Escherichia coli is one of the predominant species of facultative anaerobes in the human gut and usually harmless to the host; however, a group of pathogenic E. coli has emerged that causes diarrheal disease in humans. Referred to as Diarrheagenic E. coli (20) or commonly as pathogenic E. coli, these groups are classified based on their unique virulence factors and can only be identified by these traits. Hence, analysis for pathogenic E. coli often requires that the isolates be first identified as E. coli before testing for virulence markers. The pathogenic groups includes enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and perhaps others that are not yet well characterized (15, 20). Of these, only the first 4 groups have been implicated in food or water borne illness. Some properties and symptoms of these 4 subgroups are discussed below and summarized in Table 1.

ETEC is recognized as the causative agent of travelers' diarrhea and illness is characterized by watery diarrhea with little or no fever. ETEC infections occurs commonly in under-developed countries but, in the U.S., it has been implicated in sporadic waterborne outbreaks as well as due to the consumption of soft cheeses, Mexican-style foods and raw vegetables. Pathogenesis of ETEC is due to the production of any of several enterotoxins. ETEC may produce a heat-labile enterotoxin (LT) that is very similar in size (86 kDa), sequence, antigenicity, and function to the cholera toxin (CT). ETEC may also produce a heat stable toxin (ST) that is of low molecular size (4 kDa) and resistant to boiling for 30 min. There are several variants of ST, of which ST1a or STp is found in E. coli isolated from both humans and animals, while ST1b or STh is predominant in human isolates only. The infective dose of ETEC for adults has been estimated to be at least 10⁸ cells; but the young, the elderly and the infirm may be susceptible to lower levels. Because of its high infectious dose, analysis for ETEC is usually not performed unless high levels of E. coli have been found in a food. Also, if ETEC is detected, levels should also be enumerated to assess the potential hazard of the contaminated food. Production of LT can be detected by Y-1 adrenal cell assays (20) or serologically by commercial reverse passive latex agglutination assay and ELISA (see Appendix 1). The production of ST can also be detected by ELISA or by infant mouse assay (26). Both LT and ST genes have also been sequenced and PCR (28, 30) and gene probe assays (see chapter 24) are available. Analysis of colonies on plating media using gene probe/colony hybridization also allows enumeration of ETEC in foods.

EIEC closely resemble Shigella and causes an invasive, dysenteric form of diarrhea in humans (6). Like Shigella, there are no known animal reservoirs; hence the primary source for EIEC appears to be infected humans. Although the infective dose of Shigella is low and in the range of 10 to few hundred cells, volunteer feeding studies showed that at least 10⁶ EIEC organisms are required to cause illness in healthy adults. Unlike typical E. coli, EIEC are non-motile, do not decarboxylate lysine and do not ferment lactose, so they are anaerogenic. Pathogenicity of EIEC is primarily due its ability to invade and destroy colonic tissue. The invasion phenotype, encoded by a high molecular weight plasmid, can be detected by invasion assays using HeLa or Hep-2 tissue culture cells (6, 18) or by PCR and probes specific for invasion genes (see chapter 24).

EPEC causes a profuse watery diarrheal disease and it is a leading cause of infantile diarrhea in developing countries. EPEC outbreaks have been linked to the consumption of contaminated drinking water as well as some meat products. Through volunteer feeding studies the infectious dose of EPEC in healthy adults has been estimated to be 10⁶ organisms. Pathogenesis of EPEC involves intimin protein (encoded by eae gene) that causes attachment and effacing lesions (11); but it also involves a plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables localized adherence of bacteria to intestinal cells (27). Production of EAF can be demonstrated in Hep-2 cells and the presence of eae gene can be tested by PCR assays (20).

EHEC are recognized as the primary cause of hemorrhagic colitis (HC) or bloody diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome (HUS). EHEC are typified by the production of verotoxin or Shiga toxins (Stx). Although Stx1 and Stx2 are most often implicated in human illness, several variants of Stx2 exist. There are many serotypes of Stx-producing E. coli (STEC), but only those that have been clinically associated with HC are designated as EHEC. Of these, O157:H7 is the prototypic EHEC and most often implicated in illness worldwide (2, 10, 14, 20). The infectious dose for O157:H7 is estimated to be 10 - 100 cells; but no information is available for other EHEC serotypes. EHEC infections are mostly food or water borne and have implicated undercooked ground beef (2, 10), raw milk (23), cold sandwiches (14), water (25), unpasteurized apple juice (1) and sprouts and vegetables (3, 13). EHEC O157:H7 are phenotypically distinct from E. coli in that they exhibit slow or no fermentation of sorbitol and do not have glucuronidase activity (see chapter 4. LST-MUG for details); hence, these traits are often used to isolate this pathogen from foods. The production of Stx1 and Stx2 can be tested by cytotoxicity assays on vero or HeLa tissue culture cells or by commercially available ELISA or RPLA kits (see Appendix 1). Gene probes (see chapter 24) and PCR assays specific for stx1 and stx2 and other trait EHEC markers are also available (9, 12) (and see below).

Table 1. Some properties and symptoms associated with pathogenic E. coli subgroups.

	ETEC	EPEC	EHEC	EIEC
Toxin	LT/STa	-	Shiga or Vero toxin (Stx or VT)	-
Invasive	-	-	-	+
Intimin	-	+	+	-
Enterohemolysin	-	-	+	-
Stool	Watery	Watery, Bloody	Watery, very bloody	Mucoid, bloody
Fever	Low	+	-	+
Fecal leukocytes	-	-	-	+
Intestine involved	Small	Small	Colon	Colon, lower small
Serology	various	O26, O111 & others	O157:H7, O26, O111 & others	various
IDb	High	High	Low	High
a LT, labile toxin; ST, stable toxin. b ID, infective dose.

Isolation and Identification of Pathogenic Escherichia coli
- Except EHEC of serotype O157:H7

Since pathogenic E. coli are identified based on its unique virulence properties, the analytical procedure for these pathogens in foods generally requires the isolation and identification of the organisms as E. coli before testing for the specific virulence traits. Following is a general procedure for enrichment and isolation of pathogenic E. coli from food (17).

A. Equipment and materials

1. Balance, > 2 kg with 0.1 g sensitivity
2. Blender, Waring or equivalent model with low speed operation at 8000 rpm, with 1 liter glass or metal jar
3. Incubators, 35 ± 0.5°C and 44 ± 1°C
4. Petri dishes 20 x 150 mm
5. Pipets, Pasteur
6. pH test paper, range 6.0-8.0

B. Media

1. Tryptone phosphate (TP) broth (M162)
2. Brain heart infusion (BHI) broth (M24)
3. Levine's eosin-methylene blue (L-EMB) agar (M80)
4. MacConkey agar (M91)
5. Triple sugar iron (TSI) agar (M149)
6. Blood agar base (BAB) (M21)
7. Tryptone (tryptophane) broth (M164)
8. Bromcresol purple broth (M26) supplemented individually with 0.5% (w/v) of each: glucose, adonitol, cellobiose, sorbitol, arabinose, mannitol, and lactose
9. Urea broth (M171)
10. Lysine decarboxylase broth, Falkow (M87).
11. Potassium cyanide (KCN) broth (M126)
12. MR-VP broth (M104)
13. Indole nitrite medium (tryptic nitrate) (M66)
14. Acetate agar (M3)
15. Mucate broth (M105)
16. Mucate control broth (M106)
17. Malonate broth (M92)
18. Koser's citrate broth (M72)

C. Reagents, inorganic, organic, and biological

1. Sodium bicarbonate solution, 10%, aqueous (sterile) (R70)
2. ONPG (o-nitrophenyl-ß-D-galactopyranoside) disks (R53)
3. Phosphate buffered saline solution, (sterile) (PBS) (R60), or Butterfield's Phosphate-buffered dilution water (BPBW) (R11).
4. Kovac's reagent (R38)
5. VP reagents (R89)
6. Oxidase test reagent (R54)
7. Nitrite detection reagents (R48)
8. Mineral oil, heavy sterile (R46)
9. Gram stain reagents (R32)

D. Enumeration.

Refrigerate samples promptly after receipt. Do not freeze except to hold frozen products until just prior to analysis. Analyze samples as soon as possible. If enumeration is required, prepare a homogenate of 25 g in 225 mL of PBS or BPBW. Perform decimal dilutions (in PBS or BPBW) from the homogenate and direct plate onto MacConkey agar to yield isolated colonies. After incubating the plates for 20 h at 35°C, perform colony lifts and hybridization with specific gene probes for virulence genes (see chapter 24). This enumeration procedure is most effective if E. coli constitutes at least 10% of the microbial growth on isolation agars and it is present at a level of >1,000 cells/g.

E. Enrichment for Pathogenic E. coli

The approach recommended here permits qualitative determination of the presence of pathogenic E. coli. Aseptically weigh 25 g of sample into 225 ml of BHI broth (dilution factor of 1:10). If necessary, sample size may deviate from 25 g depending on availability of the sample, as long as the diluent is adjusted proportionally. Blend or stomach briefly. Incubate the homogenate for 10 min at room temperature with periodic shaking then allow the sample to settle by gravity for 10 min. Decant medium carefully into a sterile container and incubate for 3 h at 35°C to resuscitate injured cells. Transfer contents to 225 mL double strength TP broth in a sterile container and incubate 20 h at 44.0 ± 0.2°C. After incubation, streak to L-EMB and MacConkey agars. Incubate these agars for 20 h at 35°C.

F. Selection.

Typical lactose-fermenting colonies on L-EMB agar appear dark centered and flat, with or without metallic sheen. Typical colonies on MacConkey agar appear brick red. Lactose non-fermenting biotypes on both agars produce colorless or slightly pink colonies.

NOTE: EIEC do not ferment lactose and there may also be atypical non-lactose fermenting strains in the other pathogenic E. coli groups; therefore, as many as 20 colonies (10 typical and 10 atypical) should be picked for further characterization.

G. Conventional Biochemical Screening and identification (7, 22)

Use the procedures described in Chapter 4 for biochemical and morphological identification of E. coli. However, because many enteric bacteria can also grow in the TP enrichment broth, plus anaerogenic, non-motile and slow or lactose non-fermenting strains of E. coli must also be considered, additional tests may need to be performed. Some of these new or modified reactions are discussed here.

1. Primary screening. Transfer suspicious colonies to TSI agar, BAB slant, tryptone broth, arabinose broth, and urea broth. Incubate 20 h at 35°C. Reject H₂S-positive, urease-positive, arabinose non-fermenting, and indole-negative strains. To test for the ONPG reaction, suspend growth from TSI in 0.85% saline to give detectable turbidity. Add an ONPG-impregnated disk and incubate 6 h at 35°C. Yellow color indicates positive reaction. Reject ONPG-negative, aerogenic cultures. Some Alkalescens-Dispar strains (i.e., anaerogenic Escherichia) are ONPG-negative.
2. Secondary screening (48 h incubation at 35°C unless otherwise specified). To identify cultures, test additional reactions shown in Table 1, Chapter 4, to subdivide Escherichia spp. Since it is not known whether these additional species are of pathogenic significance to humans, strains giving typical reactions for E. coli should be further investigated. To differentiate E. coli from Shigella, examine anaerogenic, non-motile, slow lactose fermenters for lysine decarboxylase, mucate, and acetate reactions. Shigella sonnei, which may grow in the same enrichment conditions, is anaerogenic and non-motile. It also produces a negative indole reaction and shows slow or non-fermentation of lactose. The biochemical-physiological characteristics of E. coli are summarized in Table 2, chapter 4.
3. Alternatively, use API20E or the automated VITEK biochemical assay to identify the organism as E. coli.

H. Tests for Enterotoxigenic E. coli (ETEC)

When E. coli levels in foods exceed 10⁴ cells/g, perform enumeration for ETEC by colony hybridization analysis using DNA probes for LT and ST (Chapter 24). If biological activity assays are necessary, LT can be detected by the Y-l tissue culture test (20) and ST can be detected by the infant mouse test (20) (For detailed procedures of these assays, see Chapter 4, BAM, Edition 8, Revision A /1998). There are also commercially available RPLA and ELISA tests to detect LT and ST toxins (Appendix 1) as well as PCR assays (30).

I. Tests for Enteroinvasive E. coli (EIEC)

If an isolate is suspected to be EIEC, the invasive potential of the isolates may be tested by the Sereny test or the Guinea pig keratoconjunctivitis assay (20) (For detailed procedures on Sereny test, see Chapter 4, BAM, Edition 8, Revision A /1998). Invasive potential of the isolates can also be determined by the HeLa tissue culture cell assay as described (18), or with the in vitro staining technique using acridine orange to stain intracellular bacteria in HeLa monolayers (19). Alternatively, since the invA gene sequence of EIEC closely resembles that of Shigella, DNA probe (Chapter 24) and PCR (Chapter 28) assays for Shigella will also work for EIEC.

Caution: Since both EIEC and Shigella will give positive probe and PCR reactions, it is critical that the organisms are identified first as E. coli.

J. Tests for Enteropathogenic E. coli (EPEC)

EPEC strains are identified based on 3 key traits: attachment and effacing lesion (A/E), localized adherence on cells and the lack of Shiga toxin (Stx) production. This last trait is also used to distinguished strains of EPEC from EHEC. Phenotypically, A/E and localized adherence are tested using Hep-2 or HeLa tissue cells. Absence of Stx can be determined using tests outlined for EHEC (see below). There are also PCR and probes for the EAF plasmid that encodes for localized adherence and the eae gene that encodes for the intimin that causes the A/E phenotype. Caution: There are several variants of eae gene and some EPEC strains carry eae variants identical to EHEC serotypes; hence, these tests will detect strains from both pathogenic groups. For specific virulence assays for EPEC, see Nataro and Kaper, 1998 (20) for detailed procedures.

K. Enrichment and isolation of E. coli Serotype O157:H7 from Foods

An enrichment/isolation procedure using EHEC Enrichment broth (EEB) followed by plating on TCSMAC agar has been developed for detecting O157:H7 in foods. Both the enrichment and the selective medium contain several anti-microbial reagents that effectively suppress normal flora growth and interference. Comparative analysis showed that the EEB-TCSMAC method was superior to the mTSB-HC agar (chapter 24) method in the recovery of O157:H7 bacteria in contaminated and seeded foods examined (29).

L. Isolation of O157:H7 with Tellurite-Cefixime-Sorbitol MacConkey (TC SMAC) agar

Sorbitol MacConkey (SMAC) agar is effectively used in clinical testing for isolating O157:H7 from bloody stool samples. However, SMAC is not very selective and therefore, less effective for food testing, because normal flora bacteria can easily outgrow O157:H7 strains that may be present. Similarly, the HC agar developed for food analysis, though slightly more selective, is also susceptible to normal flora overgrowth. By addition of potassium tellurite and cefixime to SMAC, a very selective TC SMAC agar was developed for isolation of E. coli O157:H7. Caution: Although most non-O157:H7 E. coli ferment sorbitol, about 6% of the isolates do not (7). These atypical strains along with other sorbitol non-fermenting bacteria such as Morganella and Hafnia appear identical to O157:H7 colonies on TC SMAC agar. Hence, confirmatory tests must be performed to distinguish these from the O157:H7 isolates. For additional information on the TCSMAC procedure, contact , 425-483-4873, FDA, Bothell, WA.

M. E. coli O157:H7 Media Preparation

EHEC Enrichment Broth (EEB) - same as mTSB (M156) but without novobiocin. Instead, add the following filter-sterilized antibiotics after autoclaving and tempering.

Cefixime*	0.0125 mg/L
Cefsulodin	10.00 mg/L
Vancomycin	8.00 mg/L
*Available from Dynal Inc., Lake Success, NY (800) 638-9416.

NOTE: The level of cefixime shown above has been reduced to ¼ strength from that described in BAM, Edition 8, Revision A /1998, because the original concentration of 0.05 mg/L was found to be inhibitory to the growth of O157:H7 positive control strains in the absence of competing microflora.

Tellurite Cefixime Sorbitol-MacConkey agar (TC SMAC) - Prepare Sorbitol MacConkey Agar (M139) and add the following filter-sterilized additives after autoclaving and tempering:

Potassium tellurite*	2.5mg/L
Cefixime*	0.05mg/L
*Available from Dynal Inc., Lake Success, NY (800) 638-9416.

N. E. coli O157:H7 Enrichment Procedure

1. Weigh 25 g of food into 225 mL of EEB, blend or stomach briefly as necessary.
2. Incubate at 37 ± 0.5°C with shaking for 24 h (see O.1. below).

NOTE: An abbreviated 6 h incubation may suffice in some instances to enable rapid isolation of O157:H7 from foods, but the 24 h enrichment was found to be more sensitive and therefore is recommended.

OPTIONAL. Additional selective enrichment by use of immunomagnetic separation (IMS) has been found to be useful in the analysis of some foods, particularly those with high levels of competing microbial flora (29). Anti-O157 immunomagnetic beads (Dynabeads) are available commercially (Dynal Inc., see address above). Perform IMS on 1 mL of enrichment broth, following manufacturer's instructions. Cell-bound Dynabeads are plated on TC SMAC agar as described below.

O. E. coli O157:H7 Isolation Procedure

1. After incubation, spread plate 0.1 mL of appropriately diluted enrichment broth to TCSMAC agar plates to obtain 100-300 isolated colonies. Streak one loop-full of the enrichment to one additional TCSMAC plate. Incubate plates at 35-37°C for 18 - 24 h.
2. Sorbitol-fermenting bacteria appear as pink to red colonies on TC SMAC. Typical O157:H7 colonies are colorless or neutral/gray with a smoky center and 1-2 mm in diameter. Pick up to 5 typical O157:H7 colonies from TC SMAC onto TSAYE (M153) plates and incubate at 35°C for 18 - 24 h.

P. E. coli O157:H7 Presumptive Screening

1. Spot indole - Spot some growth from TSAYE to a filter wetted with Kovac's reagent. E. coli O157:H7 are indole-positive. Discard indole-negative strains.
2. L-EMB and MUG - Test indole-positive isolates by streak plating some growth from TSAYE onto EMB agar and another TSAYE plate. On the latter, place a ColiComplete disc (Biocontrol, Bellview, WA) in the area of heaviest streak. Similarly prepare another TSAYE plate using a known MUG-positive E. coli strain as positive control. Incubate the plates overnight at 35-37°C. On EMB, E. coli O157:H7 should appears as typical E. coli colonies showing dark centers with or without metallic green sheen. Examine the TSAYE plates under long wave UV [365 nm] light. The positive control should be MUG-positive as evidenced by blue fluorescence around the disc that is indicative of ß-glucuronidase activity on MUG. Strains of E. coli O157:H7 are MUG-negative. Eliminate isolates that give positive MUG reactions.

Q. E. coli O157:H7 Confirmation

Test the above presumptive-positive isolates with the following phenotypic and serological tests to confirm the isolates as E. coli O157:H7.

1. Using growth from TSAYE plate, perform API20E or VITEK to identify the isolates as E. coli.
2. Test for the presence of the O157 antigen using commercial O157 antiserum. Both Prolex E. coli O157 Latex Test Reagent kit (Pro-Lab Diagnostics, Round Rock, TX, 800-522-7744) and RIM E. coli O157:H7 Latex Test (Remel, Lenexa, KS, 800-255-6730) give satisfactory results. The RIM E. coli O157:H7 Latex Test kit also includes the H7 latex reagent. The O157 positive isolates should also be tested with the H7 reagent. If the isolate is H7 positive, it is good evidence that the isolate is of the O157:H7 serotype. Do not use H7 latex reagent without testing first with the O157 reagent. Caution: Use single colonies for latex agglutination tests, as the use of excessive colony growth (i.e.: sweeps) may result in false positive reactions due to autoagglutination (24).

   NOTE: If the isolate is O157 positive but is H7 negative, it may be a toxigenic, non-motile variant of O157:H7 serotype (8); therefore, test the isolate for the presence of stx1 and stx2 genes by DNA probe (see, Chapter 24) or preferably by PCR (12) to establish shiga toxigenic potential of the isolate. Use growth from the TSAYE plate in step 1 to prepare DNA templates for PCR analysis.

Additional Information - There is also O157:H7-specific DNA probe and multiplex PCR (5P PCR) (9) assays that can specifically detect O157:H7 serotype and its toxigenic non-motile variants. The probe is directed at the +93 base mutation in the uidA gene that encodes for ß-glucuronidase. The mutation is highly conserved in O157:H7 and its toxigenic, non-motile variants; therefore specific only for O157:H7 strains of public health concern. The 5P PCR couples primers to the uidA with primers specific for stx1, stx2, gamma variant of eae that is found in O157:H7 and the ehxA enterohemolysin genes to form an assay that simultaneously detect these virulence traits in Stx-producing E. coli and determines whether it is of O157:H7 serotype. The stx gene PCR primers used in this assay are also different from those described in LIB 3811; hence, this assay can be used to verify toxigenic potential of isolates, should the results of the LIB 3811 PCR assay be inconclusive. For information on the probe and 5P PCR, contact , 301-436-1650, CFSAN, FDA, College Park, MD. For protocol on using the probe, see Chapter 24. For information purposes only, a partial list of commercially available rapid methods for detecting E. coli O157:H7 is shown in Appendix 1.

References

1. Centers for Disease Control and Prevention. 1996. Outbreak of Escherichia coli O157:H7 infections associated with drinking unpasteurized commercial apple juice. Morbid. Mortal. Weekly Rep. 45:44.

2. Centers for Disease Control and Prevention. 1993. Update: Multistate outbreak of Escherichia coli O157:H7 infections from hamburgers-Western United States, 1992-1993. Morbid. Mortal. Weekly Rep. 42:258-263.

3. Como-Sebetti, K., K. S. Reagan, S. Alaire, K. Parrott, C. M. Simonds, S. Hrabowy et al. 1997. Outbreaks of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts- Michigan and Virginia, June-July 1997. Morbid. Mortal. Weekly Rep. 46:741-744.

4. Doyle, M.P. and J.L. Schoeni. 1987. Isolation of Escherichia coli O157:H7 from retail fresh meats and poultry. Appl. Environ. Microbiol. 53:2394-2396.

5. Doyle, M. P. and V. V. Padhye. 1989. Escherichia coli, p. 235-277. In M. P. Doyle (ed.), Foodborne Bacterial Pathogens, Marcel Dekker, Inc. New York, NY.

6. DuPont H.L., S.B. Formal, R.B. Hornick, M.J. Snyder, J.P. Libonati, D.G. Sheahan, E.H. LaBrec and J.P. Kalas. 1971. Pathogenesis of Escherichia coli diarrhea. N. Engl. J. Med. 285:1-9.

7. Ewing, W.H. 1986. Edwards and Ewing's Identification of Enterobacteriaceae, 4th ed. Elsevier, New York.

8. Feng, P., P.I. Fields, B. Swaminathan, and T.S. Whittam. 1996. Characterization of non-motile variants of Escherichia coli O157 and other serotypes by using an antiflagellin monoclonal antibody. J. Clin. Microbiol. 34:2856-2859.

9. Feng P. and S.R. Monday. 2000. Multiplex PCR for detection of trait and virulence factors in enterohemorrhagic Escherichia coli serotypes. Mol. Cell. Probes. 14:333-337.

10. Griffin, P.M. and R.V. Tauxe. 1991. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohemorrhagic E. coli and the associated hemolytic uremic syndrome. Epidemiol. Rev. 13:60-98.

11. Hicks, S., G. Frankel, J. B. Kaper, G. Dougan, and A. D. Phillips. 1998. Role of intimin and bundle-forming pili in enteropathogenic Escherichia coli adhesion to pediatric intestinal tissue in vitro. Infect. Immun. 66:1570-1578.

12. Hill, W.E, K.C. Jinneman, P.A. Trost, J.L Bryant, J. Bond and M.M. Wekell. 1993. Multiplex polymerase chain reaction detection of Shiga-like toxin genes in Escherichia coli. FDA LIB 3811.

13. Itoh, Y., Y. Sugita-Konishi, F. Kasuga, M. Iwaki, Y. Hara-Kuda, N. Saito, Y. Noguchi, H. Konuma, and S. Kumagai. 1998. Enterohemorrhagic Escherichia coli O157:H7 present in radish sprouts. Appl. Environ. Microbiol. 64:1532-1535.

14. Karmali, M. A. 1989. Infection by verotoxin-producing Escherichia coli. Clin Microbiol. Rev. 2:15-38.

15. Levine, M. M. 1987. Escherichia coli that cause diarrhea: enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J. Infect. Dis. 155:377-389.13.

16. McGowan, K. L., E. Wickersham, and N. A. Strockbine. 1989. Escherichia coli O157:H7 from water. (Letter). Lancet. I:967-968.

17. Mehlman, I. J. 1984. Coliforms, fecal coliforms, Escherichia coli and enteropathogenic E. coli.p. 265-285. In M. L. Speck (ed.), Compendium of Methods for the Microbiological Examination of Foods, 2nd ed. American Public Health Assoc. Washington, D.C.

18. Mehlman I.J., A. Romero, J.C. Atkinson, C. Aulisio, A.C. Sanders, W. Campbell, J. Cholenski, J. Ferreira, E. Forney, K. O'Brian, M. Palmieri, and S. Weagant. 1982. Detection of invasiveness of mammalian cells by Escherichia coli: collaborative study. J Assoc.Off. Anal. Chem. 65:602-7.

19. Miliotis, M. D. and P. Feng. 1993. In Vitro staining technique for determining invasiveness in foodbrone pathogens. FDA LIB 3754.

20. Nataro, J. P. and J. B. Kaper. 1998. Diarrheagenic Escherichia coli. Clin. Microbiol. Rev. 11:132-201.

21. Neill, M. A., P. I. Tarr, D. N. Taylor, and A. F. Trofa. 1994. Escherichia coli, p. 169-213. In Y. H. Hui, J. R. Gorham, K. D. Murell, and D. O. Cliver (ed.), Foodborne Disease Handbook, Marcel Dekker, Inc. New York, NY.

22. Orskov, F. 1984. Escherichia, p. 420-423. In N. R. Krieg and J. G. Holt (ed.) Bergey's Manual of systematic Bacteriology, vol. 1 Williams and Wilkins Co., Baltimore, MD.

23. Riley, L.W., R. S. Remis, S.D. Helgerson, H.B. McGee, J.G. Wells, B. R. Davis, R. J. Herbert, G.S. Olcott, L.M. Johnson, N. T. Hargett, P.A. Blake, and M. L. Cohen. 1983. Hemorrhagic colitis associated with a rare Escherichia coli serotype O157:H7. N. Engl. J. Med. 308:681-685.

24. Sowers, E.G., J.G. Wells, and N.A. Strockbine. 1996. Evaluation of commercial latex reagents for identification of O157 and H7 antigens of Escherichia coli. J. Clin. Microbiol. 34:1286-1289.

25. Swerdlow, D. L., B. A. Woodruff, R. C. Brady, P. M. Griffin, S. Tippen, H. D. Donnell, Jr., E. Geldreich, B. J. Payne, A. Neyer, J. G. Wells, K. D. Greene, M. Bright, N. Bean, and P. A. Blake. 1992. A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann. Intern. Med. 117:812-819.

26. Thompson M.R., H. Brandwein, M. LaBine-Racke, and R.A. Giannella. 1984. Simple and reliable enzyme-linked immunosorbent assay with monoclonal antibodies for detection of Escherichia coli heat-stable enterotoxins. J. Clin. Microbiol. 20:59-64.

27. Tobe, T., T. Hayashi, C-G. Han, G. K. Schoolnik, E. Ohtsubo, and C. Sasakawa. 1999. Complete DNA sequence and structural analysis of the enteropathogenic Escherichia coli adherence factor. Infect. Immun. 67:5455-5462.

28. Tsen, H-Y., W-R. Chi, and C-K. Lin. 1996. Use of novel polymerase chain reaction primers for the specific detection of heat-labile toxin I, heat-stable toxin I and II enterotoxigenic Escherichia coli in milk. J. Clin. Microbiol. 59:795-802.

29. Weagant, S. D., J. L. Bryant, and K. C. Jinneman. 1995. An improved rapid technique for isolation of Escherichia coli O157:H7 for foods. J. Food Prot. 58:7-12.

30. Weagant, S. D., K. C. Jinneman, and J. H. Wetherington. 2000. Use of multiplex polymerase chain reaction for identification of enterotoxigenic Escherichia coli. FDA LIB 4227.

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Hypertext Source: Bacteriological Analytical Manual, 8th Edition, Revision A, 1998. Chapter 4.
*Authors: Peter Feng, Stephen D. Weagant
Revised: 2002-September.

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Chapter 4: Enumeration of Escherichia coli and the Coliform Bacteria

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