Foodborne Gastroenteritis Caused by Escherichia coli 647 Table 27–3 Some Cases of EC O157:H7 Gastroenteritis from Drinking and Recreational Waters in the United States Year State Vehicle No of Cases 1995 1995 1995 1995 1996 1996 Minnesota Illinois Minnesota Wisconsin Georgia Minnesota Spring water Lake Lake Lake Pool Lake 33 12 8 18 Source: MMWR Morb Mort Wkly Rep 47, SS-5, 1998 The numbers of cases of EC O157:H7 recorded by the U.S Centers for Disease Control and Prevention (CDC) for the years 1994–1997 are as follows: 1420, 2139, 2741, and 2555, respectively, for 1994, 1995, 1996, and 1997.12 In 1997, 1167 or 45.7% occurred in the months of July, August, and September The gastroenteritis cases from drinking and recreational waters in the United States for 1995–1996 are summarized in Table 27–3 The outbreak that occurred in Scotland in 1996 is an example of what can happen when improper food handling and poor cleanup practices occur There were around 500 cases (279 laboratory confirmed) that were traced to at least six foods, all from a single butcher shop.3 All of the confirmed cases were caused by Stx2 toxin-producing strains In Japan, there were 29 outbreaks of EC O157:H7 human cases for the years 1991–1995 In 1996, there were 11,826 food-associated cases and 12 deaths caused by EC O157:H7.54 The 1996 outbreak traced to white radish sprouts accounted for >9000 cases and three deaths The existence of this organism in Northern Ireland is uncommon in cattle and human foods, and the case rates for Northern Ireland, England/Wales, and Scotland for 1997 were 1.8, 2.1, and 8.2/100,000.96 In the United States, the rate for 1997 was 2.3, and 2.7 and 2.8 for 1996 and 1998, respectively Enteroinvasive E coli (EIEC) These strains generally not produce enterotoxins as ETECs, but they enter and multiply in colonic epithelial cells and then spread to adjacent cells in a manner similar to the shigellae.14 Prior to the 1970s, some of these organisms were referred to as “paracolons.” Like the shigellae, EIECs possess 140-MDa enteroinvasive plasmids (pINV) that are quite similar to those found in Shigella flexneri and are essential for their invasiveness (see Chapter 26) Plasmidless strains are not invasive The classic EIEC strains are also Sereny positive Members of this group have a predilection for the colon, and bloody or nonbloody but voluminous diarrhea is a consequence Dysentery is rare, and the very young and very old are the most susceptible members of the population The incubation period is between and 48 hours with an average of 18 hours.55 Some of the serotypes that include EIEC strains are listed in Table 27–1 At least one, O167, contains both EIEC and ETEC strains.32 Some of the early foodborne outbreaks are summarized in Table 27–4 The earliest recorded occurred in England in 1947 among school children, and salmon was the apparent food vehicle.38 Although foods are a proven source for this syndrome, person-to-person transmission is known EIEC strains have been isolated from persons with travelers’ diarrhea, and they have been shown to be common in diarrheal stools from children.81 648 Modern Food Microbiology Table 27–4 Table 27–4 Synopses of the Earliest Known Foodborne Gastroenteritis Cases Caused by Pathogenic E coli (Taken from the Literature) Year Location Food/Source 1947 1961 1963 1966 1967 1971 1980 1981 1982 England Rumania Japan Japan Japan United States∗ Wisconsin Texas Oregon Salmon (?) Substitute coffee drink Ohagi Vegetables Sushi Imported cheeses Food handler Not identified Ground beef ∗ In 14 states † LT = heat labile No of Victims/ No of Risks 47/300 10/50 17/31 244/435 835/1736 387/? 500/>3000 282/3000 26/? Toxin/Strain Type Serotype EIEC EPEC EIEC EIEC ? EIEC ETEC ETEC(LT)† EHEC O124 O86:B7; H34 O124 O124 O11(?) O124:B17 O6:H16 O25:H+ O157:H7 enterotoxin Enteropathogenic E coli (EPEC) These strains generally not produce enterotoxins, although they can cause diarrhea They exhibit localized adherence to tissue culture cells and autoagglutinate in tissue culture medium They possess adherence factor plasmids that enable adherence to the intestinal mucosa After colonizing the intestinal mucosa, attachment–effacement (att–eff, A/E) lesions are produced The process starts upon initial contact and is believed to be aided by a plasmid-encoded bundle-forming pilus (see Chapter 22) EPEC-secreted proteins (Esps) block phagocytosis and lead to cytoskeletal rearrangement and tyrosine phosphorylation of Tir (see Chapter 22) When Tir binds with the outer membrane protein intimin, the attachment is intimate, resulting in destruction of brush border microvilli and formation of pedestals (see Chapter 22 for more on pathogenic mechanisms) The A/E phenomenon appears to be the most important virulence factor of EPEC strains.84 EPEC strains not produce detectable quantities of Stxs Some EPEC serotypes are listed in Table 27–1 First characterized in 1955, EPEC strains cause diarrhea in children generally under year of age Enterotoxigenic E coli (ETEC) These strains attach to and colonize the small intestine by means of fimbrial colonization factor antigens (CFAs) There are four types of CFA—I, II, III, and IV—and they have been cloned and sequenced.80 CFAs are plasmid encoded, generally on the same plasmid that encodes the heat-stable enterotoxin (see below), and they are not produced under 20◦ C Once attached, they produce either one or two enterotoxins Some of the ETEC serotypes are listed in Table 27–1 In a study of ETEC strains from 109 patients, the strains that produced both ST and LT were more restricted in O:K:H serotypes than those that produced only one of these toxins.57 These toxins are further characterized below Unlike EPEC strains, which cause diarrhea primarily in the very young, ETEC strains cause diarrhea in both children and adults These strains are among the leading causes of travelers’ diarrhea The ETEC disease syndromes are rarely accompanied by fever, and the diarrhea is sudden It has been estimated that 108 –1010 cfu are necessary for diarrhea by an ETEC strain in adult humans.60 Foodborne Gastroenteritis Caused by Escherichia coli 649 The Enterotoxins One of the E coli enterotoxins is heat-labile (LT) and the other is heat-stable (STa or ST-1, and STb or ST-II) The LT toxin is destroyed at 60◦ C in about 30 minutes, whereas ST toxins can withstand 100◦ C for 15 minutes The LT toxin is a protein with a molecular weight of about 91 kDa,18 and it possesses enzymatic activity similar to that of the cholera toxin (CT) Whereas CT is exported from the cytoplasm to the outside of producing cells, LT is deposited into the periplasm of producing cells Further, antisera to CT neutralize LT and immunization with CT induces protection against both CT and LT challenges These enterotoxins are produced early in the growth phase of producing strains, with the maximum amount of ST produced after hours of growth in one study in a Casamino acids yeast extract medium containing 0.2% glucose.47 In a synthetic medium, ST appeared as early as hours, but maximal production required 24 hours with aeration.8 Although LT and ST appear to be produced under all conditions that allow cell growth, the release of LT from cells in enriched media was favored at a pH of 7.5–8.5.59 LT toxin is composed of two protomers: A, with a molecular weight of about 25.5 kDa, which when nicked with trypsin becomes an enzymatically active A1 polypeptide chain of 21 kDa linked by a disulfide bond to an A2 -like chain; and B, which has a molecular weight of about 59 kDa and consists of five noncovalently linked individual polypeptide chains.23 LTB is the binding subunit, whereas LTA stimulates the adenylate cyclase system LTA and LTB have immunological properties similar to subunits A and B of the Vibrio cholerae toxin.46 LTh and LTp designate human and porcine strains, respectively STa is methanol soluble and elicits a secretory response in infant mice It is an 18–19 amino acid acidic peptide that contains three disulfide bonds and has a molecular weight of 1972 Da It stimulates particulate intestinal guanylate cyclase STa has been chemically synthesized.43 STb is methanol insoluble and is primarily of swine origin It is the most prevalent toxin associated with diarrheagenic isolates of porcine origin, and it affects the small intestine and the ligated ileum of weaned piglets, and also the mouse intestinal loop when a protease inhibitor is added.95 The STb gene (estB) has been sequenced and cloned.48 The trypsin-sensitive STb toxin is synthesized as a 71-amino acid polypeptide that is later cleaved to yield the active 48-amino acid molecule with four cysteine residues that pass through the inner membrane to the periplasm Although its mode of action is yet unclear, it has been shown to stimulate the synthesis of prostaglandin E2 35 Its receptor cell in mouse intestinal cells is a protein with a molecular weight of 25 kDa.34 Mode of Action of Enterotoxins ETEC gastroenteritis is caused by the ingestion of 106 –1010 viable cells per gram that must colonize the small intestines and produce enterotoxin(s) The colonizing factors are generally fimbriae or pili The syndrome is characterized primarily by non-bloody diarrhea without inflammatory exudates in stools The diarrhea is watery and similar to that caused by V cholerae Diarrhea results from enterotoxin activation of intestinal adenylate cyclase, which increases cyclic ,5 -adenosine monophosphate (cAMP) With regard to LT, the B protomer mediates binding of the molecule to intestinal cells LT binds to gangliosides, especially monosialogangliosides (GM1 ).23 CT also binds to GM1 ganglioside, and CT and LT are known to share antigenic determinants among corresponding protomers, although they not cross-react Upon binding, the A polypeptide chain (of the A protomer) catalyzes ADP ribosylation of a G protein that activates adenylate cyclase and induces increases in intracellular cAMP Regarding ST, STa binds irreversibly to a specific high-affinity nonganglioside receptor and initiates a transmembrane signal to activate particulate guanylate cyclase, and triggers the production of 650 Modern Food Microbiology intracellular cyclic guanosine monophosphate (cGMP) The increased levels of mucosal cGMP lead to loss of fluids and electrolytes ST differs from CT in that only the particulate form of intestinal guanylate cyclase is stimulated by ST.28 STa differs from LT in that the former stimulates guanylate cyclase, whereas the latter and CT activate adenylate cyclase STb elevates luminal 5-hydroxytryptamine and prostaglandin E2 , both of which are mediators of intestinal secretions STb does not activate guanylate cyclase, and genes controlling its production have been mapped56 and subcloned from its plasmid and sequenced.70 The mechanisms of Shiga, Stx1, Stx2, Stx2e, and the castor bean protein, ricin, are the same They are N -glycosidases that cleave a specific adenine residue from the 28S subunit of eukaryotic rRNA, leading to the inhibition of protein synthesis.62,92 Foodborne and Waterborne Outbreaks Some of the earliest known foodborne outbreaks caused by ETEC and other strains are summarized in Table 27–4 Regarding the virulence groups, it may be noted that EIEC was first confirmed as the cause of a foodborne outbreak in 1947, and the first in the United States occurred in 1971 An EPEC strain was confirmed as the cause of a foodborne outbreak in 1961, an ETEC in 1980, and EHEC in 1982 The first well-documented outbreak of human disease by an ETEC was a waterborne outbreak that occurred in a national park in the state of Oregon in 1975 There were about 2200 victims who drank improperly chlorinated water The causative ETEC strain was O6:H16 PREVENTION In general, the prevention/avoidance of foodborne illness by E coli can be achieved by observing the factors noted in the last section of Chapter 23 However, because of the consequences to young children, special precautions need to be observed The heat sensitivity of these organisms is such that cases should not occur when foods are properly cooked In the case of ground beef, the recommendation is that it should be cooked to 160◦ F (71.1◦ C), or that the core temperature be brought to a minimum of 155◦ F (58.3◦ C) for at least 15 seconds and that the juices are clear (1993 recommendation of the U.S Food and Drug Administration Food Code) Because of unevenness of hamburger patties, cooking at 155–160◦ F (58.3–71.1◦ C) provides a measure of safety Once cooked, hamburgers as well as other meats should not be held between 40◦ and 140◦ F for more than 3–4 hours Although the largest recorded foodborne outbreak was associated with ground beef, all raw meat, poultry, seafood; and some fruits and vegetables should be considered possible vehicles for hemorrhagic colitis TRAVELERS’ DIARRHEA E coli is well established as one of the leading causes of acute watery diarrhea that often occurs among new arrivals in certain foreign countries Among Peace Corps volunteers in rural Thailand, 57% of 35 developed the syndrome during their first weeks in the country, and 50% showed evidence of infection by ETEC strains In 1976, a shipboard outbreak of gastroenteritis was shown to be caused by serotype O25:K98:NM that produced only LT Similar strains have been recovered from other victims of travelers’ diarrhea in various countries along with EPEC and ST-producing strains Among other organisms associated with this syndrome are rotaviruses, noroviruses, Entamoeba histolytica, Yersinia enterocolitica, Giardia lamblia, Campylobacter jejuni/coli, Shigella spp and possibly Aeromonas hydrophila, Klebsiella pneumoniae, and Enterobacter cloacae Foodborne Gastroenteritis Caused by Escherichia coli 651 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