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Escherichia coli

Escherichia coli, the most significant species in the genus Escherichia, is recognized as an important potential human pathogen. It is a gram-negative bacillus, so commonly isolated from colon flora that it is used as a

primary marker of fecal contamination in water purification.

On certain selective and differential media, such as MacConkey or eosin-methylene blue (EMB) agars, E coli

has a distinctive morphology. It usually presents as a lactose-positive (pink) colony with a surrounding area of

precipitated bile salts on MacConkey agar (Figure 20-1).

On EMB agar, it presents with a green metallic sheen. Most strains of E coli are motile and generally possess adhesive fimbriae and sex pili and 0, H, and K antigens. E coli 0 groups have shown remarkable cross-reactivity with 0 antigens from other members of the Enterobacteriaceae, most notably the Shigella. This is one of the reasons the Escherichia and the Shigella are grouped together in the tribe Escherichieae.

Serotyping for both 0 and H antigens is often useful in identification of strains, particularly those associated with serious enteric disease. The K antigen often masks the 0 antigen during bacterial agglutination by specific antiserum. Some E coli K antigens are identical to capsular antigens of other species. The Kl antigen has been found to be identical to the capsular antigen found on group B Neisseria meningitidis, suggesting a role for K antigens in virulence.

Characteristically E. coli stains are associated with the following properties: Fermentation of glucose, lactose, trehalose and xylose Indole production from tyrptophane Glucose fermentation by the mixed acid pathway, methyl red positive and Voges-Proskauer negative Does not produce HzS, DNase, urease, or phenylalamine

Deaminase Cannot use citrate as a sole carbon source.


Uropathogenic Escherichia coli

First described by Theodore Escherich in 1885, E coli was initially considered a harmless member of the colon resident flora. Since then, E. coli has been associated with a wide range of clinical syndromes, from UTls, to

central nervous system infections, to diarrheal diseases.

E. coli is widely recognized as the most common cause of UTls in humans. The E coli strains that cause UTls usually originate in the large intestine as resident or transient flora and may exist either as the predominant Ecoli population or as a small part of the E coli strains in the large intestine. Moreover, strains causing lower UTls and acute pylonephritis in immunocompetent hosts are different from those causing disease in the urinary tracts of individuals who are either compromised by urinary tract defects or by instrumentation such as placement of catheters. E coli strains that cause acute pyelonephritis in immunocompetent hosts have been shown to be the dominant resident E coli in the colon. They belong to a few serotypes and are resistant to the antibacterial activity of human serum. Conversely, isolates from immunocompromised hosts consist of a wide variety of strains.

Strains that cause UTls are able to do so because they produce factors that allow them to attach to the urinary epithelial mucosa. The primary virulence factor associated with the ability of E coli to cause UTls is pili, which allow uropathogenic strains to adhere to epithelial cells and not be washed out with urine flow. Other factors contribute to the virulence of uropathogenic E. coli, for example, cytolysins and aerobactins. Cytolysins (often also characterized as hemolysins) can kill immune effector cells and inhibit the phagocytosis and chemotaxis of certain white blood cells. Aerobactin allows the bacterial cell to chelate iron; free iron is generally unavailable within the host for use by bacteria.

Gastrointestinal Pathogens

Escherichia coli may cause several different GI syndromes (fable 20-4). Based on definitive virulence factors, clinical manifestation, epidemiology, and different 0 and H serotypes, there are five major categories of diarrheagenic E. coli: enterotoxigenic (ETEC), enteroinvasive (EIEC), enteropathogenic

(EPEC), enterohemorrhagic (EHEC), and enteroadherent, which includes the diffusely adherent (DAEC) and

the enteroaggregative (EaggEC) strains. The serotypes associated with these categories and the features associated with the intestinal infections produced by these strains are summarized in Chapter 34.

Enterotoxigenic Escherichia coli

The ETEC strains are associated with diarrhea of infants and adults in tropical and subtropical climates, especially in developing countries, where it is one of the major causes of infant bacterial diarrhea. In the United States and other Western industrialized nations, ETEC diarrhea is the most common cause of a diarrheal disease, sometimes referred to as traveler’s diarrhea, or turista. Travelers from industrialized countries often become infected with ETEC when they visit developing nations. ETEC infection commonly is spread via consumption of contaminated food or water. Poor hygiene, reduced availability of sources of potable water, and inadequate sanitation are major contributing factors in the spread and transmission of this disease. A high infective dose (l06 to 1010 organisms) is necessary to initiate disease in an immunocompetent host. Protective mechanisms such as stomach acidity have been described as inhibiting colonization and initiation of disease; those suffering from achlorhydria (deficiency of hydrochloric acid within the stomach) seem to be at higher risk than are normal individuals.

Colonization of ETEC on the proximal small intestine has been recognized as being mediated by fimbriae

that permit ETEC to bind to specific receptors on the intestinal microvilli. Once ETEC strains are established, they may release one or both of two toxins into the small intestine. They produce a heatlabile toxin (LT), which is similar in action and amino acid sequence to cholera toxin from Vibrio cholerae. The LT consists of two fragments (A and B), which follow the A/B model of bacterial toxins, where A is the enzymatically active portion. During infection the A portion activates cellular adenylate cyclase, causing a rise in the conversion of adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP).

The B moiety, or binding portion, confers the specificity; the B portion binds to the GM1ganglioside of the intestinal mucosa, thereby providing entry for the A portion. The consequence of accumulation of cAMP is hypersecretion of both electrolytes and fluids into the intestinal lumen, resulting in watery diarrhea similar to cholera. In contrast the heat-stable toxin (ST) stimulates guanylate cyclase, causing increased production of cyclic guanosine monophosphate (cGMP), accumulation of which also causes hypersecretion. The usually self-limiting disease caused by ETEC is characterized by watery diarrhea, nausea, abdominal cramps, and low-grade fever. Mucosal penetration and invasion do not appear to be part of ETEC disease.

Diagnosis of ETEC infection is made primarily by the characteristic presenting symptoms and the isolation of solely lactose fermenting organisms on differential media. Testing for toxins or colonizing factors is performed by research and reference laboratories, and its use is not justified in the clinical laboratory for diagnostic purposes. Enzyme-labeled oligonucleotide probes have been reported to detect ETEC in fecal specimens, but this method is undergoing further testing to determine its efficacy. ETECinfections must be differentiated, however, from other diarrheal illnesses which may appear similar (see Chapter



Enteroinvasive Escherichia coli

EIEC differs greatly from EPEC and ETEC strains. Enteroinvasive strains produce dysentery with direct penetration, invasion, and destruction of the intestinal mucosa. This diarrheal illness is very similar to that produced by Shigellaspp. The EIEC infections seem to occur in adults and children alike. Direct transmission of EIECfrom person to person via the fecal-oral route has been reported. The clinical infection is characterized by fever, severe abdominal cramps, malaise, and watery dirarrhea.

The organisms may be easily misidentified because of their similarity to the shigellae. EIECstrains may be nonmotile and do not ferment lactose; cross-reaction between shigellae and EIEC 0 antigens has been reported.

EIEC isolates may be mistaken for nonpathogenic E. coli; although EIEC do not decarboxylate lysine, more than 80% of E. coli strains do decarboxylate lysine. For these reasons, cases of diarrheal illness resulting from EIEC may be underreported.

Although EIEC and Shigella have been found to be morphologically similar and present similar clinical disorders, the infective dose of EIEC necessary to produce disease is much higher than that of shigellae.

The enteroinvasiveness of EIEC has to be demonstrated for definitive identification. The tests currently available to determine the invasive property of EIEC are not widely performed in most clinical microbiology laboratories. The Sereny test, which determines the organisms’ ability to produce keratoconjunctivitis in the guinea pig, is one of the assays previously used to determine the virulence of both shigellae and EIEC.

DNAprobes to identify EIEChave been compared with the Sereny test with comparable results. It is also possible to detect invasiveness using monolayer cell cultures with human epithelial-2 (HEp-2) cells. Recently, DNA probes for EIEC have become commercially available; these kits are used to screen stool samples, eliminating the need for other tests to identify EIEC.


Enteropathogenic Escherichia coli

Whereas the EPEC strain has been known to cause infantile diarrhea since the 1940s, its pathogenic role has remained controversial over the last few decades.

Certain 0 serogroups of EPECwere identified in the late 1960s and 1970s as a cause of diarrhea, but only certain H antigenic types within each 0 serogroup were connected to the intestinal infections. 0 serogrouping could not, however, differentiate these E. coli strains from strains of normal flora. In 1978, Levine et al.

attempted to settle the dispute concerning the pathogenic role of EPEC by challenging volunteers with EPEC strains that lacked the toxins of ETEC and the invasivenessof EIEC. The study showed that these EPEC strains caused distinct diarrhea. Subsequent studies further showed the adhesive property of EPEC strains, a characteristic not seen in ETEC or EIEC strains.

Diarrheal outbreaks caused by EPEC have occurred

in hospital nurseries and daycare centers, but cases in adults are rarely seen. The illness is characterized by low-grade fever, malaise, vomiting, and diarrhea. The stool typically contains large amounts of mucous, but apparent blood is not present. Detection of diarrheal illness attributable to EPEC depends primarily on the index of suspicion of the clinician. In cases of severe diarrhea in children younger than 1 year, infection with EPEC should be suspected. Serologic typing with pooled antisera may be performed to identify EPEC serotypes, but this is generally used for epidemiologic studies rather than for diagnostic purposes.


Enterohemorrhagic Escherichia coli

In 1982 the 0157:H7 strain of E. coli was first recognized during an outbreak of hemorrhagic diarrhea and colitis.

The EHEC strain serotype 0157:H7 has since been associated with hemorrhagic diarrhea, colitis, and hemolyticuremic syndrome (HUS). HUS is characterized by low platelet count, hemolytic anemia, and kidney failure.

The classic illness caused by EHEC produces a watery diarrhea that progresses to bloody diarrhea with abdominal cramps, a low-grade fever, or an absence of fever. The stool contains no leukocytes, which distinguishes it from dysentery caused by Shigella spp. Or EIEC infections.The infection is potentially fatal, especially in young children and elderly persons in nursing homes. Processed meats, such as undercooked hamburgers served at fast-food restaurants, unpasteurized dairy products and apple cider, and bean sprouts have all been implicated in the spread of infection. E. coli 0157:H7 produces two cytotoxins: verotoxin I and verotoxin II. Verotoxin I is a phage-encoded cytotoxin

identical to the Shiga toxin (Stx) produced by Shigella dysenteriae type I. This toxin produces damage to vero cells (African green monkey kidney cells)- hence the term verotoxin. It also reacts with and is

neutralized by the antibody against Stx. In contrast, verotoxin II is not neutralized by antibody to Stx. Verotixin II is biologically similar to, but immunologically different from, both Stx and verotoxin I. These toxins have also been reported under the name Shiga-like toxins but are most likely to be found in the literature as Shiga toxin I (Stxl) and Shiga toxin 2 (Stx2); E. coli strains that produce these toxins are also called Shiga toxigenic E. coli (STEC). Several different STECstrains have been identified; 0157:H7 is only the first to have been widely reported. Any of the STEC serotypes can cause clinical syndromes similar to that produced by 0157:H7 E. coli. Box 20-2 lists non-0157:H7 EHEC isolated from patients with bloody diarrhea, hemorrhagic colitis, or HUS.

In the laboratory, verotoxin producing E. coli may be identified by one of three methods: Stool culture on highly differential medium, with subsequent serotyping Detecting the verotoxin in stool filtrates Demonstration of a fourfold or greater increase in verotoxin-neutralizing antibody titer Stool culture for E. coli 0157:H7 may be perfomed using MacConkey agar containing sorbitol (SMAC) instead of lactose. E. coli 0157:H7 does not ferment sorbitol in 48 hours, a characteristic that differentiates it from most other E. coli. The use of this differential medium facilitates the primary screening of E. coli 0157:H7, which ordinarily would not be distinguishable from other E. coli on lactose-containing MacConkey or other routine enteric agar. E. coli

0157:H7 appears colorless on sorbitol-MacConky agar.

Although isolation of other nonsorbitol-fermenting organisms may occur in up to 15% of cultures, E. coli 0157:H7, when present, produces heavy growth. A latex agglutination test for rapid presumptive detection of E. coli 0157:H7 has also been reported useful and is commercially available; isolates must be tested with the negative control to detect nonspecific agglutination.

The commercially available MUGassay (4-methylumbelliferyl f)-D-glucuronide) is a biochemical test that can be used to screen for E. coli 0157:H7, in addition to sorbitol fermentation. E. coli 0157:H7 rarely produces

the enzyme f)-glucuronidase, wherease 92% of other strains do. If the enzyme is present, MUG is cleaved and

a fluorescent product is formed. Sorbitol-negative colonies are subsequently subcultured for serotyping using E. coli 0157:H7 antiserum. Enzyme-linked immunosorbent assay (ELISA) or latex ENTERoBAcrERIACEAE

· CHAPTER 20 511

agglutination can be used to detect the 0157 antigen.

This somatic antigen, which is usually the target in the commercial assays, may present a problem with regard to specificity because other enteric bacteria may produce false-positive results. It is therefore important to confirm the identification of MUG-negative or sorbitolnegative colonies as E. coli isolates. A latex test to detect H7 antigen is now available as well. When testing colonies taken directly from the SMAC plate, the test for the H7 antigen may be initially negative. It is helpful to grow these isolates in motility media first to enhance flagella production and agglutination with the latex particles. Following the serotyping, isolates are tested for the presence of Stx.

Investigative reports have shown that all E. coli 0157:H7 strains produce high levels of cytotoxins, and STEC strains may be detected using cell culture assays with Vero cells. Because other toxins present in diarrhea stools may produce similar cytopathic effects, this test must be verified with specific antitoxins to Stx1 and Stx2. Free verotoxins present in stool specimens have been detected in samples that yielded negatve culture results.

It was previously reported that hemorrhagic colitis patients shed the organisms for only brief periods;

nevertheless, verotoxins may still be detected in the stool. An approved ELISA test from Meridian Diagnostics, Inc. (Cincinnati, Ohio) is able to detect Stx even in bloody stools, although not all patients have bloody stools. Gene amplification assays like those available in Europe from SY-LAB(Gerate GmbH, Austria) may be useful in detecting STEC strains. A fourfold increase in verotoxin-neutralizing antibody titer has been demonstrated in patients with HUS and in whom verotoxin or verotoxin-producing E. coli has been detected.


Enteroadherent Escherichia coli

Enteroadherent E. coli strains are generally associated with two kinds of human disease: diarrheal syndromes and UTls. There are two strains of enteroadherent E. coli: DAEC and EggEc. DAEC are associated with both

UTls and diarrheal disease. The uropathogenic DAEC strains are closely associated with cystitis in children

and acute pyelonephritis in pregnant women. They also seem to be associated with chronic or recurring UTls. Several strains of DAEC have been associated with pediatric diarrheal disease, particularly in developing nations.

EaggEC causes diarrhea by adhering to the surface of the intestinal mucosa. These strains are found to adhere to HEp2 cells, packed in an aggregative “stacked-brick” pattern on the cells and between the cells by means of a fimbria. These organisms produce watery diarrhea, vomiting, dehydration, and occasionally abdominal pain, mostly in children. The symptoms typically persist for 2 or more weeks.


Extraintestinal Infections

Escherichia coli remains one of the most common causes of septicemia and meningitis among neonates, accounting for about 40%of the cases of gram-negative meningitis. Similar infections resulting from this organism are uncommon in older children. The newborn usually acquires the infection in the birth canal just before or during delivery, when the mother’s vagina is heavily colonized. Infection may also result if contamination of the amniotic fluid takes place.

The strains associated with diarrheal disease appear to be distinct from those associated with neonatal sepsis or meningitis. The capsular antigen Kl present in certain strains of E. coli has been the most

documented virulence factor associated with neonatal meningeal infections. E. coli Kl is also immunochemically identical to the capsular antigen of group B N. meningitidis. The association of Kl antigen was established when E. coli strains possessing the capsular Kl antigen were isolated from neonates with septicemia or meningitis. Fatality rates for infants with meningitis caused by E. coli were higher than those for infants infected with non-Kl strains.

In addition to the neonatal population, E. coli remains as a clinically significant isolate in blood cultures from adults. E. coli bacteriemia in adults may result primarily from a genitourinary tract infection or from a GIsource.


Other Escherichia Species

Escherichia hermannii, formerly called E. coli atypical or enteric group II, is a yellow-pigmented organism that has been isolated from cerebrospinal fluid (CSF), wounds, and blood. Reports of isolating E. hermannii from foodstuffs such as raw milk and beef, the same sources of E. coli 0157:H7, have been published. However, its clinical significance is not fully established.

The newest species added to this genus, Escherichia vulneris, has been isolated from humans with infected wounds. More than half of the strains of E. vulneris also produce yellow-pigmented colonies. Figure 20-2 compares the colonial morphology of E. hermannii with that of E. vulneris. The other members of the tribe

Escherichieae, the shigellae, will be discussed in the enteric pathogens portion of the chapter

table 20-4 box 20-2 figure 20-1 figure 20-2 table 20-2 table 20-3.