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Bacterial Flora of Koi (anabas Testudineus) Harvested from Ponds and Their Antibiogram

Begum Safia Zaman
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. Enamul Hoq
Bangladesh Fisheries Research Institute, Mymensingh-2202, Bangladesh

Umme Kulsum
Bangladesh Fisheries Research Institute, Mymensingh-2202, Bangladesh

Mst. Minara Khatun
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Ariful Islam
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Shadia Sharmin
Department of Microbiology and Hygiene, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Fish is known to harbour bacteria of public health importance. Aquatic environments of ponds are known to influence the bacterial loads of the harvested fish. The present work was undertaken to determine total viable count (TVC) and isolation and identification of bacteria from Koi fish, mud and water samples of two selected ponds managed and owned by the Bangladesh Fisheries Research Institute (BFRI) and two ponds located at Muktagacha, Mymensingh, managed by private owners. Fish (n=16), mud (n=16) and water (n=16) samples were collected from all 4 ponds. Samples were cultured on plate count agar to determine TVC. Fish harvested from Muktagacha ponds had statistically higher bacterial count (8.44 ± 0.04 log10 CFU/ml) when compared to BFRI ponds (7.92±0.17 log10 CFU/ml) (p≤ 0.05). Similarly, highest TVC was found in mud and water samples of Muktagacha ponds (6.87±0.73 and 7.41±0.04 log10 CFU/ml, respectively) compared to mud and water samples of BFRI ponds (5.04 ±0.07 and 5.40±0.09 log10 CFU/ml, respectively). Samples were inoculated onto appropriate selective media for isolation of bacteria. Total 257 bacterial isolates representing five genera were identified: Pseudomonas spp. (21.40%), Aeromonas spp. (33.46%), Vibrio spp. (14.78%), Salmonella spp. (21.40%) and E. coli (8.94%). Antibiotic sensitivity assay showed multidrug resistant profiles of Pseudomonas sp., Aeromonas sp., Salmonella sp. and E. coli. These bacteria are known to cause food borne illness in humans and spoilage of fish.

Keywords: Total viable count, Koi fish, Antibiotic sensitivity assay, Multidrug resistant bacteria

Location: BFRI and Muktagacha ponds

Start Date: 00-01-2013

End Date: 00-06-2013

Program Area: Pest Management

Commodity: Koi/Climbing

Objectives:

The objectives of this study were (i) to determine bacterial loads and identify bacteria from pondraised Koi and (ii) to determine their antibiotic resistant profiles.

Methodology:

Collection of samples: Fish (n=16), mud (n=16) and water (n=16) samples were aseptically collected from BFRI and Muktagacha ponds from January to June 2013. Fishes were caught by using a net. A total of 48 tissue samples comprised of skin (n=16), gills (n=16) and intestine (n=16) were aseptically collected from 16 fish. The average body weight of fish of BFRI ponds was 65±10.80 g and Muktagacha ponds was 73.75±2.5 g. Samples were collected between 10.00 and 10.30 am at monthly interval during March, April and May 2013. Fish, mud and water samples were placed in sterile plastic bags, petridishes and test tubes, respectively. Samples were transferred to an ice box and transported directly to the Bacteriological laboratory of the Department of Microbiology and Hygiene for microbiological analysis. Total viable count: Skin, gill and intestine of fish samples were minced and grinded together in 1% peptone water. 0.5 ml of fish, mud and water samples were transferred to 4.5 ml of 1% peptone and a 10-fold serial dilutions were prepared upto 10-8. One ml of each of 10-fold diluted sample was spreaded onto plate count agar and incubated at 37°C for 24-48 hrs. The mean number of colony (30-300) of three plates in a particular dilution was multiplied by the dilution factor to determine TVC which was expressed as mean logarithm of the number of colony forming units (log10CFU/ml). Isolation of bacteria: Skin, gills and intestine contents of fish, mud and water samples were enriched separately in the nutrient broth at 37 °C for 24 hrs. The overnight enriched broth was streaked onto various selective media such as: Salmonella-Shigella (SS) agar (for Salmonella spp.), Eosine methylene blue (EMB) agar (for E. coli), MAeromonas (MA) agar (for Aeromonas spp.), Acetamide agar (AC) agar (for Pseudomonas spp.), Thiosulfate citrate bile salt sucrose (TCBS) agar (for Vibrio spp.) and incubated at 37 °C for 24 hrs. Single colony was further subcultured until pure culture of bacteria was obtained. Identification of bacteria: Colony characteristics of bacteria such as: shape, size, surface texture, edge, elevation and colour observed in pure culture, Gram’s staining and biochemical tests (Sugar fermentation, Methyl red, Voges-Proskauer and Indole production tests) were used for identification of bacteria (Cheesbrough, 1985). Antibiotic sensitivity test: One isolate randomly selected from five genera were tested for antimicrobial drug susceptibility against five commonly used antibiotics such as: ampicillin (10 µg/disc), chloramphenicol (30 µg/disc), ciprofloxacin (5 µg/disc), gentamicin (10 µg/disc) and cefalexin (30 µg/disc) by disk diffusion or Kirby-Bauer method (Bauer et al., 1966). Results of antibiotic sensitivity tests were recorded as sensitive, intermediate and resistant according to the guidelines of Clinical Laboratory Standard Institute (CLSI, 2007). Statistical analysis: Total viable count (TVC) of fish, mud and water samples of BFRI and Muktagacha ponds were analyzed for statistical significance using student’s t test. A p value of ≤ 0.05 was considered to be statistically significant.

Source of Information: Microbes and Health, June 2013, 2(1):8-11

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Data of this study suggest that Koi harbours multidrug resistant bacteria and TVC of Koi fish was found to be higher than the recommended value which are important issues from the public health point of view. In order to protect public health farmers should embrace standard operating practices as applicable to fish farming. People working in the fish farms should be educated and well trained on the maintenance of good hygienic practices including regular disinfection of catching gears or working equipment, and brief immersion of caught fishes in brine water to reduce the microbial load on the fish before storing at cold temperature or sold to the public.

Knowledge Management: Journal