stem (GeneAmp PCR System 2400). PCR products were visualized by ethidium bromide staining on 1.5% agarose gel electrophoresis. PCR reactions has been carried out by following the series of standardizing experimental protocol as annealing temperature, concentration of MgCl2, template DNA, Taq DNA polymerase, dNTP’s and primers. The PCR reaction components consists of 200 mm dNTP, 20 pmoles of primer, 2 units of Taq DNA polymerase enzyme, assay buffer with working concentration of 1.5 mM MgCl2, 20 - 30 ng template DNA in an assay volume of 25 mL. The 16S rRNA gene of the PCR products were sequenced using these universal primers: forward primer 27F; 5’-AGA GTT TGA TCC TGG CTC AG-3’ and reverse primer 1492R; 5’-GGT TAC CTT GTT ACG ACT T-3’ (Chromous Biotech Ltd., Bengaluru, India) [26] .

2.4.3. Analysis of 16S rDNA PCR Product

Thermal cycling was performed with GeneAmp PCR System 2400. Amplification reactions were performed in a 25 μL volume, containing: 20 mmol/L Tris-HCl (pH = 8.4), 50 mmol/L KCl, 2.0 mmol/L MgCl2, 200 umol/L of dNTPs, 1 umol/L of each primer, 30 ng of genomic DNA and 1.5 U of Taq DNA polymerase. The temperature profile was as follows: initial denaturation at 95˚C x× 3 min, 35 cycles of denaturation at 94˚C × 1 min, annealing at 55˚C × 1 min, and extension at 72˚C × 2 min, and final extension at 72˚C × 3 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels and stained with ethidium bromide. 100 bp ladder was used for evaluating the size of amplicons. The DNA bands were visualized and documented using a Gel documentation system (Biorad gel documentation system 2000).

2.5. Determination of Virulence Characteristics of Aeromonas hydrophila

2.5.1. Determination of Enterotoxin Production by Test Isolates

Enterotoxin activity was determined by the suckling mouse test according to the method employed by [27] and [28] . The test organisms were cultured in tryptone soy broth supplemented with 0.6% (w/v) yeast extract and brain heart infusion broth and incubated for 24 h at 37˚C in a shaker incubator at 300 rpm. Cell-free supernatant was obtained by centrifuging the culture at 3000 rpm for 30 min followed by filtration of the supernatant using a 0.45 µm Millipore filter. Cell-free supernatant (100 µL) mixed with 0.001% Evans blue dye (Merck, Darmstadt, Germany) was inoculated into a 2-4-day old mice intragastrically using a gastric tube. After 3 h, the mice were sacrificed by inhalation. The bowels were removed and weighed. Test isolates were considered positive if the ratio of intestinal weight to the remaining body weight of the mice inoculated with the strain was greater than 0.080.

Enterotoxin production = intestinal weight of inoculated mice ( kg ) remaining body weight without bowels (kg)

2.5.2. Determination of Hemolysin Production by Test Isolates

Hemolysis test was carried out as described by [29] . Blood agar was prepared by adding 5% to 10% of sterile sheep blood to sterile tryptic soya agar. The test isolate was inoculated on the blood agar and incubated at 28˚C for 24 - 48 h. The plates were then observed for hemolysis. Complete clearing of the blood red coloration in agar immediately surrounding growth indicated a positive result (β-hemolysis), while no change in the agar immediately surrounding growth indicated a negative result.

2.5.3. Lipase Production

Lipase detection was performed on Tween 20 agar [30] . Isolates of Aeromonas hydrophila was cultured on tween 20 agar, composed of peptone, 10 g/L; NaCl, 5 g/L; CaCl2∙2H2O, 0.1 g/L; agar-agar, 20 g/L; tween 20, 10 mL. the plate was incubated for at 28˚C for 24 to 48 h. Clear zones of precipitation on the tween 20 agar plate confirmed lipase production.

2.5.4. Biofilm Production by Test Isolates

Biofilm or slime production by the test organism was determined using the method of [31] and [32] . The medium (1L) was prepared using a composition of brain heart infusion broth; 37 g/L, sucrose; 50 g/L, agar No 1 (Oxoid); 10 g/L and Congo red dye or stain (BDH Ltd.); 0 8 g/L and distilled water; 1 L. Congo red stain was prepared separately and alone as a concentrated aqueous solution and autoclaved at 121˚C for 15 min. The sterile congo red stain was then added to the prepared, sterile agar with a temperature of 55˚C. Plates of the medium were inoculated and incubated aerobically for 24 h at 37˚C. A positive result was indicated by black colonies with a dry crystalline consistency. Non-slime producers usually remained pink, though occasional darkening at the centre of the colonies was observed. An indeterminate result was indicated by a darkening of the colonies but with the absence of a dry crystalline colonial morphology.

3. Results

Fifteen (15) out of the fifty (50) organisms isolated from fresh water catfish were Gram negative, motile, non-spore formers, tested positive to oxidase, catalase, indole, methyl red, gelatin hydrolysis and urease tests and were considered to belong to the genus Aeromonas. The PCR product with band size of 1.2 kb molecular gene size was confirmed as Aeromonas hydrophila.

Aeromonas hydrophila was more prevalent in the gut of fishes as compared to the other organs of the fish with the highest percentage occurrence of 26.7% as shown in Table 1.

Most of the isolates produced haemolysin, amylase, lipase, biofilm, and enterotoxin as shown in Table 2.

Figure 1 shows the occurrence of Aeromonas hydrophila in the different parts and organs of C. gariepinus and I. punctatus. The gut of C. gariepinus had the highest microbial count of Aeromonas hydrophila while A. hydrophila occurred most in the gut and skin of I. punctatus.

4. Discussion

Previous reports on the investigation of aeromoniasis among fishes, poultry and

Table 1. Frequency of occurrence of Aeromonas hydrophila in various fish organs.

Table 2. Virulence characteristics of Aeromonas hydrophila isolated from fresh catfish.

Key: CG1/I, CG1/L, CG2/I, KeCG2/G, CG2/L, CG1/K, CG1/G, CG2/S, CG2/K, CG1/G= Aeromonas hydrophila isolated from different parts (I―Intestine, L―Liver, G―Gut, K―Kidney, S―Skin) of Clarias gariepinus. IP2/G, IP1/G, IP2/S, IP1/I, IP1/S = Aeromonas hydrophila isolated from different parts (I―Intestine, G―Gut, S―Skin) of Ictalurus punctatus.

Figure 1. Occurrence of Aeromonas hydrophila isolated from C. gariepinus and I. punctatus.

human population were based on clinical signs and much effort has not been directed at isolating and characterizing Aeromonas sp. from fishes or man in Nigeria [2] [33] .

Aeromonas hydrophila had the lowest and highest occurrence in the liver and gut of the pond-raised catfishes respectively. The predominance of A. hydrophila in the gut of fishes may be attributed to the presence of A. hydrophila in contaminated water in which the fish lives and feeds which then infects the gastrointestinal organs of the fish.

The high occurrence of Aeromonas hydrophila reported in this study warns on public food safety problem in Nigeria. In a similar study by [34] in Zaria, Nigeria, Aeromonas species were isolated from the GIT, gills and skins of various fishes, from both fresh, sewage, chlorinated and non-chlorinated water. The occurrence of Aeromonas hydrophila in the intestine, kidney, liver, skin and gut of fishes is in agreement with studies carried out by [35] [36] [37] . In most parts of Nigeria, people handle fish with bare hands and sometimes consume improperly cooked fish which may promote the horizontal transfer of the organism from the fish to man.

This study shows that majority of the Aeromonas isolated produced hemolysins and enterotoxins. These toxins are responsible for lethality, hemolysis and enterotoxigenicity. Their production by organisms found in food signals public health concern. The secretion of these extracellular proteins including enterotoxin, haemolysin and aerolysin are associated with bacterial virulence. Previous studies have shown that hemolytic toxins, haemolysin A and aerolysin A contribute to the virulence of A. hydrophila in fish and human host [38] .

The result of this study implies that majority of the Aeromonas tested produced lipase. Lipase production may provide nutrients and may also constitute virulence factors by interacting with human leukocytes or by affecting several immune system functions through free fatty acids generated by lipolytic activity [39] .

Biofilm production is a very important virulent characteristic exhibited by Aeromonas and the isolates used in this study all produced biofilms. Biofilms enhances adherence of the organism to specific host tissues and thereby produce invasive microbial colonies and diverse illness [40] . Biofilms enhance stability and protect Aeromonas against external factors such as antibiotics [41] . Biofilms provide cell nutrients in higher concentrations than the surrounding environment via the nutrient-rich solute retained in the interstitial region of the extracellular polymeric matrix [42] [43] . Biofilms act as reservoirs in which some aeromonads are able to persist for several years and emerge later in favorable conditions [44] .

5. Conclusion

This study showed that Aeromonas hydrophila occurred in various organs of the fish samples examined and also exhibited several virulent characteristics. Fresh catfish contaminated with Aeromonas may cause toxicity and other gastrointestinal infection to man which may be lethal especially if the toxins produced are in high concentrations. It is therefore recommended that fresh catfish be cooked thoroughly before consumption. Fish handlers should ensure their hands are properly washed after catching the fishes from the river as their hands could serve as a vector introducing the organism to their gastrointestinal tract especially when used in consuming ready to eat foods.

Source of Funding

This research study was self-funded.

Cite this paper
Fowoyo, P. and Achimugu, F. (2019) Virulence of Aeromonas hydrophila Isolated from Fresh Water Catfish. Journal of Biosciences and Medicines, 7, 1-12. doi: 10.4236/jbm.2019.71001.

[1]   Carrasco, G.N., Marcos, J.Y., Salazar, M.S., Moral, C.H., Castillo, J.A. and Soriano, A.C. (1997) RFLP-PCR Analysis of the aroA Gene as a Taxonomic Tool for the Genus Aeromonas. FEMS Microbiology Letters, 156, 199-204.

[2]   Igbinosa, I.H., Igumbor, E.U., Aghdasi, F., Tom, M. and Okoh, A.I. (2012) Emerging Aeromonas Species Infections and Their Significance in Public Health. The Scientific World Journal, 2012, 1-13.

[3]   Mishra, S.K. and Agrawal, D. (2012) A Concise Manual of Pathogenic Microbiology. John Wiley & Sons, New York.

[4]   Percival, S.L., Yates, M.V., Williams, D., Chalmers, R. and Gray, N. (2013) Microbiology of Waterborne Diseases: Microbiological Aspects and Risks. Academic Press, New York.

[5]   Soon, J.M. and Baines, R. (2013) Managing Food Safety Risks in the Agri-Food Industries. CRC Press, Boca Raton.

[6]   Hossain, M.J., Sun, D., McGarey, D.J., Wrenn, S. and Alexander, L.M. (2014) An Asian Origin of Virulent Aeromonas hydrophila Responsible for Disease Epidemics in United States-Farmed Catfish. mBio, 5, e00848-14.

[7]   Gauthier, D.T. (2015) Bacterial Zoonoses of Fishes: A Review and Appraisal of Evidence for Linkages between Fish and Human Infections. The Veterinary Journal, 203, 27-35.

[8]   Clemence, M.A. and Guerrant, R.L. (2016) Infections and Intoxications from the Ocean: Risks of the Shore. In: Schlossberg, D., Ed., Infections of Leisure, 5th Edition, American Society of Microbiology, 1-54.

[9]   Batra, P., Mathur, P. and Misra, M.C. (2016) Aeromonas sp.: An Emerging Nosocomial Pathogen. Journal of Laboratory Physicians, 8, 1.

[10]   Mouton, M. and Botha, A. (2012) Cutaneous Lesions in Cetaceans: An Indicator of Ecosystem Status. INTECH Open Access Publisher.

[11]   Kaiser, L. and Surawicz, C.M. (2012) Infectious Causes of Chronic Diarrhoea. Best Practice and Research Clinical Gastroenterology, 26, 563-571.

[12]   Assiri, A.M.A. (2012) Acute Gastroenteritis in Infants and Children In: Elzouki, A.Y., et al., Eds., Textbook of Clinical Pediatrics, Springer Berlin Heidelberg, 1847-1860.

[13]   Berger, S. (2016) Aeromonas and Marine Vibrio, Global Status. GIDEON Informatics Inc., Los Angeles.

[14]   Sichewo, P.R., Gono, R.K., Muzondiwa, J. and Mungwadzi, W. (2014) Isolation and Identification of Pathogenic Bacteria in Edible Fish: A Case Study of Rural Aquaculture Projects Feeding Livestock Manure to Fish in Zimbabwe. International Journal of Current Microbiology and Applied Sciences, 3, 897-904.

[15]   Abbey, S.D. and Etang, B.B. (1988) Incidence and Biotyping of Aeromonas Species from the Environment. Microbios, 56, 228-229.

[16]   Nma, O.N. and Oruese, O.M. (2013) Bacteriological Quality of Street Vended Ready-to-Eat Fresh Salad Vegetables Sold in Port Harcourt Metropolis, Nigeria. Academia Arena, 5, 65-75.

[17]   Beaz-Hidalgo, R. and Figueras, M.J. (2013) Aeromonas sp. Whole Genomes and Virulence Factors Implicated in Fish Disease. Journal of Fish Diseases, 36, 371-388.

[18]   Atsanda, N.N., Agbede, S.A. and Onyia, L.U. (2000) Prevalence of Clinostomum Infection in Different Species and Sexes of Fish in Ponds. Journal of Tropical Veterinary Medicine, 18, 854-859.

[19]   Reed, W., Buchard, J., Hopson, L. and James, I. (1967) Fish and Fisheries in Northern Nigeria. Ministry of Agriculture, Northern Nigeria, 15-25.

[20]   Mailafia, S. (2003) Studies on Aeromonas species Isolated from Fishes in Zaria, Nigeria. M.Sc. Thesis, ABU, Zaria, 4-5.

[21]   Cowan, S.T. and Steel, K.J. (1974) Cowan and Steels Manual for the Identification of Medical Bacteria. 2nd Edition, Cambridge University Press, Cambridge, 28-106.

[22]   Cottral, G. (1978) Enteric Organisms. In: Manual of Standardized Methods for Veterinary Microbiology, Comstock Publishing Associates, Itaca, London, 52-63.

[23]   Fawole, M.O. and Oso, B.A. (2004) Laboratory Manual of Microbiology. Revised Edition, Spectrum Books Ltd., Ibadan, 127.

[24]   Cheesbrough, M. (2006) District Laboratory Practice in Tropical Countries. Cambridge University Press, Cambridge, 416.

[25]   Sarkar, A., Saha, M. and Roy, P. (2012) Identification and Typing of Aeromonas hydrophila through 16S rDNA-PCR Fingerprinting. Journal of Aquaculture Research and Development, 3, 146.

[26]   Stackebrandt, E., Murray, R.G.E. and Truper, H.G. (1988) Proteobacteria Classis Nov., a Name for the Phylogenetic Taxon That Includes the “Purple Bacteria and Their Relatives”. International Journal of Systematic Bacteriology, 38, 321-325.

[27]   Burke, V., Robinson, J., Berry, R.J. and Gracey, M. (1981) Detection of Enterotoxins of Aeromonas hydrophila by a Suckling-Mouse Test. Journal of Medical Microbiology, 14, 401-408.

[28]   Yano, T., Martins, L.M. and Marquez, R.F. (2002) Incidence of toxic Aeromonas Isolated from Food and Human Infection. FEMS Immunology and Medical Microbiology, 32, 237-242.

[29]   Janda, J.M. (2001) Aeromonas and Plesiomonas. In: Molecular Medical Microbiology, Academic Press, San Diego, 1237-1270.

[30]   Collee, J.G., Fraser, A.G., Marmino, B.P. and Simons, A. (1996) Mackin and McCartney Practical Medical Microbiology. 14th Edition, The Churchill Livingstone, Inc., London.

[31]   Freeman, D.J., Falkiner, F.R. and Keane, C.T. (1989) New Method for Detection of Slime Production by Coagulase Negative Staphylococci. Journal of Clinical Pathology, 42, 872-874.

[32]   Illanchezian, S.J., Sathishkumar, S.M. and Saritha, V. (2010) Virulence and Cytotoxicity of Seafood Borne Aeromonas hydrophila. Brazilian Journal of Microbiology, 41, 978-983.

[33]   Karimi, R.D. (2015) The Bacterial Flora of Tilapia (Oleochromis niloticus) and Catfish (Clarias Gariepinus) from Earthen Ponds in Sagana Fish Farm and Masinga Dam. Doctoral Dissertation, Kenyatta University, Kahawa.

[34]   Yakubu, S.E., Olanike, O.O. and Wong, C.M.Z. (2005) Survey of Aeromonas hydrophila from Tilapia zillii in Zaria Dam. Journal of scientific Research, 2, 59-63.

[35]   Hu, M., Wang, N., Pan, Z.H., Lu, C.P. and Liu, Y.J. (2012) Identity and Virulence Properties of Aeromonas Isolates from Diseased Fish, Healthy Controls and Water Environment in China. Letters in Applied Microbiology, 55, 224-233.

[36]   Reyes-Becerril, M., Angulo, C. and Ascencio, F. (2015) Humoral Immune Response and TLR9 Gene Expression in Pacific Red Snapper (Lutjanus peru) Experimentally Exposed to Aeromonas veronii. Fish and Shellfish Immunology, 42, 289-296.

[37]   Bogwald, J. and Dalmo, R.A. (2014) Gastrointestinal Pathogenesis in Aquatic Animals. Aquaculture Nutrition: Gut Health, Probiotics and Prebiotics. Wiley, Oxford, 53-74.

[38]   Aslani, M.M. and Hamzeh, H.S. (2004) Characterization and Distribution of Virulence Factors in Aeromonas hydrophila Strains Isolated from Fecal Samples of Diarrheal and Asymptomatic Healthy Persons in Ilam, Iran. Iran Biomedical Journal, 8, 199-203.

[39]   Timpe, J.M., Holm, M.M., Vanlenberg, S.L., Basrur, V. and Lafontaine, E.R. (2003) Identification of a Moraxella catarrhalis Outer Membrane Protein Exhibiting Both Adhesion and Lipolytic Activities. Infections and Immunity, 71, 4341-4350.

[40]   Maniati, M., Petinaki, E. and Maniatis, K.A.N. (2005) Antimicrobial Susceptibility of Aeromonas sp., Vibrio sp. and Plesiomonas shigelloides Isolated in the Philipines and Thailand. International Journal of Antimicrobial Agents, 25, 345-353.

[41]   Peterson, B.W., He, Y., Ren, Y., Zerdoum, A., Libera, M.R., Sharma, P.K. (2015) Viscoelasticity of Biofilms and Their Recalcitrance to Mechanical and Chemical Challenges. FEMS Microbiology Reviews, 39, 234-245.

[42]   Tsuchiya, Y., Ikenaga, M., Kurniawan, A., Hiraki, A., Arakawa, T. and Kusakabe, R. (2009) Nutrient-Rich Microhabitats within Biofilms Were Synchronized with the External Environment. Microbes and Environments, 24, 43-51.

[43]   Tsuchiya, Y., Eda, S., Kiriyama, C., Asada, T. and Morisaki, H. (2016) Analysis of Dissolved Organic Nutrients in the Interstitial Water of Natural Biofilms. Microbial Ecology, 72, 85-95.

[44]   Kühn, I., Allestam, G., Huys, G., Janssen, P., Kersters, K., et al. (1997) Diversity, Persistence and Virulence of Aeromonas Strains Isolated from Drinking Water Distribution Systems in Sweden. Applied and Environmental Microbiology, 63, 2708-2715.