Major project Undertaken:

Under the supervision of Dr. Agniswar Sarkar

Department of Bioscience, JIS University,
81, Nilgunj Road, Agarpara, Kolkata- 700 109

Title of the Project: Isolation and Molecular Characterization of Escherichia Coli from
fish for maintaining consumption Quality.

Escherichia coli is one of the most well recognized pathogens of humans and animals.
E. coli is a gram-negative rod that is a member of family Enterobacteriaceae. The ability of
some strain of E. coli to cause disease was known in the early 20th century and infant diarrhea
was one of the first illnesses recognized to be caused by E. coli. Toxigenic E. coli strains have
been isolated and identified from number of food, fish and meat items. There are four major
classes of disease caused by E. coli and they have distinct patterns of illness as well as different
virulent factors. The most common is the Enteropathogenic E. coli (EPEC), associated with
infant diarrhea. Other virulent groups include Enteroinvasive E. coli (EIEC), Enterotoxigenic
E. coli (ETEC) and Enterohaemorrhagic E. coli (EHEC). EHEC possess virulence factors, in
addition to verocytotoxin production, that are considered to be important in severe human
disease. A common problem encountered with fish, meat and food products is contamination
with microbiological pathogens. In fact, there have been several cases of rejections of
consignments at international markets due to contamination of food and food-products with
pathogenic and toxigenic E. coli. To ensure the quality of fish and fish products, it is essential
that the number of these pathogens are minimized to zero level and diagnostic tests so
employed only to detect these pathogens. The application and usefulness of such
microbiological and molecular techniques in detection and characterization of E. coli isolates
have been evaluated in the present investigation.
Identification criteria used are production of gas from glucose (and other sugars) and
fermentation of lactose to acid and gas within 48 h at 35oC (coliforms) and 44.5oC (fecal
coliforms and E. coli as a coliform). With all Indian Major Carps (Rohu, Catla and Mrigal),
isolated bacteria were incubated at 44.5oC (rather than 45.5oC). Some E. coli strains may be
only weakly lactose-positive (delayed lactose fermentation) or even lactose-negative.
In the present study 10 E. coli isolates were obtained when analyzed on mFC agar at
45.5oC and confirmed by growth on EMB agar at 37oC, showing typical colonies with green
metallic sheen. These isolates were used for genomic DNA extraction and samples were
electrophoresed on 0.8% agarose gel. These DNA were used in RAPD-PCR analysis and
multiple amplification of DNA bands was visualized by using OPA primers (OPA1-OPA-20).
Polymorphic banding patterns were observed. The gels were scanned using Gel Documentation
System (BioRad Gel Doc 2000) and analyzed using Bio1D software. Banding pattern, base
pair analysis of each band, Dendogramic relationship and Similarity matrix using Dice
Coefficient were analyzed. Results indicated polymorphism and genomic variation among E.
coli isolates obtained from fish. To maintain quality of the fish on the basis of the presence of
E. coli, further study is required.