ANNUAL REVIEWS
Further
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Copyright
1975. All rights reserved
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
HUMAN DIARRHEAL DISEASE
+1660
CAUSED BY ENTEROTOXIGENIC ESCHERICHIA COLI R. Bradley Sack Department of Medicine. The Johns Hopkins University School of Medicine and Baltimore City Hospitals. Baltimore. Maryland 21224
CONTENTS INTRODUCTION..
..............................................................
333
E. COLI AS A NORMAL INHABITANT OF THE GASTROINTESTINAL TRACT OF HUMANS.............................................................
E. COLI-ASSOCIATED DIARRHEAS IN HISTORICAL PERSPECTIVE............... THE RECOGNITION OF
E. COLI
334 335
ENTEROTOXIN-MEDIATED DIARRHEA IN
HUMANS............................................................. CHARACTERIZATION OF E. COLI ENTEROTOXINS .
............................. Heat-Stable and Heat-Labile Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 338 Cultural Conditions for Production of Enterotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal and Cell Models for Detection of E. coli Enterotoxins. . . . . . . . . . . . . . . . . . . 339 Physiological Effects 0/ E. coli Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 Cellular and Biochemical Effects of E. coli Enterotoxins. . . . . . . . . . . . . . . . . . . . . . . . 342 Purification of E. coli Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 344 Genetic Control of Enterotoxin Production in E. coli..... . . . . . . . . . . . . . . . . . . . . . . Relationship of E. coli Serotypes to Enterotoxin Production . .. .. . . . . . . . . . . . . . . . . 345 346 Importance 0/ K Antigens to Pathogenicity of Animal Enterotoxigenic E. coli... . . . . 347 Immunological Studies 0/ E. coli Enterotoxins ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Epidemiologic Studies 0/ Enterotoxigenic E. coli .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Enterotoxin Assays in Parenteral Isolates of E. coli .... . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis and Clinical Spectrum of Disease Produced by Enterotoxigenic E. coli. 350 Laboratory Diagnostic Methods ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 Possibilities for Immunological Control of Disease Due to Enterotoxigenic E. coli ... 350 351 Areas for Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
336 338
INTRODUCTION
Diarrheal disease is one of the leading causes of death in children under five years of age who live in developing countries (33) and is a significant cause of adult morbidity in these areas. At present there are no specific means of preventing this disease other than long-term programs of improved water supply and sanitation. 333
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
334
SACK
Part of the inability to cope with this problem is the general lack of understanding of the etiology of diarrheal illnesses. Bacteriological studies of diarrhea in different geographic areas have all given essentially the same result: the vast majority of cases (60-80%) are associated with no known recognized enteropathogen. As the result of recent studies of Escherichia coli, however, our ignorance in this area should diminish, and new approaches to disease control may become apparent. E. coli, when confined to their usual ecological niche in the intestinal tract of man, have been generally regarded as "normal flora," worthy only of a benign report from the bacteriology laboratory. The single recognized exception is found in young children under two years of age with diarrhea; their fecal E. coli can be identified as belonging to a small number of enteropathogenic serotypes. In this clinical setting these E. coli have been presumed to be responsible for the diarrheal illness. Al though this information is of little use to the physician for therapeutic purposes, it has been of epidemiologic value in recognizing nursery outbreaks of diarrhea caused by single serotypes of E. coli. Prior to the late 1960s, our understanding of E. coli as an enteropathogen was limited to the rather rigidly defined but poorly understood situation mentioned above. This concept rather abruptly changed following studies of the pathogenesis of cholera in which the importance of an enterotoxin in mediating diarrheal disease was recognized, thereby leading to investigation of noncholera patients with similar clinical features. During the past approximately six years, the recognized role of enterotoxigenic E. coli in producing human diarrheal disease has expanded to encompass a wide clinical spectrum, ranging from mild travelers' diarrhea to severe choleralike illness and involving essentially all age groups, from the nursery to geriatric populations. Much of the present information on human enterotoxigenic E. coli is a direct outgrowth of slightly earlier parallel studies in veterinary medicine that have clearly documented the role of E. coli in producing severe diarrheal disease in the young of large domestic animals. The detailed consideration of these aspects of animal E. coli diarrheal disease, however, is outside the scope of this review; only those studies directly related to human enterotoxigenic E. coli are discussed. Also omitted from this review are other related bacteria (and their products) and the more recently discovered viruses known to cause diarrheal diseases. These include the invasive Shigellalike E. coli (90), Clostridium perfringens (17), Bacillus cereus (86), and their enterotoxins, as well as the filterable agents like the Norwalk agent (5) and others more recently discovered (91), including orbiviruses (4a). This review therefore deals particularly with events of the past seven years, not necessarily exhaustively, but in a way that may bring some order out of a presently confusing and yet incomplete story, as evidenced by previous short reviews of this topic (3, 28, 34, 85).
E COLI AS A NORMAL INHABITANT OF THE GASTROINTESTINAL TRACT OF HUMANS Within a few days after birth infants acquire E. coli. largely from the mother and other intimate environmental surroundings; these quickly become established in the
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
HUMAN DIARRHEAL DISEASE
335
large bowel as the predominant aerobic inhabitant. For reasons not completely understood, these organisms selectively occupy only the distal part of the intestinal tract, and in this location are relatively permanent, usually non-disease-related, and in some ways beneficial to the host (30). Some of the partially understood mecha nisms for keeping E coli and other normal large bowel bacteria out of the proximal small intestine include (a) the acid barrier of the stomach, which is normally hostile to ingested fecal organisms, (h) the normal rapid transit time of the proximal bowel, and (c) the presence of antibodies in the secretory contents of this area. Whenever any of these normal defense mechanisms are impaired, colonization of the small bowel with enteric organisms may occur, resulting in diverse syndromes of diarrhea and malabsorption, exemplified most dramatically by the blind loop syndrome (30). Although E coli is undoubtedly less important than other enteric organisms, such as Bacteroides, in producing these overgrowth syndromes, it is at the least a marker of disordered ecology in the intestinal tract. E. COLI-ASSOCIATED DIARRHEAS IN HISTORICAL PERSPECTIVE
The first reports of E. coli-associated diarrheal disease came from nursery epidemics in the mid 1940s (6, 29, 55, 88). Prior to this time E coli was not recognized as a pathogen as long as it was confined to the gastrointestinal tract. During these nursery outbreaks, mortality rates were high, often as much as 50% (29); autopsy studies showed minimal inflammation of the small bowel, somewhat edematous mucosa, and an absence of ulceration. Bacteriologically, specimens of small intestine and stool showed a predominantly E. coli flora belonging to common serotypes that could be identified with specific antisera (6, 29, 54, 88). One of the early and widely recognized enteropathogenic isolates, originally designated as D433 (88), was later classified as serotype 0111, the designation by which we now recognize it. Although these serotypes could be identified in low frequency from normal children and adults (6), they were found in as high as 80-100% of children with diarrhea and were thus epidemiologically incriminated as the cause of diarrhea and designated enteropatho genic. With the investigation of more epidemics, the number of enteropathogenic serotypes grew, until at present there are approximately 20--25 recognized, depend ing upon the geographical areas involved (66, 75). In addition to this epidemiologic evidence for pathogenicity, volunteer experi ments in both adults (25, 51, 55) and a child (65) with two of these serotypes (0111, B4; and 055, B�) confirmed that the ingestion of large numbers of these organisms regularly resulted in diarrhea and that the strains could be recovered in large numbers from the feces. As these serotypes were identified, investigators were attempting to elucidate the virulence factors of these organisms in animal models. In studies done in India, De and co-workers (10) investigated E. coli isolated from patients with choleralike illness, and from children with infantile diarrhea, in the rabbit ileal loop. These workers found strains of E. coli that gave an ileal loop reaction identical to that given by Vibrio cholerae. They did not, however, test culture filtrates, or look further for "enterotoxins. " They concluded that "antigenically identical strains may be
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
336
SACK
either toxigenic or nontoxigenic, " and suspected that "serology will give no final proof of pathogenicity of a strain " (10). Later studies in rabbit ileal loops by Taylor et al (89) also showed a poor correla tion between the serotype of the E coli isolate and the ileal loop secretory response, but a good correlation with the origin of the strain, i.e. whether they came from children with or without diarrhea. These studies also suggested that not all enteropa thogenic serotypes possessed the virulence factor for this animal model. In one study (87) these investigators identified an enterotoxinlike material in chloroform-killed whole cultures that resulted in positive rabbit ileal loop reactions. This material was extremely labile, however, and was not characterized further. Other than the work of Taylor, De, and their co-workers, there were few studies of diarrhea-producing E coli until fairly recently. One such investigation (16) showed, as had a previous one (60), that certain of the enteropathogenic serotypes were clearly invasive, resulting in a Shigellalike picture in rabbit ileal loops. THE RECOGNITION OF E. COLI ENTEROTOXIN-MEDIATED DIARRHEA IN HUMANS
In 1967 the first reports of enterotoxigenic E coli isolated from young animals with diarrhea appeared (56, 81). Over the next three years, it became clear that these organisms were responsible for a major portion of severe diarrheal disease in young domestic animals, were associated with a specific K antigen (50, 83), and produced two enterotoxins (79), one heat labile, and the other heat stable, and both plasmid controlled (42, 80). Details of these studies are given later. Approximately one year later (1968) in Calcutta, studies were being made in adult patients who had choleralike illness, but from whom no V. cholerae could be isolated (31, 73). The c1inic�1 picture of these patients upon admission was indistinguishable from that of cholera, including the production of rice-water stool, profound dehy dration, and shock. The only clear differential feature was the relative brevity of diarrhea; the stool output usually ceased within 24--30 hr of admission (without antibiotic therapy) and was thus significantly less than that seen in cholera patients. Intubation studies during the acute phase of diarrhea (within 16-24 hr of admission) demonstrated a large population of E coli (107-109/ml) in the proximal small bowel, and these organisms were often of only one or two serotypes; during con valescence these organisms disappeared from the small bowel. Furthermore, the physiological abnormalities in fluid and electrolyte movement in the small bowel were similar in kind, although less in magnitude, to those seen in cholera (2). Thus both the bacteriological and physiological changes in the small bowel were strikingly similar to those of cholera. Using cultural techniques similar to those used in production of V. cholerae enterotoxin, it was then possible to demonstrate a heat labile, nondialyzable, immunogenic enterotoxin from these small bowel isolates (73). Paired sera from these patients demonstrated a marked rise in anti-E coli en terotoxin titers in the majority. (74). Additional studies in volunteers (18) with similar enterotoxigenic strains (from other sources in Southeast Asia) directly dem onstrated the diarrheagenic capabilities of these enterotoxigenic E. coli in man. A
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
Table
I
Clinical data from patients with enterotoxigenic E. coli diarrheaa E.
coli
Du ration of
Duration of
Total Stool
in Jejunum
Enterotoxigenic
Diarrhea before
Diarrhea after
Output in
(log IO/ml)
Jejunal
Hospitalization
Hospitalization
During Acute
(years)
Hospital
E. coli
Number
Age
(hours)
(hours)
(liters)
Diarrhea
lsolatesb
Serotypes
Acute
ConvaJescent
142
32
15
14
2.6
>6.0
10/10
078:HlI (all)
12
3.1
6.5
5/5
085:H7 (all)
9 09
7.0
1/5
078:HII
107 52 30
300
Patient's Identification
210
22
8
230
60
15
197c
60
3
0.3
4
24
5.7
7.3
a AU patients were admitted with severe saline depletion and acidosis; none were treated with antibiotics.
bEnterotoxigenic strains/total tested. C Patient aJso had
V. choierae isolated from
small bowel.
5/5
E. E. coli
078:HI
(4)
016:H39 (I)
coli Antitoxin
Titers (units/ml)
769
:I: c:: a:: )z '='
�
:;:tl :;:tl :I:
�
t""'
52 CIl
�
CIl tTl
w W -.J
338
SACK
summary of the clinical and bacteriologic data from four of the patients from Calcutta is shown in Table 1. Significantly, one of these patients also simultaneously harbored V. cholerae in the upper small bowel. CHARACTERIZATION OF E. COLI ENTEROTOXINS
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
Heat-Stable and Heat-Labile Enterotoxins
Although the heat-stability characteristics of animal E. coli enterotoxins were de scribed as early as 1970 (79), there remained some confusion regarding this property in human strains until about 1973. As a result, it is difficult to interpret some of the early physiologic and immunologic data, in which crude enterotoxin preparations that undoubtedly contained mixtures of both enterotoxins were used. Human enterotoxigenic E. coli are also now known to produce at least two general types of enterotoxins: heat-stable (ST) and heat-labile (LT). In fact, almost all human strains studied to date have been shown to produce both types. Whether or not more of one type than the other is produced by any given strain can be controlled to some extent by altering the cultural conditions. Vigorous aeration by shaking during incubation usually increases the amount of ST produced, whereas still cultures with a large surface volume ratio seem to result in more LT production (R. B. Sack, unpublished observations). Recently, however, strains that produce only ST or only LT have been isolated from humans (R. B. Sack, D. A. Sack, M. Merson, G. Morris, unpublished observa tions), thereby allowing observation of all combinations of enterotoxin production. The relative importance of these strains has yet to be determined. The definition of LT and ST is still somewhat arbitrary; generally enterotoxin that withstands 100°C for 15 min is considered heat stable. Most LT preparations are destroyed by temperatures considerably less than this, i.e. 60°C for 30 min. The different physiologic, immunologic, and genetic aspects of these two E. coli en terotoxins are discussed later. Cultural Conditions for Production of Enterotoxin
enterotoxins can be produced in a variety of media. One of the earliest to be tried was Syncase (27) because of its marked success in cholera enterotoxin production. This semisynthetic medium allows the organisms to elaborate both ST and LT, depending on the aeration of the culture. Other media have been success fully used, including peptone dialysate broth (58), and semisynthetic media with yeast extract supplementation (24). The carbon sources in the media do not appear to be critical. Cultures are usually grown at 37°C. Although toxin production also occurs at 30°C, there seems to be no advantage to these lower incubation temperatures. Enterotoxin appears in the medium during the log phase of growth and can be detected in as little as 8 hr (48, 73). Peak concentrations are reached in approxi mately 24 hr, then plateau over the next few days (73). Crude enterotoxin preparations can be obtained from cultures by centrifugation, dialysis of the supernate, and lyophilization or concentration by membrane filtra tion. Lyophilized LT preparations are stable at refrigerator or room temperature for E. coli
339
HUMAN DIARRHEAL DISEASE
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
at least six months (R. B. Sack, unpublished data). Liquid LT preparations may be stored at 5°C, although some loss of activity occurs within a period of weeks. Enterotoxigenic E coli isolates have been kept on nutrient agar slants and stabs with infrequent subculture for up to six years without loss of the ability to produce enterotoxin. This characteristic may not be as stable in all strains, however, and there have been some reports of strains losing their enterotoxin-producing capacity after repeated subculture (R. B. Sack, unpublished data). Animal and Cell Models for Detection of E coli Enterotoxins
There- are a large number of models available for enterotoxin assays. Most were originally used for studies of V. cholerae enterotoxin and have been adapted for E coli. Unfortunately, none are truly in vitro assays; all require a biologically active system. A summary of the models and the enterotoxins they detect is given in Table 2. The adult rabbit ileal loop (10), which is probably the most widely used intestinal assay for cholera toxin, is also used extensively to assay E coli enterotoxin. The response to LT is essentially identical to thai to V. cholerae enterotoxin. Fluid accumulation is maximal at about 18 hr, with a ratio of volume (in milliliters) to length (in centimeters) of about 2.0-2.5, depending upon the rabbit size. Figure 1, which illustrates an assay for E coli antitoxin, shows control loops for V. cholera e and E. coli enterotoxins that appear identical. Figure 2 shows a titration of crude enterotoxin preparations of three E. coli isolates, as compared to V. cholerae, Inaba Table 2 Biological assays for enterotoxins of E. coli and V. choleraea E. coli
Assay Rabbit ileal loop (18 hr)b (6-7 hr) Infant rabbit (6-7 hr) Infant mouse (4 hr) Rabbit skin Mouse adrenal cells Chinese hamster ovarian cells Rat epididymal fat cells Mouse thymocytes Dog jejunal Thiry-Vella loops (1-2 hr) (4-16 hr)
Heat-Stable Enterotoxin
E. coli
Heat-Labile Enterotoxin
Enterotoxin
V. cholerae
+
+
+
+
+
+
+
+
+
±c
±
+
+
+
+
+
+
±c
+
+
+
+
+
+
a Ability of assay to detect these three enterotoxin preparations is indicated by a positive or negative sign. . bTime at which assay is terminated. c Differing results in different laboratories.
340
SACK
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
"100
E. coli antitoxin titration in the 18 hr rabbit ileal loop model, showing neutraliza
Figure J
tion of a standard E. coli enterotoxin (LT) by increasing dilutions of antisera. Antisera and
enterotoxin were incubated I hr at 37°C prior to injection into the loop. Dilutions less than 1:300 completely neutralized the enterotoxin effect; preimmune sera at 1:10 dilution did not
neutralize the effect. Positive (V. cholerae and E. coli LT enterotoxin) and negative [phos phate-buffered saline (PBS)] controls are included, which demonstrate appropriate responses.
2.4
� « a: J: f
Further
Quick links to online content
Copyright
1975. All rights reserved
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
HUMAN DIARRHEAL DISEASE
+1660
CAUSED BY ENTEROTOXIGENIC ESCHERICHIA COLI R. Bradley Sack Department of Medicine. The Johns Hopkins University School of Medicine and Baltimore City Hospitals. Baltimore. Maryland 21224
CONTENTS INTRODUCTION..
..............................................................
333
E. COLI AS A NORMAL INHABITANT OF THE GASTROINTESTINAL TRACT OF HUMANS.............................................................
E. COLI-ASSOCIATED DIARRHEAS IN HISTORICAL PERSPECTIVE............... THE RECOGNITION OF
E. COLI
334 335
ENTEROTOXIN-MEDIATED DIARRHEA IN
HUMANS............................................................. CHARACTERIZATION OF E. COLI ENTEROTOXINS .
............................. Heat-Stable and Heat-Labile Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338 338 Cultural Conditions for Production of Enterotoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Animal and Cell Models for Detection of E. coli Enterotoxins. . . . . . . . . . . . . . . . . . . 339 Physiological Effects 0/ E. coli Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342 Cellular and Biochemical Effects of E. coli Enterotoxins. . . . . . . . . . . . . . . . . . . . . . . . 342 Purification of E. coli Enterotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344 344 Genetic Control of Enterotoxin Production in E. coli..... . . . . . . . . . . . . . . . . . . . . . . Relationship of E. coli Serotypes to Enterotoxin Production . .. .. . . . . . . . . . . . . . . . . 345 346 Importance 0/ K Antigens to Pathogenicity of Animal Enterotoxigenic E. coli... . . . . 347 Immunological Studies 0/ E. coli Enterotoxins ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348 Epidemiologic Studies 0/ Enterotoxigenic E. coli .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349 Enterotoxin Assays in Parenteral Isolates of E. coli .... . . . . . . . . . . . . . . . . . . . . . . . . Pathogenesis and Clinical Spectrum of Disease Produced by Enterotoxigenic E. coli. 350 Laboratory Diagnostic Methods ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350 Possibilities for Immunological Control of Disease Due to Enterotoxigenic E. coli ... 350 351 Areas for Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
336 338
INTRODUCTION
Diarrheal disease is one of the leading causes of death in children under five years of age who live in developing countries (33) and is a significant cause of adult morbidity in these areas. At present there are no specific means of preventing this disease other than long-term programs of improved water supply and sanitation. 333
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
334
SACK
Part of the inability to cope with this problem is the general lack of understanding of the etiology of diarrheal illnesses. Bacteriological studies of diarrhea in different geographic areas have all given essentially the same result: the vast majority of cases (60-80%) are associated with no known recognized enteropathogen. As the result of recent studies of Escherichia coli, however, our ignorance in this area should diminish, and new approaches to disease control may become apparent. E. coli, when confined to their usual ecological niche in the intestinal tract of man, have been generally regarded as "normal flora," worthy only of a benign report from the bacteriology laboratory. The single recognized exception is found in young children under two years of age with diarrhea; their fecal E. coli can be identified as belonging to a small number of enteropathogenic serotypes. In this clinical setting these E. coli have been presumed to be responsible for the diarrheal illness. Al though this information is of little use to the physician for therapeutic purposes, it has been of epidemiologic value in recognizing nursery outbreaks of diarrhea caused by single serotypes of E. coli. Prior to the late 1960s, our understanding of E. coli as an enteropathogen was limited to the rather rigidly defined but poorly understood situation mentioned above. This concept rather abruptly changed following studies of the pathogenesis of cholera in which the importance of an enterotoxin in mediating diarrheal disease was recognized, thereby leading to investigation of noncholera patients with similar clinical features. During the past approximately six years, the recognized role of enterotoxigenic E. coli in producing human diarrheal disease has expanded to encompass a wide clinical spectrum, ranging from mild travelers' diarrhea to severe choleralike illness and involving essentially all age groups, from the nursery to geriatric populations. Much of the present information on human enterotoxigenic E. coli is a direct outgrowth of slightly earlier parallel studies in veterinary medicine that have clearly documented the role of E. coli in producing severe diarrheal disease in the young of large domestic animals. The detailed consideration of these aspects of animal E. coli diarrheal disease, however, is outside the scope of this review; only those studies directly related to human enterotoxigenic E. coli are discussed. Also omitted from this review are other related bacteria (and their products) and the more recently discovered viruses known to cause diarrheal diseases. These include the invasive Shigellalike E. coli (90), Clostridium perfringens (17), Bacillus cereus (86), and their enterotoxins, as well as the filterable agents like the Norwalk agent (5) and others more recently discovered (91), including orbiviruses (4a). This review therefore deals particularly with events of the past seven years, not necessarily exhaustively, but in a way that may bring some order out of a presently confusing and yet incomplete story, as evidenced by previous short reviews of this topic (3, 28, 34, 85).
E COLI AS A NORMAL INHABITANT OF THE GASTROINTESTINAL TRACT OF HUMANS Within a few days after birth infants acquire E. coli. largely from the mother and other intimate environmental surroundings; these quickly become established in the
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
HUMAN DIARRHEAL DISEASE
335
large bowel as the predominant aerobic inhabitant. For reasons not completely understood, these organisms selectively occupy only the distal part of the intestinal tract, and in this location are relatively permanent, usually non-disease-related, and in some ways beneficial to the host (30). Some of the partially understood mecha nisms for keeping E coli and other normal large bowel bacteria out of the proximal small intestine include (a) the acid barrier of the stomach, which is normally hostile to ingested fecal organisms, (h) the normal rapid transit time of the proximal bowel, and (c) the presence of antibodies in the secretory contents of this area. Whenever any of these normal defense mechanisms are impaired, colonization of the small bowel with enteric organisms may occur, resulting in diverse syndromes of diarrhea and malabsorption, exemplified most dramatically by the blind loop syndrome (30). Although E coli is undoubtedly less important than other enteric organisms, such as Bacteroides, in producing these overgrowth syndromes, it is at the least a marker of disordered ecology in the intestinal tract. E. COLI-ASSOCIATED DIARRHEAS IN HISTORICAL PERSPECTIVE
The first reports of E. coli-associated diarrheal disease came from nursery epidemics in the mid 1940s (6, 29, 55, 88). Prior to this time E coli was not recognized as a pathogen as long as it was confined to the gastrointestinal tract. During these nursery outbreaks, mortality rates were high, often as much as 50% (29); autopsy studies showed minimal inflammation of the small bowel, somewhat edematous mucosa, and an absence of ulceration. Bacteriologically, specimens of small intestine and stool showed a predominantly E. coli flora belonging to common serotypes that could be identified with specific antisera (6, 29, 54, 88). One of the early and widely recognized enteropathogenic isolates, originally designated as D433 (88), was later classified as serotype 0111, the designation by which we now recognize it. Although these serotypes could be identified in low frequency from normal children and adults (6), they were found in as high as 80-100% of children with diarrhea and were thus epidemiologically incriminated as the cause of diarrhea and designated enteropatho genic. With the investigation of more epidemics, the number of enteropathogenic serotypes grew, until at present there are approximately 20--25 recognized, depend ing upon the geographical areas involved (66, 75). In addition to this epidemiologic evidence for pathogenicity, volunteer experi ments in both adults (25, 51, 55) and a child (65) with two of these serotypes (0111, B4; and 055, B�) confirmed that the ingestion of large numbers of these organisms regularly resulted in diarrhea and that the strains could be recovered in large numbers from the feces. As these serotypes were identified, investigators were attempting to elucidate the virulence factors of these organisms in animal models. In studies done in India, De and co-workers (10) investigated E. coli isolated from patients with choleralike illness, and from children with infantile diarrhea, in the rabbit ileal loop. These workers found strains of E. coli that gave an ileal loop reaction identical to that given by Vibrio cholerae. They did not, however, test culture filtrates, or look further for "enterotoxins. " They concluded that "antigenically identical strains may be
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
336
SACK
either toxigenic or nontoxigenic, " and suspected that "serology will give no final proof of pathogenicity of a strain " (10). Later studies in rabbit ileal loops by Taylor et al (89) also showed a poor correla tion between the serotype of the E coli isolate and the ileal loop secretory response, but a good correlation with the origin of the strain, i.e. whether they came from children with or without diarrhea. These studies also suggested that not all enteropa thogenic serotypes possessed the virulence factor for this animal model. In one study (87) these investigators identified an enterotoxinlike material in chloroform-killed whole cultures that resulted in positive rabbit ileal loop reactions. This material was extremely labile, however, and was not characterized further. Other than the work of Taylor, De, and their co-workers, there were few studies of diarrhea-producing E coli until fairly recently. One such investigation (16) showed, as had a previous one (60), that certain of the enteropathogenic serotypes were clearly invasive, resulting in a Shigellalike picture in rabbit ileal loops. THE RECOGNITION OF E. COLI ENTEROTOXIN-MEDIATED DIARRHEA IN HUMANS
In 1967 the first reports of enterotoxigenic E coli isolated from young animals with diarrhea appeared (56, 81). Over the next three years, it became clear that these organisms were responsible for a major portion of severe diarrheal disease in young domestic animals, were associated with a specific K antigen (50, 83), and produced two enterotoxins (79), one heat labile, and the other heat stable, and both plasmid controlled (42, 80). Details of these studies are given later. Approximately one year later (1968) in Calcutta, studies were being made in adult patients who had choleralike illness, but from whom no V. cholerae could be isolated (31, 73). The c1inic�1 picture of these patients upon admission was indistinguishable from that of cholera, including the production of rice-water stool, profound dehy dration, and shock. The only clear differential feature was the relative brevity of diarrhea; the stool output usually ceased within 24--30 hr of admission (without antibiotic therapy) and was thus significantly less than that seen in cholera patients. Intubation studies during the acute phase of diarrhea (within 16-24 hr of admission) demonstrated a large population of E coli (107-109/ml) in the proximal small bowel, and these organisms were often of only one or two serotypes; during con valescence these organisms disappeared from the small bowel. Furthermore, the physiological abnormalities in fluid and electrolyte movement in the small bowel were similar in kind, although less in magnitude, to those seen in cholera (2). Thus both the bacteriological and physiological changes in the small bowel were strikingly similar to those of cholera. Using cultural techniques similar to those used in production of V. cholerae enterotoxin, it was then possible to demonstrate a heat labile, nondialyzable, immunogenic enterotoxin from these small bowel isolates (73). Paired sera from these patients demonstrated a marked rise in anti-E coli en terotoxin titers in the majority. (74). Additional studies in volunteers (18) with similar enterotoxigenic strains (from other sources in Southeast Asia) directly dem onstrated the diarrheagenic capabilities of these enterotoxigenic E. coli in man. A
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Table
I
Clinical data from patients with enterotoxigenic E. coli diarrheaa E.
coli
Du ration of
Duration of
Total Stool
in Jejunum
Enterotoxigenic
Diarrhea before
Diarrhea after
Output in
(log IO/ml)
Jejunal
Hospitalization
Hospitalization
During Acute
(years)
Hospital
E. coli
Number
Age
(hours)
(hours)
(liters)
Diarrhea
lsolatesb
Serotypes
Acute
ConvaJescent
142
32
15
14
2.6
>6.0
10/10
078:HlI (all)
12
3.1
6.5
5/5
085:H7 (all)
9 09
7.0
1/5
078:HII
107 52 30
300
Patient's Identification
210
22
8
230
60
15
197c
60
3
0.3
4
24
5.7
7.3
a AU patients were admitted with severe saline depletion and acidosis; none were treated with antibiotics.
bEnterotoxigenic strains/total tested. C Patient aJso had
V. choierae isolated from
small bowel.
5/5
E. E. coli
078:HI
(4)
016:H39 (I)
coli Antitoxin
Titers (units/ml)
769
:I: c:: a:: )z '='
�
:;:tl :;:tl :I:
�
t""'
52 CIl
�
CIl tTl
w W -.J
338
SACK
summary of the clinical and bacteriologic data from four of the patients from Calcutta is shown in Table 1. Significantly, one of these patients also simultaneously harbored V. cholerae in the upper small bowel. CHARACTERIZATION OF E. COLI ENTEROTOXINS
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
Heat-Stable and Heat-Labile Enterotoxins
Although the heat-stability characteristics of animal E. coli enterotoxins were de scribed as early as 1970 (79), there remained some confusion regarding this property in human strains until about 1973. As a result, it is difficult to interpret some of the early physiologic and immunologic data, in which crude enterotoxin preparations that undoubtedly contained mixtures of both enterotoxins were used. Human enterotoxigenic E. coli are also now known to produce at least two general types of enterotoxins: heat-stable (ST) and heat-labile (LT). In fact, almost all human strains studied to date have been shown to produce both types. Whether or not more of one type than the other is produced by any given strain can be controlled to some extent by altering the cultural conditions. Vigorous aeration by shaking during incubation usually increases the amount of ST produced, whereas still cultures with a large surface volume ratio seem to result in more LT production (R. B. Sack, unpublished observations). Recently, however, strains that produce only ST or only LT have been isolated from humans (R. B. Sack, D. A. Sack, M. Merson, G. Morris, unpublished observa tions), thereby allowing observation of all combinations of enterotoxin production. The relative importance of these strains has yet to be determined. The definition of LT and ST is still somewhat arbitrary; generally enterotoxin that withstands 100°C for 15 min is considered heat stable. Most LT preparations are destroyed by temperatures considerably less than this, i.e. 60°C for 30 min. The different physiologic, immunologic, and genetic aspects of these two E. coli en terotoxins are discussed later. Cultural Conditions for Production of Enterotoxin
enterotoxins can be produced in a variety of media. One of the earliest to be tried was Syncase (27) because of its marked success in cholera enterotoxin production. This semisynthetic medium allows the organisms to elaborate both ST and LT, depending on the aeration of the culture. Other media have been success fully used, including peptone dialysate broth (58), and semisynthetic media with yeast extract supplementation (24). The carbon sources in the media do not appear to be critical. Cultures are usually grown at 37°C. Although toxin production also occurs at 30°C, there seems to be no advantage to these lower incubation temperatures. Enterotoxin appears in the medium during the log phase of growth and can be detected in as little as 8 hr (48, 73). Peak concentrations are reached in approxi mately 24 hr, then plateau over the next few days (73). Crude enterotoxin preparations can be obtained from cultures by centrifugation, dialysis of the supernate, and lyophilization or concentration by membrane filtra tion. Lyophilized LT preparations are stable at refrigerator or room temperature for E. coli
339
HUMAN DIARRHEAL DISEASE
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
at least six months (R. B. Sack, unpublished data). Liquid LT preparations may be stored at 5°C, although some loss of activity occurs within a period of weeks. Enterotoxigenic E coli isolates have been kept on nutrient agar slants and stabs with infrequent subculture for up to six years without loss of the ability to produce enterotoxin. This characteristic may not be as stable in all strains, however, and there have been some reports of strains losing their enterotoxin-producing capacity after repeated subculture (R. B. Sack, unpublished data). Animal and Cell Models for Detection of E coli Enterotoxins
There- are a large number of models available for enterotoxin assays. Most were originally used for studies of V. cholerae enterotoxin and have been adapted for E coli. Unfortunately, none are truly in vitro assays; all require a biologically active system. A summary of the models and the enterotoxins they detect is given in Table 2. The adult rabbit ileal loop (10), which is probably the most widely used intestinal assay for cholera toxin, is also used extensively to assay E coli enterotoxin. The response to LT is essentially identical to thai to V. cholerae enterotoxin. Fluid accumulation is maximal at about 18 hr, with a ratio of volume (in milliliters) to length (in centimeters) of about 2.0-2.5, depending upon the rabbit size. Figure 1, which illustrates an assay for E coli antitoxin, shows control loops for V. cholera e and E. coli enterotoxins that appear identical. Figure 2 shows a titration of crude enterotoxin preparations of three E. coli isolates, as compared to V. cholerae, Inaba Table 2 Biological assays for enterotoxins of E. coli and V. choleraea E. coli
Assay Rabbit ileal loop (18 hr)b (6-7 hr) Infant rabbit (6-7 hr) Infant mouse (4 hr) Rabbit skin Mouse adrenal cells Chinese hamster ovarian cells Rat epididymal fat cells Mouse thymocytes Dog jejunal Thiry-Vella loops (1-2 hr) (4-16 hr)
Heat-Stable Enterotoxin
E. coli
Heat-Labile Enterotoxin
Enterotoxin
V. cholerae
+
+
+
+
+
+
+
+
+
±c
±
+
+
+
+
+
+
±c
+
+
+
+
+
+
a Ability of assay to detect these three enterotoxin preparations is indicated by a positive or negative sign. . bTime at which assay is terminated. c Differing results in different laboratories.
340
SACK
Annu. Rev. Microbiol. 1975.29:333-354. Downloaded from www.annualreviews.org Access provided by New York University - Bobst Library on 02/02/15. For personal use only.
"100
E. coli antitoxin titration in the 18 hr rabbit ileal loop model, showing neutraliza
Figure J
tion of a standard E. coli enterotoxin (LT) by increasing dilutions of antisera. Antisera and
enterotoxin were incubated I hr at 37°C prior to injection into the loop. Dilutions less than 1:300 completely neutralized the enterotoxin effect; preimmune sera at 1:10 dilution did not
neutralize the effect. Positive (V. cholerae and E. coli LT enterotoxin) and negative [phos phate-buffered saline (PBS)] controls are included, which demonstrate appropriate responses.
2.4
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