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Isolation and Characterization of Escherichia Coli in Ready-to-eat Foods Vended in Islamic University, Kushtia

S Biswas
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh

M A K Parvez
Department of Microbiology, Jahangirnagar University, Savar, Dhaka

M Shafiquzzaman
Department of Biotechnology and Genetic Engineering, Islamic University, Kushtia 7003, Bangladesh

S Nahar
Department of Microbiology, Jahangirnagar University, Savar, Dhaka

M N Rahman
Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka

Abstract/ Executive Summary:

Context: Escherichia coli is shed in the feces of warm-blooded animals and humans and thus potential for public health. Detection and characterization of E. coli in the ready-to-eat (RTE) foods concerns due to their presence indicate fecal contamination of the food. Objective: To identify, characterize and RFLP pattern analysis of E. coli isolated from RTE foods vended in Islamic University campus, Kushtia. Materials and Methods: Fifty samples from four types of consumed foods in six student halls of residence, some temporary restaurants of Islamic University, Kushtia were assessed for bacterial contamination by standard methods. Identification and characterization of E. coli isolates were performed using IMViC tests. Genomic DNA was used to perform RFLP pattern analysis. Results: Thirty-seven out of 50 (74%) examined samples of RTE foods had E. coli contamination. The highest number of E. coli was isolated from vegetable-oriented RTE foods (90.90%) and fish, meat and cereals samples were also significantly E. coli positive. RFLP profiling of two E. coli isolates were observed. Conclusion: The results of this study provide evidence that some RTE foods had unsatisfactory levels of contamination with E. coli. Thus street vended RTE food could be important potential vehicles for food-borne diseases. Molecular characterization may be exploited to identify food-borne pathogen among different species.

Keywords: Ready-to-eat foods, Escherichia coli, RFLP pattern

Location: Islamic university campus, Kushtia, Bangladesh

Start Date: 00-04-2009

End Date: 00-07-2009

Program Area: Food Safety and Security

Commodity: Nutrition

Objectives:

The objective of this study was to determine the occurrence of E. coli in different types of RTE food samples collected from Islamic University campus, Kushtia, and to observe RFLP pattern.

Methodology:

Sample collection: Approximately 300 g of each fifty samples of RTE food of 4 categories (22 samples of cereals, 10 samples of meat, 7 samples of fish and 11 samples of vegetables) were collected between April 2009 and July 2009 from 6 student halls of residence and some temporary restaurants of Islamic university campus, Kushtia, Bangladesh. All samples were transported in sterile container to the laboratory and were tested within 24 h of collection. Ten grams of each food sample was mixed with one eighth-strength Ringers Solution (Oxoid, Basingstoke, UK). The sample was homogenized with a blender (electric) at 6000 rpm for 5- 10 minutes (10-1 dilution), followed by serial dilutions up to 10-6 dilution. Standard coliform test and isolation of E. coli: Coliform counts were determined using the most probable number (MPN) method (Cappuccino and Sherman 1999, Harley and Prescott 2002). Fermentation tubes with the appropriate quantity (10 ml) of lauryl tryptose broth medium were distributed with different strengths. The tubes were inoculated with 10 ml, 1 ml with 10 ml, and 1 ml and 0.1 ml amount of sample and incubated at 37°C for 24 h. All tubes of the presumptive test producing gas after 24 h of incubation, was further tested for confirmation. A loopful inoculum from each culture showing the production of acid and gas was transferred to Brilliant Green Bile Broth (Oxoid) and incubated for 48 h at 37°C and 44.5°C. Gas production at 44.5°C confirms the presence of fecal coliform. Streaking on the eosine methylene blue (EMB) agar plate was done for further confirmation that was performed according to Cappuccino and Sherman (1999). One or more plates containing EMB agar medium were streaked from presumptive positive test tubes in such a way that discrete colonies may appear. The plates were incubated at 37°C for 24 h. Typical nucleated colonies with or without metallic sheen indicate positive results. A maximum of five suspected E. coli colonies from each sample (based on colony size, morphology and metallic green sheen on their surfaces) were selected. Further biochemical tests were done for the identification of Escherichia coli according to Cappuccino and Sherman (1999) and Coyle et al. (1985). IMViC test was performed to distinguish between E. coli and Enterobacter aerogenes (Cappuccino and Sherman 1999). Two isolates identified as E. coli were further characterized based on DNA polymorphism by RFLP. Genomic DNA extraction: Standard and improved phenol-chloroform method was used to extract genomic DNA (Neumann et al. 1992) with a few changes. Different samples were pre-treated properly for the collection of organism cell. In regard to pure bacteria culture, 1.4 ml bacteria suspension was collected by centrifugation at 16.1 rcf for 10 min at 4°C; precipitation was mixed well with 0.4 ml TE (10 mmol-l Tris-HCl, 1 mmol-l EDTA, and pH 8.0). After adding 50 µl of 10% SDS and 50 µl proteinase K, pellete was incubated at 42°C for 20 min in water bath. All nucleic acid came out of solution soon after adding 0.15 ml of 5M NaCl and 0.25 ml of ice cold isopropanol. The supernatant was transferred to a new tube and 0.7 vol of phenol/chloroform (1: 1) was added. After gentle mixing, the mixture was centrifuged at 16.1 rcf for 10 min at 4°C. The upper phase was transferred to a new tube containing 2/3 vol of isopropanol. The mixture was cooled at -20°C for 30 min and then centrifuged at 16.1 rcf for 10 min at 4°C. The resulting pellet was dissolved with 100 µl TE buffer after washing twice with 70% ice cold ethanol. The quantity and quality of the purified DNA were determined by measuring at A260 and by calculating the ratio of A260/A280, respectively by spectrophotometer. Finally, agarose gel electrophoresis of extracted DNA was performed using 0.8% UltraPureTM agarose (Invitrogen). Restriction analysis: To get total 20 µl of volume, 1 µl of genomic DNA was mixed with 15 µl of distilled water and 2 µl of enzyme-assay buffer and finally with 2 µl of restriction enzyme (GeNei TM) i.e. BamHI (10 U-µl ), EcoRI (20 U-µl ), EcoRV (10 U-µl ), HindIII (20 Uv) and SacI (10 U-µl ) added in different tubes. After incubation for 1 h at 37°C, complete digestion was checked on 3% agarose gel with using 100 bp-1 kb reference ladders.

Source of Information: J. bio-sci. 18: 99-103, 2010 ISSN 1023-8654

Article Link (Online Journal): http://www.banglajol.info/index.php/JBS/index

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

RTE foods vended in the Islamic University campus, Kushtia had unsatisfactory levels of contamination with E. coli. The unhygienic practice may reveal the risk factors associated with the contamination of post-processing food. Food-borne disease is an urgent public health problem and requires raid intervention. RFLP pattern analysis might be useful for molecular detection of pathogenic organisms among different species if coupled with PCR. However, further detailed scientific study is necessary to develop rapid and easy detection methods of the pathogenic organism in food.

Knowledge Management: Journal