World Journal of Science and Technology 2012, 2(11):42-47 ISSN: 2231 2587 Available Online: www.worldjournalofscience.

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Isolation of pathogenic microorganism from Ulsoor lake, Bangalore, India

Jabez W. Osborne*, Ankita kumari, Juhi Sinha, S. Megha Swamy, Shivangi, Shama Parween and M. Rohini Kumar School of Bioscience and Technology, VIT University, Vellore-632014, Tamil Nadu, India Abstract Lake Ulsoor located in Bangalore, India, is one of the largest in Karnataka. It was used for recreational purpose in the past but recently it is highly polluted because of excess of industrialization. Amount of pollutant and various microorganisms present in this lake is to be explored. So it was a need to isolate and enumerate the microorganisms in Ulsoor Lake to know its harmful effect among the local population. In this study pathogenic bacterial cultures were isolated and identified from Ulsoor Lake. Samples were collected from Ulsoor Lake and were inoculated onto various media like Salmonella Shigella Agar, Mannitol Salt Agar and MacConkey Agar for isolation of pathogenic bacterium like Salmonella, Shigella, E.coli etc. The cultures were isolated, purified and maintained in respective media. The pure cultures obtained were further characterized morphologically and biochemically. The further study deals with the analysis of various toxic compounds present in the sample. Non-pathogenic bacterium will be isolated for detoxifying the toxic compound present in the water and sediments of Ulsoor Lake. Keywords: Enterobacteriaceae, Ulsoor Lake, Salmonella Shigella Agar. INTRODUCTION Water having vast content of microbial diversity with valuable product in them here our aim of the study is to find out the different species of microbes in the water so we have taken the sample from Lake Ulsoor which is present in middle of the city Bangalore (Krovacek et al., 2000). Pathogens majorily cause of human illness with many numbers of cases, 325,000 hospitalizations, and 5200 deaths in the United States approx. every year and also much higher rates in the developed countries (Poleg et al., 2006). In the water distribution systems, a peculiar microbial ecology is to be defined, considering the importance of different physical, chemical and biotic variables, like temperature, type of pipe surface, nutrient levels, presence of disinfectant chemicals, biofilms, predatory protists and their endosymbionts (Greub et al., 2010). In the water distribution system formatiom of biofilm can easily occur if the water stands inside of the distribution system for an extended period even if disinfectant residual is present and the environment is oligotrophic. (Lee el al., 2010). The contamination of water sources affects all aspects of life from heath care to limitations of commercial water use. We cannot use the same murky water for cleaning as we do for consumption, because it causes health problems and would deteriorate quickly, causing a large chain of disastrous events. The accurate reasons and mechanisms, why and how a switch from a free-living status to a colonizing and invading pathogenic status occur, are still not clearly understood (Gianinazzi et al., 2009). Many types of microbes live naturally in soil rocks and parts of sub-surface ecosystem and this increases the level of microorganism in water of
Received: Sept 10, 2012; Revised: Oct 13, 2012; Accepted: Nov 24, 2012. *Corresponding Author Jabez W. Osborne School of Bioscience and Technology, VIT University, Vellore-632014, Tamil Nadu, India Tel: +91-9894204309; Email: jabezosborne@vit.ac.in

lake or well etc. The quality of this water is basically related to point pollution sources like treated wastewater effluents and to non-point sources like storm-water run-off (Ferguson et al., 1996; Georges et al., 2001; Chapman, 1996). In this aquatic environment, pathogenic bacteria, often associated with suspended particulate matter, are grazed by protozoa (Georges et al., 2001) and may undergo different stresses that lead to loss of culturability and viability (viable but nonculturable) (Munro et al., 1995; Oliver et al., 1995; Roszak and Colwell, 1987). Many water-borne outbreaks have been traced, whereby drinking water has been accidentally contaminated by untreated waste (Fricker and Crabb, 1998; Gasser and Donoghue, 1999). Presently, the techniques used to isolate or detect the contaminating agents present in water differ enormously according to the type of microorganisms and matrices, generating time and economical losses (Brassard et al., 2011). The family Enterobacteriaceae comprises of gram negative facultative anaerobic, non sporing forming bacteria (Mary et al., 2001). Member of this family as follows: Escherichia, Salmonella, Shigella, Klebsiella, Erwinia, Enterobacter, Proteus and Citrobacter. Many Gram-negative bacteria produces membrane vesicles (MV). During normal growth are capable of killing other bacteria or microorganism in the surrounding medium (Kadurugamuwa and Beveridge, 1996; Li et al., 1998; Mary et al., 2001). Pathogens possesses several pathogenic properties like production of heat-stabile and heat-labile toxin, cytotoxin, hemolysin, haemagglutinin and many other potential virulence factors (Khardori, 1988; Tsukamoto et al., 1978; Binns et al., 1984; Gardner et al., 1987; Hostacka et al., 1982; Gardner et al., 1990; Krovacek et al., 2000) .In addition to bacteria two other type of pathogenic microorganism can affect water, viruses and protozoa. The disease causing organisms are usually shed by infected person via faeces. Like Giardia lamblia and Cryptosporidium having large size of protozoan cyst but occurrence is very rare. Disease like polio, hepatitis can be caused. Coliform bacteria live in soil enter into water supplies and then disposed to streams or lakes. When they enter into individuals body

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from contaminated sources and cause various disease. E. coli is one species of coliform bacteria. Its presence in water indicates that sewage material may be present and if sewage is present, more harmful disease causing organisms present. MATERIALS AND METHODS Sampling sites and Sample collection Water samples were collected from the different areas of the Ulsoor Lake on 30th July 2011. Water and sediment samples were collected from four different areas. These samples were collected in sterile bags and sterile bottles aseptically. Samples were processed within 48 hours. The sample was inoculated (0.1 ml) onto MacConkey agar, salmonella shigella agar, and mannitol motility agar at pH 7.8. Plates were incubated aerobically in incubator at 37 C for 24 hours.

groups based on Gram staining (Vetrivelkalai et al., 2010; Gottstein et al., 2009). The eleven phenotypic characterization test like the Gram staining, catalase production and oxidase production tests were performed (Lee et al., 2010) Morphology characterization For morphology of microorganisms the following tests were performed: Gram staining, Endospore staining, Hanging drop technique. Biochemical characterization For this also the following tests were done for conformation of microorganisms: Catalase test, Oxidase test, Indole test, Citrate test, Methyl red test, Voges Proskauer, Triple Sugar Iron Test, Mannitol Motility Test. RESULTS Isolation of pathogenic and non-pathogenic microorganism In MacConkey agar plate from mother culture six colonies were isolated shown in figure 2. From Salmonella Shigella Agar plate seven colonies were isolated shown in figure 3. And from Mannitol Salt Agar plate also seven colonies were isolated shown in figure 4.

Fig 1. Bangalore Ulsoor Lake Map

Culture Media Used for Isolation MacConkey agar is widely used for lactose fermenting gram negative microorganism found in faeces could be further distinguished by their action on other carbohydrate incorporated either singly or in combination in bile salt neutral red media which inhibit the growth of gram positive bacteria. Incubation required for the growth of the organisms like Escherichia Coli, Enterobacter and Klebsiella etc. is 24-48 hours at 36 2 C. Non lactose fermenting bacteria such as Proteus spp., Salmonella, Shigella cannot utilize lactose and will use peptone instead. Salmonella Shigella Agar (SS Agar) is highly selective, differential media used primarily for the isolation of non-lactose fermenting organisms and Salmonella and Shigella. Bile salt, brilliant green, sodium thiosulphate and citrate are the selective agents. Incubation required for the growth of organism is 35 C for 24 hours. Manitol Salt Agar (MSA) is also highly selective and differential semi solid media used for detection of motility. The high concentration of salt (75%) selects for members of the genus Staphylococcus like Serratia marcescens, since they can tolerate high saline levels. It also contains sugar mannitol and the pH indicator phenol red. Incubation required for the growth of organism is 37 C for 24 hours. Characterization of Isolates The isolates were characterized morphologically and biochemically. The isolates were initially divided into two broad
Fig 2. Isolates in MacConkey Agar plates

Fig 3. Isolates in Salmonella Shigella Agar plates

Fig 4. Isolates in Mannitol Salt Agar plates

Morphological characterization Gram staining: In two plates of MacConkey agar media six culture

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were present. After staining five gram negative rods and one gram positive rods were found. In gram negative, one aerobic spore forming and one rod in chain were found. In two plates of SS Agar seven cultures were present. In which gram negative rods shaped organisms were found. Similarly, two plates of MSA, seven cultures were present in which four were gram positive where one of the culture is pleomorphic and four were gram negative organisms were found. Hanging drop technique: In two plates of MacConkey agar media six culture were present. After performing motility test three were found to be motile and three were non motile. In two plates of SS Agar seven cultures were present. In which all organism were motile. Similarly, two plates of MSA, seven cultures were present in which five organisms were motile and two organisms were non motile. Biochemical characterization: Catalase test: All the cultures present on MacConkey agar were found to be catalase positive. In Salmonella Shigella Agar, four organisms were found to be catalase positive and three were catalase negative. In Mannitol Salt Agar six were found to be catalase positive and one was found to be catalase negative. Oxidase test: In MacConkey agar five cultures were found to be oxidase positive and one was found to be oxidase negative. In SS Agar four cultures were found positive and three cultures were found oxidase negative organisms. In MSA all the cultures were found oxidase negative organisms. Indole test: Except Vibrio and E. coli strains, all cultured organisms were found indole negative (figure 5). Citrate test: Except E. coli, all other cultured organisms were found to be citrate positive (figure 5). Methyl red test: In MacConkey agar two cultures were found MR positive and four were found MR negative organisms. In Salmonella Shigella Agar all cultures were found to be MR negative organisms. In Mannitol Salt Agar only two cultured organisms were found to be MR positive and five other culture were found to be MR negative (figure 5).

positive and rest were found VP negative. In Salmonella Shigella Agar two cultures were found to be VP positive and all other were found VP negative. Likewise in Mannitol Salt Agar three cultures were found to be VP positive and four other cultures were found to be VP negative (figure 5). Triple sugar iron test: In MacConkey agar two cultures were found with acidic butt and H2S production, one with alkaline butt with gas production and rest were only acidic. In Salmonella Shigella Agar four were found with acidic butt with H2S and gas production and rest were alkaline. In Mannitol Salt Agar all cultures were found with alkaline butt (figure 6). Mannitol motility test: In MacConkey agar four of the cultures were found to be turbid with upliftment and two were non turbid. In Salmonella Shigella Agar four of the cultures were found to be turbid with upliftment and three of them show no turbidity. In Mannitol Salt Agar three cultures shows tubidity and four cultures shows no turbidity.

Fig 5. IMViC tests

Fig 6. Triple Sugar Iron tests

Voges Proskauer test: In MacConkey agar only one culture is VP

Table 1. Biochemical tests of isolates of MacConkey agar plates

World Journal of Science and Technology 2012, 2(11):42-47 Table 2. Biochemical tests of isolates of Salmonella Shigella Agar plates

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Table 3. Biochemical tests of isolates of Mannitol Motility Agar plates

DISCUSSION The present work is a novel attempt to screen, isolate and identify microorganisms that may exhibit a pathogenic potential from different sites of lake. Water bodies selected were directly related to human activities (i.e. boating, site viewing, fishing) (Gottstein, et al, 2009). The assembly of microbes that were found in the Lake Ulsoor sample indicates that the lake has a diverse population of pathogens and potentially a complete ecosystem (DElia et al.,2008). It is necessary to assess the relative importance of animal wastes and human sewage effluent as contributors of waterborne microorganisms for individual water catchment areas. 20 cultures of a total of 4 field isolates were established during this study. All isolates were characterized based on morphological and biochemical test (Gottstein et al., 2009). Information of microorganisms density, was got from effective catchment sampling strategies, this can assist in making decisions regarding the implementation of potential control measures. A lot of efforts have been made in reaching the current level of understanding of microorganisms and the waterborne route of infection and quite high frequency of occurrence in human beings, livestock and the environment has forged multidisciplinary links

among those who are really interested in safeguarding public health (Vetrivelkalai et al., 2010). These are needed for a better understanding of both the epidemiology of endemic waterborne disease and the occurrence. Impacts of pathogens on humans There are various reports mentioning the possible diseases caused by pathogens transmitted in water. The strains of Proteus, Pseudomonas, Enterobacter, Klebsiella, Citrobacter and E. coli are well known to cause Urinary Tract Infection. Diarrhea can be resultant from the infections of strains of Proteus, Salmonella and E.coli. Septicaemia caused by Proteus, Pseudomonas, Enterobacter and Serratia. Meningitis caused by Enterobacter, Serratia and Citrobacter. Respiratory tract infection caused by Staphylococcus and Serratia (Ananthnarayan and Paniker, 2000). This study reports the presence of these harmful pathogens in the lake water which is at the center of the metro-like city. CONCLUSION From the survey we are able to isolate and identify the various

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microorganisms which can be potential candidates for being pathogens thriving in the water of ulsoor lake at the hot spot of Bengaluru. These pathogens might play a major role in causing epidemic outbreaks among the local population. ACKNOWLEDGEMENT The authors would like to thank the program manager Dr. Pragasam Viswanathan for his support throughout the project and also lab assistants of SBST. REFERENCES [1] Watanabe, K. and Baker Paul, W., 2000. Environmentally Relevant Microorganisms. Journal Of Bioscience And Bioengineering 89(1): 1-11. [2] Jeenaa, M.I., Deepaa P., Rahimana K.M., Shanthia R.T. and Hathab, A.A.M., 2006. Risk assessment of heterotrophic bacteria from bottled drinking water sold in Indian markets. Int. J. Hyg. Environ.-Health 209 : 191196. [3] Rashid, N., Kikuchi, H., Ezaki, S., Atomi, H. and Imanaka, T., 1999. Isolation and Characterization of Psychrotrophs Environments from Subterranean. Journal Of Bioscience and BioengineeringVol. 87(6) : 746-751. [4] Lee, J., Lee, C.S. , Hugunin, K.M. , Maute, C.J. and Dysko, R.C., 2010. Bacteria from drinking water supply and their fate in gastrointestinal tracts of germ-free mice: A phylogenetic comparison study. Water Research 44 : 5050 5058. [5] Ferguson, C.M., Ggote, B.G., Ashbolt, N.J. and Stevenson, I.M.,1996. Relationships between indicators and water quality in an estuarine system, Wat. Res. 30 : 20452054. [6] Georges, Petit, M., Theate, C. and Servais, P. 2001. Distribution of coliforms in the Seine river and estuary (France) studied by rapid enzymatic methods and plate counts, Estuaries 24 : 9941002. [7] Chapman, D. (Ed.), 1996. Water Quality Assessments: A Guide to the Use of Biota, Sediments and Water in Environmental Monitoring, E & FN Spon, London/New York. [8] Munro, P.M., Flatau, G.N. and Clement, R.L. and Gauhier, M.J.,1995. Influence of the RpoS (KatF) sigma factor on maintenance of viability and culturability of Escherichia coli and Salmonella typhimurium in seawater, Appl. Environ. Microbiol. 61 : 18531858 [9] Oliver, J.D., Hite, F., McDougald, D., Andon, N.D.and Simpson, L.M.1995. Entry into, and resuscitation from the VBNC state by Vibrio vulnificus in a estuarine environment, Appl. Environ. Microbiol. 61 : 26242630. [10] Roszak, D.B. and Colwell, R.R., 1987. Survival strategies of bacteria in the natural environment, Microbiol. Rev. 51 : 365 379. [11] Brassard, J., Guvremont, E., Gagn, M.J. and Lamoureux, L. 2011. Simultaneous recovery of bacteria and viruses from contaminated water and spinach by a filtration method. International Journal of Food Microbiology 144 : 565568. [12] Corsaro, D., Pages, G.S., Catalan, V., Loret, J.F. and Greub, G., 2010. Biodiversity of amoebae and amoeba-associated

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