Aquaculture International

, Volume 27, Issue 5, pp 1315–1330 | Cite as

Antimicrobial activities of chitosan nanoparticles against pathogenic microorganisms in Nile tilapia, Oreochromis niloticus

  • Nashwa Abdel-RazekEmail author


Nanotechnology is a recent unique technique generally used for nutrition and therapy purposes among others. In this respect, the present study was carried out to evaluate the antimicrobial activity of chitosan nanoparticles (CNP) against various microorganisms (fungi and bacteria) isolated from diseased or health Nile tilapia, Oreochromis niloticus. The CNP was prepared based on the ionic gelation of chitosan with tripolyphosphate anion and its mean size was 35 nm with a narrow size distribution and zeta potential of 61.2 mV. The lethal dose of pathogenic bacterial isolates for Nile tilapia was successfully standardized. Clinical signs including weakness, slower movement, swimming closer to the surface, fin hemorrhages, and red patches at the gut regions were observed. Enlargement of spleen followed by tissue necrosis along with signs of hemorrhagic septicemia was also seen in infected fish. Fungal and bacterial isolates were exposed to different CNP doses and it is noticed that CNP inhibited all examined fungal and bacterial isolates in a dose-dependent manner. However, high CNP doses (80 μg/ml) gave highest inhibition zones where Aspergillus flavus, Mucor sp., and Candida sp. were more susceptible, whereas Aspergillus niger, A. fumigatus, and Fusarium sp. were more resistant. Similarly, largest inhibition zones of tested bacteria were obtained at high CNP dose (20 μg/ml). And Aeromonas sobria, A. hydrophila, and Pseudomonas aeruginosa were the most susceptible bacterial strain; meanwhile, Staphylococcus aureus and Pseudomonas fluorescens were the most resistant ones. The minimal inhibitory concentration of CNP against the examined bacteria ranged from 0.156 to 2.5 μg/ml causing their minimal counts. The transmission electron microscope images revealed that CNP showed antagonistic action against A. hydrophila causing disruption of cell membranes and the leakage of cytoplasm. In a practical experiment, Nile tilapia fed dietary CNP at levels of 0.0 and 1.0 g/kg diet for 3 weeks and post-challenged with different pathogenic bacteria via intraperitoneal injection. It is noticed that fish fed a CNP-enriched diet showed less mortality with all bacterial strains (6.7–20%), while, fish fed a CNP-free diet showed highest mortality (66.7–100%). The dietary CNP protected Nile tilapia efficiently against A. hydrophila, A. sobria, and Streptococcus agalactiae infections with relative level of protection (RLP) value of 93.3%, while the RLP against Staphylococcus aureus was 70.0%.


Chitosan nanoparticles Antimicrobial activity Pathogenic fungi Pathogenic bacteria Nile tilapia 


Funding information

This study was funded and supported by the Central Laboratory for Aquaculture Research (CLAR), Abbassa, Abu-Hammad, Sharkia, Egypt.

Compliance with ethical standards

Conflict of interest

The author declares that she has no conflict of interest.

Ethical approval

The author declares that she followed all guidelines for the care and use of fish in the present study.


  1. Abdel-Ghany HM, Salem ME-S (2019) Effects of dietary chitosan supplementation on farmed fish; a review. Rev Aquac.
  2. Abdel-Tawwab M, Abdel Razek N, Abdel-Rahman AM (2019) Immunostimulatory effect of dietary chitosan nanoparticles on growth performance of Nile tilapia, Oreochromis niloticus (L.). Fish Shellfish Immunol 88:254–258CrossRefGoogle Scholar
  3. Akmaz S, Adjgüzel ED, Yasar M, Erguven O (2013) The effect of Ag content of the chitosan-silver nanoparticle composite material on the structure and antibacterial activity. Adv Mater Sci Eng.
  4. Allan CR, Hardwiger LA (1979) The fungicidal effect of chitosan on fungi of varying cell wall composition. Exp Mycol 3:285–287CrossRefGoogle Scholar
  5. Amer MSMI (2002) Antimicrobial activity of some species of blue green algae (Cyanobacteria). M.Sc., Botany Dep., Fac. Sci., Tanta Univ, Egypt.Google Scholar
  6. Avadi MR, Sadeghi AMM, Tahzibi A, Bayati KH, Pouladzadeh M, Zohuriaan-Mehr MJ, Rafiee (2004) Optimized synthesis and characterization of N-Triethyl chitosan Tehrani, M. Eur Polym J 40: 1355–1361.Google Scholar
  7. Bauer AW, Kirby WM, Sherris JC, Turk M (1966) Antibiotic susceptibility by a standardized single disk method. Amer J Clin Pathol 45:493–496CrossRefGoogle Scholar
  8. Benhabiles MS, Salah R, Lounici H, Drouiche N, Goosen MFA, Mameri N (2012) Antibacterial activity of chitin, chitosan and its oligomers prepared from shrimp shell waste. Food Hydrocoll 29:48–56CrossRefGoogle Scholar
  9. Cabello FC, Godfrey HP, Tomova A, Ivanova L, Dölz H, Millanao A et al (2013) Antimicrobial use in aquaculture re-examined: its relevance to antimicrobial resistance and to animal and human health. Environ Microbiol 15:1917–1942CrossRefGoogle Scholar
  10. Cabello FC, Godfrey HP, Buschmann AH, Dölz HJ (2016) Aquaculture as yet another environmental gateway to the development and globalisation of antimicrobial resistance. Lancet Infect Dis 16:127–133CrossRefGoogle Scholar
  11. Cha S, Lee J, Song C, Lee K, Jeon Y (2008) Effects of chitosan coated diet on improving water quality and innate immunity in the olive flounder (Paralithchys olivaceus). Aquaculture 278:110–118CrossRefGoogle Scholar
  12. Chen CZS, Cooper SL (2002) Interactions between dendrimerbiocides and bacterial membranes. Biomaterials 23:3359–3368CrossRefGoogle Scholar
  13. Du L, Liu W (2012) Occurance, fat, and ecotoxicity of antibiotics in agro-ecosystem. A review. Agron Sustain Dev 32:309–327CrossRefGoogle Scholar
  14. El-Sayed HS, Barakat KM (2016) Effect of dietary chitosan on challenged Dicentrarchus labrax post larvae with Aeromonas hydrophila. Russ J Mar Biol 42:501–508CrossRefGoogle Scholar
  15. Eweis M, Elkholy SS, Elsabee MZ (2006) Antifungal efficacy of chitosan and its thiourea derivatives upon the growth of some sugar-beet pathogens. Int J Biol Macromol 38:1–8CrossRefGoogle Scholar
  16. Florio D, Gustinelli A, Caوٴara M, Turci F, Quaglio F et al (2009) Veterinary and public health aspects in tilapia (Oreochromis niloticus) aquaculture in Kenya, Uganda and Ethiopia. Ittiopatologia 6:51–93Google Scholar
  17. Gopalakannan A, Arul V (2006) Immunomodulatory effects of dietary intake of chitin, chitosan and levamisole on the immune system of Cyprinus carpio and control of Aeromonas hydrophila infection in ponds. Aquaculture 255:179–187CrossRefGoogle Scholar
  18. Goy RC, Britto D, Assis OG (2009) A review of the antimicrobial activity of chitosan. Polimeros 19:241–247CrossRefGoogle Scholar
  19. Guo Z, Ren J, Dong F, Wang G, Li P (2013) Comparative study of the influence of active groups of chitosan derivatives on antifungal activity. J Appl Polym Sci 127:2553–2556CrossRefGoogle Scholar
  20. Hadwiger LA, Kendra DG, Fristensky BW, Wagoner W (1981) Chitin in nature and technology. In: Muzzarelli RAA, Jeuniaux C, Gooday GW (eds) Chitosan both activated genes in plants and inhibits RNA synthesis in fungi. Plenum, New York, p 584Google Scholar
  21. Harikrishnan R, Kim JS, Balasundaram C, Heo MS (2012) Immunomodulatory effects of chitin and chitosan enriched diets in Epinephelus bruneus against Vibrio alginolyticus infection. Aquaculture 326–329:46–52CrossRefGoogle Scholar
  22. Helander IM, Nurmiaho-Lassila E-L, Ahvenainen R, Rhoades J, Roller S (2001) Chitosan disrupts the barrier properties of the outer membrane of Gram-negative bacteria. Int J Food Microbiol 71:235–244CrossRefGoogle Scholar
  23. Hernandez-Lauzardo AN, Bautista-Banos S, Velazquez-del Valle MG, Mendez-Montealvo MG, Sanchez-Rivera MM, Bello-Perez LA (2008) Antifungal effects of chitosan with different molecular weights on in vitro development of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. Carbohydr Polym 73:541–547CrossRefGoogle Scholar
  24. Iqbal Z, Saleemi S (2013) Isolation of pathogenic fungi from a freshwater commercial fish Catla catla. Sci Int 25:851–855Google Scholar
  25. Jeon YJ, Park PJ, Kim SK (2001) Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydr Polym 44:71–76CrossRefGoogle Scholar
  26. Kaplan SL, Assaâd Silaa B, Baha Eddine A, Rihab BA, Semia EC, Ali B, Rafik B (2016) Chitin and chitosan from the Norway lobster by-products: antimicrobial and anti-proliferative activities. Int eJ Biological Macromolecules 47:341–345Google Scholar
  27. Kinner NE, Balkwill DL, Bishop PL (1983) Light and electron microscopic studies of microorganisms growing in rotating biological contactor bio films. Appl Environ Microbiol 45(5):1659–1669Google Scholar
  28. Kong M, Chen XG, Liu CS, Liu CG, Meng XH, Yu LJ (2008) Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E.coli. Colloids Surf B: Biointerfaces 65:197–202CrossRefGoogle Scholar
  29. Lam TD, Hoang VD, Lien LN, Thinh NN, Dien PG (2006) Synthesis and characterization of chitosan nanoparticles used as drug. J Chem 44:105–109Google Scholar
  30. Larone DH (1987) Medically important fungi: a guide to identification. American Society for Microbiology, Medical, pp 230Google Scholar
  31. Lo G, Higueras L, Gavara R, Herna P (2013) Silver ions release from antibacterial chitosan films containing in situgene rated silver nanoparticles. J Agric Food Chem 61:260 267CrossRefGoogle Scholar
  32. Martin MV (1979) Germ tube formation by oral strains of Candida albicans. J Med Microbiol 12:187–193CrossRefGoogle Scholar
  33. Másson M, Holappa J, Hjalmarsdottir MR, Unarsson OV, Nevalainen T, Jarvinen T (2008) Antimicrobial activity of piperazine derivatives of chitosan. Carbohydrate Polymers 74: 566–571Google Scholar
  34. Melaku H, Lakew M, Alemayehu E, Wubie A, Chane M (2017) Isolation and identification of pathogenic fungus from African catfish (Clarias gariepinus) eggs and adults in national fishery and aquatic life research center hatchery, Ethiopia. Fish Aqua J 8:213. Google Scholar
  35. Miller LC, Tainter ML (1944) Estimation of LD50 and its error by means of log probit graph paper. Proc Soc Exp Biol Med 57:261CrossRefGoogle Scholar
  36. Mohy-Eldin SM, Soliman EA, Hashem AI, Tamer TM (2008) Antibacterial activity of chitosan chemically modified with new technique. Trends Biomater Atrif Organs 22:121–133Google Scholar
  37. Moussa S, Ibrahim A, Okba A, Hamza H, Opwis K, Schollmeyer E (2011) Anticandidal action of fungal chitosan against Candida albicans. Int J Biol Macromol 48:736–741CrossRefGoogle Scholar
  38. Muktar Y, Tesfaye S, Tesfaye B (2016) Present status and future prospects of fish vaccination: a review. J Veterinar Sci Technol 7:2CrossRefGoogle Scholar
  39. Nester E W, Anderson D G, Roberts E, Pearshall, N N, Nester M T (2004) Microbiology a human prospect. Published by Boston McGraw-Hill 2e004 Accessed 30 July 2004
  40. Panyam J, Labhasetwar V (2003) Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv Drug Deliv Rev 55(3):329–347CrossRefGoogle Scholar
  41. Qi L, Xu Z, Jiang X, Hu C, Zou X (2004) Preparation and antibacterial activity of chitosan nanoparticles. Carbohydr Res 339:2693–2700CrossRefGoogle Scholar
  42. Refai M (1987) Isolation and identification of fungi. Fac. Vet. Mid., Cairo University, Cairo, EgyptGoogle Scholar
  43. Refai M, Abdel Halim MM, Afify MMH, Youssef H, Marzouk M (1987) Studies on aspergillomycosis in catfish (Clarias lasera). Allgemeine Pathologic and pathologische Anatomic. Tagung der Deutachen Veterinar—Medizinischen Gesellschaft. der Europeischen Gesellschaft fur Vet. Pathol. 63: 1–12Google Scholar
  44. Refai MK, Laila AM, Amany KM, Shimaa E-SMA (2010) The assessment of mycotic settlement of freshwater fishes in Egypt. J Am Sci 6(11):594–602Google Scholar
  45. Sarbon NM, Sandanamsamy S, Kamaruzaman SFS, Ahmad F (2015) Chitosan extracted from mud crab (Scylla olivicea) shells: physicochemical and antioxidant properties. J Food Sci Technol 52:4266–4275CrossRefGoogle Scholar
  46. Schnurch B (2000) Chitosan and its derivatives: potential excipients for peroral peptide delivery systems. Int J Pharm 194:1–13CrossRefGoogle Scholar
  47. Shaalan M, Saleh M, El-Mahdy M, El-Matbouli M (2016) Recent progress in applications of nanoparticles in fish medicine. Nanomedicine 12:701–710CrossRefGoogle Scholar
  48. Shaheen, AA (1986) Mycoflora of some freshwater fish. M.V.Sc. Thesis, Fac. Vet. Med., Zagazic Univ, Zagazig, EgyptGoogle Scholar
  49. Shahidi F, Arachchi JKV, Jeon YJ (1999) Chitosan modification and pharmaceutical/biomedical applications. Trends Food Sci Technol 10:37–51CrossRefGoogle Scholar
  50. Song C, Yu H, Zhang M, Yang Y, Zhang G (2013) Physicochemical properties and antioxidant activity of chitosan from the blowfly Chrysomya megacephala larvae. Int J Biol Macromol 60:347–354CrossRefGoogle Scholar
  51. Srivastava RC (2009) Fish mycopathology. Today and Tomorrow’s Printers and Publishers, New Dehli, p 103Google Scholar
  52. Stossel P, Leuba JL (1984) Effect of chitosan, chitin and some amino-sugars on growth of various soil borne phytopathogenic fungi. Phytopathology Z 111:82–90CrossRefGoogle Scholar
  53. Sudarshan NR, Hoover DG, Knorr D (1992) Antibacterial action of chitosan. Food Biotechnol 6:257–272CrossRefGoogle Scholar
  54. Tang ESK, Huang M, Lim LY (2003) Ultrasonication of chitosan and chitosan nanoparticles. Intern J Pharm 265:103–114CrossRefGoogle Scholar
  55. Vinagradov SV, Bronich TK, Kabanov AV (2002) Nano-sized cationic hydrogels for drug delivery: preparation, properties and interactions with cells. Adv Drug Deliv Rev 54:223–233CrossRefGoogle Scholar
  56. Wang SH, Chen JC (2005) The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. Fish Shellfish Immunol 19:191–204CrossRefGoogle Scholar
  57. Yaghobi N, Hormozi F (2010) Multistage deacetylation of chitin: kinetics study. Carbohydr Polym 81:892–896CrossRefGoogle Scholar
  58. Younes I, Rinaudo M (2015) Chitin and chitosan preparation from marine sources: structure, properties and applications. Mar Drugs 13:1133CrossRefGoogle Scholar
  59. Younes I, Sellimi S, Rinaudo M, Jellouli K, Nasri M (2014) Influence of acetylation degree and molecular weight of homogeneous chitosan on antibacterial and antifungal activities. Intern J Food Microbiol 185:57–63CrossRefGoogle Scholar
  60. Zhang W, Zhang J, Jiang Q, Xia W (2012) Physicochemical and structural characteristics of chitosan nanopowders prepared by ultrafine milling. Carbohydr Polym 87:309–313CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Department of Fish Health and ManagementCentral Laboratory for Aquaculture ResearchSharkiaEgypt

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