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Title: Characterization of bacterial spp in commercially available drinking water of
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Bangladesh
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MD. Salahuddin1*, Ashit Kumar Paul2*, M. Salahuddin3, Napolean Bonaparte4 Abdus
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Samad5, M. Bahanur Rahman1 and M. Shahidur Rahman Khan1
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Running title: Public health sanitation of commercially available drinking water.
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Author and Co-authors:
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1. Md. Salahuddin
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Department of Microbiology and Hygiene,
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Faculty of Veterinary Science,
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Bangladesh Agricultural University,
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Mymensingh-2202, Bangladesh
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2. Ashit Kumar Paul*
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Department of Medicine and Surgery,
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Faculty of Animal Science and Veterinary Medicine,
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Patuakhali Science and Technology University,
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Barisal-8210, Bangladesh
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3. M. Salahuddin
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Department of Biological Sciences,
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Sungkyunkwan University,
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Gyeonggi-do, 440-746, South Korea
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4. Napolean Bunaparte
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School of Biotechnology,
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Suranaree University of Technology,
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Korat 30000, Thailand
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5. Md. Abdus Samad
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Veterinary Surgeon
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Upazila Livestock Office,
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Department of Livestock services,
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Ministry of Fisheries and Livestock,
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Gaibandha, Bangladesh
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6. Dr. M. Bahanur Rahman
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Department of Microbiology and Hygiene,
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Faculty of Veterinary Science,
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Bangladesh Agricultural University,
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Mymensingh-2202, Bangladesh
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7. Dr. M. Shahidur Rahman Khan
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Department of Microbiology and Hygiene,
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Faculty of Veterinary Science,
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Bangladesh Agricultural University,
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Mymensingh-2202, Bangladesh
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Corresponding author:
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1. Ashit Kumar Paul*
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Department of Medicine and Surgery,
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Faculty of Animal Science and Veterinary Medicine,
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Patuakhali Science and Technology University,
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Barisal-8210, Bangladesh
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Email: akpaul2008@gmail.com
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Fax: +88-04427-56112
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2. Md. Salahuddin
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Department of Microbiology and Hygiene,
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Faculty of Veterinary Science,
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Bangladesh Agricultural University,
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Mymensingh-2202, Bangladesh
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Email: md.salahuddin84@yahoo.com
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Fax: +88-04427-56112
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Abstract
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An investigation was carried out focusing on the characterization Bacterial spp in
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commercially available drinking water of Bangladesh. This study was conducted to
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characterize the Bacteria species in culturally, biochemically, pathogenically and antibiotic
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sensitivity analysis. A total of 50 bottled water samples of 10 manufacturing companies were
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collected from the market. Out of these 50 samples 4 (8%) were found to be positive for
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Staphylococcus aureus. All isolates fermented glucose, maltose, mannitol, lactose and sucrose
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with production of acid but not fermented salicin, raffinose, or inulin. On the other hand, these
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isolates showed Indol test negative but Voges-Proskaure test and Methyl-Red test were
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positive. All these isolates were coagulase positive and pathogenic to mice. All isolates were
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highly sensitive to Enrofloxacin and Nor-floxacin, moderately sensitive to Ciprofloxacin and
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Ampicillin, less sensitive to Pefloxacin and resistant to Furazolidon. Based on the present
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study it might be concluded that the investigated bacteria species may be Staphylococcus
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aureus. This is the preliminary report focusing on public health importance. Therefore it
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needs further characterization using serological and molecular techniques.
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Key words: Bacterial spp, Commercial bottled water, Public health
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Introduction
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Water is a molecular compound of Hydrogen and Oxygen, with molecular formula
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H2O. Water has a number of roles in living organisms such as solvent, temperature buffer,
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metabolite, living environment etc. Water is one of the most important things on earth. Every
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living thing needs water for its survival. However, infectious diseases caused by pathogenic
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bacteria, viruses and protozoa, are the most common and widespread health risk associated
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with drinking water. Controlling the risks related to these pathogens is a permanent challenge
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for the water industry (Guillot and Francois, 2009) with involving public health importance.
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The control of microorganisms is critical for the prevention and treatment of diseases,
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however, the increasing number and variety of drug-resistant pathogens is a serious public
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health problem (Prescott et al., 2005).
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Since the extensive bacteriological studies on the occurrence and distribution of
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microbes of public health significance have been made which have revealed that most
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portions of the world’s population are suffering major epidemics of water borne diseases (Ali,
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2003).
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Bottled water is prepared from underground or surface water. So, if proper sanitary
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measures are not employed they may harbor potential microbes (Amer et al., 2008). During
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traveling most of the people drink bottled water. The enteric bacilli may contaminate to this
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water through the leakage and improper quality control done by manufacturing companies.
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Bacterial contamination cannot be detected by sight, smell or taste. The Environmental
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Protection Agency (EPA) requires that all public water suppliers regularly test for coliform
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bacteria and deliver water that meets the EPA standards. The hygienic problems of bottled
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drinking-water are emphasized, especially on microbial contamination (Zhao et al., 2004).
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Bottled water, because it is defined as a “food” under federal regulations, is under the
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authority of the Food and Drug Administration (FDA) while the EPA—under much stricter
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standards—regulates tap water. Thus, bottled water, depending upon the brand, may actually
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be less clean and safe than tap water. The EPA mandates that local water treatment plants
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provide city residents with a detailed account of tap water’s source and the results of any
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testing, including contaminant level violations. Bottled water companies are under no such
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directives. Also, while municipal water systems must be test for harmful microbiological
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content in water several times a day, bottled water companies are required to test for these
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microbes only once a week.
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Bacterial species associated with tap water, river water, pond water and drinking water
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were characterized and counted by Ali (2003) and Amer et al., (2008). Bacterial quality was
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studied associated with commercial fruit juice by Chung et al., (2003). But the
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characterization of Bacterial species in commercially available drinking water is not done yet
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in aspect of Bangladesh. Therefore, the present study has been undertaken to isolate and
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identify the bacterial species in commercially available bottle water of Bangladesh and their
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antibiotic resistance pattern.
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Materials and methods
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The study was conducted in the Bacteriological Laboratory of the Department of
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Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural
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University (BAU), Mymensingh, Bangladesh.
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Collection of samples
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Different water samples were used for the characterization of bacterial species. The
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list of commercially available bottled drinking water collected from the local markets is
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mentioned in table 1. After collection of water samples were transported to the laboratory for
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detail microbiological investigation.
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Preparation of various bacteriological culture media
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Nutrient broth
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Nutrient broth was prepared by dissolving 13 grams of dehydrated nutrient broth
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(HiMedia, India) in to 1000 ml of distilled water and was sterilized by autoclaving at 121ºC
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under 15 lb pressure per square inch for 15 minutes. Then the broth was dispensed into tubes
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(10 ml/tube).
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Selenite broth
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In 1000 ml of cold distilled water, 23 grams of dehydrated selenite broth (HiMedia,
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India) was added and heated up to boiling to dissolve it completely. The solution was then
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shaken well and 5 ml of solution poured into sterile test tubes, stopper with cotton plugs and
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sterilized in a boiling water bath or free flowing steam for 10 minutes and used for cultural
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characterization.
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Plate count agar
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Plate count agar was prepared by dissolving 23.5 grams of plate count agar powder
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(HiMedia, India) was suspended in 1000 ml of cold distilled water in a flask and heated upto
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boiling to dissolve it completely. The medium was then sterilized by autoclaving at 15 pounds
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pressure and 121 º C for 15 minutes. After autoclaving, the medium was poured into each
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sterile Petridish and allowed to solidify.
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Nutrient agar
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Twenty eight grams of nutrient agar powder (HiMedia, India) was suspended
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in 1000 ml of cold distilled water in a flask and heated to boiling for dissolving the medium
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completely. The medium was then sterilized by autoclaving. After autoclaving, the medium
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was poured into each sterile Petridish and allowed to solidify.
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MacConkey’s agar
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In1000 ml of distilled water 51.5 grams of MacConkey agar base (HiMedia, India)
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powder was added in a flask and heated until boiling to dissolve the medium completely. The
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medium was then sterilized by autoclaving at 15 pounds pressure and 121 º C for 15 minutes.
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After autoclaving the medium was put into water bath at 45 º-50 º C to decrease the
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temperature. Then medium was poured in 10 ml quantities in sterile glass Petridishes
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(medium sized) and in 15 ml quantities in sterile glass Petridishes (large sized) to form thick
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layer therein. To accomplish the surface be quite dry, the medium was allowed to solidify for
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about 2 hours with the covers of the Petridishes partially removed.
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Eosine methylene blue (EMB) agar
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Thirty six grams powder of EMB agar base (HiMedia, India) was suspended in 1000
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ml of distilled water. The suspension was heated to boil for few minutes to dissolve the
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powder completely with water. The medium was autoclaved for 30 minutes to make it sterile.
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After autoclaving the medium was put in to water bath at 45ºC to cool down its temperature at
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40ºC. From water bath 10-20 ml of medium was poured in to small and medium sized sterile
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petridishes to make EMB agar plates.
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Brilliant green agar
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According to the direction of manufacturer (HiMedia, India) 58 grams of dehydrated
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medium was suspended in 1000 ml distilled water and heated for boiling to dissolve the
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medium completely. The medium was sterilized by autoclaving. After autoclaving the
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medium was put in to water bath of 45ºC to decrease its temperature.
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Blood agar
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Forty grams of blood agar base (HiMedia, India) was suspended in 1000 ml of
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distilled water and heated for boiling to dissolve completely. The base was then autoclaved
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and cooled at 50ºC using water bath. Then sheep blood collected aseptically was added at the
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rate of 5-7% of base. The medium was then poured in 20 ml quantities in to 15×100 mm
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petridishes and allowed to solidify.
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Salmonella-Shigella agar
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Sixty three grams of Salmonella-Shigella agar base (HiMedia, India) powder was
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added to 1000 ml of distilled water in a flask and heated until boiling to dissolve the medium
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completely. The medium was put into water bath at 50 º C to decrease the temperature. Then
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medium was poured in 10 ml quantities in sterile glass Petri dishes (medium sized) and in 15
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ml quantities in sterile glass Petridishes (large sized) to form thick layer therein. To
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accomplish the surface be quite dry, the medium was allowed to solidify for about 2 hours
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with the covers of the Petridishes partially removed.
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Triple sugar iron (TSI) agar slant
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Sixty five grams of dehydrated medium (Difco, USA)was mixed with 1000 ml cold
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distilled water in a flask and heated for boiling to dissolve the medium completely. The solution
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was distributed in tubes which were plagued with cotton. The tubes were then sterilized by
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autoclaving and slanted in such a manner as to allow a generous butt.
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Sugar solutions
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The medium consists of 1% peptone water to which fermentable sugars were added.
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Peptone water was prepared by adding 1 gram of Bacto peptone (Difco, USA) and 0.5 grams of
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sodium chloride in 100 ml distilled water, boiled for 5 minutes, adjusted to pH 7.6 by phenol
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red (0.02%) indicator, cooled and then filtered through filter paper. The solutions were then
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dispensed in 5 ml amount into cotton plugged test tubes containing invertedly placed Durham’s
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fermentation tubes. Then the sugars, dextrose (MERCK, India), maltose (s.d. fiNE-CHEM
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Ltd.), lactose (BDH, England), sucrose (MERCK, India) and mannitol (PETERSTOL
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TENBEG) used for fermentation were prepared separately as 10 percent solutions in distilled
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water (10 grams sugar was dissolved in 100 ml of distilled water). A little heat was necessary to
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dissolve the sugar. These were then sterilized by autoclaving for 15 minutes.
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The sugar solutions were sterilized in Arnold’s steam sterilizer at 100ºC for 30
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minutes for three consecutive days. An amount of 0.5 ml of sterile sugar solution was added
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aseptically in each culture tubes containing 4.5ml sterile peptone water. The sugar solutions
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were incubated at 37ºC for 24 hours to check sterility. These solutions were used for
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biochemical test.
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Methyl-Red solution
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The indicator MR solution was prepared by dissolving 0.1 gram of Bacto methyl-red in 300
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ml of 95% alcohol and diluted to 500 ml with the addition of distilled water.
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Voges-Proskauer solution
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Alpha-naphthol solution:
Alpha-naphthol solution was prepared by dissolving 5 grams of 1-naphthol in 100 ml
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of 95% ethyl alcohol.
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Potassium hydroxide solution:
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Potassium hydroxide (KOH) solution was prepared by adding 40 grams of potassium
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hydroxide crystals in 100 ml of cold distilled water.
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Kovac’s reagent
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This solution was prepared by dissolving 25 ml of concentrated Hydrochloric acid in
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75 ml of Amyl alcohol and to this mixture 5 grams of paradimethyl-amino-benzyldehyde
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crystals were added. This was then kept in a flask equipped with rubber cork for future use.
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Methyl Red and Voges–Proskauer (MR-VP) broth
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A quantity of 3.4 gm of MR-VP medium (HiMedia, India) was dissolved in 250 ml of
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distilled water, distributed 2 ml quantities in test tube and then autoclaved.
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Phosphate buffered saline (PBS)
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For preparation of phosphate buffered saline, 8 gm of sodium chloride (NaCl), 2.89
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gm of disodium hydrogen phosphate (Na2HPO4.12H2O), 0.2 gm of potassium chloride (KCl)
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and 0.2 gm of potassium hydrogen phosphate (KH2PO4) were suspended in 1000 ml of
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distilled water. The solution was heated to dissolve completely and pH was adjusted with the
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help of pH meter.
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Sterilization and storage of media
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The sterility of the medium was checked by incubating at 37 º C for overnight. The
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contamination free medium was used for cultural characterization or stored at 4º C in
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refrigerator for future use.
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Processing of samples
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Proper care was taken during processing of samples for inoculation. Water bottles
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were first wiped with alcohol. Sterile pipette was used to collect water from plastic bottle. The
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cover of plastic bottle was opened carefully inside the hood. All commercially available
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bottled water was inoculated in the Petri dishes containing agar media.
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Bacterial isolation and identification
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The isolation and identification of bacteria form different water samples were based
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on the morphology and staining (gram’s staining) characteristics, colony characteristics,
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hemolytic activities, motility test and Biochemical tests i.e. sugar fermentation tests, catalase
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test, indole test, MR-VP test and coagulase test
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One hundred microliter of the processed sample was inoculated into nutrient agar
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media and EMB (Eosin Methylene Blue) agar media by spread plate technique. The
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inoculated media were incubated at 37ºC for overnight in an incubator. Different types of
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bacterial colonies were isolated in pure cultures.
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Detection of morphology of bacteria by Gram’s staining method
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Different types of bacterial colonies found from each water samples were selected for
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Gram’s staining. The Gram’s staining method was performed for each individual colony as
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per method described by Merchant and Packer (1976). The staining properties of isolated
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bacteria were studied. The procedures are as follows.
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A small colony was picked up from cultural media with a sterile bacteriological loop,
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smeared on separate glass slide and fixed by gently heating. Crystal violet was then applied
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on each smear to stain for two minutes and then washed with running water. Few drops of
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Gram’s iodine was then added to act as mordent for one minute and then again washed with
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running tap water. Acetone alcohol was then added (act as decolorizer) for few seconds. After
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washing with water, safranin was added as counter stain and allowed to stain for 2 minutes.
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The slides were then washed with water, blotted and dried in air and then examined under
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microscope with high power objective (100X) using immersion oil.
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Securing pure culture of isolated bacteria
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For identification, individual colony of different types was selected from primary
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culture for securing pure culture. A single colony was then picked up by a sterile platinum
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loop and subculture was performed in fresh nutrient agar media. After that plate was labeled,
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incubated at 37C for 24 hours. The colonies were examined for the specific size and shape.
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Again Gram’s staining was performed.
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Hemolytic activity
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To determine the hemolytic property of isolated bacteria, the colonies of bacteria were
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inoculated on to BA media and incubated at 37° C for 24 hours. Various types of hemolysis
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were observed after the development of bacterial colony on the BA.The hemolytic pattern of
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the bacteria was categorized according to the types of hemolysis produced on BA and this was
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made as per recommendation of Buxton and Fraser (1977).
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Motility test using Hanging drop slide
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The motility test was performed according to the method described by Cowan (1985)
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to differentiate motile bacteria from the non- motile one. Before performing the test, a pure
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culture of the organism was allowed to grow in NB. One drop of broth culture was placed on
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the cover slip and it was placed invertedly over the concave depression of the hanging drop
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slide to make hanging drop preparation. Vaseline was used around the concave depression of
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the hanging drop slide for better attachment of the cover slip and to prevent evaporation of
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fluid by air current. The hanging drop slide was then examined carefully under the 100 power
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objective of a compound microscope using immersion oil. The motile and non- motile
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organisms were identified by observing the motility in contrasting with Brownian movement
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of the bacteria.
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Biochemical tests
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All biochemical tests were performed according to the technique of Cheesbrough
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(1985).
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Isolation and identification of Staphylococcus spp.
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Staphylococcus spp. was isolated on the basis of the morphology, cultural
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characteristics and biochemical characteristics. The colonies of Staphylococci were round,
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glistening, convex, smooth opaque and golden-yellowish on NA and BA. They were Gram-
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positive cocci arranged in clusters and catalase positive. Beta () hemolysis was produced by
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most of the strains on BA. The coagulase test was performed for the identification of the
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pathogenic Staphylococcus spp. from the nonpathogenic one (Buxton and Fraser (1977).
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Catalase test
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This test was used to differentiate bacteria, which produce the enzyme catalase, such
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as Staphylococci, from that non catalase one such as, Streptococci. To perform this test, a
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small colony of good growth pure culture of test organism was smeared on a slide. Then one
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drop of catalase reagent (3%H2O2) was added on the smear. The slide was observed for
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bubble formation. Formation of bubble within few seconds was the indication of positive test
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while the absence of bubble formation indicated negative result (Cheesbuough, 1985).
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Coagulase test
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For the coagulase test, rabbit Plasma was used. Undiluted 0.5 rabbit plasma was mixed
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separately in two different test tubes containing an equal volume of 10 Staphylococcal
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cultured broths and incubated at 37C. The tubes were examined after 3-6 fours of mixing
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cultured broth for detecting the presence of clots of plasma and the result was recorded
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according to the standard method described by (Cowan, 1985). The negatives tubes were left
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at room temperature for overnight and then re-examined. A simple slide coagulase test
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(Carter, 1986) was also performed for the bacterial isolates. In this case 1-2drop diluted
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plasma was mixed with an equal volume of freshly cultured broth of a particular organisms on
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a slide and examined under microscope for the occurrence any coagulation. Pathogenic
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Staphylococcus is characterized by coagulase positive reaction.
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Pathogenicity test of Staphylococcus spp
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To perform this test adult mouse were used to observe the lethal infection initiated
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with viable organism, namely, toxic manifestations, leukopenia, and death. To study the
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pathogenicity of the isolated organisms, 2 groups of adult mice each consisting of 2 mice
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were selected, the group of mice were numbered as Group-A (Test group) and Group-B
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(Control group). The isolates of Staphylococcus spp. were first grown on NA from stock
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culture. A small colony from NA was added to the 5 ml of nutrient broth. The broth was
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incubated at 37ºC for 24 hrs aerobically. A dose of 0.5 ml of culture was administered
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intraperitoneally with the help of sterile syringe and needle to each mouse of the test group.
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Control group consisting the same number of mice were inoculated with sterile nutrient broth.
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The amount of inoculums and the route of inoculation were similar to those of the test group
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of animals. All the mice were kept isolated and under observation for 24-72 hrs. Mice that
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died during observation period were subjected to postmortem examination in order to re-
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isolate the organism.
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Antibiotic sensitivity tests
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This method was allowed for the rapid determination of the efficacy of the drugs by
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measuring the diameter of the zone of inhibition that resulted from different diffusion of the
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agent into the medium surrounding the disc (Cheesebrough, 1985). In vitro antibiotic
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sensitivity tests were done using disc diffusion test following the method described by Bauser
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(Bauser et al., 1966). 1-2 ml or freshly growing broth culture were poured on to NA, EMB
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agar or BGA and spread uniformly. Antibiotic discs were placed apart on the surface of the
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inoculated plates aseptically with the help of a sterile forceps and incubated at 370C for 24
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hrs. After incubation the plates were examined and the diameters of the zone of inhibition
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were measured. Depending on the area of the zone diameters for individual antibiotic was
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recorded as highly sensitive, moderately sensitive, less sensitive and resistant.
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RESULTS
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Bacterial species isolated from commercially available Bottled drinking water
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Bacterial species isolated from commercially available bottled water were described in
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table 2.
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Isolation and identification of Staphylococcus spp
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Cultural examination
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Nutrient broth:
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Nutrient broth was inoculated separately with commercially available bottled water
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incubated at 37º C for 24 hours. The growth of bacteria was indicated by the presence of
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turbidity.
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Nutrient agar:
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Nutrient agar plates were streaked separately with the organisms revealed the growth
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of bacteria after 24 hours of incubation at 37º C aerobically and was indicated by the growth
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of circular, small, smooth, convex and gray-white or golden-yellowish colonies.
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Gram’s staining
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Light microscopic examination after Gram’s staining revealed Gram-positive spherical
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cell arranged in pairs, tetrads and clusters.
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Biochemical tests
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Sugars fermentation test:
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The isolates fermented five basic sugars (dextrose, maltose, lactose, sucrose and
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mannitol) with production of acid which followed the description of Merchant and Packer
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(1967). The change of colour from reddish to yellow indicated acid production.
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Coagulase test
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The isolates were coagulase positive and isolated organism was Staphylococcus
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aureus which was similar to the description of Brook et al. (2002)
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Catalase test
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The isolates were catalase positive.
Pathogenicity tests
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Pathogenicity test was conducted with the Staphylococcus spp. isolated from Bottled
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water. The organism was inoculated intraperitoneally (IP) in two adult mice and another two
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mice were kept as control. The results are given in Table 8. The isolates of Staphylococcus
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spp. caused death of the mice within 48 hours in the test group and were categorized as
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pathogenic. Characteristic lesions in different organs were produced following the
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experimental inoculation of Staphylococcus spp. in adult mice. The intestine was distended.
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No other signs were detected in the visceral organs. Death of mice might be caused by neural
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toxicity.
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Antibiotic sensitivity and resistance pattern of isolated bacteria
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Out of total isolates four Staphylococcus spp., irrespective of source were tested for
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the antibiotic sensitivity and resistance against different antibiotics. The results of sensitivity
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against antibiotic discs (zone of inhibition) were recognized as resistant (-), less sensitive (+),
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moderately sensitive (++) and highly sensitive (+++). The results of antibiotic sensitivity and
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resistance pattern are given in table 4.
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Antibiotic sensitivity and resistance pattern of Staphylococcus spp.
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Among the 4 isolates of Staphylococcus spp. 66.67% were highly sensitive to
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Ampicillin, Amoxycillin, Gentamicin, Furazolidon, Nor-floxacin and Pefloxacin and 33.33%
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to ciprofloxacin. 100.00% isolates were moderately sensitive to Gentamicin, 66.67% to
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Amoxycillin and Furazolidon and 33.33% to Enrofloxacin and Pefoxacin. Among the
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organisms 100.00% were less sensitive to Furazolidon, 66.67% to Amoxicillin, Gentamicin
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and Pefloxacin and 33.33% to Ampicillin, Ciprofloxacin and Nor-floxacin.100.00% isolates
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were resistant to Amoxicillin, Gentamicin and Furazolidon, 66.67% to Pefloxaci and 33.33%
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to ciprofloxacin.
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Discussion
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Present study was undertaken to isolates, identify and characterize bacterial spp from
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commercially available bottle drinking water in Bangladesh. The characterization procedure
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included cultural studies, morphological, staining properties, biochemical test and antibiotic
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sensitivity test. Pathogenicity test of isolated Staphylococcus spp was done to determine their
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ability to produce diseases. Sensitivity and resistance pattern of isolated organisms to
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different antibacterial agents, available in the market were also a part of the study. Triplicate
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tests were done for all of the samples in all techniques.
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Bacteria isolated from commercially available bottled water were Staphylococcus
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aureus. More than 8% water sample was found positive for Staphylococcus spp.
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Staphylococcus spp was found pathogenic. Presence of these bacteria is alarming for human
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health. Ibekwe et al. (2004) isolated Staphyloccus from waste water in the accumulation pond
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and final discharge point of Nigerian Bottling Company PLC in Owerri, Nigeria.
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Staphylococcus spp were isolated from bottled mineral water by Tsai and Yu (1997),
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Okaqbue et al. (2002) and by Abd El-Salam et al. (2008)
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Coagulase test was performed using a total of 4 Staphylococcus spp to determine
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whether the organism is pathogenic or non-pathogenic. It was found that the isolated
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Staphylococcus spp were coagulase positive i.e. it was pathogenic Staphylococcus aureus.
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Brooks et al., (2002) described that Staphylococcus aureus produces coagulase.
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For the bacteria isolated from water samples, the pathogenicity test of Staphylococcus
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spp were done in mice because these organisms produce clinical disease in man, livestock and
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poultry. The organisms found to be pathogenic for adult mice.
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Among the 4 isolates of Staphylococcus spp. 66.67% were highly sensitive to
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Ampicillin, Amoxycillin, Gentamicin, Furazolidon, Nor-floxacin and Pefloxacin and 33.33%
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to ciprofloxacin. 100.00% isolates were moderately sensitive to Gentamicin, 66.67% to
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Amoxycillin and Furazolidon and 33.33% to Enrofloxacin and Pefoxacin. Among the
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organisms 100.00% were less sensitive to Furazolidon, 66.67% to Amoxicillin, Gentamicin
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and Pefloxacin and 33.33% to Ampicillin, Ciprofloxacin and Nor-floxacin.100.00% isolates
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were resistant to Amoxicillin, Gentamicin and Furazolidon, 66.67% to Pefloxaci and 33.33%
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to ciprofloxacin. Gentilini et al. (2002) stated that resistance was detected in 34 (27.6%), 4
433
(3.2%), 7 (5.7%), and 6 (4.8%) isolates for penicillin, oxacillin, erythromycin, and pirlimycin,
434
respectively. To our knowledge, this is the first study in Bangladesh regarding the
435
commercially packaging bottle water. Furthermore research is promising to verify all of the
436
commercial drinking water with molecular intervention.
437
438
Public health significance
439
It might be concluded that the presence of microorganisms in water has veterinary
440
public health importance because it might play direct or indirect role for the transmission of
441
various enteric and water borne diseases in man and animal. Among the different companies
442
bottled water them except “Everest mineral water” were found pathogen free. The isolated
443
bacteria were Staphylococcus aureus which was found pathogenic. Among the eight
18
444
antibiotics, enrofloxacin, norfloxacin and ciprofloxacin were found highly effective against
445
most of the isolated bacteria.
446
Table 1. Bottled drinking Water samples
Trade
Name of the manufacturer
Name
Mum
Partex Beverage Ltd. Jangaliapara, Bangla Bazar, Gazipur, Bangladesh.
Fresh
United Mineral water and PET Industries Ltd. Sonargaon, Narayangong.
Jibon
City PET industries Ltd. Konapara, Demra, Dhaka, Bangladesh.
Acme
The Acme agrovet and Beverage Ltd. Thamri. Dhaka. Bangladesh.
Pran
Pran Beverage Ltd. Property Hieghts, Dhaka-1203, Bangladesh.
Everest
Everest Drinks and Dairy products Ltd. Tejgaon , Dhaka- 1208, Bangladesh.
Boss
Gazipur Beverage. Rajendrapur, Sripur, Gazipur, Bangladesh.
Shanti
Dhaka washa, Mirpur. Dhaka-1217, Bangladesh.
Niagra
Jessor Corporation Ltd. Matuail, Zatrabari, Dhaka. Bangladesh
Spa
Akij Food and Beverage Ltd. Krisnapur, Dhaka, Bangladesh.
447
448
449
450
451
452
453
454
455
456
19
457
Table 2. Bacterial species isolated from Bottled water
Name of manufacturing No.
of
samples Positive samples n Name of Bacterial
company
examined
(%)
isolates
Acme
5
0 (0.0)
nil
Fresh
5
0 (0.0)
nil
Everest
5
4 (80.0)
Staphylococcus spp.
Spa
5
0 (0.0)
nil
Mum
5
0 (0.0)
nil
Niagra
5
0 (0.0)
nil
Shanti
5
0 (0.0)
nil
Pran
5
0 (0.0)
nil
Jibon
5
0 (0.0)
nil
Boss
5
0 (0.0)
nil
458
459
460
461
462
463
464
465
466
467
468
20
469
Table 3: Demonstration of the biochemical reactivity pattern of Staphylococcus spp.
470
isolated from different sources
Fermentation reaction with five basic sugars
Catalase
Coagulase
DX
ML
L
S
MN
test
test
A
A
A
A
A
+
+
471
DX = Dextrose; ML = Maltose; L = Lactose; S = Sucrose; MN = Mannitol; A = Acid
472
production + = Positive test; - = Negative test
473
474
Table 4. Antibiotic sensitivity and resistance pattern
Name of isolates
AMP
AML
CIP
CN
ENR
FR
NOR
PEF
+
+
++
+
+++
+
+
+
+ ++
+
+ ++
-
++
+
+++
++
++
+
++
+
+ ++
-
++
+
+++
-
++
-
+++
-
+++
+
Staphylococcus
spp
(n= 4).
475
ENR = Enrofloxacin, CIP = Ciprofloxacin, PEF = Pefloxacin, CN = Gentamicin, AMP =
476
Ampicillin, NOR = Norfloxacin, AML = Amoxycillin, FR = Furazolidone
477
478
479
480
481
482
483
484
21
485
Table 5. Antibiotic sensitivity and resistance pattern in percent
% of isolated strains sensitive/ resistance to various
Name
of
Sensitivity/
Sample
bacteria
antibiotics
resistance
AMP AML CIP CN
04
ENR FR
100.0 33.33 100.0 0.0
NOR PEF
Resistance
0.0
100.0 0.0
66.67
Less
33.33 66.67 33.33 66.67 0.00 100.00 33.33 66.67
sensitive
Staphylococcus
Moderately 0.0
66.67 0.0
100.00 33.33 66.67 0.0
33.33
spp.
sensitive
Highly
66.67 66.67 33.33 66.67 0.0
66.67 66.67 66.67
sensitive
486
ENR = Enrofloxacin, CIP = Ciprofloxacin, PEF = Pefloxacin, CN = Gentamicin, AMP =
487
Ampicillin, NOR = Norfloxacin, AML = Amoxycillin, FR = Furazolidone
488
489
490
491
492
493
22
Culture of Staphylococcus spp. Culture of Staphylococcus Culture
on Nutrient agar showing round, spp.
smooth,
glistening,
on
opaque, showing
Nutrient
round,
of
Staphylococcus
agar spp in Blood agar media
smooth, showing hemolysis.
convex, anorphous, edge entire glistening,
opaque,
and of golden-yellow colour anorphous, edge entire and
colonies.
of
golden-yellow
colour
colonies.
Antibiotic sensitivity test of
Postmortem
Carbohydrate
lesion
fermentation
Staphylococcus spp.
showing
distended
test of Staphylococcus spp.
stomach and intestine.
showing fermentation of all
sugar with produciton of acid.
Figure 1. Results of different test
494
495
496
23
497
Acknowledgements
498
The authors express their cordial gratitude and thanks to the Bacteriological Laboratory,
499
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh
500
Agricultural University, Mymensingh, Bangladesh for financial support and providing scope
501
for this research.
502
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503
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