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Antibiogram and Plasmid Profile Analysis of Isolated Escherichia Coli from Broiler and Layer

Masuder Rahman
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh.

Bytul M. Rahman
Department of Pharmacy, University of Rajshahi, Rajshahi-6505, Bangladesh.

Bahanur Rahman
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

A total of 17 Escherichia coli isolated from 24 fresh faecal samples of broiler and layer were screened for their antibiograms and plasmid profiles. The overall recovery rate of E. coli from faecal samples was 70.83%. All E. coli strains were analyzed to determine their susceptibility patterns to 8 commonly used antibiotics (ampicillin, cephradine, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, tetracycline and sulphamethoxazole) belonging to different groups. From the antibiogram study it was revealed that 87.50% E. coli isolated from broiler were resistant to both ampicillin and sulphamethoxazole. Only 37.50% broiler isolates were highly sensitive to gentamicin and 50% isolates to chloramphenicol. All the E. coli isolates of layer were completely resistant (100%) to sulphamethoxazole and about fifty 5% of the isolates (55.55%) were resistant to both streptomycin and tetracycline. E. coli isolated from layer were found to be highly sensitive (44.44%) to chloramphenicol and 66.66% were also highly sensitive to gentamicin. Plasmid profile of 17 isolates was analyzed by 0.8% agarose gel electrophoresis. A total of 8 different plasmid bands of different size were estimated by eye comparing to reference marker. The estimated size of the bands were 3.25, 5.20, 6.00, 8.00, 15.0, 30.0, 33.5 and 38.0 kbp. Plasmid profile analysis of the isolated E. coli revealed that the isolates carrying multiple plasmids which might be the cause of various degrees of antibiotic resistant. The plasmids were distributed at random in the isolated E. coli strains and there was no remarkable interrelationship between antibiotic resistance and plasmid present. In most of the cases, strains having similar plasmid bands but confer resistant to different antibiotics. In some cases, isolates showed resistance to antibiotics but did not harbor any plasmid indicating that chromosomal DNA may carry the genes that confer resistance to antibiotics.

Keywords: Antibiogram, Plasmid profile, Broiler, Layer

Location: Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh.

Start Date: 00-07-2005

End Date: 00-12-2005

Program Area: Pest Management

Commodity: Poultry

Objectives:

1. To find out the correlation between antibiotic sensitivity pattern and plasmid profile of the poultry E. coli isolates and also

2. To find out the effective antibiotic(s) against E. coli.

Methodology:

This study was conducted during the period of July to December’ 2005 in the Department of Microbiology and Hygiene and in the Central laboratory, Bangladesh Agricultural University. A total of 24 fresh faecal samples were collected randomly from four poultry farms adjacent to Bangladesh Agricultural University (BAU), Mymensingh, Bangladesh. All samples 100 g were collected in sterile containers. Faecal samples were collected with the help of sterile cotton bud and transferring the buds immediately to sterile nutrient broth in sterile screw capped test tubes. At each time of collection, precaution was taken to prevent or avoid cross-contamination of samples. After collection of the samples, they were transported to the laboratory as soon as possible in an insulated foam box with ice to maintain a temperature ranging from -4 to 6°C and bacteriological analyzes were performed within 4 h of collection. Primary culture was done in nutrient broth and then pure cultures were obtained using McConkey agar and Eosine Methylene Blue (EMB) agars. The pour-plate technique was followed to get the pure culture of E. coli. The colony characters were observed and staining was performed by Gram’s methods. Isolates yielding similar biochemical tests (indole test, Methyl Red (MR) test, Veges Proskauer (VP) test, citrate test, catalase test and sugar fermentation) to the standard E. coli strain, ATCC 25922 were identified as E. coli and selected for further testing. Sensitivity of E. coli isolates to different antimicrobial agents was determined in vitro by employing a modified disk diffusion test of the Kirby-Bauer method. Antibiotics used in this study are ampicillin, cephradine, chloramphenicol, ciprofloxacin, gentamicin, streptomycin, tetracycline and sulphamethoxazole. Cartridges of antimicrobial-containing discs were obtained from Mast Diagnostics (Merseyside, UK), stored between 4 and 20°C and allowed to come to room temperature prior to use. Isolates were subcultured from the bank onto Miller's LB agar and incubated for 18-24 h before being transferred to 5 mL sterile 0.9% saline to match the 0.5 MacFarland standard (Remel, Kansas). A sterile cotton-tipped swab was used to streak air-dried Mueller-Hinton II plates within 15 min of adjustment of turbidity. Subsequently, antimicrobial discs were added and plates were incubated aerobically at 35±2°C for 16-18 h. The diameter of the zones of inhibition surrounding the antimicrobial discs was measured to the nearest mm. Isolates were deemed resistant only when the zone of inhibition was less than or equal to the resistance breakpoint recommended by the guidelines of NCCLS (2002). Quality control was performed as recommended using E. coli strain ATCC 25922. Smaller circular plasmid DNA molecules were extracted from the huge chromosomal DNA for analysis on DNA. The selected bacterial strain (single colony) was grown overnight in LB broth at 37°C with aeration using the orbital shaker. The 1.5 mL overnight culture was taken in eppendorf tube for plasmid DNA extraction. The plasmid DNA from E. coli isolates was extracted through Mini alkaline lysis by SDS. The plasmid DNA extracted from E. coli isolates were compared to known molecular weight standards (Super mix DNA ladder, ranging from 0.5 to 33.5 kbs, Bangalore Genei Pvt. Ltd., India). Electrophoresis was carried out in a horizontal gel apparatus (Max submarine, Agarose gel unit, Model He 99). The method followed for agarose gel electrophoresis was as described.

Source of Information: Research Journal of Microbiology, 3: 82-90, 2008, ISSN 1816 - 4935

Article Link (Online Journal): DOI: 10.3923/jm.2008.82.90; URL: http://scialert.net/abstract/?doi=jm.2008.82.90

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the study, it was found that most of the isolates were resistant to five antibiotics such as ampicillin, ciprofloxacin, streptomycin, sulphamethoxazole and tetracycline. From the plasmid profile analysis it was revealed that some isolates carried multiple plasmids, some carried single plasmid and some carrying no plasmids. The plasmids were distributed at random in the isolated E. coli strains. E. coli isolates contained single or multiple plasmids bands and showed multiple drug resistance patterns. These multiple drug resistance patterns of the E. coli isolates of this study might be due to drug resistance gene (s) carried out by the different plasmids. In most of the cases, strains having similar antibiotic sensitivity patterns but showed different plasmid patterns. An isolate like BR-4 showed resistance to 5 antibiotics but did not harbor any plasmid. This supposition has been supported by the finding that the plasmidless strains may also be resistant to one or more antibiotics. Therefore, there was no noticeable correlation between antibiotic resistance patterns and plasmid patterns.

Knowledge Management: Journal