Pathogenic E. coli

Pathogenic E. coli (page 1) 

(This chapter has 4 pages) 



E. coli O157:H7. Phase contrast image of cells immobilized on an agar-coated slide. William Ghiorse, Department of Microbiology, Cornell University, Ithaca, New York. Licensed for use by ASM Microbe Libraryhttp://www.microbelibrary.org

Escherichia coli

Theodor Escherich first described E. coli in 1885, as Bacterium coli commune,which he isolated from the feces of newborns. It was later renamed Escherichia coli, and for many years the bacterium was simply considered to be a commensal organism of the large intestine. It was not until 1935 that a strain ofE. coli was shown to be the cause of an outbreak of diarrhea among infants.

The GI tract of most warm-blooded animals is colonized by E. coli within  hours or a few days after birth. The bacterium is ingested in foods or water or obtained directly from other individuals handling the infant. The human bowel is usually colonized within 40 hours of birth. E. coli can adhere to the mucus overlying the large intestine. Once established, an E. coli strain may persist for months or years. Resident strains shift over a long period (weeks to months), and more rapidly after enteric infection or antimicrobial chemotherapy that perturbs the normal flora. The basis for these shifts and the ecology of Escherichia coli in the intestine of humans are poorly understood despite the vast amount of information on almost every other aspect of the organism's existence. The entire DNA base sequence of the E. coli genome has been known since 1997.

E. coli is the head of the large bacterial family, Enterobacteriaceae, the enteric bacteria, which are facultatively anaerobic Gram-negative rods that live in the intestinal tracts of animals in health and disease. The Enterobacteriaceae are among the most important bacteria medically. A number of genera within the family are human intestinal pathogens (e.g. Salmonella, Shigella, Yersinia). Several others are normal colonists of the human gastrointestinal tract (e.g.Escherichia, Enterobacter, Klebsiella), but these bacteria, as well, may occasionally be associated with diseases of humans.

Physiologically, E. coli is versatile and well-adapted to its characteristic habitats. It can grow in media with glucose as the sole organic constituent. Wild-type E. coli has no growth factor requirements, and metabolically it can transform glucose into all of the macromolecular components that make up the cell. The bacterium can grow in the presence or absence of O₂. Under anaerobic conditions it will grow by means of fermentation, producing characteristic "mixed acids and gas" as end products. However, it can also grow by means of anaerobic respiration, since it is able to utilize NO₃, NO₂ or fumarate as final electron acceptors for respiratory electron transport processes. In part, this adapts E. coli to its intestinal (anaerobic) and its extraintestinal (aerobic or anaerobic) habitats.

E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity, etc., in a number of very remarkable ways considering it is a unicellular organism. For example, it can sense the presence or absence of chemicals and gases in its environment and swim towards or away from them. Or it can stop swimming and grow fimbriae that will specifically attach it to a cell or surface receptor. In response to change in temperature and osmolarity, it can vary the pore diameter of its outer membrane porins to accommodate larger molecules (nutrients) or to exclude inhibitory substances. With its complex mechanisms for regulation of metabolism the bacterium can survey the chemical contents in its environment in advance of synthesizing any enzymes that metabolize these compounds. It does not wastefully produce enzymes for degradation of carbon sources unless they are available, and it does not produce enzymes for synthesis of metabolites if they are available as nutrients in the environment.

E. coli is a consistent inhabitant of the human intestinal tract, and it is thepredominant facultative organism in the human GI tract; however, it makes up a very small proportion of the total bacterial content. The anaerobicBacteroides species in the bowel outnumber E. coli by at least 20:1. however, the regular presence of E. coli in the human intestine and feces has led to tracking the bacterium in nature as an indicator of fecal pollution and water contamination. As such, it is taken to mean that, wherever E. coli is found, there may be fecal contamination by intestinal parasites of humans.

Unstained cells of E. coli viewed by phase microscopy. about 1000X magnification. CDC.

Escherichia coli in the Gastrointestinal Tract

The commensal E. coli strains that inhabit the large intestine of all humans and warm-blooded animals comprise no more than 1% of the total  bacterial biomass. 

The E. coli flora is apparently in constant flux. One study on the distribution of different E. coli strains colonizing the large intestine of women during a one year period (in a hospital setting) showed that 52.1% yielded one serotype, 34.9% yielded two, 4.4% yielded three, and 0.6% yielded four.  The most likely source of new serotypes of E. coli is acquisition by the oral route. 

To study oral acquisition, the carriage rate of E. coli carrying antibiotic-resistance plasmids (R factors) was examined among vegetarians, babies, and nonvegetarians. It was assumed that nonvegetarians might carry more E. coliwith R factors due to their presumed high incidence in animals treated with growth-promoting antimicrobial agents. However, omnivores had no higher an incidence of R-factor-containing E. coli than vegetarians, and babies had more resistant E. coli in their feces than nonvegetarians. No suitable explanation could be offered for these findings. Besides, investigation of the microbial flora of the uninhabited Krakatoa archipelago has shown the presence of antibiotic-resistantE. coli associated with plants. 

The bottom line seems to be that most of us have more than one strain of E. coliin our gut, and intestinal strains tend to displace one another about three or four times a year.

Pathogenesis of E. coli

Over 700 antigenic types (serotypes) of E. coli are recognized based on O, H, and K antigens. At one time serotyping was important in distinguishing the small number of strains that actually cause disease. Thus, the serotype O157:H7 (O refers to somatic antigen; H refers to flagellar antigen) is uniquely responsible for  causing HUS (hemolytic uremic syndrome). Nowadays, particularly for diarrheagenic strains (those that cause diarrhea) pathogenic E. coli are classified based on their unique virulence factors and can only be identified by these traits. Hence, analysis for pathogenic E. coli usually requires that the isolates first be identified as E. coli before testing for virulence markers.

Pathogenic strains of E. coli are responsible for three types of infections in humans: urinary tract infections (UTI), neonatal meningitis, and intestinal diseases (gastroenteritis). The diseases caused (or not caused) by a particular strain of E. coli depend on distribution and expression of an array of virulence determinants, including adhesins, invasins, toxins, and abilities to withstand host defenses. These are summarized in Table 1 and applied to the discussion of pathogenic strains E. coli below.

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Table 1. Summary of the Virulence Determinants of Pathogenic E. coli 

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Adhesins
CFAI/CFAII
Type 1 fimbriae
P fimbriae
S fimbriae
Intimin (non-fimbrial adhesin)
EPEC adherence factor

Invasins
hemolysin
Shigella-like "invasins" for intracellular invasion and spread 

Motility/chemotaxis
flagella
Toxins
LT toxin
ST toxin
Shiga toxin
cytotoxins
endotoxin (LPS) 

Antiphagocytic surface properties
capsules
K antigens
LPS 

Defense against serum bactericidal reactions
LPS
K antigens 

Defense against immune responses
capsules
K antigens
LPS
antigenic variation 

Genetic attributes
genetic exchange by transduction and conjugation
transmissible plasmids
R factors and drug resistance plasmids
toxin and other virulence plasmids
siderophores and siderophore uptake systems
pathogenicity islands

Urinary Tract Infections

Uropathogenic E. coli (UPEC) cause 90% of the urinary tract infections (UTI) in anatomically-normal, unobstructed urinary tracts. The bacteria colonize from the feces or perineal region and ascend the urinary tract to the bladder. Bladder infections are 14-times more common in females than males by virtue of the shortened urethra. The typical patient with uncomplicated cystitis is a sexually-active female who was first colonized in the intestine with a uropathogenic E. colistrain. The organisms are propelled into the bladder from the periurethral region during sexual intercourse. With the aid of specific adhesins they are able to colonize the bladder.

The adhesin that has been most closely associated with uropathogenic E. coli is the P fimbria (or pyelonephritis-associated pili [PAP]). The letter designation is derived from the ability of P fimbriae to bind specifically to the P blood group antigen which contains a D-galactose-D-galactose residue. The fimbriae bind not only to red cells but to a specific galactose dissaccharide that is found on the surfaces uroepithelial cells in approximately 99% of the population.

The frequency of the distribution of this host cell receptor plays a role in susceptibility and explains why certain individuals have repeated UTI caused byE. coli. Uncomplicated E. coli UTI virtually never occurs in individuals lacking the receptors.

Uropathogenic strains of E. coli possess other determinants of virulence in addition to P fimbriae. E. coli with P fimbriae also possess the gene for Type 1 fimbriae, and there is evidence that P fimbriae are derived from Type 1 fimbriae by insertion of a new fimbrial tip protein to replace the mannose-binding domain of Type 1 fimbriae. In any case, Type 1 fimbriae could provide a supplementary mechanism of adherence or play a role in aggregating the bacteria to a specific manosyl-glycoprotein that occurs in urine.

Uropathogenic strains of E. coli usually produce siderophores that probably play an essential role in iron acquisition for the bacteria during or after colonization. They also produce hemolysins which are cytotoxic due to formation of transmembranous pores in host cell membranes. One strategy for obtaining iron and other nutrients for bacterial growth may involve the lysis of host cells to release these substances. The activity of hemolysins is not limited to red cells since the alpha-hemolysins of E. coli also lyse lymphocytes, and the beta-hemolysins inhibit phagocytosis and chemotaxis of neutrophils.

Another factor thought to be involved in the pathogenicity of the uropathogenic strains of E. coli is their resistance to the complement-dependent bactericidal effect of serum. The presence of K antigens is associated with upper urinary tract infections, and antibody to the K antigen has been shown to afford some degree of protection in experimental infections. The K antigens of E. coli are "capsular" antigens that may be composed of proteinaceous organelles associated with colonization (e.g., CFA antigens), or made of polysaccharides. Regardless of their chemistry, these capsules may be able to promote bacterial virulence by decreasing the ability of antibodies and/or complement to bind to the bacterial surface, and the ability of phagocytes to recognize and engulf the bacterial cells. The best studied K antigen, K-1, is composed of a polymer of N-acetyl neuraminic acid (sialic acid), which besides being antiphagocytic, has the additional property of being an antigenic disguise. 

Neonatal Meningitis

Neonatal meningitis affects 1/2,000-4,000 infants. Eighty percent of E. colistrains involved synthesize K-1 capsular antigens (K-1 is only present 20-40% of the time in intestinal isolates).

E. coli strains invade the blood stream of infants from the nasopharynx or GI tract and are carried to the meninges.

The K-1 antigen is considered the major determinant of virulence among strains of E. coli that cause neonatal meningitis. K-1 is a homopolymer of sialic acid. It inhibits phagocytosis, complement, and responses from the host's immunological mechanisms. K-1 may not be the only determinant of virulence, however, as siderophore production and endotoxin are also likely to be involved.

Epidemiologic studies have shown that pregnancy is associated with increased rates of colonization by K-1 strains and that these strains become involved in the subsequent cases of meningitis in the newborn. Probably, the infant GI tract is the portal of entry into the bloodstream. Fortunately, although colonization is fairly common, invasion and the catastrophic sequelae are rare.

Neonatal meningitis requires antibiotic therapy that usually includes ampicillin and a third-generation cephalosporin. 

Lysis of a dividing pair of E. coli cells in the presence of a beta-lactam antibiotic. Some beta lactam antibiotics, such as  ampicillin and cephalosporin, are effective in the treatment of meningitis caused by strains of E. coli (above). The beta lactam antibiotics prevent cell wall synthesis and assembly in the bacterium. When the bacterium grows in the presence of the antibiotic, the cell wall becomes progressively weaker and weaker, so the the organism eventually ruptures or "lyses", pouring out its cytoplasmic contents as shown here.

Intestinal Diseases Caused by E. coli

As a pathogen, E. coli is best known for its ability to cause intestinal diseases. Five classes (virotypes) of E. coli that cause diarrheal diseases are now recognized: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enteroaggregative E. coli (EAEC). Each class falls within a serological subgroup and manifests distinct features in pathogenesis. A summary of the characteristics of diarrheagenic strains of E. coli is given in Table 2 at the end of this article.

Enterotoxigenic E. coli (ETEC)
ETEC is an important cause of diarrhea in infants and travelers in underdeveloped countries or regions of poor sanitation. In the U.S., it has been implicated in sporadic waterborne outbreaks, as well as due to the consumption of soft cheeses, Mexican-style foods and raw vegetables. The diseases vary from minor discomfort to a severe cholera-like syndrome. ETEC are acquired by ingestion of contaminated food and water, and adults in endemic areas evidently develop immunity. The disease requires colonization and elaboration of one or more enterotoxins. Both traits are plasmid-encoded.

ETEC may produce a heat-labile enterotoxin (LT) that is similar in molecular size, sequence, antigenicity, and function to the cholera toxin (Ctx). It is an 86kDa protein composed of an enzymatically active (A) subunit surrounded by 5 identical binding (B) subunits. It binds to the same identical ganglioside receptors that are recognized by the cholera toxin (i.e., GM1), and its enzymatic activity is identical to that of the cholera toxin.

ETEC may also produce a heat stable toxin (ST) that is of low molecular size and resistant to boiling for 30 minutes. There are several variants of ST, of which ST1a or STp is found in E. coli isolated from both humans and animals, while ST1b or STh is predominant in human isolates only. The ST enterotoxins are peptides of molecular weight about 4,000 daltons. Their small size explains why they are not inactivated by heat. ST causes an increase in cyclic GMP in host cell cytoplasm leading to the same effects as an increase in cAMP. ST1a is known to act by binding to a guanylate cyclase that is located on the apical membranes of host cells, thereby activating the enzyme. This leads to secretion of fluid and electrolytes resulting in diarrhea.

The infective dose of ETEC for adults has been estimated to be at least 10⁸ cells; but the young, the elderly and the infirm may be susceptible to lower numbers.

ETEC adhesins are fimbriae which are species-specific. For example, the K-88 fimbrial Ag is found on strains from piglets; K-99 Ag is found on strains from calves and lambs; CFA I, and CFA II, are found on strains from humans. These fimbrial adhesins adhere to specific receptors on enterocytes of the proximal small intestine.

Symptoms ETEC infections include diarrhea without fever. The bacteria colonize the GI tract by means of a fimbrial adhesin, e.g. CFA I and CFA II, and are noninvasive, but produce either the LT or ST toxin. <>


Enteroinvasive E. coli (EIEC)
EIEC closely resemble Shigella in their pathogenic mechanisms and the kind of clinical illness they produce. EIEC penetrate and multiply within epithelial cells of the colon causing widespread cell destruction. The clinical syndrome is identical to Shigella dysentery and includes a dysentery-like diarrhea with fever. EIEC apparently lack fimbrial adhesins but do possess a specific adhesin that, as inShigella, is thought to be an outer membrane protein. Also, like Shigella, EIEC are invasive organisms. They do not produce LT or ST toxin.

There are no known animal reservoirs of EIEC. Hence the primary source for EIEC appears to be infected humans. Although the infective dose of Shigella is low (in the range of 10 to few hundred cells), volunteer feeding studies showed that at least 10⁶ EIEC organisms are required to cause illness in healthy adults. Unlike typical E. coli, EIEC are non-motile, do not decarboxylate lysine and do not ferment lactose. Pathogenicity of EIEC is primarily due to its ability to invade and destroy colonic tissue. The invasion phenotype, encoded by a high molecular weight plasmid, can be detected by PCR and probes for specific for invasion genes.


Enteropathogenic E. coli (EPEC)
EPEC induce a profuse watery, sometimes bloody, diarrhea. They are a leading cause of infantile diarrhea in developing countries. Outbreaks have been linked to the consumption of contaminated drinking water as well as some meat products.  Pathogenesis of EPEC involves a plasmid-encoded protein referred to as EPEC adherence factor (EAF) that enables localized adherence of bacteria to intestinal cells and a non fimbrial adhesin designated intimin, which is an outer membrane protein that mediates the final stages of adherence. They do not produce ST or LT toxins.

Adherence of EPEC strains to the intestinal mucosa is a very complicated process and produces dramatic effects in the ultrastructure of the cells resulting in rearrangements of actin in the vicinity of adherent bacteria. The phenomenon is sometimes called "attachment and effacing" of cells. EPEC strains are said to be "moderately-invasive",  meaning they are not as invasive as Shigella, and unlike ETEC or EAEC, they cause an inflammatory response. The diarrhea and other symptoms of EPEC infections probably are caused by bacterial invasion of host cells and interference with normal cellular signal transduction, rather than by production of toxins.

Through volunteer feeding studies the infectious dose of EPEC in healthy adults has been estimated to be 10⁶ organisms.

Some types of EPEC are referred to as diffusely adherent E. coli (DAEC), based on specific patterns of adherence. They are an important cause of traveler's diarrhea in Mexico and in North Africa.

Enteroaggregative E. coli (EAEC)
The distinguishing feature of EAEC strains is their ability to attach to tissue culture cells in an aggregative manner. These strains are associated with persistent diarrhea in young children. They resemble ETEC strains in that the bacteria adhere to the intestinal mucosa and cause non-bloody diarrhea without invading or causing inflammation. This suggests that the organisms produce an enterotoxin of some sort. Recently, a distinctive heat-labile plasmid-encoded toxin has been isolated from these strains, called the EAST (EnteroAggregative ST) toxin. They also produce a hemolysin related to the hemolysin produced by E. coli strains involved in urinary tract infections. The role of the toxin and the hemolysin in virulence has not been proven. The significance of EAEC strains in human disease is controversial.

 
Enterohemorrhagic E. coli (EHEC)
EHEC are recognized as the primary cause of hemorrhagic colitis (HC) or bloody diarrhea, which can progress to the potentially fatal hemolytic uremic syndrome (HUS). EHEC are characterized by the production of verotoxin orShiga toxins (Stx). Although Stx1 and Stx2 are most often implicated in human illness, several variants of Stx2 exist.

There are many serotypes of Stx-producing E. coli , but only those that have been clinically associated with HC are designated as EHEC. Of these, O157:H7 is the prototypic EHEC and most often implicated in illness worldwide. The infectious dose for O157:H7 is estimated to be 10 - 100 cells; but no information is available for other EHEC serotypes. EHEC infections are mostly food or water borne and have implicated undercooked ground beef, raw milk, cold sandwiches, water, unpasteurized apple juice and vegetables

EHEC are considered to be "moderately invasive". Nothing is known about the colonization antigens of EHEC but fimbriae are presumed to be involved. The bacteria do not invade mucosal cells as readily as Shigella, but EHEC strains produce a toxin that is virtually identical to the Shiga toxin. The toxin plays a role in the intense inflammatory response produced by EHEC strains and may explain the ability of EHEC strains to cause HUS. The toxin is phage encoded and its production is enhanced by iron deficiency.

E. coli O157:H7 Transmission EM. American Society for Microbiology

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Table 2. Diarrheagenic E. coli: virulence determinants and characteristics of disease

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ETEC
fimbrial adhesins e.g. CFA I, CFAII, K88. K99
non invasive
produce LT and/or ST toxin
watery diarrhea in infants and travelers; no inflammation, no fever 

EIEC
nonfimbrial adhesins, possibly outer membrane protein
invasive (penetrate and multiply within epithelial cells)
does not produce shiga toxin
dysentery-like diarrhea (mucous, blood), severe inflammation, fever

EPEC
non fimbrial adhesin (intimin)
EPEC adherence factor (EAF) enables localized adherence of bacteria to intestinal cells
moderately invasive (not as invasive as Shigella or EIEC)
does not produce LT or ST; some reports of shiga-like toxin
usually infantile diarrhea; watery diarrhea with blood, some inflammation, no fever; symptoms probably result mainly from invasion rather than toxigenesis

EAEC
adhesins not characterized
non invasive
produce ST-like toxin (EAST) and a hemolysin
persistent diarrhea in young children without inflammation or fever

EHEC
adhesins not characterized, probably fimbriae
moderately invasive
does not produce LT or ST but does produce shiga toxin
pediatric diarrhea, copious bloody discharge (hemorrhagic colitis), intense inflammatory response, may be complicated by hemolytic uremia 

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END OF CHAPTER 

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