Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens.

Diarrheagenic Escherichia coli

Since their initial recognition 20 years ago, Shiga toxin-producing Escherichia coli (STEC) strains have emerged as an important cause of serious human gastrointestinal disease, which may result in life-threatening complications such as hemolytic-uremic syndrome. Food-borne outbreaks of STEC disease appear to be increasing and, when mass-produced and mass-distributed foods are concerned, can involve large numbers of people. Development of therapeutic and preventative strategies to combat STEC disease requires a thorough understanding of the mechanisms by which STEC organisms colonize the human intestinal tract and cause local and systemic pathological changes. While our knowledge remains incomplete, recent studies have improved our understanding of these processes, particularly the complex interaction between Shiga toxins and host cells, which is central to the pathogenesis of STEC disease. In addition, several putative accessory virulence factors have been identified and partly characterized. The capacity to limit the scale and severity of STEC disease is also dependent upon rapid and sensitive diagnostic procedures for analysis of human samples and suspect vehicles. The increased application of advanced molecular technologies in clinical laboratories has significantly improved our capacity to diagnose STEC infection early in the course of disease and to detect low levels of environmental contamination. This, in turn, has created a potential window of opportunity for future therapeutic intervention.

Pathogenesis and Diagnosis of Shiga Toxin-Producing Escherichia coli Infections

Verocytotoxin (VT)-producing Escherichia coli (VTEC) are a newly recognized group of enteric pathogens which are increasingly being recognized as common causes of diarrhea in some geographic settings. Outbreak studies indicate that most patients with VTEC infection develop mild uncomplicated diarrhea. However, a significant risk of two serious and potentially life-threatening complications, hemorrhagic colitis and the hemolytic uremic syndrome, makes VTEC infection a public health problem of serious concern. The main reservoirs of VTEC appear to be the intestinal tracts of animals, and foods of animal (especially bovine) origin are probably the principal sources for human infection. The term VT refers to a family of subunit exotoxins with high biological activity. Individual VTEC strains elaborate one or both of at least two serologically distinct, bacteriophage-mediated VTs (VT1 and VT2) which are closely related to Shiga toxin and are thus also referred to as Shiga-like toxins. The holotoxins bind to cells, via their B subunits, to a specific receptor which is probably the glycolipid, globotriosyl ceramide (Gb3). Binding is followed by internalization of the A subunit, which, after it is proteolytically nicked and reduced to the A1 fragment, inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28S ribosomal ribonucleic acid. The mechanism of VTEC diarrhea is still controversial, and the relative roles of locally acting VT and "attaching and effacing adherence" of VTEC to the mucosa have yet to be resolved. There is increasing evidence that hemolytic uremic syndrome and possibly hemorrhagic colitis result from the systemic action of VT on vascular endothelial cells. The role of antitoxic immunity in preventing the systemic complications of VTEC infection is being explored. Antibiotics appear to be contraindicated in the treatment of VTEC infection. The most common VTEC serotype associated with human disease is O157:H7, but over 50 different VT-positive O:H serotypes have now been identified. The best strategies for diagnosing human VTEC infection include testing for the presence of free VT in fecal filtrates and examining fecal cultures for VTEC by means of deoxyribonucleic acid probes that specify genes encoding VT1 and VT2. Both methods are currently confined to specialized laboratories and await commercial development for wider use. In the meantime, most laboratories should continue to screen for the most common human VTEC serotype, O157:H7, using a sorbitol-containing MacConkey medium.

Infection by verocytotoxin-producing Escherichia coli.

Forty pediatric patients with idiopathic hemolytic uremic syndrome (HUS) were investigated for evidence of infection by Verotoxin-producing Escherichia coli (VTEC). Fecal VTEC (belonging to at least six different O serogroups including O26, O111, O113, O121, O145, and O157) or specifically neutralizable free-fecal Verotoxin (VT) or both were detected in 24 (60%) patients but were not detected in 40 matched controls. Ten of 15 of the former developed fourfold or greater rises in VT-neutralizing antibody titers, as did six other patients who were negative for both fecal VTEC and VT. A total of 30 (75%) patients had evidence of VTEC infection by one or more criteria. We concluded that a significant association exists between idiopathic HUS and infection by VTEC. The detection of free-fecal VT was the most important procedure for the early diagnosis of this infection because, in our study, VTEC were never isolated in the absence of fecal VT, whereas fecal VT was often present even when VTEC were undetectable.

The Association Between Idiopathic Hemolytic Uremic Syndrome and Infection by Verotoxin-Producing Escherichia coli

Two strains of Campylobacter jejuni ingested by 111 adult volunteers, in doses ranging from 8 x 10(2) to 2 x 10(9) organisms, caused diarrheal illnesses. Rates of infection increased with dose, but development of illness did not show a clear dose relation. Resulting illnesses with strain A3249 ranged from a few loose stools to dysentery, with an average of five diarrheal stools and a volume of 509 mL. Infection with strain 81-176 was more likely to cause illness, and these illnesses were more severe, with an average of 15 stools and 1484 mL of total stool volume. All patients had fecal leukocytes. The dysenteric nature of the illness indicates that the pathogenesis of C. jejuni infection includes tissue inflammation. Ill volunteers developed a serum antibody response to the C. jejuni group antigen and were protected from subsequent illness but not infection with the same strain.

https://www.jstor.org/stable/pdfplus/30136650.pdf

Experimental Campylobacter jejuni Infection in Humans

Strains of Escherichia coli previously implicated or proven to be causes of diarrhea were examined for production of a toxin similar to that of Shigella dysenteriae type 1 (Shiga). Organisms grown in an iron-depleted broth were lysed by pressure disruption followed by ultracentrifugation. Saline-dialyzed extracts were tested for cytotoxic effects on HeLa cells that were neutralizable with antiserum to Shiga toxin. Among the 13 E. coli strains so analyzed, 11 made a Shiga-like cytotoxin in levels ranging from trace (two avirulent isolates) to amounts equivalent to S. dysenteriae type 1 (two noninvasive strains that did not make E. coli heat-labile or -stable enterotoxins but were isolated from infants with diarrhea). As with extracts of Shiga toxin, lysates of these E. coli strains that produced high levels of Shiga-like toxin were enterotoxic for rabbits, paralytic and lethal for mice, and inhibited protein synthesis in HeLa cells. Thus, these data suggest that Shiga-like toxin may be another heretofore undiscovered factor in the pathogenesis of diarrhea caused by some E. coli strains.

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Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

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Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

Shigella dysenteriae 1 (Shiga) toxin was purified from whole-cell lysates by antitoxin affinity column chromatography, radioiodination, and Sephacryl S-200 gel filtration of 125I-labeled affinity column eluates. Two chromatographic peaks were observed. The percentage of radioactivity in peak I samples immunoprecipitated with antitoxin ranged from 95 to 100%. A pool of samples from this first peak contained over 90% of the HeLa-cell-cytotoxic units applied to the column and was enterotoxic for rabbit ileal loops and lethal for rabbits. This radiolabeled material migrated as a single cytotoxic band after nondenaturing polyacrylamide gel electrophoresis, but formed three bands, of 33,000, 29,000, and 4,000 to 7,000 daltons, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, material estimated as 7,000 daltons by Bio-Gel P-10 chromatography could be generated by treatment of S-200 peak I samples with 8 M urea. Pooled fractions from the second S-200 peak were separable into several low-molecular-weight peaks on a P-10 column. One of these P-10 peaks (7,000 daltons) was 27% immunoprecipitable with antitoxin. These data indicate that three of the known biological activities of Shiga toxin are associated with a 33,000-dalton substance which can be dissociated into 29,000- and 4,000- to 7,000-dalton components.

Gerald D. LaVeck

Papers

Production of Shigella dysenteriae Type 1-Like Cytotoxin by Escherichia coli

Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

Characterization of Shigella dysenteriae 1 (Shiga) toxin purified by anti-Shiga toxin affinity chromatography.