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Virulence genes contributing to Aeromonas hydrophila pathogenicity in Oreochromis niloticus

  • Helmy Mohamed El-Bahar
  • Nadia Gabr AliEmail author
  • Ibrahim Mohamed Aboyadak
  • Samy Abd El Salam Khalil
  • Madiha Salah Ibrahim
Original Article
  • 27 Downloads

Abstract

Bacterial diseases are the main cause of high economic loss in aquaculture, particularly gram-negative bacteria. This study was conducted for the isolation and identification of Aeromonas and Pseudomonas spp. from diseased fish. Twenty-two Aeromonas and sixteen Pseudomonas isolates were recovered from diseased Nile tilapia (Oreochromis niloticus) raised in eight earthen ponds in Elhox, Metoubes, Kafrelsheikh, Egypt. The recovered isolates were further identified using PCR as 22 Aeromonas hydrophila, 11 Pseudomonas aeruginosa, and 5 Pseudomonas fluorescens isolates. The 22 A. hydrophila isolates were screened for the presence of four virulence genes. Sixteen of the isolates (72.72%) were positive for the aerolysin gene (aer); 4 (18.18%) harbored the cytotoxic enterotoxin gene (act); and 2 (9.09%) carried the hemolysin A gene (hylA) while the cytotonic heat–stable enterotoxin gene (ast) was absent from all the tested isolates. The pathogenicity test indicated the direct relationship between the mortality percentage and the genotype of the tested A. hydrophila isolates as the mortality rates were 63.3 and 73.3% for isolates with two virulence genes (aer+ & act+, and aer+ and hylA+, respectively), followed by 40, 53.3, and 56.6% for isolates with only one virulence gene (hylA, act, and aer, respectively) and 20% for isolates lacking virulence genes. Based on the sensitivity test, the multi-antibiotic resistance profiles were as follows: 90.9% of the A. hydrophila isolates were sensitive to florfenicol and doxycycline; then 68.18% were susceptible to oxytetracycline, norfloxacin, and ciprofloxacin; and 63.63% were susceptible to sulfamethoxazole-trimethoprim, while only 27.27 and 4.5% were sensitive to erythromycin and cephradine, respectively, and all the isolates were resistant to amoxicillin and ampicillin.

Keywords

Pseudomonas aeruginosa Pseudomonas fluorescens Antibiotic sensitivity PCR 

Notes

Compliance with ethical standards

The fish used in the current research were handled, transported, and examined following the guidelines of the National Advisory Committee for Laboratory Animals Research (NACLAR 2004) and CCAC (2005) for the care and use of fish in research, teaching, and testing which were applied by the NIOF ethical committee.

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Abd El-Kader MF, Mousa-Balabel T (2017) Isolation and molecular characterization of some bacteria implicated in the seasonal summer mortalities of farm-raised Oreochromis niloticus at Kafr El-Sheikh and Dakahlia governorates. AJVS 53(2):107–113.  https://doi.org/10.5455/ajvs.265631. https://www.ejmanager.com/mnstemps/31/31-1500399545.pdf?t=1549660439. Accessed 18 June 2018
  2. Aboyadak IM, Ali NGM, Goda AMAS, Aboelgalagel WH, Alnokrashy AME (2015) Molecular detection of Aeromonas hydrophila as the main cause of outbreak in Tilapia farms in Egypt. J Aquac Mar Biol 2(5):00045.  https://doi.org/10.15406/jamb.2015.02.00045 Google Scholar
  3. Aboyadak IM, Ali NGM, Goda AMA-S, Saad W, Salam AME (2017) Non-selectivity of R-S media for Aeromonas hydrophila and TCBS Media for Vibrio Species isolated from diseased Oreochromis niloticus. J Aquac Res Dev 8:496.  https://doi.org/10.4172/2155-9546.1000496 Google Scholar
  4. Abulhamd AT (2009) Characterization of Aeromonas hydrophila isolated from aquatic environments using phenotypic and genotyping methods. Res J Agric Biol Sci 5(6):923–931. http://www.aensiweb.net/AENSIWEB/rjabs/rjabs/2009/923-931.pdf. Accessed 20 June 2018
  5. Ali NG, Aboyadak IM, Gouda MY (2018) Rapid detection and control of Gram negative bacterial pathogens isolated from summer mortality outbreak affecting tilapia farms. J Biol Sci 19(1):24–33.  https://doi.org/10.3923/jbs.2019.24.33 Google Scholar
  6. Austin B, Austin DA (2016) Bacterial fish pathogens disease of farmed and wild fish, 6th edn. Springer International Publishing, Switzerland.  https://doi.org/10.1007/978-3-319-32674-0 Google Scholar
  7. Bektas S, Iscimen S (2016) Antibiotic resistance of Aeromonas hydrophila strains isolated from Karasu Stream (Sinop/Turkey). International Journal of Advances in Agricultural and Environmental Engineering 3(2):269–270.  https://doi.org/10.15242/IJAAEE.ER0516026
  8. Bosworth BG, Small BC (2004) Effects of transport water temperature, aerator type, and oxygen level on Channel Catfish Ictalurus punctatus fillet quality. J World Aquacult Soc 35(3):412–419Google Scholar
  9. Carnevia D, Letamendia M, Perretta A (2013) Pathogenic Gram-negative bacteria isolated from ornamental fish in Uruguay: characterization and antibiotic resistance. Bull Eur Ass Fish Pathol 33(6):181–186. https://eafp.org/download/2013-volume33/issue_6/181-Carnevia.pdf. Accessed 20 Aug 2018
  10. CCAC (2005) Guidelines on: the care and use of fish in research, teaching and testing. Canadian Council on Animal Care. 1510–130 Albert Street Ottawa on Canada, K1P 5G4. http://post.queensu.ca/~cdm2/CCACfish.pdf. Accessed 5 April 2017
  11. Chacon MR, Figueras MJ, Castro-Escarpulli G, Soler L, Guarro J (2003) Distribution of virulence genes in clinical and environmental isolates of Aeromonas spp. Antonie Van Leeuwenhoek 84:269–278Google Scholar
  12. Chopra AK, Xu XJ, Ribardo D, Gonzalez M, Kuhl K, Peterson JW, Houston CW (2000) The cytotoxic enterotoxin of Aeromonas hydrophila induces proinflammatory cytokine production and activates arachidonic acid metabolism in macrophages. Infect Immun 68(5):2808–2818Google Scholar
  13. CLSI, Clinical and Laboratory Standards Institute (2016) Document M45-A. methods for antimicrobial dilution and disk susceptibility of infrequently isolated or fastidious bacteria; approved guideline. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USAGoogle Scholar
  14. Daood N (2012) Isolation and antibiotic susceptibility of Aeromonas spp. from freshwater fish farm and farmed carp (Dam of 16 Tishreen, Lattakia). Damas Univ J Basic Sci 28(1):27–39. http://www.damascusuniversity.edu.sy/mag/asasy/images/stories/1-2012/27-39e.pdf. Accessed 15 May 2018
  15. Degiacomi MT, Iacovache I, Pernot L, Chami M, Kudryashev M, Stahlberg H, Goot FG, Peraro MD (2013) Molecular assembly of the aerolysin pore reveals a swirling membrane-insertion mechanism. Nat Chem Biol 9:623–631.  https://doi.org/10.1038/nchembio.1312 Google Scholar
  16. Emeish WFA, Mohamed HMA, Elkamel AA (2018) Aeromonas infections in African Sharptooth Catfish. J Aquac Res Development 9:548.  https://doi.org/10.4172/2155-9546.1000548. https://www.omicsonline.org/open-access/aeromonas-infections-in-african-sharptooth-catfish-2155-9546-1000548.pdf. Accessed 5 Oct 2018
  17. FAO (2017) Feeding global aquaculture growth in food and agriculture organization aquaculture newsletter No. 56, pp 2. http://www.fao.org/3/a-i7171e.pdf. Accessed 10 May 2018
  18. FAO (2018) Major species production in world aquaculture in: the state of world fisheries and aquaculture 2018 – Meeting the sustainable development goals. Rome, pp 23. http://www.fao.org/3/i9540en/I9540EN.pdf. Accessed 18 May 2018
  19. Finlay BB, Falkow S (1997) Common themes in microbial pathogenicity revisited. Microbiol Mol Biol Rev 61(2):136–169Google Scholar
  20. Furmanek-Blaszk B (2014) Phenotypic and molecular characteristics of an Aeromonas hydrophila strain isolated from the River Nile. Microbiol Res 169:547–552Google Scholar
  21. Hayati R, Hassan MD, Ong B, Abdelhadi YM, Hidayahanum NH, Sharifah RM, Nora FAM, Kuttichantran S, Alsaid M (2015) Virulence genes detection of Aeromonas hydrophila originated from diseased freshwater fishes. Adv Environ Biol 9(22):22–26. http://www.aensiweb.net/AENSIWEB/aeb/aeb/2015/Special%20IPN%20Bandung%20Sept/22-26.pdf. Accessed 5 March 2018Google Scholar
  22. Hossain MJ, Waldbieser GC, Sun D, Capps NK, Hemstreet WB, Carlisle K, Griffin MJ, Khoo L, Goodwin AE, Sonstegard TS, Schroeder S, Hayden K, Newton JC, Terhune JS, Liles MR (2013) Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in Channel Catfish. PLoS One 8(11):80943.  https://doi.org/10.1371/journal.pone.0080943
  23. Hu M, Wang N, Pan ZH, Lu CP, Liu YJ (2012) Identity and virulence properties of Aeromonas isolates from diseased fish, healthy controls and water environment in China. Lett Appl Microbiol 55:224–233.  https://doi.org/10.1111/j.1472-765X.2012.03281.x Google Scholar
  24. Iacovache I, Carlo SD, Cirauqui N, Peraro MD, Goot VD, Zuber B (2016) Cryo-EM structure of aerolysin variants reveals a novel protein fold and the pore-formation process. Nat Commun 7:12062.  https://doi.org/10.1038/ncomms12062 Google Scholar
  25. Kingombe CIB, D’Aoust J-Y, Huys G, Hofmann L, Rao M, Kwan J (2010) Multiplex PCR method for detection of three Aeromonas enterotoxin genes. Appl Environ Microbiol 76(2):425–433.  https://doi.org/10.1128/AEM.01357-09 Google Scholar
  26. Krueger CL, Sheikh W (1987) A new selective medium for isolating Pseudomonas spp. from water. Appl Environ Microbiol 53(4):895–897Google Scholar
  27. Krumperman PH (1983) Multiple antibiotic indexing of E. coli to identify high-risk sources of fecal contamination of foods. Appl Environ Microbiol 46(1):165–170Google Scholar
  28. Lafferty KD, Harvell CD, Conrad JM, Friedman CS, Kent ML, Kuris AM, Powell EN, Rondeau D, Saksida SM (2015) Infectious diseases affect marine fisheries and aquaculture economics. Annu Rev Mar Sci 7:471–496.  https://doi.org/10.1146/annurev-marine-010814-015646 Google Scholar
  29. Le TS, Nguyen TH, Vo HP, Doan VC, Nguyen HC, Tran MT, Tran TT, Southgate PC, Kurtböke DI (2018) Protective effects of bacteriophages against Aeromonas hydrophila causing Motile Aeromonas Septicemia (MAS) in striped catfish. antibiotics 7(16).  https://doi.org/10.3390/antibiotics7010016
  30. Lee C, Cho JC, Lee SH, Lee DG, Kim SJ (2002) Distribution of Aeromonas spp. as identified by 16S rDNA restriction fragment length polymorphism analysis in a trout farm. J Appl Microbiol 93:976–985.  https://doi.org/10.1046/j.1365-2672.2002.01775.x Google Scholar
  31. Lee PY, Costumbrado J, Hsu CY, Kim YH (2012) Agarose gel electrophoresis for the separation of DNA fragments. J Vis Exp (62):1–5, e3923.  https://doi.org/10.3791/3923
  32. Li J, Ni XD, Liu YJ, Lu CP (2011) Detection of three virulence genes alt, ahp and aerA in Aeromonas hydrophila and their relationship with actual virulence to zebra fish. J Appl Microbiol 110(3):823–830Google Scholar
  33. Lowry T, Smith SA (2007) Aquatic zoonoses associated with food, bait, ornamental, and tropical fish. J Am Vet Med Assoc 231:876–880.  https://doi.org/10.2460/javma.231.6.876 Google Scholar
  34. NACLAR (2004) NACLAR (2004) National Advisory Committee for Laboratory Animals Research. Guidelines on the care and use of animals for scientific purposes, pp 46–56. http://www3.ntu.edu.sg/Research2/Grants%20Handbook/NACLAR-guide%20Lines.pdf. Accessed 1 April 2017Google Scholar
  35. Nawaz M, Khan SA, Khan AA, Sung H, Tran Q, Kerdahi K, Steele R (2010) Detection and characterization of virulence genes and integrons in Aeromonas veronii isolated from catfish. Food Microbiol 27:327–331.  https://doi.org/10.1016/j.fm.2009.11.007 Google Scholar
  36. Noga EJ (2010) Fish disease diagnosis and treatment. Blackwell Publishing, USAGoogle Scholar
  37. Oliveira STL, Veneroni-Gouveia G, Costa MM (2012) Molecular characterization of virulence factors in Aeromonas hydrophila obtained from Fish. Pesqui Vet Bras 32(8):701–706Google Scholar
  38. Omar AA, Moustafa EM, Zayed MM (2016) Identification and characterization of virulence-associated genes from pathogenic Aeromonas Hydrophila strains. World Vet J 6(4):185–192Google Scholar
  39. 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 482:1–8.  https://doi.org/10.1016/j.aquaculture.2017.09.019 Google Scholar
  40. Petty BD (2016) Bacterial Diseases of Fish. In: Aiellos SE (ed) The Merck Veterinary Manual, 11th edn. Merck & Co., Inc., SE Kenilworth, NJ, USAGoogle Scholar
  41. Podobnik M, Kisovec M, Anderluh G (2017) Molecular mechanism of pore formation by aerolysin-like proteins. Philos Trans B 372:20160209.  https://doi.org/10.1098/rstb.2016.0209
  42. Rasmussen-Ivey CR, Hossain MJ, Odom SE, Terhune JS, Hemstreet WG, Shoemaker CA, Zhang D, Xu D-H, Griffin MJ, Liu Y-J, Figueras MJ, Santos SR, Newton JC, Liles MR (2016) Classification of a hypervirulent Aeromonas hydrophila pathotype responsible for epidemic outbreaks in warm-water fishes. Front Microbiol 7:1615.  https://doi.org/10.3389/fmicb.2016.01615 Google Scholar
  43. Rather MA, Willayat MM, Wani SA, Munshi ZH, Hussain SA (2014) A multiplex PCR for detection of enterotoxin genes in Aeromonas species isolated from foods of animal origin and human diarrhoeal samples. J Appl Microbiol 117:1721–1729Google Scholar
  44. Revina O, Avsejenko J, Cirule D, Valdovska A (2017) Antimicrobial resistance of aeromonas spp. isolated from the sea trout (Salmo trutta l.) in Latvia. Res Rural Dev 1:271–275Google Scholar
  45. Ribardo DA, Kuhl KR, Boldogh I, Peterson JW, Houston CW, Chopra AK (2002) Early cell signaling by the cytotoxic enterotoxin of Aeromonas hydrophila in Macrophages. Microb Pathog 32:149–163.  https://doi.org/10.1006/mpat.2001.0490 Google Scholar
  46. Sarkar A, Saha M, Roy P (2013) Detection of 232bp virulent gene of pathogenic Aeromonas hydrophila through PCR based technique: a rapid molecular diagnostic approach. Adv Microbiol 3:83–87.  https://doi.org/10.4236/aim.2013.31013 Google Scholar
  47. Scarpellini M, Franzetti L, Galli A (2004) Development of PCR assay to identify Pseudomonas fluorescens and its biotype. FEMS Microbiol Lett 236:257–260.  https://doi.org/10.1016/j.femsle.2004.05.043 Google Scholar
  48. 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 97:1077–1086.  https://doi.org/10.1111/j.1365-2672.2004.02398.x Google Scholar
  49. 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(4):1924–1935Google Scholar
  50. Son R, Rusul G, Sahilah AM, Zainuri A, Raha AR, Salmah I (1997) Antibiotic resistance and plasmid profile of Aeromonas hydrophila isolates from cultured fish, Telapia (Telapia mossambica). Lett Appl Microbiol 24:479–482Google Scholar
  51. Song H-C, Kang Y-H, Zhang D-X, Chen L, Qian A-D, Shan X-F, Li Y (2019) Great effect of porin (aha) in bacterial adhesion and virulence regulation in Aeromonas Veronii. Microb Pathog 126:269–278.  https://doi.org/10.1016/j.micpath.2018.11.002 Google Scholar
  52. Soto-Rodriguez SA, Lozano-Olvera R, Garcia-Gasca MT, Abad-Rosales SM, Gomez-Gil B, Ayala-Arellano J (2018) Virulence of the fish pathogen Aeromonas dhakensis: genes involved, characterization and histopathology of experimentally infected hybrid tilapia. Dis Aquat Org 129:107–116.  https://doi.org/10.3354/dao03247 Google Scholar
  53. Souza CF, Baldissera MD, Guarda NS, Bollick YS, Moresco RN, Brusque ICM, Roberto CV, Santos RCV, Baldisserotto B (2017) Melaleuca alternifolia essential oil nanoparticles ameliorate the hepatic antioxidant/oxidant status of silver catfish experimentally infected with Pseudomonas aeruginosa. Microb Pathog 108:61–65.  https://doi.org/10.1016/j.micpath.2017.05.016 Google Scholar
  54. Spilker T, Coenye T, Vandamme P, LiPuma JJ (2004) PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J Clin Microbiol 42(5):2074–2079Google Scholar
  55. Stratev D, Odeyemi OA (2015) Antimicrobial resistance of Aeromonas hydrophila isolated from different food sources: a mini-review. J Infect Public Health 9:535–544.  https://doi.org/10.1016/j.jiph.2015.10.006 Google Scholar
  56. Tito MT, Rodrigues ND, Coelho SD, Souza MM, Zonta E, Coelho ID (2015) Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil. Afr J Microbiol Res 9(12):863–871.  https://doi.org/10.5897/AJMR2014.7259 Google Scholar
  57. Tohamy HG, El-Manakhly E-SM, Mohamed FAS, Massoud RG (2015) Pathological evaluation of experimental Pseudomonas fluorescens infection in Nile tilapia. WJFMS 7(6):450–457. https://www.idosi.org/wjfms/wjfms7(6)15/7.pdf. Accessed 3 Aug 2018
  58. Tomas JM (2012) The main Aeromonas pathogenic factors. ISRN Microbiol 2012:256261–256222.  https://doi.org/10.5402/2012/256261 Google Scholar
  59. Trakhna F, Harf-Monteil C, AbdelNour A, Maaroufi A, Gadonna-Widehem P (2009) Rapid Aeromonas hydrophila identification by TaqMan PCR assay: comparison with a phenotypic method. Lett Appl Microbiol 49:186–190.  https://doi.org/10.1111/j.1472-765X.2009.02635.x Google Scholar
  60. Vivekanandhan G, Savithamani K, Hatha AAM, Lakshmanaperumalsamy P (2002) Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of south India. Int J Food Microbiol 76:165–168Google Scholar
  61. Wu H-j, Wang AH-J, Jennings MP (2008) Discovery of virulence factors of pathogenic bacteria. Curr Opin Chem Biol 12:1–9.  https://doi.org/10.1016/j.cbpa.2008.01.023 Google Scholar
  62. Xu XJ, Ferguson MR, Popov VL, Houston CW, Peterson JW, Chopra AK (1998) Role of a cytotoxic enterotoxin in Aeromonas-mediated infections: development of transposon and isogenic mutants. Infect Immun 66:3501–3509Google Scholar
  63. Yang W, Li N, Li M, Zhang D, An G (2016) Complete genome sequence of fish pathogen Aeromonas hydrophila JBN2301. Genome Announcement 4(1):01615–01615.  https://doi.org/10.1128/genomeA.01615-15 Google Scholar
  64. Yogananth N, Bhakyaraj R, Chanthuru A, Anbalagan T, Nila KM (2009) Detection of virulence gene in Aeromonas hydrophila isolated from fish samples using PCR technique. GJBB 4(1):51–53. http://www.idosi.org/gjbb/gjbb4%281%2909/9.pdf. Accessed 2 Sept 2018Google Scholar
  65. Yousr AH, Napis S, Rusul GRA, Son R (2007) Detection of aerolysin and hemolysin genes in Aeromonas spp. isolated from environmental and shellfish sources by Polymerase Chain Reaction. ASEAN Food J 14(2):115–122. https://www.researchgate.net/publication/237723263. Accessed 20 Aug 2018
  66. Zheng W, Cao H, Yang X (2012) Grass carp (Ctenopharyngodon idellus) infected with multiple strains of Aeromonas hydrophila. Afr JMicrobiol Res 6(21):4512–4520.  https://doi.org/10.5897/AJMR11.1405. http://www.academicjournals.org/app/webroot/article/article1380712634_Zheng%20et%20al.pdf. Accessed 30 June 2018
  67. Zhu D, Aihua L, Jianguo W, Ming L, Taozhen C, Jing H (2007) Correlation between the distribution pattern of virulence genes and virulence of Aeromonas hydrophila strains. Frontiers of Biology in China 2(2):176–179.  https://doi.org/10.1007/s11515-007-0024-4 Google Scholar

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© Springer Nature Switzerland AG 2019

Authors and Affiliations

  1. 1.Veterinary DirectorateKafrelsheikhEgypt
  2. 2.Fish Diseases Lab.National Institute of Oceanography and FisheriesAlexandriaEgypt
  3. 3.Department of Microbiology, Faculty of Veterinary MedicineAlexandria UniversityAlexandriaEgypt
  4. 4.Department of Microbiology, Faculty of Veterinary MedicineDamanhour UniversityDamanhurEgypt

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