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Frequency of Drug Resistant Escherichia Coli Isolated from Commercial Broiler Chicken in Bangladesh

Most. Nazmunnaher Khatun
Department of Microbiology and Hygiene, Sylhet Agricultural University, Sylhet, Bangladesh.

A.T.M. Mahbub-E-Elahi
Department of Microbiology and Hygiene, Sylhet Agricultural University, Sylhet, Bangladesh.

Sultan Ahmed
Department of Microbiology and Hygiene, Sylhet Agricultural University, Sylhet, Bangladesh.

Md. Shafiullah Parvej
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Sharmin Akhter
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Wahedul Karim Ansari
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Mohammad Shaokat Ali
Department of Applied Chemistry and Chemical Technology, Chittagong Veterinary and Animal Sciences University, Khulshi, Chittagong-4225, Bangladesh.

Abstract/ Executive Summary:

The study was conducted to isolate and identify Escherichia coli from naturally healthy-looking broiler chicken sold at a local market in Sylhet region of Bangladesh. The study was also focused on the investigation of drug sensitivity and resistance patterns of the isolated organisms. A total of 100 samples comprising of 80 cloacal swabs and 20 liver samples were collected from an apparently healthy broiler. Of these 35 (43.75%) cloacal swabs and 7 (35%) liver samples were found positive for E. coli. All isolates revealed the same morphological, cultural, and biochemical characteristics. All isolates possessed E. coli specific 16s rRNA gene detected by PCR using the primers ECO-1 and ECO-2. The antibiotic sensitivity test demonstrated that the isolates were multidrug-resistant against Gentamycin, Erythromycin, Penicillin, Cephalexin, Amoxicillin, Nalidixic Acid while sensitive to Ceftriaxone. The high level of antibiotic resistance in Broiler chicken of Bangladesh indicates that the widespread use of antibiotics as feed additives for growth promotion and disease prevention could have negative implications for human and animal health and the environment.

Keywords: Broiler, E. coli, Liver sample, Antibiotic resistance

Location: Department of Microbiology and Hygiene, Sylhet Agricultural University.

Start Date:

End Date:

Program Area: Animal Health and Management

Commodity: Broiler

Objectives:

To provide information on the present status of antibiotic resistance patterns in E. coli in apparently healthy broiler birds sold in Bangladesh.

Methodology:

Samples were aseptically collected from apparently healthy birds following a convenience sampling method without repetition of any birds. Sterile cotton buds were used for the collection of swab samples, and the swab was transferred to nutrient broth instantly. The liver samples were collected after the slaughtering of birds. The samples were transported to the Bacteriology Laboratory at the Department of Microbiology and Hygiene, Sylhet Agricultural University. The nutrient broth containing swab samples were incubated for 6 hours at 37°C for bacterial multiplication. After incubation samples from the nutrient broth were cultured on to EMB agar, MacConkey agar, Salmonella –Shigella agar (SS agar), Brilliant green agar. Isolated organisms with specific growth characteristics of E. coli in different culture media were subjected to sugar (dextrose, fructose, maltose, lactose, and sucrose) fermentation, MR- VP, and indole production test following the procedure mentioned by Buxton and Fraser. Bacterial DNA was extracted from the isolates using the method described by Queipo-Orton et al. with little modification. Briefly, the organisms were cultured onto nutrient broth. After overnight incubation at 37°C, 1 ml of broth culture were taken in an Eppendorf tube, centrifuged at 10000 rpm for 2 minutes, discard supernatant, and the pellet cells were mixed with 200μl of deionized water. The mixture was then heated in boiling water for 10 minutes followed by dipping into ice for 10 minutes and centrifuged at 10,000 rpm for 10 minutes. The supernatant was collected and used as template DNA in PCR. The E. coli were confirmed by PCR using primers specific to E. coli 16S rRNA gene). ECO-1 (5´- GACCTCGGTTTAGTTCACAGA-3´) and ECO- 2 (5´-CACACGCTGACGCTGACCA-3´) were used as forward and reverse primers, respectively for an amplification target sequence of 585bp. PCR reaction was performed following the procedure described by Schippa et al. with slight modification. A 25μl reaction mixture was prepared that containing 12.5 μl of 2 X PCR master mixes, 1 μl of each primer (10 pmol/ μl), 2 μl of template DNA, and 8.5 μl of nuclease-free water. After initial incubation at 95°C for 3 min, a 30-cycle amplification protocol was followed as 94°C for 45 s, 58°C for 45 s, and 72°C for 60 s, and a final extension step of 72^°C for 3 min. The PCR products were resolved in 1.5% agarose gel. After electrophoresis, the gel was stained for 10 minutes in ethidium bromide and destained with water for visualization. Antibiotic sensitivity test of isolated E. coli was performed with the standardized commercial antibiotic discs following Disc Diffusion Method. Sensitivity to antibiotics was studied on Muller Hinton agar plates (Himedia, India) with Ceftriaxone, Gentamycin, Erythromycin, Penicillin, Cephalexin, Amoxicillin, Nalidixic Acid. An amount of 0.1 mL freshly grown pure culture of E. coli was poured on agar plates and allowed to spread gently over the entire surface with a glass rod spreader. After 5 minutes, the discs were placed at a distance of about 1 cm apart and incubated at 37°C overnight. On the basis of the diameter of zones of inhibition produced around the antibiotic discs the inhibitory effect of the antibiotic on the growth of the culture was recorded and analyzed according to CLSI.

Source of Information: International Journal of Natural and Social Sciences, 2015, 2(4): 01-05

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Pathogenic E. coli may produce disease in poultry if the immunity of poultry is reduced due to various reasons. The environmental condition of the farm may be a reason for the highest prevalence of the bacterium in poultry, which has been transmitted to humans via meat. The environment inside and outside of the farm should be kept clean and the feed supply should be carefully monitored. Ceftriaxone would be the choice of the drug against E. coli. However, the findings from this work support the need for a critical review of the usage of antimicrobial agents in livestock in Bangladesh and the importance of taking concrete steps in terms of the policy to curtail the indiscriminate use of antimicrobial agents in a bid to prevent the possible adverse consequences in animal production, as well as in humans. There is an urgent need to formulate a policy and put the necessary plan in place to execute a policy targeted at the promotion of rational use of antimicrobial agents, as an important element in antimicrobial-resistant containment and the dose of antibiotics to poultry should be carefully controlled as the intermediate sensitive bacterium can gain resistance to antibiotics.

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