South As. J. Biol. Sci. 2(3): ISSN

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1 South As. J. Biol. Sci. 2(3): ISSN A Study on Hemolysin Production by Aeromonas hydrophila isolated from Infected fresh Water Fish Labeo rohita (Rogu) * 1 V.Geetha, 2 A.Michael and 1 Ram Rammohan 1 Department of Microbiology, CMS College of Science and Commerce, Coimbatore Department of PG Microbiology and Research, PSG College of Arts and Science, Coimbatore Tamilnadu, India To cite this article: V.Geetha, A.Michael and Ram Rammohan, A Study on Hemolysin Production by Aeromonas hydrophila isolated from infected fresh Water Fish Labeo rohita (Rogu). South As. J. Biol.Sci., 2(3): Abstract Fish pathogen Aeromonas hydrophila was isolated from infected fresh water fish showing hemorrhagic symptom. Isolation of Aeromonas hydrophila was done on Aeromonas selective agar base medium. Phenotypic characterisation of virulence factors such as hemolytic activity, proteolytic and lipolytic activity was performed. Aeromonas hydrophila demonstrated a very clear β hemolysis on the blood agar plate. Hemolysin was detected as a extracellular virulence factor produced by the pathogen. The effect of environmental factors such as ph and temperature on hemolysin production was also studied. The isolate showed significantly varying amounts of extra cellular protein production at different temperatures and ph. The optimal conditions for hemolysin production was found to be at 37 0 C and ph 7. The molecular weight of the secreted protein fraction was determined. The protein profile of the extra cellular fraction clearly demonstrated the presence of protein bands between 48 kda to 53 kda with a significantly prominent band of 50kDa corresponding to the virulence protein, hemolysin of Aeromonas hydrophila. Key words: Aeromonas hydrophila virulence factor characterization -hemolysin production- infected fish. *Corresponding author: V. Geetha, Assistant Professor, Department of Microbiology, CMS College of Science and Commerce, Coimbatore Mail Id: geethuvenugopal03@gmail.com 173 South As. J. Biol. Sci. 2(3): Geetha et al.

2 Introduction Aeromonas hydrophila is an opportunist human pathogen which is widely distributed in aquatic environments. Over the past decade, the emergence of Aeromonas species as bonafide human pathogens and their probable role as etiologic agents of bacterial gastroenteritis have resulted in an explosion of scientific interest in the genus. The organisms are ubiquitous in fresh and brackish water. Aeromonas infections are one of the most common bacterial diseases diagnosed in marine and cultured freshwater fish. Aeromonas hydrophila is found in diverse habitats, including soil, water and is pathogenic to warm and cold-blooded animals. Aquatic environment along with sea-food is thus important potential source for the transmission of Aeromonas hydrophila resulting in human infections. Aeromonas spp. have been involved in wound infections, sepsis, outbreaks of water, and food-borne gastroenteritis. Virulence in Aeromonas hydrophila is multi-factorial which consists of aerolysins, hemolysins, enterotoxins, and proteolytic enzymes which play significant role in pathogenesis. Aeromonas hydrophila causes a variety of diseases in both fish and human populations. The ubiquitous nature of the bacterium in aquatic environments provides significant opportunity for animals, mainly fish and amphibians to contact and ingest organisms. Aeromonas hydrophila is very toxic to many organisms because of its structure 1. Motile aeromonad of the Aeromonas hydrophila complex causes hemorrhagic septicemia in fish. This bacterium has been observed in numerous species of freshwater fish and occasionally in marine fish and in amphibians, reptiles, cattle and humans throughout the world 2. The bacterium is distributed widely in freshwater and bottom sediments containing organic material as well as in the intestinal tract of fish 3. Aeromonas hydrophila is typically recognized as an opportunistic pathogen or secondary invader; however, there have been some reports that have shown Aeromonas.hydrophila to occur as a primary pathogen 1. Some strains have been reported to be highly virulent and others non-virulent. Most cultured and wild freshwater fish are susceptible to the infection and display cutaneous hemorrhages of the fins and trunk and a hemorrhagic septicemia disease. Both A. hydrophila and A. sobria produced enterotoxins, dermonecrotic factors and hemolysins 4. Although both species exhibited hemolysis on blood agar plates at 30 C, only A. hydrophila did so at 10 C. Because these researchers were working with salmonid fish, they suggested that the hemolysis of red blood cells by A. hydrophila at temperatures comparable to those of the water in which fish live may at least partially account for the difference in virulence between A. hydrophila and A. sobria. Enterotoxins, hemolysins, proteases, hemagglutinins, and endotoxins produced by this complex of bacterial organisms have been the subject of much research South As. J. Biol. Sci. 2(3): Geetha et al.

3 Aeromonas hydrophila infection is the scourge of fresh and warm water fish farming worldwide and is considered as a significant economic problem particularly in China and India over the past decade 6. It is also believed to be a pathogen of emerging importance for humans through consuming fish and shellfish contaminated with A. hydrophila 7. Aeromonas hydrophila are psychrotrophic in nature with a multiplicity of virulence factors. They are commonly isolated from normal healthy fish, with only certain strains possessing the virulence factors necessary to induce disease 7. There are many combinations of factors involved in host susceptibility to A. hydrophila infections. In the present investigation, we have isolated and characterized.aeromonas hydrophila strain from infected freshwater fish. We have also assessed the protein profile of extracellular proteins (ECPs) and confirmed the presence of hemolysin protein. We have also studied the effect of environmental factors such as ph and temperature on hemolysin production. Materials and Methods Isolation of Aeromonas hydrophila from infected fish Infected fish sample Rohu (Labeo rohita) showing characteristic hemorrhagic lesion was purchased from local market in Coimbatore. The fish was carefully dissected under aseptic conditions for sample collection. Isolation of the pathogen was performed in accordance to Microbiological standards. Collection of abdominal fluids The fish was surface sterilized with 70% ethanol. Using a sterile syringe, the needle was carefully punched in the abdominal region and 2-3 ml of fluid was taken and inoculated in 100mL sterile Alkaline Buffered Peptone water. The flask was incubated at 37 C for 18 h. Collection of internal organs The fish was carefully dissected with a sterile blade and the abdomen was cut open. The internal organs such as liver, heart, gills and stomach were carefully dissected (25 gm of organ/ 225 ml of alkaline peptone water) and inoculated individually in alkaline peptone water. The flasks were incubated at 37 C for 18 h. Confirmation of Aeromonas hydrophila A loopful of broth from enriched alkaline peptone water was inoculated onto Aeromonas selective agar base medium and the plates were incubated at 37 C for 24 h. 175 South As. J. Biol. Sci. 2(3): Geetha et al.

4 Determination of colony characteristics and Gram staining of the test bacterial isolate followed by biochemical tests as described by Bergey s Manual of Systematic Bacteriology was also performed. Phenotypic characterization of virulence factors Five colonies of those grown on ASA (Aeromonas Selective Agar plate) were suspended in 3 ml of Mueller Hinton broth. The density of this suspension was adjusted to 0.5 of the McFarland standard (1.5X10 8 cells) µl of this suspension are added to several media or substrates for the phenotypic determination of virulence factors as described by Castro-Escarpulli 8. Hemolytic activity The strains were tested for β-hemolytic activity on agar base supplemented with 5% human erythrocytes. 5µL of each suspension was streaked onto the plates and incubated at 37 o C for 24 h as described by Castro-Escarpulli 8. Proteolytic activity Casein hydrolysis was tested on Mueller Hinton agar containing 10% (w/v) skimmed milk by streaking 10 µl of each suspension onto the plates and incubating at 37 o C 24 h as described by Castro- Escarpulli 8 Lipolytic activity Lipase activity was determined by streaking 10 µl suspension onto plates containing 0.5% tributyrin emulsified with 0.2% Triton X-100 and incubated at 37 o C for 24 h as described by Castro- Escarpulli 8. Extraction of extracellular proteins (hemolysin) The isolated organism Aeromonas hydrophila from infected fish grown on Aeromonas selective medium was inoculated into sterilized 50 ml of LB broth, and was incubated at 37 C for 24 h at 200rpm in a shaker incubator for 24 h. Following incubation, the cultures were suspended in sterile centrifuge tubes, and were centrifuged at 5000rpm at 4 C for 15 minutes. The supernatant was re-suspended into fresh centrifuge tubes. The re-suspended supernatant was treated with 4% trichloroacetic acid for precipitation of extracellular proteins. The centrifuge tubes were incubated overnight at 4 C. Following incubation, the tubes were centrifuged at rpm for 20 minutes at 4 C. The supernatant was discarded and the pellet containing extracellular proteins was resuspended in 1M Tris HCl for further analysis. Production was done based on the method described by Anju Pandey 9. Protein production assay was done in broth, following Lowry method 176 South As. J. Biol. Sci. 2(3): Geetha et al.

5 Effect of temperature and ph on hemolysin production Overnight culture of Aeromonas hydrophila in LB broth was inoculated in different conical flasks and incubated at different temperatures viz: 4 C, 22 C, 37 C, 45 C, 55 C. LB broths with varying ph ranges from 4-10 was also inoculated with Aeromonas hydrophila. The production and extraction of hemolysin was performed as described by Anju Pandey 9. Molecular weight determination of extracellular proteins The ECPs were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE) using a 12.5% (w/v) separating gel and a 5% (w/v) stacking gel according to the procedure of Laemmli (Laemmli, 1970). Molecular weight standards were run along with the samples in order to determine the molecular weight of the proteins. Results In the present study the strain isolated produced hemolytic, proteolytic, and lipolytic activities, which attribute to the virulence of Aeromonas spp(figure-1). The bacterial strain were identified based on morphological, cultural and biochemical characteristics (Table-1). The culture was checked for its hemolysin production incubated at different temperature viz 4 C, 22 C, 37 C, 45 C, 55 C and different ph ranges. The isolate showed significantly different amount of extra cellular proteins at different temperature and different ph ranges (Figures-2&3). It was interesting to note that Aeromonas strain produced different amount of protein indicating that temperature and ph as an important role to influence the hemolysin production. Increased amount of hemolysin was found to be produced at temperature 37 C (2.4mg/ml) and at ph 7 (2.8mg/ml). The protein profile of the extra cellular fraction clearly demonstrated the presence of protein bands between 48kDa to 53kDa with a significantly prominent band of 50kDa corresponding to the virulence protein, hemolysin of Aeromonas hydrophila (Figure-4). Environmental factors such as temperature and ph has a significant role in pathogenicity. Optimal temperature for the growth of bacteria is determining the maximal virulence in an organism Table -1 showing the biochemical characteristics of Aeromonas hydrophila Indole Methyl red Voges proskauer Simmon citrate Triple sugar iron agar Carbohydrate fermentation Catalase Starch hydroly Casein hydroly No gas production Mannitol, Dextrose, without gas production South As. J. Biol. Sci. 2(3): Geetha et al.

6 Figure-1: Hemolytic colonies of Aeromonas hydrophila on Blood agar plates Figure-2: Effect of temperature on hemolysin production Figure-3: Effect of ph on hemolysin production Figure 4: SDS-PAGE analysis of extracellular protein fraction. (1) Control. (2) Extracellular protein fraction from Aeromonas hydrophila showing hemolysin (50 kda). (3) Protein molecular weight marker. Conclusion Aeromonas hydrophila was isolated and was detected as a common organism infecting fish. It was not only found in the aquatic environment but also found in other habitats like soil and is pathogenic to 178 South As. J. Biol. Sci. 2(3): Geetha et al.

7 warm and cold blooded animals. Thus sea food is an important potential source for the transmission of Aeromonas hydrophila resulting in human infections such as wound infection, sepsis and food borne gastroenteritis. Another study was performed to study the effect of temperature and ph on protein production. It revealed the production of ECP was maximum at optimal temperature 37 C and at ph 7. But the organism has the ability to grow at wide range of temperature, this gives the proof that the organism can cause infection in any habitat. Aeromonas hydrophila showed lipolytic and hemolytic property and it also showed the protein profile of the extra cellular fraction clearly demonstrated the presence of protein bands with a significantly prominent band of 50kDa corresponding to the virulence protein, hemolysin of Aeromonas hydrophila. In the present study it can be concluded that enzymes or toxins are actually reflective of biological virulence and appear to enhance the disease process in-vivo. This means that the whole process of pathogenesis is a complex interaction among the host, agent and environmental determinants. Since all the small outbreaks were recorded in completely dry seasons, the environmental factors like temperature change and deterioration of water quality ph may broadly contribute to the onset of the disease by A. hydrophila.in many organisms virulence gene and its product have been identified. Therefore,a future study should be done to characterize a molecular or immunological probe to screen aeromonads in water bodies for successful aquaculture. References 1. Austin DA., McIntosh D. and Austin B., Taxonomy of fish associated Aeromonas spp., with the description of Aeromonas salmonicida subsp. smithia subsp. nov. Systematic and Applied Microbiology. 11: Bullock GL., Chen PK. and Stuckey HM., Studies of motile aeromonads isolated from diseased warm water and coldwater fishes. Abstracts of the Annual Meeting of the American Society for Microbiology, Philadelphia, Pennsylvania.pp: Sugita H., Tanaka K., Yoshinami M. and Deguchi Y., Distribution of aeromonas species in the intestinal tracts of river fish. Applied and Environmental microbiology. 61: Olivier G., Lallier R. and Lariviere S., A toxigenic profile of Aeromonas hydrophila and Aeromonas sobria isolated from fish. Canadian Journal of Microbiology. 27: Cahill MM., Virulence factors in motile Aeromonas species. Journal of Applied Bacteriology. 69: South As. J. Biol. Sci. 2(3): Geetha et al.

8 6. Karunasagar I., Rosalind GM., Karunasagar I. and Rao KG., Aeromonas hydrophila septicemia in Indian major carps in some commercial fish farms of West Godavari District, Andhra Pradesh. Curr. Sci., 58: Vivekanandhan G., Hatha AAM. and Lakshmanaperumalsamy P., Prevalence of Aeromonas hydrophila in fish and prawns from the seafood market of Coimbatore, South India. Food Microbiol., 22 (1): Castro-Escarpulliab G., Figuerasb MJ., Aguilera-Arreolaa G., Solerb L., Ferna ndez-rendo na E., Aparicioa GO., Guarrob J. and Chaco MR., Characterisation of Aeromonas spp. isolated from frozen fish intended for human consumption in Mexico. International Journal of Food Microbiology.84: Anju Pandey, Milind Naik, and Santosh Kumar Dubey Hemolysin, Protease, and EPS Producing Pathogenic Aeromonas hydrophila Strain An4 Shows Antibacterial Activity against Marine Bacterial Fish Pathogens.Journal of Marine Biology.Volume 2010 (2010), Article ID , 9 pagesdoi: /2010/ South As. J. Biol. Sci. 2(3): Geetha et al.