Probiotics and Antimicrobial Proteins

, Volume 11, Issue 1, pp 186–197 | Cite as

Functional Characterization of Probiotic Potential of Novel Pigmented Bacterial Strains for Aquaculture Applications

  • Sekar Jinendiran
  • Seenivasan Boopathi
  • Natesan SivakumarEmail author
  • Gopal Selvakumar


The bioprospecting proficient of novel pigmented probiotic strains with respect to aquaculture industry was unexplored hitherto. In this study, we investigated the probiotic potential of novel pigmented bacterial strains isolated from the indigenous soil sediments in their vicinal habitats, which were screened for their antimicrobial activity against aquatic pathogens using agar well diffusion assay. The strains namely Exiguobacterium acetylicum (S01), Aeromonas veronii (V03), and Chryseobacterium joostei (V04) were phenotypically identified and confirmed by 16S rRNA gene sequence analysis. Further characterization revealed that strains S01 and V03 survive relatively in lower pH and higher bile salt concentrations and possess good adherence ability and broad-spectrum antibiotic susceptibility. The isolate S01 exhibited the higher adhesion ability to hydrocarbons (82%) and mannose-specific adhesion (msa) gene expression. Additionally, the probiotic effects were evaluated in Artemia nauplii fed with algae supplemented with S01, V03, and V04 strains (2.7 × 107 cfu/mL) for 3 days under axenic environment. We observed a significant increase (p < 0.05) in the survival rate of Artemia nauplii treated with S01 (83 ± 5%) and V03 (55 ± 5%), whereas the survival rate was only 30 ± 0% in the untreated group. Moreover, the individual length (IL) was increased in treated group S01 (156.7 ± 2.2 μm), V03 (146.1 ± 3.4 μm), and V04 (134.4 ± 2.5 μm) compared with untreated group (116.0 ± 4.8 μm). Our results revealed that E. acetylicum S01 exhibits desirable functional probiotic attributes compared to A. veronii and C. joostei and it would be a promising probiotic strain, which can be efficiently used in the aquaculture applications.


Aquaculture Pigmented bacteria Probiotics Artemia salina Gut microbiota 



The authors wish to acknowledge DST-Science Engineering Research Board (SERB), Government of India, for the financial support under Young Scientist Scheme (SB/YS/LS-05/2014) and DST-PURSE Phase-II, M.KU and UGC-SAP of School of Biotechnology for instrumentation support. The authors wish to acknowledge Dr. B. S. Dileep Kumar, Senior Principal Scientist & Head, CSIR-National Institute for Interdisciplinary Science and Technology (NIIST), Thiruvananthapuram, for permitting to use SEM imaging facility. The authors also wish to acknowledge Mr. Abel Arul Nathan for his help in the statistical analysis.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that there is no conflict of interest.

Ethical Statement

The experiments in this study were performed in accordance with relevant national and international guidelines. This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

12602_2017_9353_MOESM1_ESM.doc (44 kb)
ESM 1 (DOC 43 kb)


  1. 1.
    Toranzo AE, Magarinos B, Romalde JL (2005) A review of the main bacterial fish diseases in mariculture systems. Aquaculture 246(1-4):37–61. CrossRefGoogle Scholar
  2. 2.
    Cordero H, Esteban MA, Cuesta A (2014) Use of probiotic bacteria against bacterial and viral infections in shellfish and fish aquaculture. INTECH Open Sci 8:239–265Google Scholar
  3. 3.
    Balcazar JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114(3-4):173–186. CrossRefGoogle Scholar
  4. 4.
    Fjellheim AJ, Klinkenberg G, Skjermo J, Aasen IM, Vadstein O (2010) Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae. Vet Microbiol 144(1-2):153–159. CrossRefGoogle Scholar
  5. 5.
    Verschuere L, Rombaut G, Sorgeloos P, Verstraete W (2000) Probiotic bacteria as biological agents in aquaculture. Microbiol Mol Biol Rev 64(4):655–671. CrossRefGoogle Scholar
  6. 6.
    Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME (2014) Expert consensus document: the International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol 11(8):506–514. CrossRefGoogle Scholar
  7. 7.
    Irianto A, Austin B (2002) Probiotics in aquaculture. J F Dis 25(11):633–642. CrossRefGoogle Scholar
  8. 8.
    Medina CO, Cortes AL, Martinez AMM (2009) Aerobic Gram-positive heterotrophic bacteria Exiguobacterium mexicanum and Microbacterium sp. in the gut lumen of Artemia franciscana larvae under gnotobiotic conditions. Curr Sci 96:120–129Google Scholar
  9. 9.
    Gunasekara R, Rekecki A, Baruah K, Bossier P, Broeck WV (2010) Evaluation of probiotic effect of Aeromonas hydrophila on the development of the digestive tract of germ-free Artemia franciscana nauplii. J Exp Mar Biol Ecol 393(1-2):78–82. CrossRefGoogle Scholar
  10. 10.
    Wu ZQ, Jiang C, Ling F, Wang GX (2015) Effects of dietary supplementation of intestinal autochthonous bacteria on the innate immunity and disease resistance of grass carp (Ctenopharyngodon idellus). Aquaculture 438:105–114. CrossRefGoogle Scholar
  11. 11.
    Aliabadi MA, Ghasemi MF, Isaazadeh KH (2014) Antimicrobial activity bioactive compounds produced by Exiguobacterium acetylicum PTCC1756 against pathogenic bacteria. Sci J Microbiol 3:55–62Google Scholar
  12. 12.
    Tasiemski A, Massol F, Cuvillier-Hot V, Boidin-Wichlacz C, Roger E, Rodet F, Fournier I, Thomas F, Salzet M (2016) Reciprocal immune benefit based on complementary production of antibiotics by the leech Hirudo verbana and its gut symbiont Aeromonas veronii. Sci Rep 5:17498CrossRefGoogle Scholar
  13. 13.
    Morelli L (2007) In vitro assessment of probiotic bacteria: from survival to functionality. Int Dairy J 17(11):1278–1283. CrossRefGoogle Scholar
  14. 14.
    Kaushik JK, Kumar A, Duary RK, Mohanty AK, Grover S, Batish VK (2009) Functional and probiotic attributes of an indigenous isolate of Lactobacillus plantarum. PLoS One 4(12):e8099. CrossRefGoogle Scholar
  15. 15.
    Zago M, Fornasari ME, Carminati D, Burns P, Suarez V, Vinderola G, Reinheimer J, Giraffa G (2011) Characterization and probiotic potential of Lactobacillus plantarum strains isolated from cheeses. Food Microbiol 28:1033e1040CrossRefGoogle Scholar
  16. 16.
    Grimm A, Cho GS, Hanak A, Dorn A, Huch M, Franz CMAP (2011) Characterization of putative adhesion genes in the potentially probiotic strain Lactobacillus plantarum BFE 5092. Pro Antimicrobial Prot 3(3-4):204–221. CrossRefGoogle Scholar
  17. 17.
    Marques A, Dinh T, Ioakeimidis C, Huys G, Swings J, Verstraete W, Dhont J, Sorgeloos P, Bossier P (2005) Effects of bacteria on Artemia franciscana cultured in different gnotobiotic environments. Appl Environ Microbiol 71(8):4307–4317. CrossRefGoogle Scholar
  18. 18.
    Cintas LM, Rodriguez JM, Fernandez MF, Sletten K, Nes IF, Hernandez PE, Holo H (1995) Isolation and characterization of pediocin L50, a new bacteriocin from Pediococcus acidilactici with a broad inhibitory spectrum. Appl Environ Microbiol 61(7):2643–2648Google Scholar
  19. 19.
    Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, Schleifer KH, Whitman WB (eds) (2009) Bergey’s manual of systematic bacteriology, vol 3, 2nd edn. Springer-Verlag, New YorkGoogle Scholar
  20. 20.
    Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22(22):4673–4680. CrossRefGoogle Scholar
  21. 21.
    Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425Google Scholar
  22. 22.
    Todorov SD, Furtado DN, Saad SMI, Tome E, Franco BDGM (2011) Potential beneficial properties of bacteriocin-producing lactic acid bacteria isolated from smoked salmon. J Appl Microbiol 110(4):971–986. CrossRefGoogle Scholar
  23. 23.
    CLSI (2011) Clinical and Laboratory Standards Institute (CLSI): Performance standards for antimicrobial susceptibility testing: twenty-first informational supplement M100–S21. Wayne, PA, USA: 30:65Google Scholar
  24. 24.
    Sorokulova IB, Pinchuk IV, Denayrolles M, Osipova IG, Huang JM, Cutting SM (2008) The safety of two Bacillus probiotics strains for human use. Digest Dis Sci 53(4):954–963. CrossRefGoogle Scholar
  25. 25.
    Sorgeloos P, Lavens P, Leger P, Tackaert W, Versichele D (1986) Manual for the Culture and use of brine shrimp Artemia in aquaculture. Artemia Reference Center, Faculty of Agriculture, State University of Ghent, Belgium, p 318Google Scholar
  26. 26.
    Decamp O, Moriarty DJW, Lavens P (2008) Probiotics for shrimp larviculture: review of field data from Asia and Latin America. Aquac Res 39:334e8CrossRefGoogle Scholar
  27. 27.
    Cruz PM, Ibanez AL, Monroy Hermosillo OA, Ramirez Saad HC (2012) Use of probiotics in aquaculture. Int Scholarly Res Network Microbiol 13:916845Google Scholar
  28. 28.
    Maeda M, Liao IC (1994) Microbial processes in aquaculture environment and their importance for increasing crustacean production. Japan Int Res Center Agric Sci 28:283–288Google Scholar
  29. 29.
    Garriques D, Arevalo G (1995) An evaluation of the production and use of a live bacterial isolate to manipulate the microbial flora in the commercial production of Penaeus vannamei postlarvae in Ecuador, 53–59. In: Browdy CL, Hopkins JS (ed) Swimming through troubled water. Proceedings of the Special Session on Shrimp Farming, Aquaculture 95. World Aquaculture Society Baton Rouge, LaGoogle Scholar
  30. 30.
    Rengpipat S, Phianphak W, Piyatiratitivorakul S, Menasveta P (1998) Effects of a probiotic bacterium on black tiger shrimp Penaeus monodon survival and growth. Aquaculture 167(3-4):301–313. CrossRefGoogle Scholar
  31. 31.
    Powedchagun P, Suzuki H, Rengpipat S (2011) Characterization of a probiotic Bacillus S11 bacterium of black tiger shrimp Penaeus monodon. Songklanakarin J Sci Technol 33:1–8Google Scholar
  32. 32.
    Shanthakumar SP, Duraisamy P, Ganga Selvanesan BCV, Ramaraj V, David BV (2015) Broad spectrum antimicrobial compounds from the bacterium Exiguobacterium mexicanum MSSRFS9. Microbiol Res 178:59–65. CrossRefGoogle Scholar
  33. 33.
    Nikoskelainen S, Salminen S, Bylund G, Ouwehand A (2001) Characterisation of the properties of human and dairy-derived probiotics for prevention of infectious diseases in fish. Appl Environ Microbiol 67:2430e–22435CrossRefGoogle Scholar
  34. 34.
    Balcazar JL, Vendrell D, Blas I, Ruiz ZI, Muzquiz JL, Girones O (2008) Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture 278:188e91CrossRefGoogle Scholar
  35. 35.
    Giri SS, Sukumaran V, Dangi NK (2012) Characteristics of bacterial isolates from the gut of freshwater fish, Labeo rohita that may be useful as potential probiotic bacteria. Pro Antimicrobial Prot 4:238e242Google Scholar
  36. 36.
    Succi M, Tremonte P, Reale A, Sorrentino E, Grazia L, Pacifico S (2005) Bile salt and acid tolerance of Lactobacillus rhamnosus strains isolated from Parmigiano Reggiano cheese. FEMS Microbiol Let 244(1):129–137. CrossRefGoogle Scholar
  37. 37.
    Berecka MP, Wasko A, Paduch R, Skrzypek T, Bartnicka AS (2014) The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus. Antonie Van Leeuwenhoek 106(4):751–762. CrossRefGoogle Scholar
  38. 38.
    Mohapatra S, Chakraborty T, Prusty AK, Das P, Paniprasad K, Mohanta KN (2012) Use different microbial probiotic in the diet of rohu, Labeorohita fingerlings: effect on growth, nutrient digestibility and retention, digestive enzyme activities and intestinal microflora. Aquacult Nutr 18(1):1–11. CrossRefGoogle Scholar
  39. 39.
    Hosoi T, Kiuchi K (2003) Handbook of fermented functional foods. CRC Press 227–245Google Scholar
  40. 40.
    Mathur S, Singh R (2005) Antibiotic resistance in food lactic acid bacteria a review. Int J Food Microbiol 105(3):281–295. CrossRefGoogle Scholar
  41. 41.
    Pridgeon JW, Klesius PH, Mu X, Song L (2011) An in vitro screening method to evaluate chemicals as potential chemotherapeutants to control Aeromonas hydrophila infection in channel catfish. J Appl Microbiol 111(1):114–124. CrossRefGoogle Scholar
  42. 42.
    Bharathi K, Akila M, Selvakumar D (2016) Evaluation of probiotic characterization of marine bacteria and its growth performance in zebrafish. Imper J Interdisci Res 2:2129–2137Google Scholar
  43. 43.
    Schrehardt A (1987) Ultrastructural investigations of the filter-feeding apparatus and the alimentary canal of Artemia. In: Sorgeloos P, Bengtson DA, Decleir W, Jaspers E (eds) Artemia research and its applications. Universa Press, WetterenGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  • Sekar Jinendiran
    • 1
  • Seenivasan Boopathi
    • 1
  • Natesan Sivakumar
    • 1
    Email author
  • Gopal Selvakumar
    • 2
  1. 1.Department of Molecular Microbiology, School of BiotechnologyMadurai Kamaraj UniversityMaduraiIndia
  2. 2.Department of MicrobiologyAlagappa UniversityKaraikudiIndia

Personalised recommendations