Aquaculture International

, Volume 28, Issue 1, pp 235–248 | Cite as

Distribution of multi-virulence factors among Aeromonas spp. isolated from diseased Xiphophorus hellerii

  • Soumya Das
  • R. Aswani
  • B. Jasim
  • K. S. Sebastian
  • E. K. Radhakrishnan
  • Jyothis MathewEmail author


Changes in water quality and environmental factors have been reported to play key roles on the health and disease susceptibility of the ornamental fish Xiphophorus hellerii (sword tail). This can further be decided by the natural distribution of pathogens with enhanced virulence properties in the aquatic system. As Aeromonas spp. are the major fish pathogens, a detailed analysis on its association with the disease of X.hellerii was conducted in the study. Here, bacterial isolation has been carried out from the surface, gill and intestine of diseased fish, and also from water samples. The twelve bacterial isolates obtained were identified biochemically and also by 16S rDNA sequence analysis as Aeromonas spp.. Among the various virulence properties screened by in vitro and PCR-based methods, most of the Aeromonas spp. were found to be positive for multiple virulence properties. Further disease challenge using the highly virulent Aeromonas veronii XhG1.2 in X. hellerii showed remarkable histopathological changes of the gill, liver, and intestine in the treated fish. This confirmed the hyper -pathogenic potential of XhG1.2 as the disease occurred within a short period of 24 h. The identification of broad range of virulence factors among the fish pathogenic bacterial isolates of aquatic environment indicates the need for periodic screening to predict the threat to aquatic life especially fish.


Xiphophorus hellerii Aeromonas Virulence factors Histopathology 


Funding information

This study is instrumentally supported by the Kerala State Council for Science, Technology, and Environment-Kerala Biotechnology Commission-Young Investigators Programme in Biotechnology (KSCSTE-KBC-YIPB) (Order no. 673/2017/KSCSTE dated 13.10.2017).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The experiment with Aeromonas sp. has been carried out with approval from institutional biosafety committee.

Supplementary material

10499_2019_456_MOESM1_ESM.doc (1.3 mb)
ESM 1 (DOC 1357 kb)


  1. Abdelhamed H, Ibrahim I, Baumgartner W, Lawrence ML, Karsi A (2017) Characterization of histopathological and ultrastructural changes in channel catfish experimentally infected with virulent Aeromonas hydrophila. Front Microbiol 8:1519PubMedPubMedCentralGoogle Scholar
  2. Ananth A, Rajan MR, Sivakumar P (2015) Isolation, identification, enzyme productivity, antibacterial activity and molecular characterization of intestinal bacteria of ornamental fish Koi Carp (Cyprinus carpio Var Koi) and it’s role on growth. Indian J Appl Res 4(7):582–585Google Scholar
  3. Austin B, Austin DA (2012) Bacterial fish pathogens; diseases of farmed and wild fish. Springer, New YorkGoogle Scholar
  4. Baldissera MD, Souza CF, Parmeggiani B, Leipnitz G, Verdi CM, Santos RV, Baldisserotto B (2018) The disturbance of antioxidant/oxidant balance in fish experimentally infected by Aeromonas caviae: relationship with disease pathophysiology. Microb Pathog 122:53–57PubMedGoogle Scholar
  5. Banerjee G, Nandi A, Ray AK (2016) Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish. Arch Microbiol 199:115–124PubMedGoogle Scholar
  6. Beaz Hidalgo R, Figueras MJ (2013) Aeromonas spp . whole genomes and virulence factors implicated in fish disease. J Fish Dis 36:371–388PubMedGoogle Scholar
  7. Casabianca A, Orlandi C, Barbieri F, Sabatini L, di Cesare A, Sisti D, Pasquaroli S, Magnani M, Citterio B (2015) Effect of starvation on survival and virulence expression of Aeromonas hydrophila from different sources. Arch Microbiol 197:431–438PubMedGoogle Scholar
  8. Chenia HY, Duma S (2016) Characterization of virulence, cell surface characteristics and biofilm-forming ability of Aeromonas spp. isolates from fish and sea water. J Fish Dis 40(3):339–350PubMedGoogle Scholar
  9. Cruz PM, Ibanez AL, Hermosillo OAM, Saad HCR (2012) Use of probiotics in aquaculture. ISRN Microbiol 2012:96845Google Scholar
  10. da Silva JLS, de Holanda Cavalcante D, de Carvalho FCT, dos Fernandes Vieira RHS et al (2017) Aquatic microbiota diversity in the culture of nile tilapia (Oreochromis niloticus) using bioflocs or periphyton: virulence factors and biofilm formation. Acta Sci Anim Sci 38(3)Google Scholar
  11. Daskalov H (2006) The importance of Aeromonas hydrophila in food safety. Food Control 17:474–483Google Scholar
  12. Dong HT, Techatanakitarnan C, Jindakittikul P, Thaiprayoon A, Taengphu S, Charoensapsri W, Senapin S (2017) Aeromonas jandaei and Aeromonas veronii caused disease and mortality in Nile tilapia, Oreochromis niloticus (L.). J Fish Dis 40(10):1395–1403PubMedGoogle Scholar
  13. Elgendy MY, Soliman WS, Abbas WT et al (2017) Investigation of some virulence determents in Aeromonas hydrophila strains obtained from different polluted aquatic environments. Jordan J Biol Sci 10:265–272Google Scholar
  14. Erova TE, Sha J, Horneman AJ, Borchardt MA, Khajanchi BK, Fadl AA, Chopra AK (2007) Identification of a new hemolysin from diarrheal isolate SSU of Aeromonas hydrophila. FEMS Microbiol Lett 275:301–311PubMedGoogle Scholar
  15. Haenen O (2017) Major bacterial diseases affecting aquaculture. 10–11Google Scholar
  16. Hamid Salari Joo, Mohammad Reza Kalbassi, Seyed Ali Johari (2018) "Hematological and histopathological effects of silver nanoparticles in rainbow trout (Oncorhynchus mykiss)—how about increase of salinity?." Environmental Science and Pollution Research 25(16):15449–15461PubMedGoogle Scholar
  17. Harikrishnan R, Balasundaram C, Heo M-S (2010) Herbal supplementation diets on hematology and innate immunity in goldfish against Aeromonas hydrophila. Fish Shellfish Immunol 28(2):354–361PubMedGoogle Scholar
  18. Hassan MA, Noureldin EA, Mahmoud MA, Fita NA (2017) Molecular identification and epizootiology of Aeromonas veronii infection among farmed Oreochromis niloticus in Eastern Province, KSA. Egypt J Aquat Res 43(2):161–167Google Scholar
  19. Heuzenroeder MW, Wong CY, Flower RL (1999) Distribution of two hemolytic toxin genes in clinical and environmental isolates of Aeromonas spp.: correlation with virulence in a suckling mouse model. FEMS Microbiol Lett 174:131–136PubMedGoogle Scholar
  20. Hu M, Wang N, Pan ZH et al (2012) Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China. Lett Appl Microbiol 55:224–233PubMedGoogle Scholar
  21. Igbinosa IH, Igumbor EU, Aghdasi F et al (2012) Emerging Aeromonas species infections and their significance in public health. Sci World J 2012:625023Google Scholar
  22. In-Young N, Kiseong J (2007) Rapid detection of virulence factors of Aeromonas isolated from a trout farm by Hexaplex-PCR. J Microbiol 45(4):297–304Google Scholar
  23. Janda JM (2002) Aeromonas and Plesiomonas. In: Sussman M (ed) Molecular Medical Microbiology. Academic, San Diego, pp 1237–1270Google Scholar
  24. Janda JM, Abbott SL (2010) The genus Aeromonas: taxonomy, Pathogenicity, and Infection. Clin Microbiol Rev 23:35–73. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jasim B, Aswathy Joseph A, John CJ, Mathew J, Radhakrishnan EK (2014) Isolation and characterization of plant growth promoting endophytic bacteria from the rhizome of Zingiber officinale. 3 Biotech 4:197–204PubMedGoogle Scholar
  26. John N, Abdulla MH (2012) Prevalence, distribution and drug resistance of motile aeromonads in freshwater ornamental fishes. Indian J Fish 59(2):161–164Google Scholar
  27. John N, Abdulla MH (2013) Distribution, extracellular virulence factors and drug resistance of motile aeromonads in fresh water ornamental fishes and associated carriage water. Int J Aquacult 3Google Scholar
  28. Kingombe CIB, Huys G, Tonolla M, Albert MJ, Swings J, Peduzzi R, Jemmy T (1999) PCR detection, characterization and distribution of virulence genes in Aeromonas spp. Appl Environ Microbiol 65:5293–5302PubMedPubMedCentralGoogle Scholar
  29. Lakshmi narayanan (2000) Histological Techniques: A Practical Manual (2nd Edition)Google Scholar
  30. Kumar S, Mukhopadhyay P, Chatterjee M, Bandyopadhyay MK, Bandyopadhyay M, Ghosh T, Samaddar D (2012) Necrotizing fasciitis caused by Aeromonas caviae. Avicenna journal of medicine, 2(4), 94PubMedPubMedCentralGoogle Scholar
  31. Loong SK, Khor CS, Jafar FL, AbuBakar S (2016) Utility of 16S rDNA sequencing for identification of rare pathogenic Bacteria. J Clin Lab Anal 30:1056–1060PubMedPubMedCentralGoogle Scholar
  32. Lucinda J, Cavalcante DDH, De FCT (2016) Acta Scientiarum Aquatic microbiota diversity in the culture of Nile tilapia (Oreochromis niloticus) using bioflocs or periphyton: virulence factors and biofilm formation. 31910:233–241Google Scholar
  33. Madden T (2013) The BLAST sequence analysis tool. In The NCBI Handbook [Internet]. 2nd edition. National Center for Biotechnology Information (US)Google Scholar
  34. Marian CM, MacRae IC (1992) Characteristics of 0/129-sensitive motile Aeromonas strains isolated from freshwater on starch-ampicillin agar. Microb Ecol 24(2):215–226Google Scholar
  35. Martha Reyes-Becerril, Guluarte C, Ceballos-Francisco D, Angulo C, Esteban MÁ (2017) Enhancing gilthead seabream immune status and protection against bacterial challenge by means of antigens derived from Vibrio parahaemolyticus. Fish & shellfish immunology, 60, 205–218Google Scholar
  36. Merino S, Gavin R, Vilches S, Shaw JG, Tomas JM (2003) A colonization factor (production of lateral flagella) of mesophilic Aeromonas spp. is in active in Aeromonas salmonicida strains. Appl Environ Microbiol 69(1):663–667PubMedPubMedCentralGoogle Scholar
  37. Palumbo SA, Maxino F, Williams AC, Buchanan RL, Thayer DW (1985) Starch-ampicillin agar for the quantitative detection of Aeromonas hydrophila. Appl Environ Microbiol 50(4):1027–1030PubMedPubMedCentralGoogle Scholar
  38. Peatman E, Mohammed H, Kirby A, Shoemaker CA, Yildirim-Aksoy M, Beck BH (2018) Mechanisms of pathogen virulence and host susceptibility in virulent Aeromonas hydrophila infections of channel catfish (Ictalurus punctatus). Aquaculture 482:1–8Google Scholar
  39. Romero J, Feijoo CG, Navarrete P (2012) Antibiotics in aquaculture – use, abuse and alternatives. In: Carvalho E (ed) Health and environment in aquaculture. InTech, Rijeka, pp 159–198 ISBN 978-953- 51-0497-1Google Scholar
  40. Sen K, Rodgers M (2004) Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: a PCR identification. J Appl Microbiol 97(5):1077–1086PubMedGoogle Scholar
  41. Senderovich Y, Ken-Dror S, Vainblat I, Blau D, Izhaki I et al (2012) A molecular study on the prevalence and virulence potential of Aeromonas spp. recovered from patients suffering from diarrhea in Israel. PLoS One 7(2):e30070PubMedPubMedCentralGoogle Scholar
  42. Sha J, Kozlova EV, Chopra AK (2002) Role of various enterotoxins in Aeromonas hydrophila-induced gastroenteritis: generation of enterotoxin gene-deficient mutants and evaluation of their enterotoxic activity. Infect Immun 70:1924–1935PubMedPubMedCentralGoogle Scholar
  43. Sha J, Galindo C, Pancholi V et al (2003) Differential expression of the enolase gene under in vivo versus in vitro growth conditions of Aeromonas hydrophila. Microb Pathog 34:195–204PubMedGoogle Scholar
  44. Sha J, Pillai L, Fadl AA, Galindo CL, Erova TE, Chopra AK (2005) The type III secretion system and cytotoxic enterotoxin alter the virulence of Aeromonas hydrophila. Infect Immun 73(10):6446–6457PubMedPubMedCentralGoogle Scholar
  45. Sha J, Wang SF, Suarez G, Sierra JC, Fadl AA, Erova TE, Foltz SM, Khajanchi BK, Silver A, Graf J, Schein CH, Chopra AK (2007) Further characterization of a type III secretion system (T3SS) and of a new effector protein from a clinical isolate of Aeromonas hydrophila–part I. Microb Pathog 43:127–146. CrossRefPubMedPubMedCentralGoogle Scholar
  46. Sihag, Sharma (2012) Probiotics: the new ecofriendly alternative measures of disease control for sustainable aquaculture. J Fish Aquat Sci 7(2):72–103Google Scholar
  47. Smillie CS, Smith MB, Friedman J, Cordero OX, David LA, Alm EJ (2011) Ecology drives a global network of gene exchange connecting the human microbiome. Nature 480:241–244PubMedGoogle Scholar
  48. Smith KF, Schmidt V, Rosen GE, Amaral-Zettler L (2012) Microbial diversity and potential pathogens in ornamental fish aquarium water. PLoS One 7(9):e39971PubMedPubMedCentralGoogle Scholar
  49. Soler L, Figueras MJ, Chacón MR, Vila J, Marco F, Martinez-Murcia AJ, Guarro J (2002) Potential virulence and antimicrobial susceptibility of Aeromonas popoffii recovered from freshwater and seawater. FEMS Immunol Med Microbiol 32(3):243–247PubMedGoogle Scholar
  50. Sreedharan K, Philip R, Singh ISB (2012) Virulence potential and antibiotic susceptibility pattern of motile aeromonads associated with freshwater ornamental fish culture systems: a possible threat to public health. Braz J Microbiol 43(2):754–765PubMedPubMedCentralGoogle Scholar
  51. Sudheesh PS, Al-ghabshi A, Al-mazrooei N, Al-habsi S (2012) Comparative pathogenomics of bacteria causing infectious diseases in fish. Int J Evol Biol 2012:16Google Scholar
  52. Sun J, Zhang X, Gao X, et al (1865) Characterization of virulence properties of Aeromonas veronii isolated from diseased Gibel carp (Carassius gibelio). 5–9. PubMedPubMedCentralGoogle Scholar
  53. Tomas JM (2012) The main Aeromonas pathogenic factors. ISRN Microbiol 2012:1–22Google Scholar
  54. Turska-Szewczuk A, Lindner B, Komaniecka I, Kozinska A, Pekala A, Choma A, Holst O (2013) Structural and immunochemical studies of the lipopolysaccharide from the fish pathogen, Aeromonas bestiarum strain K296, serotype O18. Mar Drugs 11(4):1235–1255PubMedPubMedCentralGoogle Scholar
  55. Von Graevenitz A (2007) The role of Aeromonas in diarrhea: a review. Infection 35:59–64. CrossRefGoogle Scholar
  56. Wang G, Clark CG, Liu C, Pucknell C, Munro CK, Kruk TMAC, Caldeira R, Woodward DL, Rodgers FG (2003) Detection and characterization of the Hemolysin genes in Aeromonas hydrophila and Aeromonas sobria by multiplex PCR. J Clin Microbiol 41:1048–1054. CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhang Z, Schwartz S, Wagner L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J Comput Biol 7:203–214. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Soumya Das
    • 1
  • R. Aswani
    • 1
  • B. Jasim
    • 1
  • K. S. Sebastian
    • 2
  • E. K. Radhakrishnan
    • 1
  • Jyothis Mathew
    • 1
    Email author
  1. 1.School of BiosciencesMahatma Gandhi UniversityKottayamIndia
  2. 2.Department of ZoologyGovernment CollegeKottayamIndia

Personalised recommendations