THE JOURNAL OF L\,FECTIOUS DISEASES. VOL. 135, 1\'0. 2 • FEBRUARY 1977
© 1977 by the University of Chicago. All rights reserved.
Enterotoxigenic Escherichia coli Isolated from Food R. Bradley Sack, David A. Sack, Ira Frits grskov, and Ida 0'rskov
J. Mehlman,
From the Department of Medicine, Baltimore City Hospitals, and The Johns Hopkins University School of Medicine, Baltimore, Maryland; the Division of Microbiology, the Food and Drug Administration, Washington, D.C.; and the World Health Organization Collaborative Center for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark
Enterotoxigenic Escherichia coli have recently been etiologically associated with diarrheal disease in humans of all age groups from diverse global locations [I]. The clinical spectrum of disease which these microorganisms produce encompasses diarrhea in infants, nonspecific diarrheas of childhood [2-4], severe cholera-like illness [5], and traveler's diarrhea [6, 7]. These E. coli, which grow in the small bowel, induce diarrheal illness by the production of either or both of two enterotoxins: a heat-labile enterotoxin (LT) that is antigenic and similar to cholera enterotoxin or a heat-stable enterotoxin (ST) that is of small molecular weight and nonantigenic [8, 9]. The genetic material that codes for both of these enterotoxins has been shown to reside in plasmid DNA and is transferable from enterotoxigenic to other nontoxigenic strains [10]. Although diarrheal disease in humans has been associated largely with L Tvproducing E. coli [1-7], it is now also clear that E. coli that produce only ST also cause diarrhea
to colonize the small bowel after their ingestion -in large numbers from food or water, the scanty epidemiologic evidence that is available has implicated a food source in only one study [7] and contaminated water in one other [12]. Although no studies have been reported in which enterotoxigenic E. coli have been sought extensively in water or food, these organisms were identified in river water in Whiteriver, Arizona [2] and in tank water in Bangladesh [13], areas where diarrheal disease due to enterotoxigenic E. coli was occurring, and in two of 58 miscellaneous food items examined in a recently reported study by the Food and Drug Administration, Washington, D.C. [14]. The routine examination of food items for E. coli at present is performed either for indication of fecal contamination or for identification of classical serotypes enteropathogenic in humans. The latter procedure is open to question because (1) there is no clear evidence that these organisms, outside of limited epidemic situations in infants, are important in diarrheal disease, and (2) almost no enterotoxigenic E. coli would be detected by this method [2]. In the present study enterotoxigenic E. coli of diverse nonenteropathogenic serotypes were recovered from various food sources; this observation may be of significant importance to public health.
[11]. Although enterotoxigenic E. coli are presumed Received for publication May 13, 1976, and in revised form July 28, 1976. This study was supported by contract no. DADA 17-73C-3055 from the U. S. Army. The expert technical assistance of Jean Froehlich and Adrian George is gratefully acknowlelged. The Gerontology Research Center, National Institute on Aging, provided facilities under its Guest Scientist Program. Please address requests for reprints to Dr. R. Bradley Sack, Department of Medicine, Baltimore City Hospitals. 4940 Eastern Avenue, Baltimore, Maryland 21224.
Materials and Methods
Sources of bacterial isolates.
313
A total of 240
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
Approximately 8% of 240 isolates of Escherichia coli obtained from food of animal origin in the United States were found to be enterotoxigenic, as determined in adrenal cells, rabbit ileal loops, and assays in infant mice. These organisms were of diverse serotypes that are not included among the so-called enteropathogenic serotypes and would not have been identified by usual laboratory methods. These enterotoxigenic E. coli are of potential importance to public health.
314
Sack et al.
Table 1.
Serotyping. All enterotoxin-producing strains were examined by bacterial agglutination procedures previously described [19]. The 160 0 and 50 H antisera used were the standard sera of the World Health Organization Collaborative Center for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark. Results
The food items from which the 240 strains of E. coli were isolated are listed in table 1; all foods were of animal origin. Nineteen isolates (S' were found to produce LT as measured in the adrenal cells. Of these 19 isolates, eight were found also to produce ST, as assayed in the infant mouse, and II were found to produce only LT. Of the 221 isolates that were negative in the adrenal cell assay, 38 from the different food sources were chosen randomly for testing in the infant mouse. Three (S1 ml of fluid/em of intestine (average of three loops per test) when tested as live cultures in the rabbit intestinal loop assay. The serotypes of the enterotoxigenic strains
ro)
Results of tests for production of enterotoxin by strains of Escherichia coli isolated from food.
No. of strains, origin 12, Oriental cheese 27, Danish cheese 49, Hamburger 48, French cheese 37, Seafood 39, Sausage 28, Soft cheese Total (240)
Adrenal cell-positive strains assayed in infant mice Positive 2 1 2 1 1 1 0
8
Negative 1 1 5 0 0 4 0 11
Adrenal cell-negative strains assayed in infant mice Positive 0 0 2 0 0 1 0 3
Negative
Not tested
2
7 22
3 7 7 7
8 4
38
33 40 29 25 24 180
NOTE. Nineteen strains (designated adrenal cell-postive ) produced heat-labile enterotoxin (LT). Positive results with these strains in infant mice refer to organisms that also produced heat-stable enterotoxin (ST), and negative results with these strains refer to organisms that produced only LT. Adrenal cell-negative strains did not produce LT. Positive results with these strains refer to those that produced only ST. Negative results refer to strains that did not produce enterotoxin.
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
strains of E. coli (nonenteropathogenic serotypes) were isolated from food items not known to be associated with diarrheal disease in humans. Each isolate was from a separate food sample, and none represented multiple samples from the same specific location. E. coli were isolated from different geographical areas in the United States by laboratories of the Food and Drug Administration with use of a recommended procedure [15]. Briefly, 25g of foodstuff was homogenized in 225 ml of MacConkey's broth and incubated for 20 hr at 35 C. A loopful of the culture was transferred to 30 ml of lauryl tryptose broth (Difco Laboratories, Detroit, Mich.) that was then incubated for an additional 20 hr at 44 C. The culture was then streaked onto Levine's eosin-methylene-blue agar (Difco). Colonies were identified as E. coli with use of standard techniques [16]; pure cultures were maintained on veal infusion agar slants for periods of up to two years before testing for enterotoxin. Testing for production of enterotoxin. All strains were assayed for LT production by the adrenal cell tissue culture miniplate system [17]. All strains that gave a positive reaction in the adrenal cell assay were assayed in both the IS-hr adult rabbit intestinal loop [2] and the infant mouse [1S]. In the rabbits all strains were tested both as syncase-grown live cultures and as culture filtrates [2]. At least three loops from three rabbits were used to assay each preparation. In the infant mice, only culture filtrates were used; three mice were used to assay each filtrate.
Enterotoxigenic E. coli in Food
Table 2.
315
Enterotoxigenic strains of Escherichia coli isolated from food sources. Enterotoxin assays
Source
Geographical area
Date isolated
Oriental soft cheese
San Francisco district
March 1973
Danish soft fermented cheese Hamburger (retail)
Los Angeles district
April 1973
Washington, D. C.
November 1973
*(+)
Boston
May 1973
Washintgon, D. C. Washington, D. C.
October 1973 December 1973
= production of heat-labile enterotoxin; (-) = no
Serotype
4 9 10 20 21 52 58 59 78 79 80 81 50 55 109
078:H11 015:H11 025:H? (OI47):H? 045:H? O149:HI0 08:H19 078:H11 0149:H10 0147:H? 0141:H4 08:H19 015:H11 09:H0149:HI0
173 211 212 214 216 218 210
06:H08:H0149:H19 0149:H19 0149:H19 0149:HI0 0101:H-
Adrenal cells" + + + + + + + + + + + +
+ + + + + + +
Rabbit ileal Ioop ]' 1.23 1.31 0.68 0.23 1.49 2.02 1.59 ND 1.24 0.84 1.98 0.66 ND ND 1.45 0.41 1.39 1.57 0.40 1.23 0.95 ND
(3) (3) (9) (6) (3) (3) (3) (4) (6) (7) (8)
(5) (7) (3) (3) (8) (3) (3)
Infant mouse f 0.055 0.135 0.106 0.125 0.068 0.107 0.076 0.071 0.138 0.064 0.068 0.052 0.125 0.120 0.095 0.129 0.060 0.105 0.065 0.055 0.060 0.111
production of heat-labile enterotoxin.
tData are ratios of the mean loop volume to length of intestine (ml/cm}; number of loops is in parentheses. Live cultures of all strains tested gave ratios of> 1.0. ND = not done. tData are ratios of the mean intestinal weight to remaining body weight; three mice per test were used; a ratio of >0.083 was considered to be a positive response.
are also shown in table 2. None were of classical enteropathogenic serotypes. The most common 0 serotype was 0149 (seven of 22 isolates); this serotype was associated with either HI 0 (four cases) or H 19 (three cases) flagellar an tigens. All but one of these serotypes were isolated from hamburger or sausage. Discussion
The present study demonstrates that (1) production of enterotoxin is relatively common among E. coli isolated from food and (2) such foods are therefore potentially important vehicles in the transmission of diarrheal disease of humans mediated by enterotoxigenic E. coli. Furthermore, this estimate of frequency must be considered minimal, since it is possible that some strains may have lost plasmids during storage [1].
E. coli in food have been suspected of being associated with outbreaks of gastrointestinal disease since 1903 [20]. In the 1940s, when certain serotypes of E. coli were identified as responsible for epidemic diarrheal disease in young children, it was thought important to look for these enteropathogenic serotypes in food. Although these serotypes were not found uncommonly in food [21], there have been relatively few reported outbreaks of food-borne diarrhea associated with these serotypes [22]. However, no testing of these strains for enterotoxin production has been reported. In one well-documented outbreak of diarrheal disease associated with a cheese product, a single 0 group (0124) of invasive E. coli was incriminated [23]. However, in spite of the ability to identify E. coli serologically, the number of recognized E. coli-associated diarrheal outbreaks has been small. Of 307 outbreaks of food-borne disease in
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
French soft ripened cheese Crabmeat Summer sausage
Strain no.
316
at.
or animal origin. Since the food products were of animal origin, the likelihood of contamination by strains of E. coli of animal origin must be considerable. Indeed, if the strains were of animal origin, it is not known whether they would be pathogenic for humans. In the only reported experiment with volunteers [26J, a strain of enterotoxigenic E. coli from a pig was found to be virtually nondiarrhetic in humans. On the other hand, some of the enterotoxigenic serotypes identified in this study, such as 078:Hll or 015:Hll, have also been isolated from cases of severe cholera-like diarrhea in humans [5]; this finding would suggest that these organisms may be able to cause diarrhea in humans. It seems clear that to cause disease these strains must also contain surface antigens that facilitate colonization of the small bowel. Thus far, these antigens have been well characterized only in animal strains [27J and may account for the possible species specificity of the bacteria. A recent report [28J also suggests that similar surface antigens are found in some enterotoxigenic strains in humans. It is possible that the recognition of certain uncommon serotypes as enterotoxigenic may indicate that there are clones of E. coli identifiable by surface antigens that somehow enable these organisms to colonize the small bowel and to carry plasmids for production of enterotoxin. The potential for these organisms found in food items to be involved etiologically in diarrheal disease of humans, either sporadically or in point-source outbreaks, remains. It seems reasonable to suggest that laboratories involved in isolation of E. coli from food sources should not be limited to the identification of "pathogenic" serotypes but should develop the capabilities for determination of whether the strains are enterotoxigenic.
References 1. Sack, R. B. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol.
29:333-353,1975. 2. Sack, R. B., Hirschhorn, N., Brownlee, I., Cash, R. A., Woodward, W. E., Sack, D. A. Enterotoxigenic Escherichia coli-associated diarrheal disease in Apache children. N. Engl. J. Med. 292:1041-1045,1975.
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
1973 in the United States, none were attributed to E. coli) and 180 were of unknown cause [24]. In the same year, there was a total of 1,720 cases of waterborne disease, of which none was specifically ascribed to E. coli) although 1,065 were designated as "sewage poisoning." These observations suggest either that E. coli is rarely a cause of outbreaks of food- or waterborne diarrhea or that techniques for the determination of whether E. coli may be of etiologic significance have been inadequate. Since the practice of identifying classical enteropathogenic serotypes of E. coli has now been shown to be inadequate for the identification of enterotoxigenic organisms [2], and since the plasmids that control production of enterotoxin may theoretically be found in any serotype of E. coli) the latter possibility seems most likely. A recent investigation of the serotypes of enterotoxigenic E. coli associated with diarrhea in humans from widespread geographical areas [25] indicated that some well-characterized serotypes were found to be prevalent among the strains (serotypes 06:HI6, 08:H9, 015:Hll. 078:Hll, and 078:HI2); some of these serotypes were also found in the present study. One of these serotypes, 078, is a very common cause of diarrhea in calves and septicemia in chickens, and another, 0149, is responsible for >50% of all cases of diarrhea in piglets in several countries. However, most of the above mentioned O:H serotypes and 0 groups are uncommon in the intestine of healthy humans [25]. If we assume that enterotoxigenic E. coli are found frequently in food, is there any way to assess the risk to the food-consuming public? To act as significant vehicles of transmission, the food must contain the bacteria in high enough concentrations so that an infectious dose is ingested, and to produce disease these ingested organisms must be able to colonize the small bowel of humans. It is known from experiments in volunteers that a large inoculum of enterotoxigenic E. coli (__ 109-1010 ) is probably necessary to cause diarrheal disease in normal adults [26]. U nfortunately, in the present study no information regarding concentrations of organisms in the food was obtained. With regard to colonization of the small bowel, it is unclear whether the organisms isolated in the present study were of human
Sack et
Enterotoxigenic E. coli in Food
17.
18.
19.
20. 21.
22.
23.
24.
25.
26.
27.
28.
bacteriaceae. 3rd cd. Burgess Publishing, Minneapolis, 1972. 362 p. Sack, D. A., Sack, R. B. Test for enterotoxigenic Escherichia coli using YI adrenal cells in miniculture. Infec. Immun. 11:334-336,1975. Dean, A. G., Ching, Y. C., Williams, R. G., Harden, L. B. Test for Escherichia coli enterotoxin using infant mice: application in a study of diarrhea in children in Honolulu. J. Infect. Dis. 125:407-411. 1972. 0rskov, f'., 0rskov, I. Escherichia coli O:H serotypes isolated from human blood. Prevalence of Kl antigen with technical details of 0 and H determination. Acta Pathol. Microbiol. Scand. 83 :595-600, 1975. Delepine, S. Food poisoning and epidemic diarrhea. J.A.M.A. 40:657,1903. Fantasia, L. D., Mestrandrea, L., Schrade, J. P., Yager, J. Detection and growth of enteropathogenic Escherichia coli in soft ripened cheese. J. Appl. Microbiol. 29:179-185,1975. Costin, I. D., Voiculescu, D., Gorcea, V. An outbreak of food poisoning in adults associated with serotype 086:B7:H34. J. Pathol. Microbiol. 27:68-78, 1964. Marier, R., Wells, J. G., Swanson, R. C., Callahan, W., Mehlman, I. J. An outbreak of enteropathogenic Escherichia coli foodborne disease traced to imported French cheese. Lancet 2:1376-1378,1973. Center for Disease Control. Foodborne and waterborne disease outbreaks. Annual summary Publication no. (CD) 75-8185, U. S. Department of Health, Education, and Welfare, Washington, D.C., 1973. 0rskov, F., 0rskov, I., Evans, D. J., Sack, R. B., Sack, D. A., Wadstrom, T. Special Escherichia coli serotypes among entero'toxigenic strains from diarrhea in adults and children. Med. Microbiol. Immunol. 162: 73-80, 1976. Dupont, H. L., Formal, S. B., Hornick, R. B., Snyder, M. J., Libonati, J. P., Sheahan, D. c., LaBree, E. H., Kalas, J. P. Pathogensis of Escherichia coli diarrhea. N. Engl. J. Med. 285: 1-9, 1971. Smith, H. W., Linggood, M. A. Observations on the pathogenic properties of the K88, HLY and ENT plasm ids of Escherichia coli with particular reference to porcine diarrhoea. J. Med. Microbiol. 4:467-485, 1971. Evans, D. G., Silver, R. P., Evans, D. J., Jr., Chase, D. G., Gorbach, S. L. Plasmid-controlled colonization factor associated with virulence in Escherichia coli entcrotoxigenic for humans. Infec, Immun. 12:656667,1975.
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3. Guerrant, R. L., Moore, R. A., Kirschenfeld, P. M., Sande, M. A. Role of toxigenic and invasive bacteria in acute diarrhea of childhood. K Engl. J. Med. 293:567-573, 1975. 4. Gorbach, S. L., Khurana, C. M. Toxigenic Escherichia coli: a cause of infantile diarrhea in Chicago. N. Engl. J. Med. 287:791-795,1972. 5. Sack, R. B., Gorbach, S. L., Banwell, J. G., Jacobs, B., Chatterjee, B. D., Mitra, R. C. Enterotoxigenic Escherichia coli isolated from patients with severe cholera-like disease. J. Infect. Dis. 123:378-385, 1971. 6. Gorbach, S; L., Kean, B. H., Evans, D. G., Evans, D. J., Bessudo, D. Traveler's diarrhea and toxigenic Escherichia coli. ~. Engl. J. Med. 292:933-936, 1975. 7. Merson, M. H., Morris, G. K., Sack, D. A., Wells, J. G., Creech, W., Feeley, J. C., Sack, R. B., Kapikian, A. Z., Gangarosa, E. J. Travelers' diarrhea in Mexico: a prospective study. N. Engl. J. Med. 294: 12991305, 1976. 8. Gyles, C. L. Heat-labile and heat-stable forms of the enterotoxin from E. coli strains enteropathogenic for pigs. Ann. N.Y. Acad. Sci. 176:314-322,1971. 9. Xalin, D. R., Bhattacharjee, A. L., Richardson, S. H. Cholera-like toxic effect of culture filtrates of Escherichia coli. J. Infect. Dis. 130:595-601, 1974. 10. Skerman, F. J., Formal, S. B., Falkow, S. Plasmidassociated enterotoxin production in a strain of Escherichia coli isolated from humans. Infec. Immun. 5:622-624,1972. II. Sack, D. A., Merson, M. H., Wells, J. G., Sack, R. B., Morris, G. K. Diarrhea associated with heat-stable enterotoxin-producing strains of Escherichia coli. Lancet 2:239-241,1975. 12. Center for Disease Control. Follow-up on outbreak of gastrointestinal illness at Crater Lake National Park-Oregon. Morbidity and Mortality Weekly Rep. 24 (31):261-262, 1975. 13. Ryder, R. W., Sack, D. A., Kapikian, A. Z., Rahman, A. S. M. M., Merson, M. H., Wells, J. G. Enterotoxigenic Escherichia coli and reovirus-like agent in rural Bangladesh. Lancet 1:659-663, 1976. 14. Mehlman, I. j., Fishbein, M., Gorbach, S. L., Sanders, A. C., Eide, E. L., Olson, J. C., Jr. Pathogenicity of Escherichia coli recovered from food. J. Assoc. Off. Anal. Chem. 59:67-80,1976. 15. Mehlman, I. J., Sanders, A. C., Simon, N. T., Olson, J. C., Jr. Methodology for recovery and identification of enteropathogenic Escherichia coli. J. Assoc. Off. Anal. Chem. 57:101-110,1974. 16. Edwards, P. R., Ewing, W. H. Identification of Entero-
317
© 1977 by the University of Chicago. All rights reserved.
Enterotoxigenic Escherichia coli Isolated from Food R. Bradley Sack, David A. Sack, Ira Frits grskov, and Ida 0'rskov
J. Mehlman,
From the Department of Medicine, Baltimore City Hospitals, and The Johns Hopkins University School of Medicine, Baltimore, Maryland; the Division of Microbiology, the Food and Drug Administration, Washington, D.C.; and the World Health Organization Collaborative Center for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark
Enterotoxigenic Escherichia coli have recently been etiologically associated with diarrheal disease in humans of all age groups from diverse global locations [I]. The clinical spectrum of disease which these microorganisms produce encompasses diarrhea in infants, nonspecific diarrheas of childhood [2-4], severe cholera-like illness [5], and traveler's diarrhea [6, 7]. These E. coli, which grow in the small bowel, induce diarrheal illness by the production of either or both of two enterotoxins: a heat-labile enterotoxin (LT) that is antigenic and similar to cholera enterotoxin or a heat-stable enterotoxin (ST) that is of small molecular weight and nonantigenic [8, 9]. The genetic material that codes for both of these enterotoxins has been shown to reside in plasmid DNA and is transferable from enterotoxigenic to other nontoxigenic strains [10]. Although diarrheal disease in humans has been associated largely with L Tvproducing E. coli [1-7], it is now also clear that E. coli that produce only ST also cause diarrhea
to colonize the small bowel after their ingestion -in large numbers from food or water, the scanty epidemiologic evidence that is available has implicated a food source in only one study [7] and contaminated water in one other [12]. Although no studies have been reported in which enterotoxigenic E. coli have been sought extensively in water or food, these organisms were identified in river water in Whiteriver, Arizona [2] and in tank water in Bangladesh [13], areas where diarrheal disease due to enterotoxigenic E. coli was occurring, and in two of 58 miscellaneous food items examined in a recently reported study by the Food and Drug Administration, Washington, D.C. [14]. The routine examination of food items for E. coli at present is performed either for indication of fecal contamination or for identification of classical serotypes enteropathogenic in humans. The latter procedure is open to question because (1) there is no clear evidence that these organisms, outside of limited epidemic situations in infants, are important in diarrheal disease, and (2) almost no enterotoxigenic E. coli would be detected by this method [2]. In the present study enterotoxigenic E. coli of diverse nonenteropathogenic serotypes were recovered from various food sources; this observation may be of significant importance to public health.
[11]. Although enterotoxigenic E. coli are presumed Received for publication May 13, 1976, and in revised form July 28, 1976. This study was supported by contract no. DADA 17-73C-3055 from the U. S. Army. The expert technical assistance of Jean Froehlich and Adrian George is gratefully acknowlelged. The Gerontology Research Center, National Institute on Aging, provided facilities under its Guest Scientist Program. Please address requests for reprints to Dr. R. Bradley Sack, Department of Medicine, Baltimore City Hospitals. 4940 Eastern Avenue, Baltimore, Maryland 21224.
Materials and Methods
Sources of bacterial isolates.
313
A total of 240
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
Approximately 8% of 240 isolates of Escherichia coli obtained from food of animal origin in the United States were found to be enterotoxigenic, as determined in adrenal cells, rabbit ileal loops, and assays in infant mice. These organisms were of diverse serotypes that are not included among the so-called enteropathogenic serotypes and would not have been identified by usual laboratory methods. These enterotoxigenic E. coli are of potential importance to public health.
314
Sack et al.
Table 1.
Serotyping. All enterotoxin-producing strains were examined by bacterial agglutination procedures previously described [19]. The 160 0 and 50 H antisera used were the standard sera of the World Health Organization Collaborative Center for Reference and Research on Escherichia, Statens Seruminstitut, Copenhagen, Denmark. Results
The food items from which the 240 strains of E. coli were isolated are listed in table 1; all foods were of animal origin. Nineteen isolates (S' were found to produce LT as measured in the adrenal cells. Of these 19 isolates, eight were found also to produce ST, as assayed in the infant mouse, and II were found to produce only LT. Of the 221 isolates that were negative in the adrenal cell assay, 38 from the different food sources were chosen randomly for testing in the infant mouse. Three (S1 ml of fluid/em of intestine (average of three loops per test) when tested as live cultures in the rabbit intestinal loop assay. The serotypes of the enterotoxigenic strains
ro)
Results of tests for production of enterotoxin by strains of Escherichia coli isolated from food.
No. of strains, origin 12, Oriental cheese 27, Danish cheese 49, Hamburger 48, French cheese 37, Seafood 39, Sausage 28, Soft cheese Total (240)
Adrenal cell-positive strains assayed in infant mice Positive 2 1 2 1 1 1 0
8
Negative 1 1 5 0 0 4 0 11
Adrenal cell-negative strains assayed in infant mice Positive 0 0 2 0 0 1 0 3
Negative
Not tested
2
7 22
3 7 7 7
8 4
38
33 40 29 25 24 180
NOTE. Nineteen strains (designated adrenal cell-postive ) produced heat-labile enterotoxin (LT). Positive results with these strains in infant mice refer to organisms that also produced heat-stable enterotoxin (ST), and negative results with these strains refer to organisms that produced only LT. Adrenal cell-negative strains did not produce LT. Positive results with these strains refer to those that produced only ST. Negative results refer to strains that did not produce enterotoxin.
Downloaded from http://jid.oxfordjournals.org/ at Sheffield University on September 12, 2015
strains of E. coli (nonenteropathogenic serotypes) were isolated from food items not known to be associated with diarrheal disease in humans. Each isolate was from a separate food sample, and none represented multiple samples from the same specific location. E. coli were isolated from different geographical areas in the United States by laboratories of the Food and Drug Administration with use of a recommended procedure [15]. Briefly, 25g of foodstuff was homogenized in 225 ml of MacConkey's broth and incubated for 20 hr at 35 C. A loopful of the culture was transferred to 30 ml of lauryl tryptose broth (Difco Laboratories, Detroit, Mich.) that was then incubated for an additional 20 hr at 44 C. The culture was then streaked onto Levine's eosin-methylene-blue agar (Difco). Colonies were identified as E. coli with use of standard techniques [16]; pure cultures were maintained on veal infusion agar slants for periods of up to two years before testing for enterotoxin. Testing for production of enterotoxin. All strains were assayed for LT production by the adrenal cell tissue culture miniplate system [17]. All strains that gave a positive reaction in the adrenal cell assay were assayed in both the IS-hr adult rabbit intestinal loop [2] and the infant mouse [1S]. In the rabbits all strains were tested both as syncase-grown live cultures and as culture filtrates [2]. At least three loops from three rabbits were used to assay each preparation. In the infant mice, only culture filtrates were used; three mice were used to assay each filtrate.
Enterotoxigenic E. coli in Food
Table 2.
315
Enterotoxigenic strains of Escherichia coli isolated from food sources. Enterotoxin assays
Source
Geographical area
Date isolated
Oriental soft cheese
San Francisco district
March 1973
Danish soft fermented cheese Hamburger (retail)
Los Angeles district
April 1973
Washington, D. C.
November 1973
*(+)
Boston
May 1973
Washintgon, D. C. Washington, D. C.
October 1973 December 1973
= production of heat-labile enterotoxin; (-) = no
Serotype
4 9 10 20 21 52 58 59 78 79 80 81 50 55 109
078:H11 015:H11 025:H? (OI47):H? 045:H? O149:HI0 08:H19 078:H11 0149:H10 0147:H? 0141:H4 08:H19 015:H11 09:H0149:HI0
173 211 212 214 216 218 210
06:H08:H0149:H19 0149:H19 0149:H19 0149:HI0 0101:H-
Adrenal cells" + + + + + + + + + + + +
+ + + + + + +
Rabbit ileal Ioop ]' 1.23 1.31 0.68 0.23 1.49 2.02 1.59 ND 1.24 0.84 1.98 0.66 ND ND 1.45 0.41 1.39 1.57 0.40 1.23 0.95 ND
(3) (3) (9) (6) (3) (3) (3) (4) (6) (7) (8)
(5) (7) (3) (3) (8) (3) (3)
Infant mouse f 0.055 0.135 0.106 0.125 0.068 0.107 0.076 0.071 0.138 0.064 0.068 0.052 0.125 0.120 0.095 0.129 0.060 0.105 0.065 0.055 0.060 0.111
production of heat-labile enterotoxin.
tData are ratios of the mean loop volume to length of intestine (ml/cm}; number of loops is in parentheses. Live cultures of all strains tested gave ratios of> 1.0. ND = not done. tData are ratios of the mean intestinal weight to remaining body weight; three mice per test were used; a ratio of >0.083 was considered to be a positive response.
are also shown in table 2. None were of classical enteropathogenic serotypes. The most common 0 serotype was 0149 (seven of 22 isolates); this serotype was associated with either HI 0 (four cases) or H 19 (three cases) flagellar an tigens. All but one of these serotypes were isolated from hamburger or sausage. Discussion
The present study demonstrates that (1) production of enterotoxin is relatively common among E. coli isolated from food and (2) such foods are therefore potentially important vehicles in the transmission of diarrheal disease of humans mediated by enterotoxigenic E. coli. Furthermore, this estimate of frequency must be considered minimal, since it is possible that some strains may have lost plasmids during storage [1].
E. coli in food have been suspected of being associated with outbreaks of gastrointestinal disease since 1903 [20]. In the 1940s, when certain serotypes of E. coli were identified as responsible for epidemic diarrheal disease in young children, it was thought important to look for these enteropathogenic serotypes in food. Although these serotypes were not found uncommonly in food [21], there have been relatively few reported outbreaks of food-borne diarrhea associated with these serotypes [22]. However, no testing of these strains for enterotoxin production has been reported. In one well-documented outbreak of diarrheal disease associated with a cheese product, a single 0 group (0124) of invasive E. coli was incriminated [23]. However, in spite of the ability to identify E. coli serologically, the number of recognized E. coli-associated diarrheal outbreaks has been small. Of 307 outbreaks of food-borne disease in
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French soft ripened cheese Crabmeat Summer sausage
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or animal origin. Since the food products were of animal origin, the likelihood of contamination by strains of E. coli of animal origin must be considerable. Indeed, if the strains were of animal origin, it is not known whether they would be pathogenic for humans. In the only reported experiment with volunteers [26J, a strain of enterotoxigenic E. coli from a pig was found to be virtually nondiarrhetic in humans. On the other hand, some of the enterotoxigenic serotypes identified in this study, such as 078:Hll or 015:Hll, have also been isolated from cases of severe cholera-like diarrhea in humans [5]; this finding would suggest that these organisms may be able to cause diarrhea in humans. It seems clear that to cause disease these strains must also contain surface antigens that facilitate colonization of the small bowel. Thus far, these antigens have been well characterized only in animal strains [27J and may account for the possible species specificity of the bacteria. A recent report [28J also suggests that similar surface antigens are found in some enterotoxigenic strains in humans. It is possible that the recognition of certain uncommon serotypes as enterotoxigenic may indicate that there are clones of E. coli identifiable by surface antigens that somehow enable these organisms to colonize the small bowel and to carry plasmids for production of enterotoxin. The potential for these organisms found in food items to be involved etiologically in diarrheal disease of humans, either sporadically or in point-source outbreaks, remains. It seems reasonable to suggest that laboratories involved in isolation of E. coli from food sources should not be limited to the identification of "pathogenic" serotypes but should develop the capabilities for determination of whether the strains are enterotoxigenic.
References 1. Sack, R. B. Human diarrheal disease caused by enterotoxigenic Escherichia coli. Annu. Rev. Microbiol.
29:333-353,1975. 2. Sack, R. B., Hirschhorn, N., Brownlee, I., Cash, R. A., Woodward, W. E., Sack, D. A. Enterotoxigenic Escherichia coli-associated diarrheal disease in Apache children. N. Engl. J. Med. 292:1041-1045,1975.
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1973 in the United States, none were attributed to E. coli) and 180 were of unknown cause [24]. In the same year, there was a total of 1,720 cases of waterborne disease, of which none was specifically ascribed to E. coli) although 1,065 were designated as "sewage poisoning." These observations suggest either that E. coli is rarely a cause of outbreaks of food- or waterborne diarrhea or that techniques for the determination of whether E. coli may be of etiologic significance have been inadequate. Since the practice of identifying classical enteropathogenic serotypes of E. coli has now been shown to be inadequate for the identification of enterotoxigenic organisms [2], and since the plasmids that control production of enterotoxin may theoretically be found in any serotype of E. coli) the latter possibility seems most likely. A recent investigation of the serotypes of enterotoxigenic E. coli associated with diarrhea in humans from widespread geographical areas [25] indicated that some well-characterized serotypes were found to be prevalent among the strains (serotypes 06:HI6, 08:H9, 015:Hll. 078:Hll, and 078:HI2); some of these serotypes were also found in the present study. One of these serotypes, 078, is a very common cause of diarrhea in calves and septicemia in chickens, and another, 0149, is responsible for >50% of all cases of diarrhea in piglets in several countries. However, most of the above mentioned O:H serotypes and 0 groups are uncommon in the intestine of healthy humans [25]. If we assume that enterotoxigenic E. coli are found frequently in food, is there any way to assess the risk to the food-consuming public? To act as significant vehicles of transmission, the food must contain the bacteria in high enough concentrations so that an infectious dose is ingested, and to produce disease these ingested organisms must be able to colonize the small bowel of humans. It is known from experiments in volunteers that a large inoculum of enterotoxigenic E. coli (__ 109-1010 ) is probably necessary to cause diarrheal disease in normal adults [26]. U nfortunately, in the present study no information regarding concentrations of organisms in the food was obtained. With regard to colonization of the small bowel, it is unclear whether the organisms isolated in the present study were of human
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