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Escherichia coli Associated with Hemorrhagic Colitis1
APPLIED TECHNOLOGY
Escherichia coli
Associated with Hemorrhagic Col itis 1 S.Stavric and ).1. Speirs Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health and Welfare Canada, Ottawa, Ontario K1A Ol2
Abstract
Outbreaks
The first outbreak of hemorrhagic colitis due to Escherichia coli 0157:H7 in Canada occurred in 1982. There were 31 cases with one death in a home for the aged; undercooked hamburger was the suspected vehicle. The serotype 0157 :H7 had rarely been encountered prior to 1982, but since then, sporadic and outbreak cases of hemorrhagic colitis and its complication, hemolytic uremic syndrome, have steadily increased. In Canada, the most severe of all outbreaks, with a case fatality rate of 31 %, occurred in a nursing home in 1985. In 1987, 315 cases in eight outbreaks were reported. At least six outbreaks of hemorrhagic colitis in North America have been linked to ingestion of undercooked beef and one to raw milk. E. coli 0157:H7 has been isolated from market samples of beef, pork, poultry and lamb, feces of healthy heifers and calves, and raw milk. Although E. coli 0157:H7 has been responsible for outbreaks of hemorrhagic colitis, other serotypes have also been implicated in sporadic cases, especially in children with hemolytic uremic syndrome. All isolates produce high levels of cytotoxin(s) affecting Vero cells (verotoxin). Since food of animal origin is the primary source of infection in humans, careful handling and adequate cooking of all raw meats is required to minimize or eliminate the risk of infection with E. coli 0157:H7 and other verotoxin-producing serotypes.
In 1982, an outbreak of hemorrhagic colitis occurred in Ottawa in a home for the aged (Stewart et al., 1983). Thirty-one of the 353 residents became ill with gastrointestinal symptoms and E. coli 0157:H7 was isolated from 18 of the 31 cases. In the first wave, undercooked hamburger was suspected as the vehicle; in the second wave of three cases, the spread was person-to-person. Four people were hospitalized, one died. Two outbreaks of HC, due to E. coli 0157:H7, had been reported from the United States earlier that year (Riley et al., 1983; Wells et al., 1983). Hamburgers in fast-food restaurants were suspected, and the 0157:H7 serotype was isolated from the implicated lot of meat eaten by persons in the Michigan restaurant. In the United States in 1982, besides the two outbreaks, there were 25 sporadic cases, 24 associated with hamburger consumption (Uyeyama et al., 1982). This gastrointestinal illness became known as "hamburger disease". Symptoms of HC include severe abdominal pain, watery diarrhea frequently followed by bloody diarrhea, nausea and vomiting, but little or no fever. Incubation varies from one to 14 days, with a duration of illness of about eight days. Persons in all age groups are at risk. Although generally self limiting, the young and elderly are more prone to serious complications such as HUS, thrombotic thrombocytopenic purpura (HP) and death. HUS is one of the leading causes of kidney failure in young children worldwide, with about 5% mortality reported in North America (Karmali, 1985a). Symptoms include a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. TTP is an extension of HUS in some adults and includes neurological symptoms and fever. E. coli 0157 was first isolated in 1970 from piglets with enteritis in Ireland, but this strain contained the flagellar antigen H19 instead of H7 implicated in HC (Stewart et a/., 1983). Prior to 1982, serotype 0157:H7 was rarely encountered. In Canada, from 1978 to 1982, this serotype was isolated from only six of more than 2000 diarrheal isolates (Johnson et al. , 1983). In the United States, one isolate was found among 3000 E. coli diarrheal strains collected between 1973 and 1982 (Wells et al., 1983). In the United Kingdom, prior to 1982, only one strain was
Introduction There is considerable information in the literature concerning the emergence of the new enteric pathogen Escherichia coli 0157:H7 and its involvement in hemorrhagic colitis (HO and hemolytic uremic syndrome (HUS). Several outbreaks and cases of HC and HUS have been linked epidemiologically to consumption of undercooked beef or raw milk. In addition to E. coli 0157:H 7, other serotypes have also been implicated in illness. These organisms are of great importance to food microbiologists as well as clinicians. It is the purpose of this paper to review and summarize the most pertinent data on outbreaks, pathogenicity, reservoirs and detection methods for E. coli causing HC and HUS.
, Based on a paper presented at the Health Protection Branch Workshop, "Microbial Food Poisoning and its Significance in Canada ", Ottawa, October 18-19, 1988.
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isolated from about 15,000 strains examined (Day et al., 1983). Since 1982 isolates of E. coli 0157:H7 have generally doubled annually in Canada, with 1342 reported in 1987 (Hockin et al., 1988). There is a marked seasonal variation, at least with sporadic cases, most occurring from June to September (Hockin and Lior, 1987; Pai et al., 1988). Isolates from sporadic cases have outnumbered those from outbreaks. In 1987 there were 315 outbreak-associated cases, 10 patients developed HUS and there were six deaths. Up to 1988 there have been 19 outbreaks recorded in Canada, with 518 persons affected, 29 cases of HUS and 26 deaths (Hockin and l.ior, 1987; Carter et al., 1987; Todd et al., 1988). The most severe of all outbreaks in Canada was in a nursing home in London, Ont. in 1985 (Carter et al., 1987). In this outbreak there were 73 cases with 33% of the residents and 13% of staff members affected; 12 residents developed HUS and 17 died. A sandwich meal was incriminated as the primary source of infection. Nursing homes have been the setting for eight outbreaks (six in Ontario, two in Alberta), the community for six, families for three and one each for a school trip and a girl's camp. Besides serotype 0157:H7, at least 22 other E. coli serotypes have been isolated worldwide from sporadic cases of HC or HUS (Pai et al., 1988; Karmali, 1987; Bopp et al., 1987). One of these, E. coli 0145:H- was responsible for an outbreak of acute enteritis in Japan in 1984 (Itoh et al., 1985). In general, with serotypes other than 0157:H7, the diarrhea is more prolonged and less frequently bloody. Secondary cases of E. coli 0157:H7 have been found to be less severe - diarrhea is milder and less frequently bloody (Pai et al., 1988). The first population-based study on the incidence of infection with E. coli 0157:H7 in sporadic cases over a one-year-period was conducted in the United States (MacDonald et al., 1988). Of 6485 diarrheal stool samples analyzed for this serotype, E.coli 0157:H7 was identified in 25 specimens (0.4%). Twenty-four (96%) were from patients with bloody diarrhea. Twenty of the patients had consumed undercooked beef and three drank raw milk before the onset of illness.
Pathogenicity E. coli strains associated with diarrhea are divided on the basis of their mode of action into several well established groups: enterotoxigenic (ETEC), enteroinvasive (EIEC) and enteropathogenic (EPEC). In the EPEC group, which is the most controversial, two new groups: enteroadherent (EAEC) and enterohemorrhagic (EHEC) or verotoxin (VT)producing (VTEC) strains have recently been added. ETEC strains cause a cholera-like secretory diarrhea which is induced by heat-labile and/or heat-stable enterotoxins. EIEC strains cause a dysentery-like inflammation of the large bowel; cells are invaded. EPEC strains cause diarrhea by mechanisms different from those of ETEC or EIEC. EAEC strains, as the name impl ies, adhere to entorocytes and cause mucosal damage. Strains in the EHEC or VTEC group cause HC and HUS by Cl mechanism as yet not fully understood. The name EHEC was established for E. coli 0157:H7 which usually induces a bloody diarrhea. The name VTEC is used for the various serotypes that produce VT, including those implicated in HUS. Although it is not known at present whether the verotoxins play a role in the pathogenesis of human diarrhea, their involvement in extragastrointestinal disease, such as HUS, is supported by 206/ AT
numerous data. It is thought that toxins released in the bowel are absorbed into the blood and cause endothelial damage of small blood vessels which may lead to HUS and central nervous system dysfunction (Edelman et al., 1988). All of the HC and HUS isolates to date produce high levels of one or more cytotoxins, known as verotoxins or Shiga-like toxins (SLT), which are toxic for Vero and Hela cell cultures. Four antigenically distinct VTs have been named (Cannon and Cyles, 1987). VT1 and/or VT2 appear to be the predominant VTs produced by North American HC strains. O'Brien and LaVeck (1983) have shown that VT1 is essentially identical to Shiga toxin (of Shigella dysenteriae), which is one of the most potent bacterial toxins known. Canadian scientists have made important contributions in this field. The discovery of cytotoxins in E. coli that affect Vero cells was made in the Food Directorate, HPB, Ottawa, in 1977 by Konowalchuk et al. (1977, 1978). Three different VTs were distinguished at that time by biochemical and immunological methods. Johnson et al. (1983), at the Laboratory Centre for Disease Control, Ottawa, were the first to report that E. coli 0157:H7 isolates produced VT. Karmali et al. (1983), at the Hospital for Sick Children, Toronto, discovered that VT-producing E. coli were the probable cause of idiopathic HUS in children. The modes of action of verotoxin and Shiga toxin on mammalian cells appear similar. These toxins are composed of an A subunit, molecular weight about 32,000, and five copies of a B subunit, molecular weight about 7,700. The B subunit binds to glycolipid receptors on the cell. After internalization, the A subunit is reduced enzymatically to a smaller fragment, A 1, which binds to 60S ribosomes, inhibiting protein synthesis and resulting in cell death (O'Brien and Holmes, 1987). Examination of HC patients has revealed that the intestinal areas affected are the cecum, and the ascending and transverse colon (Riley, 1987). Barium enemas have detected a "thumbprinting" pattern indicative of submucosal edema or hemorrhage. Colonoscopic examinations have shown erythema, hemorrhage and edema. Post mortem examinations of fatalities have shown focal areas of acute and chronic inflammation with congestion and hemorrhage in the lamina propria. Similar mucosal damage occurs in the cecum and colon of piglets fed E. Coli 0157:H7, but mutant strains which lack the cytotoxin-producing property are equally damaging, demonstrating that these toxins are not required for diarrhea in piglets (Tzipori et al., 1987). However, the neurological symptoms with edema disease of piglets are thought to be associated with the toxin (Marques et al., 1987). In rabbits, diarrhea and mucosal abnormalities in the cecum and colon resemble those in humans, and are induced by feeding the whole bacterium as well as VT alone (Pai et al., 1986). Intravenous injection of purified toxin resulted in diarrhea and paralysis followed by kidney failure (Barrett et al., 1988); lesions occurred in the cecum, colon, central nervous system, and kidney. The kidney lesions, however, did not resemble those seen in humans with HUS. Mice injected intraperitoneally with purified toxin developed a bloody diarrhea; lesions occurred in the colon, kidney and lymphoid tissues (Padhye et al., 1987).
Reservoirs E. coli HC is primarily a food-borne illness, but personto-person transmission also occurs, especially in institutional settings. At least six outbreaks and numerous sporadic cases in North America have been linked to ingestion of
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undercooked beef (Stewart et al., 1983; Riley et al., 1983; Wells et al., 1983 ; Hockin and Lior, 1987; MacDonald et al., 1988; Ryan et al., 1986), and one outbreak and several sporadic cases to raw milk (MacDonald et al., 1988; Duncan et al., 1987; Borczyk et al., 1987; Martin et al., 1986). Other foods implicated include sandwiches, sour cream dressing and crisp meat burritos (Carter et al., 1987; Ostroff et al., 1987), but cross-contamination of these foods from beef may have occurred. Dairy cattle are considered to be an important reservoir of E. coli 0157:H7. Isolates have been obtained in North America from raw milk, feces of healthy heifers and calves (Wells et al., 1987; Martin et al., 1986) and market samples of beef, pork, poultry and lamb (Doyle and Schoeni, 1987). One survey in the United States of feces from 899 heifers, calves and adult cows from 16 farms, a slaughter house and a stockyard revealed E. coli 0157:H7 in samples from 18 heifers or calves on nine farms and from one stockyard calf (Wells et al., 1987). All adult cows tested were negative but one of 24 raw milk samples from two farms was positive. Other VTEC serotypes were found from. all five farms examined and from the stockyard. Surveys in western United States of 599 beef cattle failed to find E. coli 0157:H7 (Martin et al., 1986). A survey of 896 retail fresh meat and poultry samples detected E. coli 0157:H7 in 0.7% of beef, 1.2% of pork, 1.5% of poultry and 2.0% of lamb from Madison, Wis., and in 29.4% of beef and 7.1 % of pork from Calgary, Alta. (Doyle and Schoeni, 1987). In southwestern Ontario, Clarke et al. (1988) showed that at slaughter, 10.5% of beef cattle, 19.5% of cull dairy cows and 3.5% of veal calves contained VTEC in their feces; four of the 600 animals had the serotype 0157:H7. In other surveys by these workers (Clarke et al., 1987); 89% of feces from healthy calves from a university research farm and 2% of milk filters from 498 dairy farms revealed VTEC of serotype other than 0157:H7. These data indicate that E. coli 0157:H7 and VTEC are widely distributed in the intestinal tract of animals and that food-products of animal origin are primary sources of human infection.
Detection Clinical diagnosis of E. coli HC has centred on detection of the serotype 0157:H7 and its sorbitol-negative property. Fecal extracts are inoculated onto a sorbitol MacConkey agar and colourless colonies at 24 hours are tested by slide agglutination against 0157 and H7 antisera. About 95% of E. coli are sorbitol-fermenters (Farmer and Davis, 1985), but among nonfermenters, 0157:H7 may account for as few as 2% (Harris et al., 1985). This procedure would not detect the other serotypes that have been implicated in cases of HC and HUS, thus it is preferable to test for VT production. Fecal filtrates may readily be screened for free VT if cell culture facilities are available. Filtrates containing cytotoxin induce a typical cytopathic effect on Vero cells (Konowalchuk et aI., 1977; Karmali, 1987). VT-positive strains are confirmed by neutralization of the cytopathic effect with antisera against the toxins. However, these antisera are not commercially available. Karmali et al. (1985b) developed a sensitive method for screening fecal cultures for verotoxin-producing E. coli employing polymyxin B to release VT from colony sweeps ("VT/PECS" procedure). This method can detect VT-producing organisms among non-VTEC in as low as a 1% concentration in feces. Studies with feces from HUS patients indicated that VT-positive colonies have often been only 5 to 20% of the total E. coli population.
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The procedures used for clinical samples are applicable to food samples, but recently-developed methods (Todd et al., 1988; Doyle and Schoeni, 1987) using hydrophobic grid membrane filters (HGMF) are more suitable. In principle, food extracts are filtered through HGMF and the membranes cultured on selective agar. Blots, or replicas of the colonies, are examined against antisera to E. coli 0157 by an enzyme-labelled immunoassay procedure. At present, DNA probes and monoclonal antibodies are being developed in several laboratories for use in detection of the cytotoxins in clinical specimens and foods. In view of the increasing frequency of cases of hemorrhagic colitis, and the wide variety of contaminated meats, careful handling and adequate cooking of all raw meats is required to minimize or eliminate the risk of infection with E. coli 0157:H7 and other VTEC, and indeed other pathogenic organisms.
References Barrett, T.)., Potter, M.E. and Wachsmuth, I.K. 1988. Pathologic effects of Shiga-like toxin 11 in rabbits. Ann. Meet. Am. soc. Microbiol. Abst. B- 87, p. 44. Bopp, CA, Greene, K.D., Downes, F.P., Sowers, E.G., Wells, ).G. and Wachsmuth, I.K. 1987. Unusual verotoxin-producing Escherichia coli associated with hemorrhagic colitis. ). Clin. Microbiol. 25: 1486. Borczyk, AA., Karmali, M.A., Lior, H. and Duncan, L.M.C 1987. Bovine reservoir for verotoxin-producing Escherichia coli 0157:H7. Lancet i:98. Carter, A.O., Borczyk, AA., Carlson, ).A.K., Harvey, B., Hockin, ).C, Karmali, M A, Krishnan, C, Korn, D.A. and Lior, H. 1987. A severe outbreak of Escherichia coli 0157:H7associated hemorrhagic colitis in a nursing home. N. Engl.). Med. 317:1496. Clarke, R.C, McEwen, SA, Gannon, V.P., Valli, V.E.O., Lior, H. and Gyles, CL. 1987. Isolation of verotoxin-producing Escherichia coli from milk filters and calves in Ontario. International Symposium and Workshop on Verocytotoxin-Producing Escherichia coli Infections. Abst. LFE-15. Clarke, R., McEwen, 5., Harnett, N., Lior, H. and Gyles, C 1988. The prevalence of verotoxin-producing Escherichia coli (VTEC) in bovines at slaughter. Ann. Meet. Am. soc. Microbiol. Abst. P-48, p. 282. Day, N.P., Scotland, s.M., Cheasty, T., Rowe, B. 1983. Escherichia coli 0157:H7 associated with human infections in the United Kingdom. Lancet i:825. Doyle, M.P. and schoeni, j.L. 1987. Isolation of Escherichia coli 0157:H7 from retail fresh meats and poultry. Appl. Environ. Microbiol. 53:2394. Duncan, L., Mai, V., Carter, A., Carlson, j.A.K., Borczyk, A. and Karmali, MA 1987. Outbreak of gastrointestinal disease - Ontario. Can. Dis. Weekly Rep. 13-2:5. Edelman, R., Karmali, M. A. and Fleming, P.A. 1988. Summary of the international symposium and workshop on infections due to verocytotoxin (Shiga-like toxin)-producing Escherichia coli. ). Infect. Dis. 157:1102. Farmer Ill, J.). and Davis, B.R. 1985. H7 antiserum-sorbitol fermentation medium: a single tube screening medium for detecting Escherichia coli 0157:H7 associated with hemorrhagic colitis. ). Clin. Microbiol. 22:620. Gannon, V.P.). and Gyles, CL. 1987. Four types of Escherichia coli verotoxins. International Symposium and Workshop on Verocytotoxin-Producing Escherichia coli Infections. Abst. sTF-15. Harris, A.A., Kaplan, R.L., Goodman, L.)., Doyle, M., Landau, W., segreti, )., Mayer, K. and Levin, S. 1985. Results of a screening method used in a 12-month stool survey for Escherichia coli 0157:H7. ). Infect. Dis. 152:775. Hockin, ). and Lior, H. 1987. Hemorrhagic colitis and hemolytic uremic syndrome caused by Escherichia coli 0157:H7 in Canada. Can. Dis. Weekly Rep. 13-45:203.
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Hockin, j., Lior, H., Stratton, F., Ratnam, S., April, N., Remis, R., Bedard, L., Carlson, J., Borczyk, A., Naus, M., Hodgkinson, j., Poffenroth, L., Roberts, A., West, R., Horsmann, G., Toth, L., Pitula-Grocott, D., Friesen, B., Robson, L. and Crawford, C 1988. Update hemorrhagic colitis due to Escherichia coli (verotoxigenic) in Canada. Can. Dis. Weekly Rep. 14-33:147. Itoh, T., Kai, A., Saito, K., Yanagawa, Y., Inaba, M., Takahashi, M., Takano, I., Matsushita, S., Kudoh, Y., Terayama, T., Ohashi, M., Karaki, K., Yamashita, H., Ikenoue, S., Satoh, H., Seki, T., Katoh, K., Hirooka, Y., Yamada, M., Akiyama, H., Ohzeki, T., Ohshiba, Y., Yamanouchi, J., Tsuchitani, H., Tsunakawa, T. and Tazaki, T. 1985. Epidemiological and laboratory investigation of an outbreak of acute enteritis associated with cytotoxin-producing Escherichia coli 0145:H-. Ann. Rep. Tokyo Metrop. Res. Lab. Public Health 36:16. Johnson, W.M., Lior, H. and Bezanson, G. S. 1983. Cytotoxic Escherichia coli 0157:H7 associated with haemorrhagic colitis in Canada. Lancet i:76. Karmali, MA, Petric, M., Lim, C, Fleming, P.C and Steele, B.T. 1983. Escherichia coli cytotoxin, haemolytic-uraemic syndrome, and haemorrhagic colitis. Lancet i:1299. Karmali, M. A. 1985a. Bacterial diarrhea: an update. Diagn. Med. May: 1. Karmali, M.H., Petric, M., Lim, C, Cheung, R. and Arbus, G.S. 1985b. Sensitive method for detecting low numbers of Verotoxin-producing Escherichia coli in mixed cultures by use of colony sweeps and polymyxin extraction of Verotoxin. J. Clin. Microbiol. 22:614. Karmali, M.A. 1987. Laboratory diagnosis of verotoxin-producing Escherichia coli infections. Clin. Microbiol. Newsletter 9:65. Konowalchuk, j., Speirs, j.1. and Stavric, S. 1977. Vero response to a cytotoxin of Escherichia coli. Infect. Immun. 18:775. Konowalchuk, J., Dickie, N., Stavric, S. and Speirs, J.1. 1978. Comparative studies of five heat-labile toxic products of Escherichia coli. Infect. Immun. 22:644. MacDonald, K.L., O'Leary, M.L Cohen, M.L., Norris, P., Wells, J.G., Noli, E., Kobayashi, J.M. and Blake, P. A. 1988. Esherichia coli 0157:H7, an emerging gastrointestinal pathogen. JAMA. 259:3567. Marques, L.R.M., Peiris, j.S.M., Cryz, S.J. and O'Brien, A.D. 1987. Escherichia coli strains isolated from pigs with edema disease produce a variant of Shiga-like toxin 11. FEMS Microbiol. Lett. 44:33. Martin, M.L., Shipman, L.D., Wells, J.G., Potter, M.E., Hedberg, K., Wachsmuth, I.K., Tauxe, R.V., Davis, J.P., Arnoldi, J. and - Tilleli, J. 1986. Isolation of Escherichia coli 0157:H7 from dairy cattle associated with two cases of haemolytic uraemic syndrome. Lancet ii: 1043. O'Brien, A.D. and LaVeck, G.D. 1983. Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli. Infect. Immun. 40:675. O'Brien, A.D. and Holmes, R.K. 1987. Shiga and Shiga-like toxins. Microbiol. Rev. 51 :206. Ostrof!. S.M., Griffin, P.M., Tauxe, R.V., Wells, J.G., Green, K.D., Lewis, J.H., Blake, P.A., and Kobayashi, J .M. 1987.
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Source tracing in an outbreak of E. coli 0157:H7-induced illness. International Symposium and Workshop on Verocytotoxin-Producing Escherichia coli Infections. Abst. CEP-8. Padhye, V.V., Beery, J.T., Kittell, F.B. and Doyle, M.P. 1987. Colonic hemorrhage produced in mice by a unique Vero cell cytotoxin from an Escherichia coli strain that causes hemorrhagic colitis. J. Infect. Dis. 155: 1249. Pai, CH., Kelly, j.K. and Meyers, G.L. 1986. Experimental infection of infant rabbits with verotoxin-producing Escherichia coli. Infect. Immun. 51: 16. Pai, CH., Ahmed, N., Lior,- H., Johnson, W.M., Sims, H.V. and Woods, D.E. 1988. Epidemiology of sporadic diarrhea due to verocytotoxin-producing Escherichia coli: a twoyear prospective study. J. Infect. Dis. 157:1054. Riley, L.W., Remis, R.S., Helgerson, S.D., McGee, H.B., Wells, j.G., Davis, B.R., Hebert, R.J., O!cott, E.S., Johnson, L.M., Hargrett, N.T., Blake, P.A., and Cohen, M.L. 1983. Hemorrhagic colitis associatod with a rare Escherichia coli serotype. N. Engl. J. Med. 308:681. Riley, L.W. 1987. The epidemiologic, clinical, and microbiologic features of hemorrhagic colitis. Annu. Rev. Microbiol. 41:383. Ryan, CA., Tauxe, R.V., Hosek, G.W., Wells, J.G., Stoesz, P.A., McFadden, H.W. Jr., Smith, P.W., Wright, G.F. and Blake, P.A. 1986. Escherichia coli 0157:H7 diarrhea in a nursing home: clinical, epidemiological, and pathological findings. J. Infect. Dis. 154:631. Stewart, P.J., Desormeaux, W. and Chene, J. 1983. Hemorrhagic colitis in a home for the aged - Ontario. Can. Dis. Weekly Rep. 9-8:29. Todd, E.CD., Szabo, RA, Peterkin, P., Sharpe, A.N., Parrington, L., Bundle, D., Gidney, MAJ. and Perry, M.B. 1988. Rapid hydrophobic grid membrane fi Iter - enzymelabelled antibody procedure for identification and enumeration of Escherichia coli 0157:H7 in foods. Appl. Environ. Microbiol. 54:2536. Tzipori, S., Karch, H., Wachsmuth, K.I., Robins-Browne, R.M., O'Brien, A. D., Lior, H., Cohen, M.L., Smithers, J. and Levine, M.M. 1987. Role of a 60-megadalton plasmid and Shiga-like toxins in the pathogenesis of infection caused by enterohemorrhagic Escherichia coli 0157:H7 in gnoto-biotic piglets. Infect. Immun. 55:3117. Uyeyama, R. R., Werner, S.B., Chin, S., Pearce, S. F., Kollip, C L., Williams, L.P. and Googins, J.A, 1982. Isolation of E. coli 0157:H7 from sporadic cases of hemorrhagic colitis - United States. Morb. Mortal. Weekly Rep. 31 :580. Wells, J.G., Davis, B.R., Wachsmuth, I.K., Riley, L.W., Remis, R.S., Sokolow, R., and Morris, G.K. 1983. Laboratory investigation of hemorrhagic colitis outbreaks associated with a rare Escherichia coli serotype. J. Cl in. Microbiol. 18:512. Wells, J.G., Shipman, L.D., Greene, K.D., Downes, F.P., Martin, M.L., Tauxe, R.V. and Wachsmuth, I.K. 1987. Isolation of Escherichia coli 01 S7:H7 and other Shiga-likeNero toxin-producing E. coli from dairy cattle. International Symposium and Workshop on Verocytotoxin-Producing Escherichia coli Infections Abst. LFE-4.
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Escherichia coli
Associated with Hemorrhagic Col itis 1 S.Stavric and ).1. Speirs Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health and Welfare Canada, Ottawa, Ontario K1A Ol2
Abstract
Outbreaks
The first outbreak of hemorrhagic colitis due to Escherichia coli 0157:H7 in Canada occurred in 1982. There were 31 cases with one death in a home for the aged; undercooked hamburger was the suspected vehicle. The serotype 0157 :H7 had rarely been encountered prior to 1982, but since then, sporadic and outbreak cases of hemorrhagic colitis and its complication, hemolytic uremic syndrome, have steadily increased. In Canada, the most severe of all outbreaks, with a case fatality rate of 31 %, occurred in a nursing home in 1985. In 1987, 315 cases in eight outbreaks were reported. At least six outbreaks of hemorrhagic colitis in North America have been linked to ingestion of undercooked beef and one to raw milk. E. coli 0157:H7 has been isolated from market samples of beef, pork, poultry and lamb, feces of healthy heifers and calves, and raw milk. Although E. coli 0157:H7 has been responsible for outbreaks of hemorrhagic colitis, other serotypes have also been implicated in sporadic cases, especially in children with hemolytic uremic syndrome. All isolates produce high levels of cytotoxin(s) affecting Vero cells (verotoxin). Since food of animal origin is the primary source of infection in humans, careful handling and adequate cooking of all raw meats is required to minimize or eliminate the risk of infection with E. coli 0157:H7 and other verotoxin-producing serotypes.
In 1982, an outbreak of hemorrhagic colitis occurred in Ottawa in a home for the aged (Stewart et al., 1983). Thirty-one of the 353 residents became ill with gastrointestinal symptoms and E. coli 0157:H7 was isolated from 18 of the 31 cases. In the first wave, undercooked hamburger was suspected as the vehicle; in the second wave of three cases, the spread was person-to-person. Four people were hospitalized, one died. Two outbreaks of HC, due to E. coli 0157:H7, had been reported from the United States earlier that year (Riley et al., 1983; Wells et al., 1983). Hamburgers in fast-food restaurants were suspected, and the 0157:H7 serotype was isolated from the implicated lot of meat eaten by persons in the Michigan restaurant. In the United States in 1982, besides the two outbreaks, there were 25 sporadic cases, 24 associated with hamburger consumption (Uyeyama et al., 1982). This gastrointestinal illness became known as "hamburger disease". Symptoms of HC include severe abdominal pain, watery diarrhea frequently followed by bloody diarrhea, nausea and vomiting, but little or no fever. Incubation varies from one to 14 days, with a duration of illness of about eight days. Persons in all age groups are at risk. Although generally self limiting, the young and elderly are more prone to serious complications such as HUS, thrombotic thrombocytopenic purpura (HP) and death. HUS is one of the leading causes of kidney failure in young children worldwide, with about 5% mortality reported in North America (Karmali, 1985a). Symptoms include a triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. TTP is an extension of HUS in some adults and includes neurological symptoms and fever. E. coli 0157 was first isolated in 1970 from piglets with enteritis in Ireland, but this strain contained the flagellar antigen H19 instead of H7 implicated in HC (Stewart et a/., 1983). Prior to 1982, serotype 0157:H7 was rarely encountered. In Canada, from 1978 to 1982, this serotype was isolated from only six of more than 2000 diarrheal isolates (Johnson et al. , 1983). In the United States, one isolate was found among 3000 E. coli diarrheal strains collected between 1973 and 1982 (Wells et al., 1983). In the United Kingdom, prior to 1982, only one strain was
Introduction There is considerable information in the literature concerning the emergence of the new enteric pathogen Escherichia coli 0157:H7 and its involvement in hemorrhagic colitis (HO and hemolytic uremic syndrome (HUS). Several outbreaks and cases of HC and HUS have been linked epidemiologically to consumption of undercooked beef or raw milk. In addition to E. coli 0157:H 7, other serotypes have also been implicated in illness. These organisms are of great importance to food microbiologists as well as clinicians. It is the purpose of this paper to review and summarize the most pertinent data on outbreaks, pathogenicity, reservoirs and detection methods for E. coli causing HC and HUS.
, Based on a paper presented at the Health Protection Branch Workshop, "Microbial Food Poisoning and its Significance in Canada ", Ottawa, October 18-19, 1988.
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isolated from about 15,000 strains examined (Day et al., 1983). Since 1982 isolates of E. coli 0157:H7 have generally doubled annually in Canada, with 1342 reported in 1987 (Hockin et al., 1988). There is a marked seasonal variation, at least with sporadic cases, most occurring from June to September (Hockin and Lior, 1987; Pai et al., 1988). Isolates from sporadic cases have outnumbered those from outbreaks. In 1987 there were 315 outbreak-associated cases, 10 patients developed HUS and there were six deaths. Up to 1988 there have been 19 outbreaks recorded in Canada, with 518 persons affected, 29 cases of HUS and 26 deaths (Hockin and l.ior, 1987; Carter et al., 1987; Todd et al., 1988). The most severe of all outbreaks in Canada was in a nursing home in London, Ont. in 1985 (Carter et al., 1987). In this outbreak there were 73 cases with 33% of the residents and 13% of staff members affected; 12 residents developed HUS and 17 died. A sandwich meal was incriminated as the primary source of infection. Nursing homes have been the setting for eight outbreaks (six in Ontario, two in Alberta), the community for six, families for three and one each for a school trip and a girl's camp. Besides serotype 0157:H7, at least 22 other E. coli serotypes have been isolated worldwide from sporadic cases of HC or HUS (Pai et al., 1988; Karmali, 1987; Bopp et al., 1987). One of these, E. coli 0145:H- was responsible for an outbreak of acute enteritis in Japan in 1984 (Itoh et al., 1985). In general, with serotypes other than 0157:H7, the diarrhea is more prolonged and less frequently bloody. Secondary cases of E. coli 0157:H7 have been found to be less severe - diarrhea is milder and less frequently bloody (Pai et al., 1988). The first population-based study on the incidence of infection with E. coli 0157:H7 in sporadic cases over a one-year-period was conducted in the United States (MacDonald et al., 1988). Of 6485 diarrheal stool samples analyzed for this serotype, E.coli 0157:H7 was identified in 25 specimens (0.4%). Twenty-four (96%) were from patients with bloody diarrhea. Twenty of the patients had consumed undercooked beef and three drank raw milk before the onset of illness.
Pathogenicity E. coli strains associated with diarrhea are divided on the basis of their mode of action into several well established groups: enterotoxigenic (ETEC), enteroinvasive (EIEC) and enteropathogenic (EPEC). In the EPEC group, which is the most controversial, two new groups: enteroadherent (EAEC) and enterohemorrhagic (EHEC) or verotoxin (VT)producing (VTEC) strains have recently been added. ETEC strains cause a cholera-like secretory diarrhea which is induced by heat-labile and/or heat-stable enterotoxins. EIEC strains cause a dysentery-like inflammation of the large bowel; cells are invaded. EPEC strains cause diarrhea by mechanisms different from those of ETEC or EIEC. EAEC strains, as the name impl ies, adhere to entorocytes and cause mucosal damage. Strains in the EHEC or VTEC group cause HC and HUS by Cl mechanism as yet not fully understood. The name EHEC was established for E. coli 0157:H7 which usually induces a bloody diarrhea. The name VTEC is used for the various serotypes that produce VT, including those implicated in HUS. Although it is not known at present whether the verotoxins play a role in the pathogenesis of human diarrhea, their involvement in extragastrointestinal disease, such as HUS, is supported by 206/ AT
numerous data. It is thought that toxins released in the bowel are absorbed into the blood and cause endothelial damage of small blood vessels which may lead to HUS and central nervous system dysfunction (Edelman et al., 1988). All of the HC and HUS isolates to date produce high levels of one or more cytotoxins, known as verotoxins or Shiga-like toxins (SLT), which are toxic for Vero and Hela cell cultures. Four antigenically distinct VTs have been named (Cannon and Cyles, 1987). VT1 and/or VT2 appear to be the predominant VTs produced by North American HC strains. O'Brien and LaVeck (1983) have shown that VT1 is essentially identical to Shiga toxin (of Shigella dysenteriae), which is one of the most potent bacterial toxins known. Canadian scientists have made important contributions in this field. The discovery of cytotoxins in E. coli that affect Vero cells was made in the Food Directorate, HPB, Ottawa, in 1977 by Konowalchuk et al. (1977, 1978). Three different VTs were distinguished at that time by biochemical and immunological methods. Johnson et al. (1983), at the Laboratory Centre for Disease Control, Ottawa, were the first to report that E. coli 0157:H7 isolates produced VT. Karmali et al. (1983), at the Hospital for Sick Children, Toronto, discovered that VT-producing E. coli were the probable cause of idiopathic HUS in children. The modes of action of verotoxin and Shiga toxin on mammalian cells appear similar. These toxins are composed of an A subunit, molecular weight about 32,000, and five copies of a B subunit, molecular weight about 7,700. The B subunit binds to glycolipid receptors on the cell. After internalization, the A subunit is reduced enzymatically to a smaller fragment, A 1, which binds to 60S ribosomes, inhibiting protein synthesis and resulting in cell death (O'Brien and Holmes, 1987). Examination of HC patients has revealed that the intestinal areas affected are the cecum, and the ascending and transverse colon (Riley, 1987). Barium enemas have detected a "thumbprinting" pattern indicative of submucosal edema or hemorrhage. Colonoscopic examinations have shown erythema, hemorrhage and edema. Post mortem examinations of fatalities have shown focal areas of acute and chronic inflammation with congestion and hemorrhage in the lamina propria. Similar mucosal damage occurs in the cecum and colon of piglets fed E. Coli 0157:H7, but mutant strains which lack the cytotoxin-producing property are equally damaging, demonstrating that these toxins are not required for diarrhea in piglets (Tzipori et al., 1987). However, the neurological symptoms with edema disease of piglets are thought to be associated with the toxin (Marques et al., 1987). In rabbits, diarrhea and mucosal abnormalities in the cecum and colon resemble those in humans, and are induced by feeding the whole bacterium as well as VT alone (Pai et al., 1986). Intravenous injection of purified toxin resulted in diarrhea and paralysis followed by kidney failure (Barrett et al., 1988); lesions occurred in the cecum, colon, central nervous system, and kidney. The kidney lesions, however, did not resemble those seen in humans with HUS. Mice injected intraperitoneally with purified toxin developed a bloody diarrhea; lesions occurred in the colon, kidney and lymphoid tissues (Padhye et al., 1987).
Reservoirs E. coli HC is primarily a food-borne illness, but personto-person transmission also occurs, especially in institutional settings. At least six outbreaks and numerous sporadic cases in North America have been linked to ingestion of
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undercooked beef (Stewart et al., 1983; Riley et al., 1983; Wells et al., 1983 ; Hockin and Lior, 1987; MacDonald et al., 1988; Ryan et al., 1986), and one outbreak and several sporadic cases to raw milk (MacDonald et al., 1988; Duncan et al., 1987; Borczyk et al., 1987; Martin et al., 1986). Other foods implicated include sandwiches, sour cream dressing and crisp meat burritos (Carter et al., 1987; Ostroff et al., 1987), but cross-contamination of these foods from beef may have occurred. Dairy cattle are considered to be an important reservoir of E. coli 0157:H7. Isolates have been obtained in North America from raw milk, feces of healthy heifers and calves (Wells et al., 1987; Martin et al., 1986) and market samples of beef, pork, poultry and lamb (Doyle and Schoeni, 1987). One survey in the United States of feces from 899 heifers, calves and adult cows from 16 farms, a slaughter house and a stockyard revealed E. coli 0157:H7 in samples from 18 heifers or calves on nine farms and from one stockyard calf (Wells et al., 1987). All adult cows tested were negative but one of 24 raw milk samples from two farms was positive. Other VTEC serotypes were found from. all five farms examined and from the stockyard. Surveys in western United States of 599 beef cattle failed to find E. coli 0157:H7 (Martin et al., 1986). A survey of 896 retail fresh meat and poultry samples detected E. coli 0157:H7 in 0.7% of beef, 1.2% of pork, 1.5% of poultry and 2.0% of lamb from Madison, Wis., and in 29.4% of beef and 7.1 % of pork from Calgary, Alta. (Doyle and Schoeni, 1987). In southwestern Ontario, Clarke et al. (1988) showed that at slaughter, 10.5% of beef cattle, 19.5% of cull dairy cows and 3.5% of veal calves contained VTEC in their feces; four of the 600 animals had the serotype 0157:H7. In other surveys by these workers (Clarke et al., 1987); 89% of feces from healthy calves from a university research farm and 2% of milk filters from 498 dairy farms revealed VTEC of serotype other than 0157:H7. These data indicate that E. coli 0157:H7 and VTEC are widely distributed in the intestinal tract of animals and that food-products of animal origin are primary sources of human infection.
Detection Clinical diagnosis of E. coli HC has centred on detection of the serotype 0157:H7 and its sorbitol-negative property. Fecal extracts are inoculated onto a sorbitol MacConkey agar and colourless colonies at 24 hours are tested by slide agglutination against 0157 and H7 antisera. About 95% of E. coli are sorbitol-fermenters (Farmer and Davis, 1985), but among nonfermenters, 0157:H7 may account for as few as 2% (Harris et al., 1985). This procedure would not detect the other serotypes that have been implicated in cases of HC and HUS, thus it is preferable to test for VT production. Fecal filtrates may readily be screened for free VT if cell culture facilities are available. Filtrates containing cytotoxin induce a typical cytopathic effect on Vero cells (Konowalchuk et aI., 1977; Karmali, 1987). VT-positive strains are confirmed by neutralization of the cytopathic effect with antisera against the toxins. However, these antisera are not commercially available. Karmali et al. (1985b) developed a sensitive method for screening fecal cultures for verotoxin-producing E. coli employing polymyxin B to release VT from colony sweeps ("VT/PECS" procedure). This method can detect VT-producing organisms among non-VTEC in as low as a 1% concentration in feces. Studies with feces from HUS patients indicated that VT-positive colonies have often been only 5 to 20% of the total E. coli population.
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The procedures used for clinical samples are applicable to food samples, but recently-developed methods (Todd et al., 1988; Doyle and Schoeni, 1987) using hydrophobic grid membrane filters (HGMF) are more suitable. In principle, food extracts are filtered through HGMF and the membranes cultured on selective agar. Blots, or replicas of the colonies, are examined against antisera to E. coli 0157 by an enzyme-labelled immunoassay procedure. At present, DNA probes and monoclonal antibodies are being developed in several laboratories for use in detection of the cytotoxins in clinical specimens and foods. In view of the increasing frequency of cases of hemorrhagic colitis, and the wide variety of contaminated meats, careful handling and adequate cooking of all raw meats is required to minimize or eliminate the risk of infection with E. coli 0157:H7 and other VTEC, and indeed other pathogenic organisms.
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