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E. coli O157:H7: Procedure for Isolation and Identification from Stool Specimens
Foodborne and Diarrheal Diseases Branch, Division of Baterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention MS C-09 1600 Clifton Rd Atlanta, GA 30333 To order call: (703)487-4650
Publication date: 08/01/1994
Table of Contents
DIAGNOSTIC CONSIDERATIONS AND SPECIMEN COLLECTION PROCEDURES
SPECIMEN HANDLING PROCEDURES
ISOLATION AND PRESUMPTIVE IDENTIFICATION PROCEDURE
H7 SEROLOGY AND TOXIN TESTING
ADDRESSES OF SOURCES LISTED
POINT OF CONTACT FOR THIS DOCUMENT:
In Vitro Diagnostic Products and Control Strains
Strains of Escherichia coli O157:H7
Procedure for Isolation and Identification of E.coli O157:H7
DIAGNOSTIC CONSIDERATIONS AND SPECIMEN COLLECTION PROCEDURESThe diagnosis of E. coli O157:H7 infection needs to be considered for all patients who present with diarrhea, especially bloody diarrhea or hemolytic uremic syndrome (HUS) (1). Stool specimens (whole stools, swabs prepared from whole stools or rectal swabs with visible fecal staining) should be collected. Ideally, specimens should be collected as close to the time of onset of diarrhea as possible; however, specimens taken even weeks after the onset of symptoms are sometimes positive (2,3). Antibiotic treatment decreases the chance of recovery of E. coli O157:H7; therefore, when follow-up specimens are being obtained, the patient should have received no antibiotic for a minimum of 48 hours before culture. (Figure 1)
SPECIMEN HANDLING PROCEDURESIdeally, stool specimens should be examined as soon as they are received in the laboratory. If whole stool specimens will not be processed immediate- ly, they should be either refrigerated or frozen at -70 C as soon as possible after collection. Refrigerated specimens should be examined within 1-2 hours. If stools cannot be examined within this time, they should be placed in transport medium. All rectal swabs should be placed immediately into transport medium. If specimens in transport medium will be examined within 2-3 days, they should be refrigerated. If specimens will not be examined within 3 days, they should be frozen immediately, preferably at -70 C. Specimens should not be refrigerated for days and then frozen, or placed in transport medium and left at room temperature.
If a transport medium will be used, any of the commercially available transport media (e.g., Cary-Blair, Stuart's, Amie's, buffered glycerol saline) are satisfactory. A swab should be completely covered by the transport medium. If the medium does not cover the swab, the swab will not be kept sufficiently moist and recovery of E. coli O157:H7 and other organisms may be compromised.
ISOLATION AND PRESUMPTIVE IDENTIFICATION PROCEDUREE. coli O157:H7 rapidly ferments lactose and is indistinguishable from most other E. coli on traditional lactose-containing media. However, unlike approximately 80% of other E. coli, nearly all isolates of E. coli O157:H7 ferment D-sorbitol slowly, or not at all. Sorbitol-MacConkey (SMAC) agar was developed to take advantage of this characteristic by substituting the carbohydrate sorbitol for lactose in MacConkey agar and is the medium of choice for isolation of E. coli O157:H7 (4).
Inoculate stool specimens onto SMAC and incubate 18-24 hours at 35-37C. Sorbitol-negative colonies will appear colorless on SMAC. Test sorbitol- negative colonies selected from SMAC with E. coli O157 antiserum or latex reagents (O157 antibody-coated latex and control latex) according to the procedures recommended by the manufacturer (5). If using O157 latex reagents, it is important to test isolates in the control latex to detect nonspecific agglutination of organisms with latex. Manufacturers of O 157 latex reagents recommend heating strains that agglutinate in the latex control reagent and then retesting them in both the O157 antibody-coated and control latex reagents. However, E. coli O157:H7 strains have not been shown to agglutinate in both the antibody-coated and control latex reagents (6). For this reason, some laboratories report isolates that agglutinate in the latex control as negative for O157 without heating and retesting the isolate.
Colonies may be tested with antisera directly from the plate, or subcultured to another nonselective medium (blood agar, for example) and tested the next day. If colonies are tested directly from the plate, O157- positive colonies should also be transferred to another medium for subsequent testing. Although it is more labor-intensive and delays results by a day, subculturmg to another medium and testing the next day offers the advantage of providing more bacterial growth on which to perform the O157 agglutination assay. The extra growth makes it easier to observe agglutination and allows repeat testing of the isolate. if necessary. Once one colony from a plate as been identified as O157-positive, no further colonies from the same plate need to be tested.
Isolates agglutinating in O157 antiserum or O157 latex reagent should be identified biochemically as E. coli, since strains of several species cross-react with O157 antiserum (7,8,9). However, because biochemical confirmation may take 24 hours or longer, an oral report of presumptive E. coli O 157 may be given before biochemical identification is completed.
Specimens from which sorbitol-negative colonies have been isolated that agglutinate in O157 antiserum or O157 latex reagent, and are biochemically E. coli, may be reported as presumptively positive for E. coli O157:H7. A preliminary written report should be issued to the clinician and to public health authorities. It may be useful to note on the laboratory report that E. coli 0157:H7 is an enteric pathogen and can cause nonbloody diarrhea, bloody diarrhea, and HUS.
H7 SEROLOGY AND TOXIN TESTINGConfirmation of E. coli O157:H7 requires identification of the H7 flagellar antigen. This is usually performed by reference laboratories, although some clinical laboratories do H7 testing. E. coli O157 strams that appear to be H7 negative in the clinical laboratory should be sent to a reference laboratory. H7 serology may be difficult since isolates often require multiple passages before the flagellar antigen is detected.
Testing for the H7 antigen as well as for the production of the Shiga-like toxins, which are associated with pathogenic strains, is available through reference laboratories. Isolates that are nonmotile or that are negative for the H7 antigen should be tested for production of the Shiga-like toxins to identify pathogenic strains. Toxin testing of E. coli O157 strains that have the H7 antigen is not necessary, because virtually all of these strains produce the Shiga-like toxins. Although they are uncommon, some strains of E. coli O157 have other H types and do not produce Shiga-like toxin; these are not recognized pathogens. A final report can be issued after H type results have been obtained. (Table 2)
MUGSome laboratories also test E. coli O157 strains for the enzyme B-glucuronidase using broth or agar medium containing the substrate 4-methylumbelliferyl-B-D-glucuronide (MUG) (10). When MUG is cleaved by this enzyme, a fluorescent product is produced that is detectable with long-wave ultraviolet light. Unlike approximately 92% or E. coli, E. coli O157:H7 and nonmotile E. coli O157 strains that produce Shiga-like toxins lack the enzyme and are MUG negative. For this reason the MUG assay used in conjunction with testing for sorbitol fermentation and agglutination in E. coli O 157 antiserum is a useful screening test for toxigenic strains of O 157. (Table 1)
- Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O 157:H7, other enterohemorrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991: 13:60-98.
- Belongia EA, Osterholm MT, Soler JT, Ammend DA, Braun JE, MacDonald KL. Transmission of Escherichia coli 0157:H7 infection in Minnesota child day-care facilities. JAMA 1993; 269:883-888.
- Pai CH, Ahmed N, Lior H, Johnson WM, Sims HV, Woods DE. Epidemiology of sporadic diarrhea due to verocytotoxin-producing Escherichia coli: a two-year prospective study. J Infect Dis 1988; 157: 1054-1057.
- March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 1986;23:869-872.
- March SB, Ratnam S. Latex agglutination test for detection of Escherichia coli serotype O157. J Clin Microbiol 1989;27: 1675-1677.
- Borczyk AA, Harnett N, Lombos M, Lior H. False-positive identification of Escherichia coli O157 by commercial latex agglutination tests. Lancet 1990;336:946-947.
- Bettelheim KA, Evangelidis H, Pearce JL, Sowers E, Strockbine NA. Isolation of a Citrobacter freundii strain which carries the Escherichia coli O 157 antigen. J Clin Microbiol 1993;31:760-761.
- Corbel MJ. Recent advances in the study of brucella antigens and their serological cross-reactions. Vet Bull 1985;55:927-942.
- Lior H, Borczyk AA. False positive identifications of Escherichia coli O157. Lancet 1987:i:333. 10.Thompson JS, Hodge DS, Borczyk AA. Rapid biochemical test to identify verocytotoxin-positive strains of Escherichia coli serotype O 157. J Clin Microbiol 1990;28:2 165-2 168.
ADDRESSES OF SOURCES LISTED
- American Type Culture Collection
10801 University Blvd.
Manassas, VA 20110-2209 (USA)
- Becton-Dickinson / BBL
PO Box 243
Cockeysville, MD 21030
410 - 771-0100
- Boehringer Mannheim Corp.
9115 Hague Road
P. O. Box 50414
Indianapolis, IN 46250-0414
- Difco Laboratories
PO Box 331058
Detroit, MI 48232-7058
800 - 521-0851
- DiMed 2956 Yorkton Blvd.
St. Paul, MN 55117
612 - 490-5350
- Gene-Trak Systems
31 New York Avenue
Framingham, MA 01701
508 - 872-3113
- Pro-Lab Inc.
2111 Sam Bass Road
Round Rock, TX 78681
800 - 522-7740
12076 Santa Fe Dr.
Lenexa, KS 66215
800 - 255-6730
- Research Organic
4353 East 49th St.
Cleveland, OH 44125
800 - 334-0144
- Sigma Chemical Company
PO Box 14508
St. Louis, MO 63178
800 - 325-3010
- Unipath / Oxoid
PO Box 691
Ogdensburg, NY 13669
800 - 567-8378
POINT OF CONTACT FOR THIS DOCUMENT:To request a copy of this document or for questions concerning this document, please contact the person or office listed below. If requesting a document, please specify the complete name of the document as well as the address to which you would like it mailed. DIVISION OF BACTERIAL & MYCOTIC DISEASES
NTIS U.S. Dept. Commerce
5285 Port Royal
Springfield, VA 22161
Table 1Table 1. In Vitro Diagnostic Products and Control Strains for the Detection and Identification of Escherichia coli O157:H7 ============================================================================ Item Source Sorbitol-MacConkey Agar: Dehydrated medium Difco Cat. No. 0079-17-7 500 gm Oxoid Cat. No. CM813 500 gm Prepared Medium(1) Becton Dickinson/BBL Cat. No. 97953 10 plates/pkg Difco Cat. No. 4260-22-1(2) 5 plates/pkg DiMed Cat. No. 50-1430 10 plates/pkg Remel Cat. No. 01-554 10 plates/pkg Cat. No. 09-548(3) 20 pour tubes (deeps)/pkg MUG (4-methylumbelliferyl-B-D-glucuronide) products: Reagent Boehringer Mannheim Corp. Cat. No. 270954 100 mg Oxoid Cat. No. BR71 Box of 10 vials (50 mg/vial) Research Organic Cat. No. 01 84 M 10 mg 50 mg 100 mg Sigma Chemical Co. Cat. No. M 9130 10 mg 100 mg Media with MUG Becton Dickinson/BBL Mac Conkey agar with MUG (prepared) Cat. No. 43-21938 20 plates/pkg MacConkey agar with MUG (dehydrated) Cat. No. 99057 500 gm Difco Nutrient agar with MUG (dehydrated) Cat. No. 0023-17-4 500 gm Gene-Trak Systems Flourocult(R) E. coli O157:H7 (dehydrated) Cat. No. 4036 500 gm Remel MacConkey agar + MUG (prepared) Cat. No. 01-554 10 plates/pkg MacConkey-sorbitol with MUG (prepared) Cat. No. 01-563 10 plates/pkg Disks with MUG Cat. No. 21-135 25 disks/vial O157 antiserum (rabbit): Difco Cat. No. 2970-47-7 3 ml (tube test) O157 latex reagents(rabbit antiserum conjugated to latex beads): Oxoid Cat. No. DR 620 100 tests (slide test) Pro-Lab Cat. No. PL070 50 test (slide test) Cat. No. PL071 100 tests (slide test) Remel* Cat. No. 24-250 50 tests (slide test) H7 antiserum (rabbit) Difco Cat. No. 2159-47-0 3 ml (tube test) H7 latex reagent (rabbit antiserum conjugated to latex beads): Remel* Cat. No. 24-250 50 tests (slide test) ---------------------------------------------------------------------------- 1 Except where noted, the shelf life of prepared sorbitol-MacConkey agar plates stored at 2-8C ranges from 4 to 12 weeks. 2 Extended shelf life medium; the shelf life of this product stored at room temperature is from 6 to 12 months. 3 Plates are prepared from pour tubes (deeps) by melting the agar and pouring plates as needed. The shelf life of this product stored at 2-8C is 4 months. =============================================================================
Table 2Table 2. Strains of Escherichia coli O157:H7 available from the American Type Culture Collection ============================================================================= ATCC No. CDC Nos. Toxin(s) State Origin Source produced* -------- --------- --------- ------ ------- ------- 43890 C984 SLT I WA Human Stool 3621-88| 43889 B1409-C1 SLT II NC Human Stool 1271-84| 35150 EDL 931 SLT I & II OR Human Stool 43894 EDL 932 SLT I & II MI Human Stool 43895 EDL 933 SLT I & II MI Food Meat 43888 B6914-MS1 SLT neg WA Human Stool 3417-86| ------------------------------------------------------------------------------- *SLT I = Shiga-like toxin I (Verocytotoxin 1); SLT= Shiga-like toxin II (Verocytotoxin 2) The above list of commercially available products for the detection and identification of Escherichia coli O157:H7 is not intended to be a complete listing of all such products and is not an endorsement of the named products by the Centers for Disease Control and Prevention. ===============================================================================
Procedure for Isolation and Identification of E.coli O157:H7