Sampling sites: A total of 250 samples were collected from Cloacal swabs of chicken, intestinal fluid of chicken, egg surface, faecal material of chicken and hand wash of chicken handlers from different poultry markets of Dhaka, Bangladesh.
Sampling from cloacal swab: Sterile swab stick moistened with sterile normal saline water was inserted in the cloacae of the chicken and placed in sterile vials.
Sample collection from intestinal fluid: The intestines were collected just after the sacrifice of chickens. Each intestine was placed separately into a sterile jar containing 500 ml of normal saline, and this suspended fluid of normal saline was used later for bacteriological analysis.
Sample of egg surface: 10 eggs collected from poultry cases just after laying were washed in 1000 ml of normal saline water and then taken into a sterile jar.
Collection of sample from faecal material: About 50 gm of fresh faecal sample was collected aseptically from poultry cases into sterile vials with the help of sterile cotton bud and 5 gm sample was transferred immediately to screw caped test tubes containing 10 ml of sterile nutrient broth.
Sample from hand wash of chicken handlers: Hands of the chicken handlers just after processing of slaughtered chickens and handling of chicken for sale were washed directly with 1000 ml of normal saline water and then taken into a sterile jar and sealed.
Transportation of sample: After collection, all the samples were transported to the laboratory immediately in an insulating foam box with ice.
Bacteriological analysis: A loop full of s elective enriched broth from previously incubated sample from cloacal swab and faecal material and 0.1 ml of sample from intestinal fluid were spread on the solid surface of Eosine Methylene Blue (EMB) agar medium (Hi - Media, India), 1.0 ml sample from intestinal fluid was placed onto sterile plates which was then mixed with sterile medium (EMB) poured into the plates after being cooled to about 42-45oC. 10-100 ml sample from egg surface and hand wash of chicken handlers was filtrated through the membrane filter (0.45 μm, Millipore, USA) which was then placed on the surface of EMB agar plates. All samples were incubated for 24 hours at 37oC in three triplication of EMB plates or filters on EMB agar for successful isolation of typical colonies. Identification was done according to Buchanan and Gibbons (1974) following a series of biochemical tests included gram staining, tests for oxidase, methyl red, Voges-Proskauer reactions, indole, citrate, catalase, urea hydrolysis, gelatin hydrolysis, lactose fermentation, nitrate reduction, casein hydrolysis and sugar fermentation. Moreover, identification of E. coli was further confirmed by latex agglutination tests using polyvalent antisera (DENKA SEIKEN Co. Ltd, Tokyo, Japan).
Drug Sensitivity Test: Single disc diffusion method (Bauer et al. 1966) was used to examine bacterial susceptibility to antimicrobial agents. A total of 13 antibiotic discs (Becton Dickinson, U.S.A.) with Streptomycin (10 μ g), Erythromycin (15 μ g), Chloramphenicol (30μ g), Ciprofloxacin (5μ g), Te tracycline (30μ g), Penicillin (10 μ g), Norfloxacin (10μ g), Riphampicin (5μ g), Neomycin (30μ g), Cefixine (5μ g), Ampicillin (10 μ g), Kanamycin (20 μ g) and Gentamicin (10μ g) were used. By the standard method of inoculation, the top of a single and well-isolated colony was touched with a sterile loop and the growth was inoculated into 2 ml of Mueller–Hinton broth. The broth culture was then allowed to incubate at 37°C for 4 hours to obtain the young culture. The turbidity of actively growing broth cultures was then adjusted to a 0.5 McFarland standard and then a sterile cotton swab was dipped into the adjusted suspension within 15 minutes and excess broth was purged by pressing and rotating the swab firmly against the inside of the tube above the fluid level. The swab was then spread evenly over the entire surface of the plate of LB agar to obtain uniform inoculums. The plates were then allowed to dry for 3 to 5 minutes. Antibiotics impregnated discs were then applied to the surface of the inoculated plates with sterile forceps. Each disc was gently pressed down onto the agar to ensure complete contact with the agar surface. Even distribution of discs and minimum distance of 24 mm from center to center were ensured. Five discs (four antibiotics discs and one blank disc as control) were placed in each petri dish. Within 15 minutes of the application of the discs, the plates were inverted and incubated at 37°C. After 16 to 18 hours of incubation, the plates were examined, and the diameters of the zones of complete inhibition to the nearest whole millimeter were measured. The zone diameter for individual antimicrobial agents was then translated into susceptible, intermediate and resistant categories according to the interpretation table of the Becton Dickinson Microbiology Company, USA.