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Enterotoxin Profiling and Antibiogram of Escherichia Coli Isolated from Poultry Feces in Dhaka District of Bangladesh

Tanvir Bashar
Department of Microbiology, Stamford University Bangladesh, Dhaka

Rashed Noor
Department of Microbiology, Stamford University Bangladesh, Dhaka

Fazle Alam Rabbi
Department of Microbiology, Stamford University Bangladesh, Dhaka

M. Majibur Rahman
Department of Microbiology, Stamford University Bangladesh, Dhaka

Mostafizur Rahman
National Tuberculosis Reference Laboratory, National Institute of Diseases of the Chest and Hospital, Bangladesh

Abstract/ Executive Summary:

Bacterial diseases remain a threat to the poultry industry and countermeasures to prevent and control them are needed to prevent production losses. With the continued threat of exotic and emerging diseases and the problem of the use of antibiotics in animal production, there is a serious and urgent need to find safe and practical alternatives to prevent or control pathogens. That,s why, our research has addressed in different poultry farms for detection of toxigenic Escherichia coli from poultry chicken feces adjacent to the Dhaka city, during the period of January 2009 to June 2009 and characterize their ability to produce enterotoxin and also examined the antibiogram of the isolates. A total of 60 fecal samples were collected and E. coli were isolated and identified by conventional cultural, biochemical and motility test.The heatstable toxins were determined by Suckling Mice Assay (SMA). The overall prevalence of E. coli was 100%. Among the isolates of E. coli, only 45% and 14% isolates showed β hemolysis and α hemolysis, respectively. Congo red binding has been used as a potential virulence marker and 69 % E. coli isolates in the present study showed congo red binder, among which, 59% isolates were hemolytic. A total of 20% and 5% isolates showed the presence of LT and ST enterotoxin in their guts by Suckling Mice assay, respectively. The antibiotic sensitivity pattern shown that the isolates were highly sensitive (100%) to imipenem, pipercillin, neomycin and ceftrixone. Results from this study revealed the high prevalence rate of E. coli isolates with variable resistance against a wide range of antimicrobial drug. It may suggest that the high resistance of E. coli to antibiotics as well as their toxigenic nature constitutes a threat not only to poultry industry of Bangladesh but also possesses a serious threat to public and animal health.

Keywords: Enterotoxin, Profiling, Antibiogram, Escherichia coli, Poultry Feces

Location: Department of Microbiology, Stamford University Bangladesh

Start Date: 00-01-2009

End Date: 00-06-2009

Program Area: Pest Management

Commodity: Poultry

Objectives:

1. Present study, focused on the detection of toxigenic E. coli and their antibiogram profiling.

Methodology:

Sample collection. A total of 250 fecal samples from poultry chickens were collected from different poultry farms adjacent to Dhaka city, using sterile cotton buds and transferring the buds immediately to sterile nutrient broth. The samples were wrapped with ice, kept in box, and transferred within 30 minutes. Identification of E. coli. The isolates of E. coli were identified by observing gross colony morphology using MacConkey agar, Eosin Methylene Blue (EMB) agar, Salmonella-Shigella (SS) agar, Gram staining properties and motility. In the basal level, these isolates were subjected to different biochemical tests such as sugar fermentation test, Indole production test, Methyl-Red and Voges- Proskauer (MR-VP) test. Pure culture of E. coli was obtained using EMB agar. Maintenance of stock culture. Nutrient agar slants were used to maintain the stock culture for each of the E. coli isolates. The E. coli isolates were introduced in the slant by streaking and were incubated at 37 °C for 24 hours. Finally, glycerol was overlaid and the culture was kept at room temperature. Hemolytic Property. The hemolytic activity of E. coli strain isolates was tested on a blood agar plate containing 5% defebrinated sheep blood. Initially, the strains were subcultured onto a nutrient agar (NA) plate and incubated at 37 °C overnight. The lower surface of the blood agar plate was marked into several squares of 0.7 cm each and labeled with the test strain number. A portion of the colony from the overnight E. coli culture on the NA plate was transferred into one chamber having a corresponding isolate number. The inoculated plates were incubated aerobically at 37 °C overnight. Following incubation, hemolytic strains were identified according to their types/extent of hemolysis by each colony onto blood agar plate. Congo red Binding. To evaluate the Congo red binding, bacteria were grown at 37º C for 18h on tryptic soy agar supplemented with 0.02% congo red and 0.15% bile salt. Positive colonies appeared red, whereas negative colonies were pale. Based on the intensity of red color, the binding was scored as +, ++, and +++. Toxin profile. Overnight broth cultures were centrifuged at 1500 rpm for 20 minutes and supernatants were collected and transferred into new vials, then gentamycin (5ug/ml) was added and kept at room temperature for overnight. The purity of the toxins were tested by streaking the supernatants on EMB agar and incubated at 37°C for 18 hours. Absence of any colony leads the supernatant to be used for detection of heat-stable (ST) toxin by Infant Suckling Mice Assay (SMA). Enterotoxigenic effect in suckling mice and determination of ST toxin by SMA. SMA was used for the detection of heat-stable toxin. Sixty Swiss Albino suckling mice of 1-3 days old were separated from their mother immediately before used and were divided into two groups A and B consisting of three mice in each group. An amount of 2.5μl crude culture supernatant containing enterotoxin were administered to the mice of group A through oral route with micropipette. Group B mice were kept as control. Mice were incubated at 37°C for 18 hours to observe toxic effects. The weight of the gut and the remaining carcass were taken and the ratio was calculated for each mouse. The average ratio of less than 0.070 was considered negative while 0.070«0.085 was considered positive for ST toxin.

Source of Information: Stamford Journal of Microbiology, July 2011. Vol. 1, Issue 1; ISSN: 2074-5346

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The study reveals that, the poultry borne bacteria could be a good source as vectors in transmitting drug resistance. Attention is to be paid for personal hygiene in processing and handling of poultry and poultry products and the excess use or abuse of antibiotics should be reduced or stopped by the judicious application of antibiotics for the safety of public health in Bangladesh.

Knowledge Management: Journal