SHIBAM Medium

Cat. no. A146 SHIBAM Medium, 15x100mm Plate, 18ml 10 plates/bag

INTENDED USE

SHIBAM (STEC Heart Infusion washed Blood Agar with Mitomycin C) Medium is recommended for use as a selective and differential growth medium for the cultivation of Shiga toxin-producing E. coli (STEC) from food and other samples. This medium can be used as a screening method for non-O157 STEC as mentioned by the Food and Drug Administration in the Bacteriological Analytical Manual (FDA BAM).

This product is not intended to be used for the diagnosis of human disease.

SUMMARY

Escherichia coli is a diverse species of bacteria that includes non-pathogenic strains as well as strains possessing a wide variety of virulence factors that allows them to cause a heterogenous spectrum of disease in humans and animals. One such group of pathogenic E. coli are those that have acquired the ability to produce Shiga-like toxins 1 and/or 2 (Stx1 and/or Stx2). This group, collectively, is referred to by several names, including enterohemorrhagic E. coli (EHEC), verotoxin-producing E. coli (VTEC), or most commonly, Shiga toxin-producing E. coli (STEC).(1-2) Cattle appear to be the major reservoir for STEC, and outbreaks of disease are generally associated with the consumption of beef or other food products that have been in contact with contaminated beef.(3-4,11) STEC infections can cause gastroenteritis ranging from watery to bloody diarrhea, and severe infection can lead to hemolytic uremic syndrome (HUS). Treatment with antibiotics can paradoxically increase the severity of the disease by stimulating E. coli to release more toxin.

Historically, serotype O157 was the most commonly documented STEC serotype. However, not all STEC belong to this serotype. Over 250 serotypes have been identified as STEC, with over 100 of these being associated with human illness. Data suggests that 50% or more of human infections are caused by non-O157 STEC strains.(19) In the United States, O26, O45, O103, O111, O121, and O145 are considered to be the "top six" non-O157 STEC serotypes, though even this list does not account for all human cases.(5-6)

Research shows that production of enterohemolysin (not to be confused with alpha-hemolysin) by E. coli is highly correlated with Shiga toxin production. Colonies that express enterohemolysin produce beta-hemolytic colonies indicated by a clear zone when grown on media containing washed erythrocytes. In 2001, Sugiyama et. al. found that of 185 STEC strains tested--including O157--97% were beta-hemolytic on SHIBAM Medium.(10) Therefore, identification of hemolytic colonies on washed sheep blood agar plates has proven to be a useful screening method for STEC.(7)

SHIBAM Medium utilizes washed sheep blood cells in a blood agar base first described by Beutin et. al. and is appropriate as a screening tool for O157 and non-O157 STEC strains.(8) In addition, the medium includes improvements made by Kimura et. al. and Sugiyama et. al. in order to improve identification of STEC isolates.(9,10) Hardy Diagnostics SHIBAM Medium complies with the Food and Drug Administration, Bacteriological Analytical Manual (FDA BAM) formulation and the medium is listed in BAM as a screening method for non-O157 STEC.(18)

FORMULA

Ingredients per liter of deionized water:*

Agar 14.0 gm
Peptones 17.0 gm
Sodium Chloride 5.0 gm
Tryptose 5.0 gm
Yeast Extract 4.0 gm
Calcium Chloride 1.47 gm
Mitomycin C 0.5 mg
Washed Defibrinated Sheep Blood 40 ml

Final pH 7.2 +/- 0.2 at 25ºC.

* Adjusted and/or supplemented as required to meet performance criteria.

STORAGE AND SHELF LIFE

Storage: Upon receipt store at 2-8ºC. away from direct light. Media should not be used if there are any signs of deterioration (shrinking, cracking, or discoloration), hemolysis, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.

PRECAUTIONS

PROCEDURE

Sample collection: Infectious material should be submitted directly to the laboratory without delay and protected from excessive heat and cold. If there is to be a delay in processing, the sample should be inoculated onto an appropriate transport media such as modified Cary Blair (Cat. no. 280505) and refrigerated until inoculation. Consult listed references for information on sample collection.(12-18)

Method of Use: Allow plates to warm to room temperature prior to use. The agar surface should be dry prior to inoculating. Inoculate the sample onto the medium as soon as possible after receipt in the laboratory. Incubate plates aerobically at 35-37ºC. for 16-24 hours. Observe plates for characteristic colony morphology and beta-hemolysis (clear zones).

INTERPRETATION OF RESULTS

STEC isolates should produce smooth, off-white colonies that are beta-hemolytic (produce a clear zone) on SHIBAM. Visualization of beta-hemolytic colonies can be facilitated by viewing plates through transmitted light or by using a light box (Cat. no. 378642000) to enhance clear zones. Most non-STEC E. coli will produce non-hemolytic or alpha-hemolytic (green zone) colonies. Species other than E. coli should be inhibited or produce non-hemolytic colonies (see Limitations for exceptions).

LIMITATIONS

Growth and/or hemolysis on SHIBAM Medium does not necessarily indicate the isolate produces Shiga-toxin, as some non-STEC strains may also produce enterohemolysin. Testing for confirmation of Shiga-toxin production should be carried out by approved methods.

E. coli ATCC 25922 may produce hemolysis on SHIBAM Medium (most likely due to production of alpha-hemolysin). E. coli strains carrying the alpha-hemolysin (α-hly) gene may demonstrate zones of hemolysis that are wider than strains that carry the enterohemolysin gene. alpha-hemolysin is most commonly found in uropathogenic E. coli and in certain ETEC, EPEC, and STEC strains. Thereofore, E. coli ATCC 8739 is recommended as the negative control for QC testing.(18)

Use of a light box (Cat. no. 378642000) will enhance visualization of clearing zones of beta-hemolysis.

Some strains of Listeria spp., Staphylococcus aureus, Salmonella enterica, Serratia marcescens, Edwardsiella tarda, and Bacillus cereus can produce beta-hemolytic growth on SHIBAM Medium. Selective pre-enrichment may be useful to limit the growth of these organisms. Gram staining, serological, and/or biochemical testing must be performed on all isolates for complete identification.

MATERIALS REQUIRED BUT NOT PROVIDED

Standard microbiological supplies and equipment such as loops, swabs, transport media such as modified Cary Blair (Cat. no. 280505), applicator sticks, other culture media, incinerators, light box (Cat. no. 378642000), and incubators, etc., as well as serological and biochemical reagents, are not provided.

QUALITY CONTROL

Test Organisms Inoculation Method* Incubation Results
Time Temperature Atmosphere
Escherichia coli O157
ATCC® 43889**
A 24hr 35°C Aerobic Growth; beta hemolysis
Escherichia coli
ATCC® 8739
A 24hr 35°C Aerobic Growth; no hemolysis

** Recommended QC strains for User Quality Control according to the CLSI document M22 when applicable

USER QUALITY CONTROL

PHYSICAL APPEARANCE

SHIBAM Agar should appear opaque, and cherry red in color.

E. coli O45 STEC colonies growing on SHIBAM Agar (Cat. no. A146). Incubated aerobically for 24 hours at 35ºC. Note zones of clear beta-hemolysis as viewed through a light box. Image credit FDA BAM.

E. coli (ATCC 8739) colonies growing on SHIBAM Agar (Cat. no. A146). Incubated aerobically for 24 hours at 35ºC. Note green alpha-hemolysis as viewed through a light box. Image credit FDA BAM.

REFERENCES

1. Paton, J.C., et al. 1998. Pathogenesis and Diagnosis of Shiga toxin-producing Escherichia coli infections. Clin. Microbiol. Rev. 11:450-479.

2. Scallan, E., et al. 2011. Foodborne illness acquired in the United States- Major Pathogens. Emerging Infect. Dis. 17:7-15.

3. Orskov, F., et al. 1987. Cattle as a reservoir of verotoxin-producing Escherichia coli O157:H7. Lancet. i:276.

4. Smith, H.R., et al. 1988. Vero cytotoxin production and presence of VT genes in Escherichia coli strains of animal origin . J. Gen. Microbiol. 134:829-834.

5. Johnson, K.E., et al. 2006. The emerging clinical importance of non-O157 Shiga toxin-producing Escherichia coli. Clin. Infect. Dis.

6. Kappeli, U., et al. 2011. Human nfections with Non-O157 Shiga toxin-producing Escherichia coli, Switzerland, 2000-2009. Emerging Infect. Dis. 17:180-185.

7. Beutin, L., et al. 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. 27:2559-2564.

8. Beutin, L., et al. 1996. Rapid detection and isolation of Shiga-like toxin (verotoxin)-producing Escherichia coli by direct testing of individual enterohemolytic colonies from washed sheep blood agar plates in the VTEC-RPLA assay . J. Clin. Micro. 34:2812-2814.

9. Kimura, N., et al. 1999. Basic study of Beutin's washed sheep blood agar plate used for selective screening of verocytotoxin-producing/enterohemorrhagic Escherichia coli (VTEC/EHEC) . Kansenshogaku Zasshi. 73:318-327.

10. Sugiyama, K., et al. 2001. Mitomycin-supplemented washed blood agar for the isolation of Shiga toxin-producing Escherichia coli other than O157:H7 . Letters Appl. Micro. 33:193-195.

11. Wallace, J.S., and K. Jones. 1996. The Use of Selective and Differential Agars in the Isolation of Escherichia coli O157 from Dairy Herds . Jour. Appl. Bacteriol.; 81:663-668.

12. Jorgensen., et al. Manual of Clinical Microbiology , American Society for Microbiology, Washington, D.C.

13. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology , C.V. Mosby Company, St. Louis, MO.

14. Isenberg, H.D. Clinical Microbiology Procedures Handbook , Vol. I, II and III. American Society for Microbiology, Washington, D.C.

15. Koneman, E.W., et al. Color Atlas and Textbook of Diagnostic Microbiology , J.B. Lippincott Company, Philadelphia, PA.

16. American Public Health Association. Standard Methods for the Examination of Dairy Products, APHA, Washington, D.C.

17. APHA Technical Committee on Microbiological Methods for Foods. 2001. Compendium of Methods for the Microbiological Examination of Foods, 4th ed. APHA, Washington, D.C.

18. U.S. Food and Drug Administration. Bacteriological Analytical Manual. AOAC, Arlington, VA.
https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm 

19. Foodborne Diseases Active Surveillance Network (FoodNet) Annual Report. 2010. Centers for Disease Control and Prevention.

ATCC is a registered trademark of the American Type Culture Collection.

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