Aquaculture International

, Volume 26, Issue 4, pp 959–980 | Cite as

In vitro and in vivo evaluation of probiotic properties of Enterobacter cloacae in Kenyi cichlid, Maylandia lombardoi

  • Nisha Rajagopalan Girijakumari
  • Kannapiran Ethiraja
  • Prabhu Narayanasamy Marimuthu


In this study, Enterobacter cloacae bacterium was isolated from curd and its antibacterial potential against the pathogen Plesiomonas shigelloides was evaluated using the freshwater ornamental fish Kenyi cichlid (Maylandia lombardoi). Among the bacterial isolates, E. cloacae exhibited tolerance to acidic pH 2 and demonstrated the highest antibacterial activity against P. shigelloides in various in vitro assays. Dietary supplementation of E. cloacae with prebiotic supplement 2% mannan oligosaccharide improved the growth performance and reduced the toxic metabolites such as nitrite in culture tank water of Kenyi cichlid. In vivo application of E. cloacae with mannan oligosaccharide significantly elevated (p < 0.05) white blood cell counts (88.47 ± 2.15 103 mm−3) and respiratory burst activity (0.243 ± 0.007) of Kenyi cichlid when challenged with P. shigelloides. Intestinal morphology of Kenyi cichlid fish treated with probiotic and prebiotic combinations showed improved intestinal architectures. The present findings confirm that the isolated bacterium E. cloacae is a potential probiotic and can be used effectively to prevent infection of P. shigelloides in freshwater ornamental fish culture.


E. cloacae Fish disease Kenyi cichlid Probiotics P. shigelloides 



The authors gratefully acknowledge University Grant Commission (UGC), India, for providing grants (UGC Major Project—40-401-2011 (SR) and 04.07.2011) to pursue this work.


  1. Abareethan M, Amsath A (2015) Characterization and evaluation of probiotic fish feed. Int J Pure Appl Zool 3(2):148–153Google Scholar
  2. Abdul Kader Mohideen MM, Selva Mohan T, Peer Mohamed S, Zahir Hussain MI (2010) Effect of probiotic bacteria on the growth rate of fresh water fish, Catla catla. Int J Biol Technol 1:113–117Google Scholar
  3. Aly SM, Ahmed YAG, Ghareeb AAA, Mohamed MF (2008) Studies on Bacillus subtilise and Lactobacillus acidophilus, as potential probiotics, on the immune response and resistance of Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fish Shellfish Immunol 25:128–136CrossRefPubMedGoogle Scholar
  4. Andrew EA, Melissa GB, Marc EF, Peter GV, Jonathan PZ, Sabino Z (2001) Diversity and detection of nitrate assimilation genes in marine bacteria. Appl Environ Microbiol 67:5343–5348CrossRefGoogle Scholar
  5. Austin B, Stobie M, Robertson PAW (1992) Citrobacter freundii: the cause of gastro-enteritis leading to progressive low level mortalities infarmed rainbow trout, Oncorhynchus mykiss walbaum, in Scotland. B Eur Assoc Fish Pat 12:166–168Google Scholar
  6. Azza M, Abd ER, Yassir AEK, Adel MES (2009) Micrococcus luteus and Pseudomonas species as probiotics for promoting the growth performance and health of Nile tilapia, Oreochromis niloticus. Fish Shellfish Immunol 27:175–180CrossRefGoogle Scholar
  7. Balcazar JL, Blas ID, Ruiz-Zarzuela I, Cunningham D, Vendrell D, Muzquiz JL (2006) The role of probiotics in aquaculture. Vet Microbiol 114:173–186CrossRefPubMedGoogle Scholar
  8. Blaxhall PC, Daisley KE (1973) Routine haematological methods for use with fish blood. Fish Biol 5:771–781CrossRefGoogle Scholar
  9. Capkin E, Altinok I (2009) Effects of dietary probiotic supplementations on prevention/treatment of yersiniosis disease. J Appl Microbiol 106:1147–1153CrossRefPubMedGoogle Scholar
  10. Cerezuela R, Meseguer J, Esteban MA (2011) Current knowledge in synbiotic use for fish aquaculture: a review. J Aquac Res Dev S1:008Google Scholar
  11. Coman MM, Verdenelli MC, Cecchini C, Silvi S, Orpianesi C, Boyko N, Cresci A (2014) In vitro evaluation of antimicrobial activity of Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502 and SYNBIO against pathogens. J Appl Microbiol 117:518–527CrossRefPubMedGoogle Scholar
  12. Cruz JM, Saraiva A, Eiras JC, Branco R, Sousa JC (1986) An outbreak of Plesiomonas shigelloides in farmed rainbow trout, Salmo gairdneri Richardson, in Portugal. B Eur Assoc Fish Pathol 6:20–22Google Scholar
  13. Dalben M, Varkulja G, Basso M, Krebs VL, Gibelli MA et al (2008) Investigation of an outbreak of Enterobacter cloacae in a neonatal unit and review of the literature. J Hosp Infect 70:7–14CrossRefPubMedGoogle Scholar
  14. Daniels C, Hoseinifar SH (2014) Prebiotic applications in shellfish. In: Ringo E, Merrifield DL (eds) Aquaculture nutrition: gut health, probiotics and prebiotics. Wiley-Blackwell scientific Publication, LondonGoogle Scholar
  15. Davoodabadi A, Mohammad MSD, Abbas RF, Masoumeh D, Mohammad kazem SY, Farzaneh AH (2015) Antibacterial activity of Lactobacillus spp. isolated from the feces of healthy infants against enteropathogenic bacteria. Anaerobe 34:3–8CrossRefGoogle Scholar
  16. Dimitroglou A, Davies SJ, Sweetman J, Divanach P, Chatzifotis S (2010) Dietary supplementation of mannan oligosaccharide on white sea bream (Diplodous sargus L.) larvae: effects on development, gut morphology and salinity tolerance. Aquac Res 41:245–251CrossRefGoogle Scholar
  17. Donaldson JR (2016) 117 Probiotics as an alternative for antimicrobials and improving energy availability. Journal of Animal Science Abstract - Symposium Abstracts: Antimicrobial Resistance Symposium. Vol 94. p 57Google Scholar
  18. Gonzalez CJ, Lopez Diaz TM, Garsia Lopez ML, Prieto M, Otero A (1999) Bacterial microfl ora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss). J Food Prot 62:1270–1277CrossRefPubMedGoogle Scholar
  19. Gram L, Melchiorsen J, Spangaard B, Huber I, Nielsen TF (1999) Inhibition of Vibrio anguillarum by Pseudomonas fluorescens AH2, a possible probiotic treatment of fish. Appl Environ Microbiol 65:969–973PubMedPubMedCentralGoogle Scholar
  20. Gupta A, Paromita G, Asha D (2014) Dietary supplementation of probiotics affects growth, immune response and disease resistance of Cyprinus carpio fry. Fish Shellfish Immunol 41:113–119CrossRefPubMedGoogle Scholar
  21. Guzman-Villanueva LT, Tovar Ramírez D, Gisbert E, Cordero H, Guardiola FA, Cuesta A, Meseguer J, Ascencio Valle F, Esteban MA (2014) Dietary administration of β-1,3/1,6- glucan and probiotic strain Shewanella putrefaciens, single or combined, on gilthead seabream growth, immune responses and gene expression. Fish Shellfish Immunol 39:34–41CrossRefPubMedGoogle Scholar
  22. Herich R, Levkut M (2002) Lactic acid bacteria, probiotics and immune system. Vet Med – Czech 47:169–180CrossRefGoogle Scholar
  23. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergey’s manual of determinative bacteriology, 9th edn. Williams & Wilkins, MarylandGoogle Scholar
  24. Hoque MZ, Akter F, Hossain KM, Rahman MSM, Billah MM, Islam KMD (2010) Isolation, identification and analysis of probiotic properties of Lactobacillus spp. from selective regional yoghurts. World J Dairy Food Sci 5:39–46Google Scholar
  25. Hu Q, Lin Q, Shi C, Fu X, Li N, Liu L, Wu S (2014) Isolation and identification of a pathogenic Plesiomonas shigelloides from diseased grass carp. Wei Sheng Wu Xue Bao 54:229–235PubMedGoogle Scholar
  26. Huu HD, Sang HM, Thanh Thuy NT (2016) Dietary β-glucan improved growth performance, Vibrio counts, haematological parameters and stress resistance of pompano fish, Trachinotus ovatus Linnaeus, 1758. Fish Shellfish Immunol 54:402–410CrossRefPubMedGoogle Scholar
  27. Huynh TG, Shiu YL, Nguyen TP, Truong QP, Chen JC, Liu CH (2017) Current applications, selection, and possible mechanisms of actions of synbiotics in improving the growth and health status in aquaculture: a review. Fish Shellfish Immunol 64:367–382CrossRefPubMedGoogle Scholar
  28. Jayalal L, Ramachandran A (2012) Export trend of Indian ornamental fish industry. Agri Biol J North Am 3:439–451CrossRefGoogle Scholar
  29. Jun JW, Kim JH, Choresca CH, Shin SP, Han JE, Jeong DS, Park SC (2011) Isolation and molecular detection of Plesiomonas shigelloides containing tetA gene from Asian arowana (Scleropages formosus) in a Korean aquarium. Afr J Microbiol Res 5:5019–5021Google Scholar
  30. Kanmani P, Sathish Kumar R, Yuvaraj N, Paari KA, Pattukumar V, Venkatesan A (2010) Comparison of antimicrobial activity of probiotic bacterium Streptococcus phocae P180, Enterococcus faecium MC13 and Carnobacterium divergens against fish pathogen. World J Dairy Food Sci 5:145–151Google Scholar
  31. Karthik R (2017) Growing demand for high-quality seafood products and production systems in aquaculture. Oceanogr Fish Open Access J 3(3):555614Google Scholar
  32. Kim DH, Austin B (2006) Innate immune responses in rainbow trout Oncorhynchus mykiss induced by probiotics. Fish Shellfish Immunol 21:513–524CrossRefPubMedGoogle Scholar
  33. Kim DH, Austin B (2008) Characterization of probiotic carnobacteria isolated from rainbow trout (Oncorhynchus mykiss) intestine. Lett Appl Microbiol 47:141–147CrossRefPubMedGoogle Scholar
  34. Krovacek K, Eriksson LM, Gonzalez-Rey C, Rosinsky J, Ciznar I (2000) Isolation, biochemical and serological characterisation of Plesiomonas shigelloides from freshwater in Northern Europe. Comp Immunol Microbiol Infect Dis 23:45–51CrossRefPubMedGoogle Scholar
  35. Kumar K, Sarma S, Chakrabarti PP, Rajesh K, Mohanty UL, Sahoo M et al (2013) Anabas (koi) farming in Sonapur, Assam. A successful demonstration. Fish Chimes 33:136–137Google Scholar
  36. Liu Z, Ke X, Lu M, Gao F, Cao J, Zhu H, Wang M (2015) Identification and pathological observation of a pathogenic Plesiomonas shigelloides strain isolated from cultured tilapia (Oreochromis niloticus). Wei Sheng Wu Xue Bao 55:96–106PubMedGoogle Scholar
  37. Maftuch M, Sanoesi E, Farichin I, Saputra BA, Ramdhani L, Hidayati S, Fitriyah N, Prihanto AA (2017) Histopathology of gill, muscle, intestine, kidney, and liver on Myxobolus sp.-infected koi carp (Cyprinus carpio). J Parasit Dis:1–7Google Scholar
  38. Meidong R, Khotchanalekha K, Doolgindachbaporn S, Nagasawa T, Nakao M, Sakai K, Tongpim S (2018) Evaluation of probiotic Bacillus aerius B81e isolated from healthy hybrid catfish on growth, disease resistance and innate immunity of Pla-mong Pangasius bocourti. Fish Shellfish Immunol 73:1–10CrossRefPubMedGoogle Scholar
  39. Mitsunobu S, Nadine AS, Arnold LD (1987) Cell-free synthesis of the dipeptide antibiotic bacilysin. J Ind Microbiol 2:201–208CrossRefGoogle Scholar
  40. Munir MB, Hashim R, Abdul Manaf MS, Mohd Nor SA (2016) Dietary prebiotics and probiotics influence the growth performance, feed utilisation, and body indices of snakehead (Channa striata) fingerlings. Trop Life Sci Res 27(2):111–125CrossRefPubMedPubMedCentralGoogle Scholar
  41. Nadirah M, Ruhil HH, Jalal KCA, Najiah M (2012) Occurrence of Plesiomonas shigelloides in cultured red hybrid tilapia (Oreochromis niloticus) from tropical rivers, east coast Malaysia. Pak J Biol Sci 15:600–603CrossRefPubMedGoogle Scholar
  42. Nawaz M, Khan AA, Khan S, Sung K, Steele R (2008) Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish. Food Microbiol 25:85–91CrossRefPubMedGoogle Scholar
  43. Nguyen TL, Park C, Kim DH (2017) Improved growth rate and disease resistance in olive flounder, Paralichthys olivaceus, by probiotic Lactococcus lactis WFLU12 isolated from wild marine fish. Aquaculture 471:113–120CrossRefGoogle Scholar
  44. Nisha RG, Rajathi V, Manikandan R, Prabhu NM (2014) Isolation of Plesiomonas shigelloides from infected cichlid fishes using 16S rRNA characterization and its control with probiotic Pseudomonas sp. Acta Sci Vet 42:1195Google Scholar
  45. Ouattara HG, Reverchon S, Niamke SL, Nasser W (2017) Regulation of the synthesis of pulp degrading enzymes in Bacillus isolated from cocoa fermentation. Food Microbiol 63:255–262CrossRefPubMedGoogle Scholar
  46. Sahoo PK, Rauta PR, Mohanty BR, Mahapatra KD, Saha JN, Rye M, Eknath AE (2011) Selection of improved resistance to Aeromonas hydrophila in Indian major carp Labeo rohita: survival and innate immune responses in first generation of resistant and susceptible lines. Fish Shellfish Immunol 31:432–438CrossRefPubMedGoogle Scholar
  47. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. In: Sambrook J, Russell D W (eds) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. p 1689Google Scholar
  48. Sanders ER (2012) Aseptic laboratory techniques: plating methods. J Vis Exp 63:e3064Google Scholar
  49. Saurav K, Raman RP, Pandey PK, Snatashree M, Abhay K, Kundan K (2013) Effect of orally administered azadirachtin on non-specific immune parameters of goldfish Carassius auratus (Linn. 1758) and resistance against Aeromonas hydrophila. Fish Shellfish Immunol 34:564–573CrossRefGoogle Scholar
  50. Secombes CJ (1990) Isolation of salmonid macrophage and analysis of their killing activity. In: Stolen JS, Fletcher TC, Anderson DP, Roberson BS, Van Muiswinkel WB (eds) Technique in fish immunology. SoS Publication, Fair Haven, pp 137–152Google Scholar
  51. Sheehan D, Hrapchek B (1983) Theory and practice of histotechnology, 2nd edn. The CV Mosby Company, St. LouisGoogle Scholar
  52. Solorzano L (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnology. Oceanography 14:799–801Google Scholar
  53. Standen BT, Rawling MD, Davies SJ, Castex M, Foey A, Gioacchini G, Carnevali O, Merrifield DL (2013) Probiotic Pediococcus acidilactici modulates both localised intestinal and peripheral-immunity in tilapia (Oreochromis niloticus). Fish Shellfish Immunol 35:1097–1104CrossRefPubMedGoogle Scholar
  54. Strickland JDH, Parsons TRA (1972) Practical hand book of sea water analysis. Bulletin. The. J Fish Res Board Can:167–311Google Scholar
  55. Strus M (1998) A new method for testing antagonistic activity of lactic acid bacteria (LAB) on selected pathogenic indicator bacteria. (in Polish; English abstract). Med Dosw Mikrobiol 50:123–130PubMedGoogle Scholar
  56. Tankoano A, Kabore D, Savadogo A, Soma A, Fogny NF, Sereme DC, Hounhouigan JD, Lingani HS (2016) Evaluation of microbiological quality of raw milk, sour milk and artisanal yoghurt from Ouagadougou, Burkina Faso. Afr J Microbiol Res 10(16):535–541CrossRefGoogle Scholar
  57. Vandepitte J, Van Damme L, Fofana Y, Desmyter J (1980) Edwardsiella tarda and Plesiomonas shigelloides. Their role as diarrhea agents and their epidemiology Bulletin de la Societe de pathologie exotique et de ses fi liales 73:139–149Google Scholar
  58. Wang F, Hangxian Y, Yizhen W (2013) Structure characterization of a fucose-containing exopolysaccaride produced by Enterobacter cloacae Z0206. Carbohyr Polym 92:503–509CrossRefGoogle Scholar
  59. Wang X, Sun Y, Wang L, Li X, Qu K, Xu Y (2017) Synbiotic dietary supplement affects growth, immune responses and intestinal microbiota of Apostichopus japonicas. Fish Shellfish Immunol 68:232–242CrossRefPubMedGoogle Scholar
  60. Zhou X, Yanbo W, Jiangtao Y, Weifen L (2010) Inhibition ability of probiotic, Lactococcus lactis, against A. hydrophila and study of its immunostimulatory effect in tilapia (Oreochromis niloticus). Int J Eng Sci Technol 2:73–80Google Scholar
  61. Zokaeifar H, Nahid B, Che RS, Mohd SK, Kamaruzaman S, Jose LB (2014) Administration of Bacillus subtilis strains in the rearing water enhances the water quality, growth performance, immune response, and resistance against Vibrio harveyi infection in juvenile white shrimp, Litopenaeus vannamei. Fish Shellfish Immunol 36:68–74CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Nisha Rajagopalan Girijakumari
    • 1
  • Kannapiran Ethiraja
    • 2
  • Prabhu Narayanasamy Marimuthu
    • 1
  1. 1.Disease Control and Prevention Lab, Department of Animal Health and ManagementAlagappa UniversityKaraikudiIndia
  2. 2.Department of Zoology, Directorate of Distance EducationAlagappa UniversityKaraikudiIndia

Personalised recommendations