qian, A. D. Effect of Bacillus velezensis on Aeromonas veronii-induced intestinal mucosal barrier function damage and inflammation in crucian carp (Carassius auratus). Front. Microbiol. 2019, 10, 2663. https://doi.org/10.3389/fmicb.2019.02663
Li, X.; Gao, X.; Zhang, S.; Jiang, Z.; Yang, H.; Liu, X.; Jiang, Q.; Zhang, X. Characterization of a Bacillus velezensis with antibacterial activity and inhibitory effect on common aquatic pathogens. Aquaculture 2020, 523, 735165. https://doi.org/10.1016/j.aquaculture.2020.735165
Zhou, P.; Chen, W.; Zhu, Z.; Zhou, K.; Luo, S.; Hu, S.; Xia, L.; Ding, X. Comparative study of Bacillus amyloliquefaciens X030 on the intestinal flora and antibacterial activity against Aeromonas of grass carp. Front. Cell. Infect. Microbiol. 2022, 12, 815436. https://doi.org/10.3389/fcimb.2022.815436
Yao, Y. Y.; Xia, R.; Yang, Y. L.; Hao, Q.; Ran, C.; Zhang, Z.; Zhou, Z. G. Study about the combination strategy of Bacillus subtilis wt55 with AiiO-AIO6 to improve the resistance of zebrafish to Aeromonas veronii infection. Fish Shellfish Immunol. 2022, 128, 447-454. https://doi.org/10.1016/j.fsi.2022.08.019
Nayak, A.; Harshitha, M.; Dubey, S.; Munang'andu, H. M.; Chakraborty, A.; Karunasagar, I.; Maiti, B. Evaluation of probiotic efficacy of Bacillus subtilis RODK28110C3 against pathogenic Aeromonas hydrophila and Edwardsiella tarda using in vitro studies and in vivo gnotobiotic zebrafish gut model system. Probiotics Antimicrob. Proteins 2024, 16(5), 1623-1637. https://doi.org/10.1007/s12602-023-10127-w
Agustina, P.; Sarjito, A. H.; Haditomo, C. Study of Bacillus methylotrophicus as a probiotic candidate bacteria with different concentrations against Aeromonas hydrophila on water as a cultivation media of tilapia (Oreochromis niloticus). IOP Conf. Ser. Earth Environ. Sci. 2019, 246(1), 012030. https://doi.org/10.1088/1755-1315/246/1/012030
Kumariya, R.; Garsa, A. K.; Rajput, Y. S.; Sood, S. K.; Akhtar, N.; Patel, S. Bacteriocins: Classification, synthesis, mechanism of action and resistance development in food spoilage causing bacteria. Microb. Pathog. 2019, 128, 171-177. https://doi.org/10.1016/j.micpath.2019.01.002
Markelova, N.; Chumak, A. Antimicrobial activity of Bacillus cyclic lipopeptides and their role in the host adaptive response to changes in environmental conditions. Int. J. Mol. Sci. 2025, 26(1), 336. https://doi.org/10.3390/ijms26010336
Olishevska, S.; Nickzad, A.; Déziel, E. Bacillus and Paenibacillus secreted polyketides and peptides involved in controlling human and plant pathogens. Appl. Microbiol. Biotechnol. 2019, 103(3), 1189-1215. https://doi.org/10.1007/s00253-018-9541-0
Chen, R.; Zhou, Z.; Cao, Y.; Bai, Y.; Yao, B. High yield expression of an AHL-Lactonase from Bacillus sp. B546 in Pichia pastoris and its application to reduce Aeromonas hydrophila mortality in aquaculture. Microb. Cell Fact. 2010, 9, 39. https://doi.org/10.1186/1475-2859-9-39
Li, L.; Hu, K.; Hong, B.; Lu, X.; Liu, Y.; Xie, J.; Jin, S.; Zhou, S.; Zhao, Q.; Lu, H.; Liu, Q.; Gao, M.; Li, X.; Fu, C.; Xu, H.; Guo, M.; Ma, R.; Zhang, H.; Qian, D. The inhibitory effect of Bacillus amyloliquefaciens L1 on Aeromonas hydrophila and its mechanism. Aquaculture 2021, 539, 736590. https://doi.org/10.1016/j.aquaculture.2021.736590
Simón, R.; Docando, F.; Nuñez-Ortiz, N.; Tafalla, C.; Díaz-Rosales, P. Mechanisms used by probiotics to confer pathogen resistance to teleost fish. Front. Immunol. 2021, 12, 653025. https://doi.org/10.3389/fimmu.2021.653025
Fuller, R. Probiotics in man and animals. J. Appl. Bacteriol. 1989, 66(5), 365-378. https://doi.org/10.1111/j.1365-2672.1989.tb05105.x
Golnari, M.; Bahrami, N.; Milanian, Z.; Rabbani Khorasgani, M.; Asadollahi, M. A.; Shafiei, R.; Fatemi, S. S.-A. Isolation and characterization of novel Bacillus strains with superior probiotic potential: Comparative analysis and safety evaluation. Sci. Rep. 2024, 14(1), 1457. https://doi.org/10.1038/s41598-024-51823-z
Altavas, P. J. dR.; Amoranto, M. B. C.; Kim, S. H.; Kang, D.-K.; Balolong, M. P.; Dalmacio, L. M. M. Safety assessment of five candidate probiotic Lactobacilli using comparative genome analysis. Access Microbiol. 2024, 6(1), 000715.v4. https://doi.org/10.1099/acmi.0.000715.v4
Hancz, C. Application of probiotics for environmentally friendly and sustainable aquaculture: A review. Sustainability 2022, 14 (22), 15479. https://doi.org/10.3390/su142215479
Liu, H.; Wang, S.; Cai, Y.; Guo, X.; Cao, Z.; Zhang, Y.; Liu, S.; Yuan, W.; Zhu, W.; Zheng, Y.; Xie, Z.; Guo, W.; Zhou, Y. Dietary administration of Bacillus subtilis HAINUP40 enhances growth, digestive enzyme activities, innate immune responses and disease resistance of tilapia, Oreochromis niloticus. Fish Shellfish Immunol. 2017, 60, 326-333. https://doi.org/10.1016/j.fsi.2016.12.003
Lee, G.; Heo, S.; Kim, T.; Na, H.-E.; Park, J.; Lee, E.; Lee, J.-H.; Jeong, D.-W. Discrimination of Bacillus subtilis from other Bacillus species using specific oligonucleotide primers for the pyruvate carboxylase and shikimate dehydrogenase genes. J. Microbiol. Biotechnol. 2022, 32(8), 1011-1016. https://doi.org/10.4014/jmb.2205.05014
Ruiz-García, C.; Béjar, V.; Martínez-Checa, F.; Llamas, I.; Quesada, E. Bacillus velezensis sp. nov., a surfactant-producing bacterium isolated from the river Vélez in Málaga, southern Spain. Int. J. Syst. Evol. Microbiol. 2005, 55(Pt 1), 191-195. https://doi.org/10.1099/ijs.0.63310-0
Islam, M. I.; Seo, H.; Redwan, A.; Kim, S.; Lee, S.; Siddiquee, M.; Song, H.-Y. In vitro and in vivo anti-Clostridioides difficile effect of a probiotic Bacillus amyloliquefaciens strain. Polish J. Microbiol. 2022, 32(1), 46-55. https://doi.org/10.4014/jmb.2107.07057
Sumpavapol, P.; Tongyonk, L.; Tanasupawat, S.; Chokesajjawatee, N.; Luxananil, P.; Visessanguan, W. Bacillus siamensis sp. nov., isolated from salted crab (Poo-Khem) in Thailand. Int. J. Syst. Evol. Microbiol. 2010, 60(10), 2364-2370. https://doi.org/10.1099/ijs.0.018879-0
Huynh, T.; Vörös, M.; Kedves, O.; Turbat, A.; Sipos, G.; Leitgeb, B.; Kredics, L.; Vágvölgyi, C.; Szekeres, A. Discrimination between the two closely related species of the operational group B. amyloliquefaciens based on whole-cell fatty acid profiling. Microorganisms 2022, 10(2), 418. https://doi.org/10.3390/microorganisms10020418
Ngalimat, M. S.; Yahaya, R. S. R.; Baharudin, M. M. A.-A.; Yaminudin, S. M.; Karim, M.; Ahmad, S. A.; Sabri, S. A Review on the biotechnological applications of the operational group Bacillus amyloliquefaciens. Microorganisms 2021, 9(3), 614. https://doi.org/10.3390/microorganisms9030614
Fan, B.; Blom, J.; Klenk, H.-P.; Borriss, R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “Operational group B. amyloliquefaciens” within the B. subtilis species complex. Front. Microbiol. 2017, 8, 22. https://doi.org/10.3389/fmicb.2017.00022
