Adaptation of Advanced technique for rapid and precise detection of Escherichia coli: O157: H7: Foodborne Pathogen

Abstract:

Immunomagnetic separation (IMS) is highly efficient technique to detect specific types of microorganism and biomolecules from a complex mixture of antibody coated magnetic beads and specific antigens. Nowadays this rapid detection method is developed for precise isolation of the food borne pathogens (like E.coli, Salmonella, Listeria). This advance technique of identification has several advantages over traditional separation methods, including its speed, sensitivity, and ability to handle low-volume or low- concentration sample.

What makes this superior to conventional methods

• Gives high throughput, rapid, and effective E.coli O157:H7 sample preparation.
• Significantly shortens sample pre-enrichment time.
• Significantly reduces overall analysis time.

Difference between E.coli and E.coli O157

E. coli is a diverse group of bacteria, mostly non- pathogenic, found in the intestines.
E. coli O157 is a pathogenic strain that produces Shiga toxins, causing severe illness like bloody diarrhea and kidney failure.

Introduction:

The Escherichia coli O157: H7 is a strain of E. coli that is a major cause of foodborne illness. The O157 refers the O antigen which is the part of the cellular antigen and H7 which are found in the Flagellar antigen. Using these two surface antigens of the E.coli, scientists are involved in detection of E.coli O157:H7 by Immunomagnetic Separation (IMS) technique. The technique of IMS is designed for rapid & selective isolation of E. coli O157 from food and water samples. Antibody coated Magnetic beads reacts with all surface antigens of E. coli O157, which are incubated in the selective broth. With an aliquot of the pre- enriched sample and the antibodies coated onto the beads will specifically bind the target antigen of O & H of E.coli O157. The bead-bacteria complexes are subsequently separated by applying a magnetic field. This complex is washed with the provided wash buffer that other antigenic molecules is washed away from the Eppendorf tubes. Then contents of the Eppendorf are inoculated on the selective agar plates to isolates and detect the E. coli O157. Materials required for IMS detection technique are Magnetic Separator, Sterile Eppendorf, Dynabeads Anti E. coli O157: H7 Thermofisher, Micropipette (10-100 μL,100-1000μL), Modified Soybean Casein Bile Broth with novobiocin, Cefixime Tellurite Sorbitol MacConkey agar (CT – SMAC), Hicrome ECO157:H7 agar, Test tubes, Glassware, Inoculation Loops, Sterile Eppendorf tubes.

Trapping a bacterium with an external magnet...!!! Is it possible?

How “trapped E. coli O157:H7” is nurtured in their bed of selective agar plates…

There are two types of selective agar plates (CT- SMAC & Hicrome ECO157:H7 agar) we are using during the isolation and detection of E. coli O157: H7 from the separated bead matrix. They appear Colorless colonies in the CT SMAC and produce purple type Pigmented colonies in the Hicrome ECO157:H7 agar plates. CT-SMAC medium is recommended for isolation of enteropathogenic Escherichia coli O157: H7, which ferments lactose but does not ferment sorbitol, hence produces colourless colonies. Crystal violet and bile salt mixture present in the medium inhibit growth of the gram-positive bacteria. The addition of cefixime and tellurite, significantly reduces the number of sorbitol non-fermenters that are to be screened during the attempted isolation of E.coli O157:H7. HiCrome EC O157:H7 Agar medium. It contains sorbitol as fermentable carbohydrate and chromogenic mixture instead of lactose and indicator dyes respectively. The chromogenic substrate is specifically and selectively cleaved by E. coli O157: H7 resulting in a dark purple to magenta colored moiety. In both medium Tryptone and yeast extract provides carbonaceous and nitrogenous compounds, long chain amino acids, vitamins and growth nutrients. Sodium chloride maintains osmotic equilibrium.

Is there any application of this technique

Big Yes...!! This technique is not limited to the detection of foodborne pathogens. Nowadays this technique implemented on the several research work of cancer biology, isolation of stem cells, purification of human proteins used for the drug discovery, transplantation of auto-immune cell leads to cure of auto immune disease. Moreover, implementation on the biological techniques like Enzyme assay, Gene therapy is under development and will be published near future.

Conclusion:

In summary, immunomagnetic separation (IMS) is a highly effective and versatile technique for isolating specific cells or molecules from a mixture. This technique offers numerous advantages, including high sensitivity, minimal sample preparation, and the ability to isolate rare target cells from complex biological samples. However, some limitations exist, such as the potential for non-specific binding, the need for high-quality antibodies, and the possible interference from sample complexity. Secondly the magnetic beads and antibodies can be relatively expensive, especially for large-scale separations. Despite these challenges, ongoing improvements in magnetic bead technology and antibody selection continue to enhance the effectiveness and versatility of immunomagnetic techniques.

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