Isolation of E. coli O157:H7 or NM in Foods

Laboratory Procedure MFLP-80
March 2008

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Health Products and Food Branch
Ottawa

1. APPLICATION

This method is applicable to the isolation of viable Escherichia coli O157 in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. This revised method replaces MFLP-80, dated February 2006.

2. DESCRIPTION

The method has been shown to produce satisfactory results with artificially-contaminated meats (including beef, veal and pork), vegetables, dairy products, spices and environmental samples as well as naturally-contaminated samples. This method can be used successfully for the detection of E. coli O157 in other foods, food ingredients and environmental samples.

3. PRINCIPLE

This method is based on the work of Dr. S. Weagant (USFDA, 8.3, 8.4 and pers. com.) with modifications by Warburton et al. (8.2 and unpublished data) as well as in-house studies conducted by HC and CFIA Laboratories. The sample is enriched in a selective broth and plated on selective agars. Presumptive positives are determined within 48 h. Confirmatory biochemical and serological tests are performed on purified colonies.

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

The media and reagents listed below are commercially available and are to be used, prepared and/or sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of most of the individual media.

1) Modified Tryptic Soy Broth with Novobiocin (mTSB-n)

2) Enterohemorrhagic E. coli (EHEC) Enrichment Broth (EEB)

3) Group 1 Agars (this group of agars have data showing more selectivity for E. coli O157 - one or two may be chosen)
Modified Hemorrhagic Coli Agar (mHC) with Tellurite and Cefsulodin
mHC Agar with CT supplement (Oxoid SR172)
BBL CHROMagar for E. coli O157 (BD)

4) Group 2 Agars (choose one from this group if only one media from group 1 has been chosen)
Modified Sorbitol MacConkey agar (TCCSMAC) with Tellurite, Cefixime, and Cefsulodin
CT-SMAC (Oxoid CR-SMAC agar base (CM1005) with CT supplement)
CR-SMAC Agar

5) Group 3 Agars (must be validated by the laboratory prior to use) Chromogenic agars, including Rainbow Agar (Biolog),

6) Trypticase Soy Agar with Yeast Extract (TSA-YE)

7) Phenol Red Sorbitol agar

8) Sorbitol MacConkey agar

9) Motility Agar

10) Urea Agar Slants

11) Purple broth with cellobiose or carbohydrate agar with cellobiose

12) K₂SO₄ (final concentration 0.5% in mTSB-n - needed for some spices and foods containing large amounts of spices)

13) Stomacher, blender, or equivalent

14) Control cultures (use ATCC cultures or equivalent), positive control: E. coli O157 (H7 or other serovars) and negative control: non-0157 E. coli

15) Dynabeads (MFLP-90) or equivalent immunomagnetic beads or concentration steps

16) O157 and H7 antisera and agglutination tests

17) Latex Agglutination Kits; for E. coli O157 (Oxoid), for O157 and H7 (Wellcolex) or validated equivalent.

18) Incubators capable of maintaining 35 and 42°C

19) M Broth

20) Gram stain reagents

21) IMVIC Reagents (see MFHPB-19)

22) MUG (4-methylumbelliferyl-β-D-glucuronide), LST-MUG broth (Oxoid), Colicomplete (MUG disks; BioControl)

23) BCIG (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) either -Na or -CHX (cyclohexylammonium) salt

24) Rapid identification kits such as Vitek, API or equivalent

25) Rapid detection kits such as VIP (MFLP- 87), Singlepath E. coli (MFLP-82), Tecra VIA (MFLP-91), Polymixin EIA (MFLP-92) or equivalent (see Appendix K of the Compendium).

26) Toxin detection kits such as Meridian Premier EHEC kit (MFLP-93 ), Duopath VT kit (MFLP-83 ) or equivalent.

7. PROCEDURE

Each sample unit may be analyzed individually or the analytical units may be combined. Carry out the test in accordance with the following instructions:

7.1 Handling of Sample Units

7.1.1 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.

7.1.2 Analyze sample units as soon as possible after their receipt in the laboratory.

7.2 Preparation for Analysis

7.2.1 Have ready sterile mTSB-n (and EHEC enrichment broth (EEB) if required).

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.3 Preparation of Sample

7.3.1 To ensure a truly representative analytical unit agitate liquids or free flowing materials until the contents are homogeneous. 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. It is recommended that a composite contain not more than five analytical units.

7.3.2 Prepare a 1:10 dilution of the food by aseptically adding 25 g or mL (or other suitable analytical unit, such as 65 g for ground beef) into 225 mL (or amount necessary to maintain the 1:10 ratio) of the enrichment broth mTSB-n (and EEB, if applicable). Stomach or blend. A second primary enrichment broth started directly in EEB should be done when there is a high bacterial load of competing organisms, during food-borne illness investigations and when otherwise deemed appropriate. (Therefore you will have 2 separate primary enrichments of 25 g each when a high bacterial load is suspected).

7.3.3 A positive and a negative control should be set up at the same time.

7.3.4 Incubate the enrichment mixture and controls for 22-24 h at 42°C.

7.3.5 Either screen the enrichment broth for E. coli O157 by using rapid detection kits (see Appendix K of the Compendium) or proceed to 7.3.6.
If a screening method is used, a negative result obtained with the corresponding rapid kit is considered the final result. If a presumptive positive result is obtained with the corresponding rapid kit, proceed with step 7.3.6 of MFLP-80 using the same enrichment broth that was determined to be presumptive positive.

7.3.6 Within 48 h, or preferably as soon as possible (see Note above), concentrate E. coli O157 from the "presumptive positive" enrichment broth(s) by immunomagnetic separation (IMS) using MFLP-90, or equivalent procedures. Proceed with 7.4 or 7.5.

7.4 Secondary Enrichment in EEB

7.4.1 After agitation of the enrichment broth, transfer 1 mL of the mTSB-n and/or EEB to 9 mL of EEB. Incubate 18-24 h at 35°C. Concentrate as per 7.3.6. Resuspend the pellet and streak following manufacturers instructions onto the selective agars and follow confirmation steps for typical colonies described below. Do not refrigerate broth, analysis must be continued at this stage. (Optional: to delay incubation of the secondary enrichment in EEB, while waiting for confirmation of initial plating, retain original mTSB for up to 48 hours, only transferring to EEB for incubation when it is determined that this step is necessary).

7.5 Selective Isolation

7.5.1 Resuspend the pellet from 7.3.6 and streak following manufacturers instructions onto at least two different agars; one or two from the Group 1 agars and one from Group 2 agars if only one group 1 agar is selected. Group 1 includes mHC (with tellurite and Cefsulodine), CHROMagar and mHC with CT supplement (Oxoid SR172). Group 2 includes TCCSMAC, CT-SMAC and CR-SMAC.

Group 1 Agars

mHC/mHC-CT: Incubate plates for 18-24 h at 42°C. Typical E. coli O157:H7 colonies appear blue. Other serovars of E. coli, including O157 (not H7) will be yellow on mHC/mHC-CT).

BD CHROMagar: Incubate plates for 18-24 h at 35°C. Typical E. coli O157:H7 colonies appear mauve against a white background. Other Gram-negative organisms will appear colourless, blue, green or blue-green.

Group 2 Agars

TCCSMAC: Incubate plates for 18-24 h at 42°C. Typical E. coli O157:H7 colonies appear colorless, bear the tint of the medium or are gray to pink with smokey centers (i.e., ferment sorbitol slowly or not at all). Other serovars of E. coli, including O157 (not H7) will be red on TCCSMAC.

CT-SMAC: Incubate plates for 18-24 h at 35°C. Typical E. coli O157:H7 colonies appear dark and magenta with a clear edge. In some cases, the agar becomes orange and the colonies have the colour of the agar with dark grey centres.

CR-SMAC: Incubate plates for 18-24 h at 35°C. Typical E. coli O157:H7 colonies appear dark maroon with clear edges. In some cases, the agar becomes a light, clear beige and colonies take on the colour of the agar with dark centres.

7.6 Confirmation Steps

7.6.1 Where the suspect colonies are well isolated and large enough, at least 10 colonies (if available) should be carried forward. O157 serology on these colonies can help in the screening process at this point however this is optional. If this optional step is used, then continue with the confirmation steps only on those colonies that are O157 positive.
Where not well isolated or the colonies are small (a slow growing serotype), streak suspect colonies onto selective agars and/or TSA-YE for purity, and then continue, as below.

7.6.2 Draw a grid on the two agars chosen from group 1 and/or 2. Inoculate at least 10 typical (if available) and isolated colonies onto the grid cells of the two agars. Incubate at the appropriate temperature and for the appropriate time of incubation for the specific agars used. Use the same isolates to inoculate tubes of purple broth containing 1% cellobiose or stab onto carbohydrate agar containing 1% cellobiose. Incubate at 35°C for 18-24 h. Proceed with the isolates that are typical on the selective agars and are negative for cellobiose and sorbitol.

See Table 2 for typical biochemical and serological reactions.

7.6.3 Confirm that the suspect isolates are E. coli with a rapid identification system such as VITEK or API.

7.6.4 Confirm isolates as O157 using latex agglutination kits or O157 anti-sera.

7.6.5 Refer to Table 1 for the additional required confirmation steps. It is preferable that A) or B) be done first.

Do either: A) VT Toxin Test (When toxin testing is required, it is recommended that a Compendium method be used such as MFLP-93 or MFLP-83 (or equivalent) that confirms the presence of toxin);

OR B) Validated PCR methods that detect VT genes;

OR C) Validated PCR method (eg. BAX or GDS or equivalent);

OR D) H7 Agglutination (complete serological testing using H7 antisera following manufacturer's instructions).

If positive, proceed as a confirmed E. coli O157:H7 or NM.

7.6.5.1 Interpretation of Results

If A) or B) is negative, then consider the isolate to be negative for E. coli O157:H7 or NM

If C) or D) is negative, perform A) or B), if they haven't been done yet. If A) or B) is positive then proceed as if the isolate is a confirmed E. coli O157:H7 or NM. If A) or B) is negative then consider the isolate to be negative for E. coli O157:H7 or NM.

7.6.6 MUG test - fluorescence (Do one of the following:)

1) Streak suspect colonies onto Phenol Red Sorbitol agar with MUG (PSRA). Incubate plates at 35°C for 22-24 h.

2) Inoculate LST broth with MUG. Incubate at 35°C for 22-24 h.

3) Inoculate TSA plate with an isolate (7.6.2) to form a lawn of bacteria. Aseptically transfer a MUG disks (Colicomplete) onto the agar plate. Incubate at 35°C for 22 - 24 h.

Check MUG reaction using UV Light at 345 nm. Ensure that positive and negative controls give proper reaction. When using test tubes ensure that the control tube does not show fluorescence.

7.6.7 Optional Confirmation Steps: For difficult identifications these optional steps may be helpful

1) Do a Gram stain

2) BCIG : Streak suspect colonies onto SMAC agar with BCIG. Incubate plates at 35°C for 22-24 h.

3) Inoculate IMViC tests and Urea slants. Incubate at 35°C for 22-24 h.

8. REFERENCES

8.1 Szabo, R.A., E. Todd, J. MacKenzie and L. Parrington. 1990. Increased sensitivity of the Rapid HGMF-ELA procedure for E. coli O157 detection in foods and bovine feces. Appl. Environ. Microbiol. 56:3546-3549.

8.2 Warburton, D.W., J.W. Austin, B.H. Harrison and G.Sanders. 1998. Survival and recovery of Escherichia coli O157:H7 in inoculated bottled water. J. Food Prot. 61(8):948-952.

8.3 Weagant, S.D., J.L. Bryant and K.G. Jinneman. 1994. An improved rapid technique for isolation of Escherichia coli O157:H7 from foods. Bulletin # 3900. Laboratory Information Bulletin (USFDA) 10(9):1-12.

8.4 Weagant, S.D., J.L. Bryant and K.G. Jinneman. 1995. An improved rapid technique for isolation of Escherichia coli O157:H7 from foods. J. Food Prot. 58(1):7-12.

8.5 Guideline No 10. Health Canada (HC) website:http://www.hc-sc.gc.ca/fn-an/legislation/guide-Id/guidelines_raw_groound_beef-directives_boeuf_hache_cru-eng.php.

Table 1. Confirmation Steps Required by Guideline No 10 for Ground Beef and Meat Products (8.5)

Based upon presently published HC methods, the following is acceptable:

Selective Plating from MFLP-80 enrichment or equivalent:

Concentrate enrichment using an IMS step and plate on selective media. Choose typical colonies and purify. Complete the following:

1) Confirm the isolate to be an E. coli by a rapid ID system for enterics such as Vitek, API or equivalent.

AND

2) Continue to confirm isolates as E. coli O157 serotypes by the following biochemical assays: as sorbitol -ve, cellobiose -ve, MUG -ve, and O157 agglutination positive (either anti-sera or latex agglutination).

[if any of these biochemicals are done via the rapid ID system or as a screening step, do NOT redo]

If the E. coli O157 isolate meets the above criteria, do one of the following combinations of assays on the same isolate. The preference is to do A) or B) first:

A) VT toxin test (must be positive by MFLP-93 or equivalent)

OR

B) positive for VT toxin genes. If positive, proceed as a confirmed E. coli O157:H7 or NM isolate.

OR

C) Confirm the ID by a validated PCR method (eg. BAX E. coli O157:H7 MP or equivalent). If positive, proceed as a confirmed E. coli O157:H7 or NM isolate.

OR

D) H7 agglutination is positive. Note: this is not recommended unless you are trained in this type of serology, due to the difficulty in resuscitating some H antigens. Also the time delay in doing the H serology, prevents compliance activity in a timely manner.

If positive, proceed as a confirmed E. coli O157:H7 or NM.

Interpretation of Results

If A) or B) is negative, then consider the isolate to be negative for E. coli O157:H7 or NM

If C) or D) is negative, perform A) or B), if they haven't been done yet. If A) or B) is positive then proceed as if the isolate is a confirmed E. coli O157:H7 or NM. If A) or B) is negative then consider the isolate to be negative for E. coli O157:H7 or NM.

Table 2. Characteristics of E. coli O157:H7

	E. coli O157:H7 ReactionsA	Other Escherichia Reactions
Gram Stain	Negative	Negative
IMViCs (see MFHPB-19) Indole	+ (Red)B	variable
Methyl Red	+ (Red)	+ (Red)
Vogues-Proskauer	- (No Color)	- (No Colour)
Citrate	- (Green or No Growth)	- (Green or No Growth)
Cellobiose	- (Purple)	- (Purple)
Sorbitol	- (Pale or No Colour)	+ (Coloured)
Urea slants	- (Pale)	- (Pale)
Pigment Production on Nutrient Agar	- (No Pigment)	- (No Pigment)
MUG Reaction	- (No fluorescenceC)	+ (Fluorescence)
BCIG Reaction	- (Pale)	+ (Coloured)
Latex Agglutination	+ (Positive)	- (Negative)
O157	+ (Positive)	- (Negative)
H7	+ (Positive)	- (Negative)
Verotoxin production (VT)	VT 1+ and/or VT 2+	VT 1 and/or VT 2+ or -