Doyle J. Evans Jr.
Dolores G. Evans
Depending on the virulence factors they possess, virulentEscherichia coli strains cause either noninflammatory diarrhea(watery diarrhea) or inflammatory diarrhea (dysentery with stoolsusually containing blood, mucus, and leukocytes).
These are Gram-negative bacilli of the familyEnterobacteriaceae. Virulent strains differ from nonvirulent Ecoli only in possessing genetic elements for virulence factors.Strains producing enterotoxins are enterotoxigenic E coli (ETEC).
Transmission is by the fecal-oral route. Pili (fimbriae) allowthe bacteria to colonize the ileal mucosa. Cytotonic enterotoxins(encoded on plasmid or bacteriophage DNA) induce watery diarrhea.Plasmid-encoded invasion factors permit invasion of the mucosa,and plasmid- or bacteriophage-encoded cytotoxic enterotoxinsinduce tissue damage; the presence of either of these factorsinduces a host inflammatory reaction with an influx oflymphocytes and resulting dysentery.
Gastric acid and intestinal transit time are importantdefenses. Specific intestinal immunoglobulin A (IgA) develops andappears to be protective.
Infection is common where sanitation is poor; both infants andsusceptible travelers to developing countries are particularly atrisk. The disease is most serious in infants.
The diagnosis is suggested by the clinical picture andconfirmed by stool culture. Serotyping and tests for virulencefactors are occasionally performed for outbreaks.
Prevention depends on sanitary measures to prevent fecal-oraltransmission; hand-washing and proper preparation of food;chlorination of water supplies; and sewage treatment anddisposal. Parenteral or oral fluid and electrolyte replacement isused to prevent dehydration. Broad-spectrum antibiotics are usedin chronic or life-threatening cases.
Escherichia coli is a common member of the normal flora of thelarge intestine. As long as these bacteria do not acquire geneticelements encoding for virulence factors, they remain benigncommensals. Strains that acquire bacteriophage or plasmid DNAencoding enterotoxins or invasion factors become virulent and cancause either a plain, watery diarrhea or an inflammatorydysentery. These diseases are most familiar to Westerners astraveler's diarrhea, but they are also major health problems inendemic countries, particularly among infants. Three groups of Ecoli are associated with diarrheal diseases. Escherichia colistrains that produce enterotoxins are called enterotoxigenic Ecoli (ETEC). There are numerous types of enterotoxin. Some ofthese toxins are cytotoxic, damaging the mucosal cells, whereasothers are merely cytotonic, inducing only the secretion of waterand electrolytes. A second group of E coli strains have invasionfactors and cause tissue destruction and inflammation resemblingthe effects of Shigella(EIEC). A third group of serotypes, calledenteropathogenic E coli (EPEC), are associated with outbreaks ofdiarrhea in newborn nurseries, but produce no recognizable toxinsor invasion factors. Figure 25-1 presents a summary of thediseases caused by virulent E coli.
FIGURE 25-1 Virulence mechanisms of E coli.
The diarrheal disease caused by ETEC is characterized by arapid onset of watery, nonbloody diarrhea of considerable volume,accompanied by little or no fever (Fig. 25-2). Other commonsymptoms are abdominal pain, malaise, nausea, and vomiting.Diarrhea and other symptoms cease spontaneously after 24 to 72hours.
FIGURE 25-2 Pathogenesis of E coli diarrheal disease.
ETEC organisms are Gram-negative, short rods not visiblydifferent from E coli found in the normal flora of the humanlarge intestine. Virulence-associated fimbriae are too small tobe seen by light microscopy. All ETEC contain plasmids, but thisis also not a distinguishing feature unless gene probe techniquesare used to detect specific virulence-associated genes on theseplasmids.
E coli organisms are serogrouped according to the presence orabsence of specific heat-stable somatic antigens (O antigens)composed of polysaccharide chains linked to the corelipopolysaccharide (LPS) complex common to all Gram-negativebacteria. O specificity is determined by sugar or amino-sugarcomposition and by the sequence of these outer polysaccharidechains. More than 170 different O-specific antigens have beendefined since Kauffmann began this method of typing E coli in1943. In normal smooth strains, which are typable, the core LPSis buried beneath the O antigen. Also occurring are untypableO-minus mutants in which the core LPS is exposed; these arecalled rough strains. There is considerable cross-reactivityamong E coli O antigens; also, many O groups of E coli arecross-reactive or identical with specific O groups of Shigella,Salmonella, or Klebsiella.
Escherichia coli serotypes are specific O-group/H-antigencombinations. The H antigens are the flagellar antigens, of whichthere are at least 56 types. Escherichia coli isolates may benonmotile and nonflagellated and hence H negative (H). H typingis important for E coli associated with diarrheal disease for tworeasons. First, a strain causing an outbreak or epidemic can bedifferentiated from the normal stool flora by its unique O:Hantigenic makeup. Second, most ETEC belong to specific serotypes(Table 25-1); this relationship facilitates their identificationeven in isolated cases. The reason for the close associationbetween specific serotypes and the production ofplasmid-determined virulence factors remains a mystery.
One plasmid-encoded but enterotoxin- and serotype-independentpilus (a long polar structure termed longus) has been reportedrecently in ETEC; like the CFAs, production of this pilus isrestricted to E coli isolated from human sources.
Most E coli isolates also produce heat-labile,surface-associated proteins antigenically unrelated to O and H.These antigens can be seen in electron micrographs as filamentousstructures called pili (fimbriae), which are much thinner andusually more rigid than flagella. Commensal E coli strainsusually produce so-called common pili, which are defined as aspecific set of an antigen. When E coli possessing common piliare mixed with erythrocytes (the standard test uses guinea pigerythrocytes), rapid hemagglutination occurs. Thishemagglutination is blocked and also reversed by millimolarconcentrations of the carbohydrate mannose.
ETEC possess specialized pili, antigenically unrelated tocommon pili, which act as ligands to bind the bacterial cells tospecific complex carbohydrate receptors on the epithelial cellsurfaces of the small intestine. Since this interaction resultsin colonization of the intestine by ETEC, with subsequentmultiplication on the gut surface, these pili are termedcolonization-factor antigens (CFAs). Most ETEC isolates produceeither CFA/I, CFA/II or CFA/IV, whereas CFA/III and anundetermined number of other CFAs occur on other particularserotypes (Table 25-1). CFA-type pili play a major role in hostspecificity; for instance, different CFAs (e.g., K88, K99, and987P) are produced by E coli that cause acute diarrhea indomestic animals.
A simple presumptive assay for CFAs on E coli is a test formannose-resistant (non-common pili) hemagglutination reactionwith either human or bovine erythrocytes. However, identificationmust be confirmed by reaction of the bacteria with antibodydirected against a specific CFA or polymerase chain reaction(PCR) assay for specific CFA genes.
Genes coding for the production of CFAs reside on the ETECvirulence plasmids, usually on the same plasmids that carry thegenes for one or both of the two types of E coli enterotoxin,heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST).Most cases of ETEC diarrhea are caused by E coli possessing a CFAand both LT and ST; fewer are caused by those possessing a CFAand only one toxin (usually LT); and the fewest are caused by Ecoli that lack a CFA and possess only ST.
Escherichia coli diarrheal disease is contracted orally byingestion of food or water contaminated with a pathogenic strainshed by an infected person. ETEC diarrhea occurs in all agegroups, but mortality is most common in infants, particularly inthe most undernourished or malnourished infants in developingnations.
The pathogenesis of ETEC diarrhea involves two steps:intestinal colonization, followed by elaboration of diarrheagenicenterotoxin(s) (Fig 25-3). ST is actually a family of toxicpeptides ranging from 18 to 50 amino acid residues in length.Those termed STa can stimulate intestinal guanylate cyclase, theenzyme that converts guanosine 5'-triphosphate (GTP) to cyclicguanosine 5'-monophosphate (cGMP). Increased intracellular cGMPinhibits intestinal fluid uptake, resulting in net fluidsecretion. Those termed STb do not seem to cause diarrhea by thesame mechanism. One method for testing suspect E coli isolatesfor ST production involves injection of culture supernatantfluids into the stomach of infant mice and seeing whetherdiarrhea ensues. Specific DNA gene probes and PCR assays havebeen developed to test isolated colonies for the presence ofgenes encoding ST and LT.
FIGURE 25-3 Cellular pathogenesis of E coli having CFApili.
The E coli LTs are antigenic proteins whose mechanism ofaction is similar to that of Vibrio cholerae enterotoxin. LTshares antigenic determinants with cholera toxin, and its primaryamino acid sequence is similar.
LT is composed of two types of subunits. One type of subunit(the B subunit) binds the toxin to the target cells via aspecific receptor that has been identified as Gm1 ganglioside.The other type of subunit (the A subunit) is then activated bycleavage of a peptide bond and internalized. It then catalyzesthe ADP-ribosylation (transfer of ADP-ribose from nicotinamideadenine dinucleotide [NAD]) of a regulatory subunit ofmembrane-bound adenylate cyclase, the enzyme that converts ATP tocAMP. This activates the adenylate cyclase, which produces excessintracellular cAMP, which leads to hypersecretion of water andelectrolytes into the bowel lumen.
LT production is demonstrable by serologic methods, testingfor diarrheagenic activity in ligated rabbit intestine, and bytesting for specific cAMP-mediated morphological changes incultured Y-1 adrenal tumor cells or Chinese hamster ovary (CHO)cells.
As in any orally transmitted disease, the first line ofdefense against ETEC diarrhea is gastric acidity. Othernonspecific defenses are small-intestinal motility and a largepopulation of normal flora in the large intestine.
Information about intestinal immunity against diarrhealdisease is still somewhat superficial. However, intestinalsecretory immunoglobulin (IgA) directed against surface antigenssuch as the CFAs and against LT appears to be the key to immunityfrom ETEC diarrhea. Passive immune protection of infants bycolostral antibody is important. Human breast milk also containsnonimmunoglobulin factors (receptor-containing molecules) thatcan neutralize E coli toxins and CFAs.
Escherichia coli diarrheal disease of all types is transmittedfrom person to person with no known important animal vectors. Theincidence of E coli diarrhea is clearly related to hygiene, foodprocessing sophistication, general sanitation, and theopportunity for contact. The geographic frequency of ETECdiarrhea is inversely proportional to the sanitation standards.Single-source outbreaks of ETEC diarrhea involving contaminatedwater supplies or food have been found in adults in the UnitedStates and Japan. Adults traveling from temperate climates tomore tropical areas typically experience traveler's diarrheacaused by ETEC. This phenomenon is not readily explained, butcontributing factors are low levels of immunity and an increasedopportunity for infection.
ETEC diarrhea is characterized by copious watery diarrhea withlittle or no fever. The diarrheal stool yields a virtually pureculture of E coli. Since the disease is self-limiting, virulencetesting of isolates and serotyping is impractical except in anoutbreak situation. Confirmation is achieved by serotyping,serologic identification of a specific CFA on isolates,demonstration of LT or ST production, and identification of genesencoding these virulence factors (Fig. 25-4).
FIGURE 25-4 Laboratory methods for isolation andidentification of ETEC.
Escherichia coli diarrheal disease is best controlled bypreventing transmission and by stressing the importance ofbreast-feeding of infants, especially where ETEC is endemic. Thebest treatment is oral fluid and electrolyte replacement(intravenous in severe cases). Antibiotics are not recommendedbecause this practice leads to an increased burden ofantibiotic-resistant pathogenic E coli and of morelife-threatening enteropathogens.
Diarrhea caused by the enteroinvasive, cytotoxic, andenteropathogenic (EPEC) strains of E coli ranges from very mildto severe. Illness is usually protracted and accompanied byfever. Infection with a few serogroups (O157, O26) ischaracterized by bloody diarrhea (hemorrhagic colitis). Infectionwith the Shigella-like serogroups presents as bacillary dysentery(i.e., abdominal pain and scanty stool containing blood andmucus).
As in the case with ETEC, these strains of E coli are notdetectably different in structure from E coli of the normalflora. The EPEC serogroups listed in Table 25-2 were the first Ecoli groups to be recognized as causative agents of diarrhea ininfants. Their status as pathogens remained controversial fordecades, mainly because the same O groups can be isolated fromhealthy contacts in outbreaks and from healthy adults. Recentwork has proven that these E coli serogroups possess an antigenicadherence factor (termed bundle-forming pilus, or BFP); the genefor BFP is carried by a plasmid termed EAE (enteroaggregativeEscherichia coli) plasmid. BFP is responsible for the initialattachment of EPEC to intestinal target cells.
A small but important group of EPEC includes serotypesO157:H7, O26:H11, and some O111 isolates. These cause epidemichemorrhagic colitis. Serotype O157:H7 is often associated withfood-borne outbreaks.
Table 25-2 lists the Shigella-like enteroinvasive E coliserotypes (i.e., those with somatic antigens reactive withspecific anti-Shigella typing serum). Also, like Shigella, theseE coli strains are non-motile and therefore H negative. Theseserogroups usually do not harbor ETEC virulence plasmids andtherefore are usually CFA negative.
Escherichia coli strains belonging to the classic EPECserogroups (Table 25-2) bind intimately to the epithelial surfaceof the intestine, usually the colon, via the adhesive BFP. Thelesion caused by EPEC consists mainly of destruction ofmicrovilli. There is no evidence of tissue invasion. Cell damageoccurs in two steps, collectively termed attaching and effacing;first is intimate contact, sometimes characterized as pedestalformation; second is loss of microvilli which is the result ofrearrangement of the host cell cytoskeleton. Loss of microvillileads to malabsorption and osmotic diarrhea. Diarrhea ispersistent, often chronic, and accompanied by fever. EPEC arenegative for ST and LT, but most strains produce relatively smallamounts of a potent Shiga-like toxin that has both enterotoxinand cytotoxin activity.
The E coli strains associated with hemorrhagic colitis(enterohemorrhagic E coli, or EHEC) most notably O157:H7, producerelatively large amounts of the bacteriophage-mediated Shiga-liketoxin. This toxin is called Vero toxin (VT), or Vero cytotoxinafter its cytotoxic effect on cultured Vero cells. Many strainsof O157:H7 also produce a second cytotoxin (Shiga-like toxin 2,or Vero toxin 2), which is similar in effect but antigenicallydifferent.
The Shigella-like E coli strains are highly virulent; oralexposure to a very small number of these invasive bacteria causessevere illness. The site of the infection is the colon, whereadherence is rapidly followed by invasion of the intestinalepithelial cells (Fig. 25-5). An acute inflammatory response andtissue destruction produce diarrhea with little fluid, muchblood, and sheets of mucus containing polymorphonuclear cells.Invasive E coli, like Shigella, causes a rapidkeratoconjunctivitis when placed on the conjunctiva of the guineapig eye (Sereny test). Virulent Sereny test-positive isolatescarry a large (usually 140-megadalton) plasmid responsible forthis property.
FIGURE 25-5 Cellular pathogenesis of invasive E coli
Host defenses against EPEC are the same as those for ETEC.These defenses are frequently deficient or lacking in the infantand the elderly, which is consistent with the epidemiology ofEPEC illness. An important example is the role of the immunesystem. Passive immune protection of infants by colostralantibody is important; breast-feeding is especially relevantwhere crowding and poor economic conditions prevail. Infectionwith these pathogens often excites an inflammatory cell responsein the intestine, as is frequently reflected in the diarrhealsymptoms.
The geographic distribution of all EPEC is generally the sameas for the ETEC, with a more severe disease in infants and youngchildren, and so EPEC are much less important in traveler'sdiarrhea. Common-source community outbreaks are rare ingeographic areas with satisfactory sanitation. However, sporadiccases are seen in the United States, Canada, and Europe, andoutbreaks occur in these areas, but most commonly inclose-contact institutions such as hospital nurseries, day-carecenters, and nursing homes.
Diagnosis is usually based on the symptomatology describedabove. Enterohemorrhagic E coli, such as serotype O157:H7, issuspected in the setting of copious bloody diarrhea without fecalleukocytes or fever, especially when symptoms include hemolyticuremic syndrome, or HUS. Escherichia coli serotyping is useful inchronic cases and in outbreaks, because identification of theagent and its antibiotic sensitivity pattern are valuable inthese situations. Testing for specific EPEC virulence factors isusually impractical because it can be done only in reference andspecialized research laboratories.
Prevention and control are generally the same as for ETEC.Intervention of the fecal-oral transmission cycle is mosteffective in institutional situations. Broad-spectrum antibioticsare recommended in chronic and/or life-threatening cases.
Blanco J, Blanco M, Gonzalez EA et al: Serotypes andcolonization factors of enterotoxigenic Escherichia coli isolatedin various countries. Eur J Epidemiol 9:489, 1993
Chapman PA, Siddons CA, Wright DJ et al: Cattle as a possiblesource of verocytotoxin-producing Escherichia coli O157infections in man. Epidemiol Infect 111:439, 1993
Donnenberg MS, Tacket CO, James SP et al: Role of the eaeAgene in experimental enteropathogenic Escherichia coli infection.J Clin Invest 92:141, 1993
Dytoc M, Sone R, Cockerill, F, III et al: Multipledeterminants of verotoxin-producing Escherichia coli O157:H7attachment-effacement. Infect Immun 61:3382, 1993
Evans DJ, Jr., Evans DG: Colonization factor antigens of humanpathogens. Current Topics Microbiol Immunol 151:129, 1990
Giron JA, Ho ASY, Schoolnik GK: An inducible bundle-formingpilus of enteropathogenic Escherichia coli. Science 254:710, 1991
Giron JA, Levine MM, Kaper JB: Longus: a long pilusultrastructure produced by human enterotoxigenic Escherichiacoli. Molec Microbiol 12:71, 1994
Spangler BD: Structure and function of cholera toxin and therelated Escherichia coli heat-labile enterotoxin. Microbiol Rev56:622, 1992
Tesh VL, O'Brien AD. Adherence and colonization mechanisms ofenteropathogenic and enterohemorrhagic Escherichia coli. MicrobPathogenesis 12:245, 1992
Wenneras C, Svennerholm AM, Ahren C, Czerkinsky C:Antibody-secreting cells in Human peripheral blood after oralimmunization with an inactivated enterotoxigenic Escherichia colivaccine. Infect Immun 60:2605, 1992
