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SOP: Detection of Enterohemorrhagic Escherichia coli O157 (EHEC) in Food Products and Food Ingredients by the VIP Gold for EHEC Method

GIFSQ — Global Institute for Food Safety & Quality

Principle

This method detects enterohemorrhagic Escherichia coli O157 (EHEC) in food products and food ingredients to determine compliance with Sections 4 and 7 of the Food and Drugs Act and/or other relevant federal regulations. It is validated for use in raw processed and raw unprocessed meats and pasteurized juices. Positive results must be confirmed with a cultural method.

VIP Gold for EHEC is a visual immunoprecipitate assay that detects enterohemorrhagic E. coli including E. coli O157:H7. Specific antibodies directed against EHEC antigens are used, and the assay is formulated to minimize cross-reactivity with many Enterobacteriaceae while maintaining sensitivity. A proprietary reagent system forms a visually apparent antigen-antibody-chromogen complex when EHEC is present. Appropriately enriched samples are added to the VIP unit; any EHEC antigens bind to the antibody-chromogen complex as it flows across a supporting membrane. When EHEC is present, the complex forms a detection line in the test sample window. Sample flow continues down the membrane to form a control line in the test verification window whether or not the sample contains EHEC.

Apparatus

  • VIP Gold for EHEC (catalog number 60036-40, BioControl Systems Inc.) — one test device per test sample; units may not be reused
  • Enrichment broth: Modified Trypticase Soy Broth and Novobiocin (mTSB-n; 20mg/L novobiocin); BioControl mEHEC broth
  • Stomacher, blender or equivalent
  • Stomacher bags
  • Incubators capable of maintaining 42 °C — It is the responsibility of each laboratory to ensure that the temperatures of the incubators or water baths are maintained at the recommended temperatures. Where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators or water baths be maintained within 0.5°C due to potential lethality of the higher temperatures on the microorganism(s) being isolated.
  • Filter tips for pipettes
  • Water bath capable of maintaining 95–105 °C or equivalent (e.g. autoclave with flowing steam, dry heater)
  • 13 X 100 mm test tubes and caps
  • Control cultures: Positive control E. coli O157:H7 or E. coli O157:NM (e.g. ATCC 35150); Negative control E. coli (not O157) (e.g. ATCC 25922)
autoclave

Reagents

The method does not formulate a general-purpose reagent list; the enrichment media named above are prepared as the method directs:

  • mEHEC media: pre-warm the appropriate volume (225–1500 mL per sample) of sterile deionized water at 42°C overnight. On day of use, aseptically transfer the appropriate amount of mEHEC media into the pre-warmed sterile water — use 7.1 g of mEHEC media for every 225 mL sterile water, maintaining this ratio for larger volumes of water (e.g. use 15.8 g of mEHEC media for 500 mL sterile water). Mix to dissolve the powder. Use prepared medium within 6 h. Media must be pre-warmed to 42± 1°C prior to sample addition.
  • Alternative preparation: mEHEC media can be prepared in advance and autoclaved — add 31.6 g media per liter of deionized water, stir to dissolve the powder, dispense into desired volume and autoclave at 121°C for 15 minutes. Media must be pre-warmed to 42 ± 1°C prior to sample addition.
  • mTSB-n (20mg/L): pre-warm media to 42± 1°C — for pasteurized juices.

Media formulations

  • mTSB-n (20 mg/L Novobiocin)
  • To prepare: add novobiocin to mTSB to a final concentration of 20 mg/L; pre-warm to 42 ± 1 °C before use

Sample preparation

  • Handling of sample units: in the laboratory prior to analysis, except for shelf-stable foods, keep sample units refrigerated or frozen depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. Analyze sample units as soon as possible after their receipt in the laboratory.
  • Preparation for analysis: clean the surface of the working area with disinfectant; prepare the BioControl mEHEC media for raw meat or sterile mTSB-n for pasteurized juices as directed under Reagents above.
  • Preparation of sample: to ensure a truly representative analytical unit, if the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload, the analytical units may be combined for analysis to a maximum of 375 g for raw meats or 325 g for pasteurized juices.
  • Each sample unit may be analyzed individually or the analytical units may be combined.

Procedure

Raw meat products (65–375 g samples)

Aseptically weigh the sample into pre-warmed mEHEC — stomach, vortex, or homogenize as required for thorough mixing
↓
Maintain the ratio of 1 portion sample to 4 portions mEHEC media (i.e. 375 g sample in 1500 mL mEHEC media)
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Incubate samples for 10–24 h at 42°C
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Proceed to sample inactivation

Sample inactivation (applicable to mEHEC enrichment only)

Gently mix the enriched test portion and allow food particles to settle
↓
Transfer 4.0 mL of enriched broth to a test tube
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Inactivate microorganisms at 95–105 °C for 10 min in a water bath, autoclave or dry heater
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Store inactivated tubes for up to 4 days at 2–8 °C prior to testing
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Proceed to the VIP Gold assay

Pasteurized juices

Aseptically weigh 25 g sample into 225 mL of pre-warmed mTSB-n — stomach, vortex, or homogenize as required for thorough mixing
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Incubate samples for 18–28 h at 42 °C; incubate analytical units greater than 25 g for 22–24 hours
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Proceed to the VIP Gold assay (sample inactivation is not used for the juices protocol); presumptive positive enrichment broths may be retained for up to 3 days under refrigeration for confirmation if required

Performing the VIP Gold assay

Open the sealed pouch containing VIP Gold units and remove the sheet of test devices — one device is necessary for each test sample; break away the necessary number of devices
↓
Units may not be reused — immediately reseal unused units in the pouch containing desiccant; store at room temperature (15–30 °C)
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Gently shake the enrichment broth and allow food particles to settle — enriched broths should be equilibrated to 25–37 °C prior to running the test; do not mix inactivated test broths from the raw meat protocol
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Transfer 0.1 mL of inactivated test broth for the raw meat protocol or 0.1 mL of enriched broth for the pasteurized juices protocol to the sample addition well — avoid transferring particulate matter to the device (sample particles may affect flow; it is recommended that filter tips be used when pipetting the sample)
↓
Incubate the device at room temperature (15–30 °C) for 15–20 min for the 10 h raw meat protocol, or for 10 min for the 18 h pasteurized juices protocol

Reading results

Examine the VIP Gold unit for the presence of distinct detection lines in both the test sample and test verification zones — lines should be dark when contrasted with the white background and should extend across the zone; the intensity of the test sample and test verification lines may differ
↓
– Positive: lines are present in the test sample zone and in the test verification zone
– Negative: a line is present in the test verification zone and no line is seen in the test sample zone
– Invalid: absence of a test verification line — repeat the test; if the result remains invalid, contact BioControl Technical Services
↓
Examine the test unit at the specified time — beyond this time faint lines may develop because of non-specific color development and should be disregarded

Confirmation of positive VIP samples

Presumptive positive samples must be confirmed culturally as described in MFHPB-10 — perform immunomagnetic separation on the enrichment broths (non-deactivated, as applicable) and inoculate onto at least two selective agars as described in MFHPB-10 (Isolation of Escherichia coli O157:H7/NM from Foods and Environmental Surface Samples: https://gifsq.com/food-analysis/sop-isolation-of-escherichia-coli-o157-h7-nm-from-foods-and-environmental-surface-samples/).

Precautions

  • The laboratory supervisor must ensure that completion of the analysis described in this method is carried out in accordance with the International Standard referred to as ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories.
  • It is the responsibility of the laboratory to ensure equivalency if any variations of the media formulations listed here are used (either product that is commercially available or made in-house).

References

  • Health Canada. 2016. MFLP-87: Detection of Enterohemorrhagic Escherichia coli O157 (EHEC) in Food Products and Food Ingredients by the VIP Gold for EHEC Method. Volume 3. Compendium of Analytical Methods. This revised method replaces MFLP-87, dated June 2005. Health Products and Food Branch.
  • Microbiological Methods Committee. 2014. Isolation of Escherichia coli O157:H7 or NM from foods and environmental surface samples (MFHPB-10). In: Volume 2. The Compendium of Analytical Methods.