ECOLI

ENTEROPATHOGENIC ESCHERICHIA COLI
Hin-chung Wong
Department of Microbiology
Soochow University
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1. INTRODUCTION
2. HEAT-LABILE ENTEROTOXINS
2.1. General Characteristics
2.2. Gene and Regulation
2.3. Mode of Action
3. HEAT-STABLE ENTEROTOXINS
3.2. Mode of Action and Regulation
4. ENTEROTOXIN PLASMIDS
5. SHIGA-LIKE TOXINS
5.1. Purification and Structure
5.2. Mode of Action
5.3. Production and Regulation
5.4. Genetics
5.5. Role in Disease
6. HEMOLYSINS
6.1. Production and Purification
6.2. Characteristics
6.3. Genetics
6.4. Role in Virulence and Mode of Action
7. ADHERENCE
7.1. In Enterotoxigenic E. coli
7.2. In Enteropathogenic E. coli
7.3. In Enterohemorrhagic E. coli
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8. INVASIVENESS
9. DETECTION
9.1. Using glucuronidase assay
9.2. Animal Tissue Culture
9.3. Animal Assays
9.4. Immunological Methods
9.5. Enzymatic bio-nanotransduction
9.6. Nucleic Acid Probes
9.7. Using polymerase chain reaction
10. REFERENCES
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1. INTRODUCTION
Escherichia coli is part of the normal large-bowel flora of humans and

animals. Although most strains of E. coli are non-pathogenic in the intestine,
some can produce diarrhea and by a number of distinct mechanisms.
E. coli is usually considered to be an opportunistic pathogen which

constitutes a large portion of the normal intestinal flora of humans. This
organism can, however, contaminate, colonize, and subsequently cause infection
of extraintestinal sites and is a major cause of septicemia, peritonitis, abscesses,
meningitis, and urinary tract infections in humans (Cavalieri et al., 1984).
E. coli was first incriminated as an enteropathogen in 1945, responsible for an

outbreak of infantile diarrhea. Enteropathogenic E. coli (EEC) has been
associated with diarrhea in developing countries and localities having poor
sanitation. In the developed countries, EEC has been historically associated
primarily with infantile diarrhea, but it was later recognized that adults also may
suffer from the illness. E. coli are also enteropathogenic in animals (Wasteson et
al., 1988; Weinstein et al., 1988b). E. coli O157:H7 causes severe illneses
(hemorrhagic) and it does possess distinguishing characteristics, e.g. does not
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ferment sorbitol with 24 h, does not possess α-glucuronidase activity, and does
not grow well at all at 44-45.5C (Doyle, 1991).
There are several subgroups of EEC: (A) enterotoxigenic (ETEC), (B)

enteroinvasive (EIEC), (C) hemorrhagic (EHEC), and (D) enteropathogenic
(EPEC) strains. The serogrouping and virulence factors associated with
different EEC groups are shown in (Table 1) (39). Some authors classify them
into six different pathotypes: ETEC, EIEC, EPEC, enteroaggregative E. coli,
diffusely adherent E. coli, and Shiga toxin-producing E. coli (STEC)
(Hernandes et al., 2009).
In 1995, the enteropathogenic E. coli (EPEC) pathotype is divided into two

groups, typical EPEC (tEPEC) and atypical EPEC (aEPEC). The property that
distinguishes these two groups is the presence of the EPEC adherence factor
plasmid (pEAF), which is only found in tEPEC. aEPEC strains are emerging
enteropathogens that have been detected worldwide. The large variety of
serotypes and genetic virulence properties of aEPEC strains from nonclassical
EPEC serogroups makes it difficult to determine which strains are truly
pathogenic (Hernandes et al., 2009).
Humans are thought to be the principal if not the only reservoir of toxigenic
and invasive strains of E. coli, contaminating foods via contact with food or via
contact of processing equipment with water contaminated by human feces. In
contrast, animals are reservoirs of the hemorrhagic strain (O157:H7); hence,
foods of animal origin may become contaminated via slaughter procedures or
post-processing recontamination. However, when E. coli is isolated from foods,
pathogenic serotypes are usually absent or represent a very low percentage of
the total isolates.
2. HEAT-LABILE ENTEROTOXINS
Probably the most common type of EEC strains is the enterotoxigenic type.
Heat-labile (LT) and heat-stable (ST) enterotoxins are produced.
Two partially cross-reacting antigenic variants of plasmid-coded LT,

designated LTh and LTp, have been described in E. coli. LTh is associated with
3
E. coli isolates from humans, and LTp is associated with E. coli isolates from
pigs. The LT family from restricted geographical region exhibited a segregated
pattern of dissemination that was revealed by a restriction enzyme site
polymorphism (Vinal and Dallas, 1987).
Another heat-labile enterotoxin was discovered in extracts of E. coli SA53, a

strain isolated from water buffalo. It activated adenylate cyclase. Hyperimmune
antisera prepared against LTh and LTp or CT do not neutralize the crude LT-like
toxin in Y1 adrenal cell assays. The structural genes for this LT-like toxin are
not located on plasmids and are probably located on the bacterial chromosome.
The structural gene for LT-like toxin does not have extensive nucleotide
sequence homology with the genes encoding the A and B polypeptides of LTp.
Subcloning and minicell experiments indicated that the toxin is composed of
two polypeptide subunits that are encoded by two genes. The two toxin subunits
exhibited mobilities on PAGE gels that are similar to those of CT and LT (Fig. 1)
(Pickett et al., 1986).
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It is proposed by Pickett et al. that the LTp and LTh (antigenic variants of LT
will both be included in serogroup I and should be designated LTp-I and LTh-I
and the LT-like toxin will be the prototype for serogroup II enterotoxins and
should be renamed LT-II. Two distinct members of the LT-II family, LT-IIa and
LT-IIb, are now known, and both have A and B subunits which are similar in
size to those of CT and LT-I (Pickett et al., 1986; Pickett et al., 1987).
2.2. Gene and Regulation
The LT gene was cloned into E. coli and two proteins of molecular weights
11,500 (B subunit) and 25,500 (A subunits) were produced (Dallas et al., 1979).
The LT A subunit structureal gene (eltA) was sequenced and the amino acid
sequence deduced (Spicer and Noble, 1982). It starts with methionine, ends with
leucine, and comprises 254 amino acids. The computed molecular weight of LT
A is 29,673. The A subunit genes of CT and LT (LT-I) are 78.6% homologous,
and the B subunit genes are 78% homologous. The NH2-terminal regions
exhibit the highest degree of homology (91%) as compared with CT subunit A,
while the COOH-terminal region, containing the sole cystine residue in each
toxin is less conserved (52%). Alignment of homologous residues in the
COOH-terminal regions of LT A and CT A indicates that a likely site for
proteolytic cleavage of LT A is after Arg residue 188 (Spicer and Noble, 1982).
The gene of LT-IIa was studied. It is organized in a transcriptional unit

similar to those of CT and LT-I. The A subunit gene of LT-IIa was found to be
57% homologous with the A subunit gene of LTh-I and 55% homologous with
the A gene of CT. Most of the homology derived from the region of the A gene
which encodes the A1 fragment. The B gene of LT-IIa was not homologous with
the B gene of LTh-I or CT (Pickett et al., 1987).
The LT-IIb gene was also cloned and analysed. The A genes of LT-IIa and
LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57%
homology with the A genes of CT and LT-I. The B subunits of LT-IIa and LT-IIb
differ from the LT-I in their carbohydrate-binding specificities. The B genes of
LT-IIa and LT-IIb were 66% homologous, but neither had significant homology
with the B genes of CT and LT-Is. The A subunits of the heat-labile enterotoxins
also have limited homology with other ADP-ribosylating toxins, including
pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A
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(Pickett et al., 1989).
The carboxy-terminal domain of EtxB (encodes B subunit) mediates A

subunit-B subunit interaction. The gene encoding the B subunit of LT was
mutated at its 3' end by targeted addition of random nucleotide sequences. The
functional and structural properties of the gene products were analysed, that
these mutants were defective in their ability to associate stably with A subunits
and form holotoxin (Sandkvist et al., 1987).
2.3. Mode of Action
LT from E. coli is a protein of approximately 86,000 daltons that consists of

one A polypeptide and five B polypeptides held together by noncovalent bonds.
LT is closely related to cholera enterotoxin (CT) in structure, antigenicity, and
mode of action. Both LT and CT bind to ganglioside GM1 receptors on
eukaryotic target cells via their B subunits. The A subunit of LT like CT
undergoes a proteolytic cleavage that produces two fragments designated A1
and A2. The A1 fragment catalyzes the NAD-linked ADP ribosylation of a
regulatory subunit of adenylate cyclase in the plasma membrane of eucaryotic
target cells, resulting in stimulation of adenylate cyclase activity. The activation
of adenylate cyclase in mucosal cells in the small intestine causes secretion of
fluid and electrolytes into the lumen and produces watery diarrhea.
The LT-II, similar to CT and LT-I, increases cAMP by activating adenylate

cyclase through the GTP-dependent ADP-ribosylation of specific membrane.
Fibroblasts incubated with LT-II had an increased cAMP content (Fig. 2) as well
as a fourfold elevation of membrane adenylate cyclase activity (Fig. 3). In
membranes, activation of cyclase by toxin was enhanced by NAD, GTP, and
dithiothreitol. The effect of LT-II on intact fibroblasts or membranes was
increased by trypsin treatment of toxin (Chang et al., 1987).
6
7
The B subunit of LTh (Human) is also hemagglutinating. Very strong
hemagglutination of both neuraminidase- and pronase-treated human
erythrocytes was induced by the B subunit of LTh. Different blood groups
reacted differently to such enhancement (Sugii et al., 1988; Sugii and Tsuji,
1989; Sugii and Tsuji, 1990). Combining site of the B subunit may gain access
to the receptor exposed on erythrocytes more easily by enzyme treatment.
Neuraminidase and pronase are suppose to convert major gangliosides to GM1
and/or expose masked receptors for the B subunit. Both CT and LT strongly
react with ganglioside GM1.
Separation of ETEC bacteria from target intestinal epithelial monolayers by

semipermeable filters prevented activation of adenylate cyclase suggesting that
pathogen-host cell contact is required for efficient toxin delivery. Likewise, a
non-motile strain bearing a mutation in the flagellar fliD gene was deficient in
delivery of LT relative to the ETEC prototype. Although LT secretion via the
type II secretion system (T2SS) was responsive to a variety of environmental
factors, neither toxin release nor delivery depended on transcriptional activation
of genes encoding LT or the T2SS (Dorsey et al., 2006).
Enterotoxigenic E. coli LT-induced diarrhea is the leading cause of infant death

in developing countries. Ginger significantly blocked the binding of LT to
cell-surface receptor G M1 (Fig. 4), resulting in the inhibition of fluid
accumulation in the closed ileal loops of mice (Fig. 5).
Biological-activity-guided searching for active components showed that
zingerone (vanillylacetone) was the likely active constituent responsible for the
antidiarrheal efficacy of ginger. Further analysis of chemically synthesized
zingerone derivatives revealed that compound 31 (2-[(4-methoxybenzyl)oxy]
benzoic acid) significantly suppressed LT-induced diarrhea in mice via an
excellent surface complementarity with the B subunits of LT(Fig. 6) (Chen et al.,
2007b). Similar inhibition effect was found in the extract of Chaenomeles
speciosa fruits (寒梅) (Chen et al., 2007a).
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3. HEAT-STABLE ENTEROTOXINS
The heat-stable enterotoxins are low-molecular-weight, heat-stable,

nonantigenic proteins which do not cause intestinal secretion by activation of
adenylate cyclase. At least two types have been described, one with biological
activity in suckling mice and piglets (STa, or named as STh, or ST-I) due to
stimulation of particulate intestinal guanylate cyclase and a second which
induces secretion by an unknown mechanism only in piglets (STb, or known as
STp, or ST-II).
Generally STs are known to be small peptide toxins consisting of 18 (STb) or

19 (STa) amino acids. The different STa (M.W. about 2,000) from different
animal origins are remarkably homogeneous. The amino acid composition of
STa of porcine, bovine, and human origins were identical and consisted of 10 of
the 18 amino acids usually present in proteins. Six of the 18 amino acids were
half-cystines which appear to be present as three disulfide bonds in the native
form of the toxin. These disulfide bonds are important for toxic activity.
STb is a heat-stable enterotoxin which does not cause intestinal fluid

secretion in the suckling mouse as STa does, but does cause intestinal fluid
secretion in pig intestinal loop assays. It is insoluble in methanol, while STa is
methanol-soluble.
Since STs are small peptides and are nonantigenic, fusion proteins (e.g. STa
with outer membrane protein C, etc.) (Saarilahti et al., 1989) or synthetic ST
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peptide conjugated with ovalbumin (Frantz et al., 1987) could be use as the
immunoprophylactic agents against diarrhea caused by STs. Nontoxic fusion
protein of LT was also contructed (Clements, 1990).
In swine, the most common and severe enterotoxigenic E. coli (ETEC)

infections are caused by strains that express K88 F4+ fimbriae, heat-labile
enterotoxin (LT), heat-stable enterotoxin b (STb), and enteroaggregative E. coli
heat-stable toxin 1. Previous studies based on a design that involved enterotoxin
genes cloned into a nontoxigenic fimbriated strain have suggested that LT but
not STb plays an important role in dehydrating diarrheal disease in piglets <1
week old and also enhances bacterial colonization of the intestine. In a study,
these two toxins were examined in terms of importance for piglets >1 week old
with the construction of isogenic single- and double-deletion mutants and
inoculation of 9-day-old F4ac receptor-positive gnotobiotic piglets. Based on
the postinoculation percent weight change per h and serum bicarbonate
concentrations, the virulence of the STb- mutant (Delta estB) did not
significantly differ from that of the parent. However, deletion of the LT genes
(Delta eltAB) in the STb(-) mutant resulted in a complete abrogation of weight
loss, dehydration, and metabolic acidosis in inoculated pigs, and LT
complementation restored the virulence of this strain. These results support the
hypothesis that LT is a more significant contributor than STb to the virulence of
F4(+) ETEC infections in young F4ac receptor-positive pigs less than 2 weeks
old (Erume et al., 2008).
3.2. Mode of Action and Regulation
The enteropathogenic strains (O8:KX105) which colonized the small

intestine of piglets produced STb and LT enterotoxins, wheras the
nonenteropathogenic strains produced the STb. In another report, a
hypertoxigenic E. coli strain which produced 17- to 60-fold more ST than
laboratory ETEC. This strain rapidly induced severe form of scours in calves
and piglets (Saeed et al., 1986).
The action of STa stimulated the cGMP level in epithelial cell. Accumulation
of cGMP by STa was synergistically enhanced by phorbol esters which are
direct activators of protein kinase but not guanylate cyclase (Weikel et al.,
1990).
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The cysteine residues were substituted in vivo by oligonucleotide-directed
site-specific mutangenesis to dissociate each disulfide bond and examined the
biological activitites of the resulting mutants (Fig.7, 8). All three disulfide
bonds formed at fixed positions are required for full expression of the biological
activity of STb. It has some fexibilities in its conformation to exert toxic
activity and that the role of each disulfide bond in exerting toxic activity is not
quite the same (Okamoto et al., 1987).
The STs share biologically active sequences which reside in the C-terminal 13
amino acid residues. Substitution of the asparagines residue at position 11 of
STb by other amino acids resulted in significant decrease in enterotoxic
activities, although the conformation was not changed (Okamoto et al., 1988).
The amino acid sequences and disulfide bonds of the heat-stable enterotoxins of
E. coli, Yersinia enterocolitica, and Vibrio cholerae non-O1 are shown in Fig. 9
(Okamoto et al., 1988; Okamoto et al., 1987).
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Analogs of ST were made, including the native 18-amino-acid ST, the
14-amino-acid carboxy terminus of this native peptide with a proline at position
12, and the 14-amino-acid carboxy terminus of in which the proline at position
12 was substituted with glycine (Table 1). Each analog bound to the receptor in
a dose-dependent fashion, native ST with the highest adherence (Fig. 10).
Similarly, these peptides maximally activated particulate guanylate cyclase and
stimulate intestinal secretion in suckling mice, and native ST with the highest
potency (Fig. 11, Table 2). It demonstrats that the four amino-terminal residues
contribute significantly to the potency of these peptides. In addition, the turn
imposed by the proline residue at position 12 is not absolutely required for
receptor occupancy or activation of the biochemical cascade that results in
intestinal secretion. However, it significantly increases the potency of the toxin
(Waldman and O'Hanley, 1989).
13
14
The action of STb in the intestine of pig is still not consistant. It is susceptible
to trypsin degradation and that variable amounts of trypsin-like activity in swine
jejuna are responsible for inconsistent responses to STb in jejunal loops of
swine (Whipp, 1987). When endogenous protease activity was blocked with
trypsin inhibitor, STb evoked a dose-dependent secretory response in infant
mice and jejunal loops of rats and pig (Whipp, 1990).
The gene determined the nucleotide sequence for STb has been cloned. The
only open reading frame with STb activity encoded for a 71-amino acid protein.
It began with methionine-lysine-lysine followed by 8 to 20 hydrophobic amino
acids and had a calculated molecular weight of 5,090.
The STb structural gene (estA) was cloned into high-expression vector
pKC30 downstream from the strong PL promoter and the expression was
studied (Lawrence et al., 1990). 10-20-fold increase in mRNA was produced by
the recombinant strain.
Two types of STs, STI and STII, have been found. Both STs are synthesized
as precursor proteins and are then converted to the active forms with
intramolecular disulfide bonds after being released into the periplasm. The
active STs are finally translocated across the outer membrane through a tunnel
made by TolC. However, it is unclear how the active STs formed in the
periplasm are led to the TolC channel. Several transporters in the inner
membrane and their periplasmic accessory proteins are known to combine with
TolC and form a tripartite transport system. Pulse-chase experiments using E.
coli BL21(DE3) mutants in which various transporter genes (acrAB, acrEF,
emrAB, emrKY, mdtEF, macAB, and yojHI) had been knocked out and analyzed
the secretion of STs in those strains. The results revealed that the extracellular
secretion of STII was largely decreased in the macAB mutant and the toxin
15
molecules were accumulated in the periplasm, although the secretion of STI was
not affected in any mutant. The periplasmic stagnation of STII in the macAB
mutant was restored by the introduction of pACYC184, containing the macAB
gene, into the cell (Fig. 12). These results indicate that MacAB, an ATP-binding
cassette transporter of MacB and its accessory protein, MacA, participates in the
translocation of STII from the periplasm to the exterior. Therefore, the
MacAB-TolC system may capture the periplasmic STII molecules and exports
the toxin molecules to the exterior (Yamanaka et al., 2008).
4. ENTEROTOXIN PLASMIDS
Enterotoxin plasmids from classical strains (frequently associated with

diarrhea, e.g. O6, O25, O27, O128, and O159) did not transfer by conjugation
from clinical isolates, whereas those from rare strains (rarely associated with
diarrhea, e.g. O7, O17, O80, O98, O139, and O153) transferred almost always
from the clinical isolates by conjugation. Analyses of enterotoxin plasmids by
restriction endonucleases and hybridization with the enterotoxin probes revealed
that the strains with the same O serotype and toxigenicity carry closely related
enterotoxin plasmids. These results suggest that classical strains resulted from
the dissemination of ancestral clones which received enterotoxin plasmids long
ago, while the rare strains acquired the enterotoxin plasmids recently by
conjugation and have not yet been spread to the same degree as the ancestral
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clones.
5. SHIGA-LIKE TOXINS
Enteropathogenic E. coli (EPEC, e.g. O26:H11, O128:B12, O138:K81) and

enterohemorrhagic E. coli (EHEC, e.g. O157:H7) are not enteroinvasive and do
not produce the classical heat-stable and heat-labile enterotoxins. A cytotoxin
known as Shiga-like toxin was demonstrated in EPEC and EHEC. A cytotoxin
which could be neutralized by anti-shiga toxin is known as Shiga-like toxin I
(SLT-I), and the non-neutralizable one is known as shiga-like toxin II (SLT-II)
(Downes et al., 1988; O'Brien and Holmes, 1987). A total of 9% of
LT-II-producing isolates from cows and buffalo hybridized with DNA probes
for genes coding for SLT-II (Seriwatana et al., 1988b). In Thailand,
SLT-producing E. coli was isolated from 9% of market beef specimens, from 8
to 28% of fresh beef specimens at slaughterhouses, and from 11 to 84% of fecal
specimens from cattle (Suthienkul et al., 1990). Vero cell cytotoxin E. coli
O157:H7 was isolated from 3.7% of beef, 1.5% of port, 1.5% of poultry, and
2% of lamb samples in Canada (Doyle and Schoeni, 1987).
The SLT-I is also known as verotoxin 1 and the SLT-II is also known as
verotoxin 2. E. coli producing large among of SLTs are also named as VTEC
(Verotoxin producing E. coli)(Smith and Scotland, 1988).
Iron is known to depress Shiga toxin production by Shigella dysenteriae 1,

and temperature has been shown to regulate several genes required for Shigella
invasiveness and also expression of virulence plasmid in Yersinia. Iron also
suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not
demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I
genes (Table 3). Temperature had no effect on SLT-I synthesis (Weinstein et al.,
1988a).
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5.1. Purification and Structure
The SLT-I and SLT-II have been purified to homogeneity. The bacteria grown
in iron-depleted medium were disrupted by French press and the toxins purifed
by anti-shiga toxin affinity chromatography or by conventional biochemical
methods. The SLT-I A subunit has a MW 32,200 and the SLT-I B subunit has a
MW of 7,700 (O'Brien and Holmes, 1987).
The SLT-II was purified from E. coli strain containing the cloned toxin genes
on recombinant plasmid. Purification was accomplished by a series of column
chromatography techniques including monoclonal antibody affinity
chromatography (against SLT-II). The SLT-II consisted of A and B subunits with
apparent molecular weights of 32,000 and 10,200, respectively. Also an A
subunit of M.W. 25,000 was also observed and identified by immunoblot
analysis (Downes et al., 1988). A variant of SLT-II was purified and bands of
molecular weights of 33,000, 27,500, and 7,500 were identified to be A, Aa, and
B subunits, respectively. Electrophoresis under nonreducing conditions resulted
in disappearance of the 27,000 band (MacLeod and Gyles, 1990).
By analogy with Shiga toxin, the most likely A-to-B subunit ratio for SLT-I is
1:5. Then the MW of holotoxin is about 70,000 (O'Brien and Holmes, 1987).
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5.2. Mode of Action
The purified SLTs have the same biological activities as and comparable
specific activities to purified Shiga toxin. The molecular basis is probably the
catalytic inactivation of 60S ribosomes in toxin-sensitive (receptor-expressing)
cells (Fig. 14) (O'Brien and Holmes, 1987). The receptor for SLT-I and SLT-II
has been identified and is the same as for Shiga toxin; it is a globotriosyl
ceramide containing a galactose-à-(1->4)-galactose-á-(1->4)-glucose ceramide
(Smith and Scotland, 1988).
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But experiment in gnotobiotic piglets showed that SLTs is not essential for
expression of virulence by EHEC strains O157:H7 (Tzipori et al., 1987).
5.3. Production and Regulation
Production of SLT is activated in iron-limited media. By a gene fusion

experiment, the sltA and sltB genes are regulated by a fur locus. A gene fusion
between the promoter and proximal portion of the SLT gene with gene for
bacterial alkaline phosphatase was made. Growth in low-iron conditions
resulted in a 13- to 16-fold increase in alkaline phosphatase activity. In the
presence of a null mutation in the fur locus, however, alkaline phosphatase
activity was constitutively high regardless of the iron concentration. These data
indicate negative regulation of the slt operon by the fur gene product
(Calderwood and Mekalanos, 1987).
5.4. Genetics
Phage conversion is responsible for controlling the production of several

important bacterial toxins, including diphtheria toxin, streptococcal
erythrogenic toxin, botulinum toxin, and staphylococcal enterotoxin A.
Production of SLTs was also shown to be determined by specific phages in
selected strains of both EPEC and EHEC serotypes isolated from humans (Fig.
15) (Smith and Scotland, 1988). In contrast, production of SLT in E. coli strains
isolated from pigs was not found to be controlled by phages. The organization
and expression of the operon for SLT-I are shown schematically in (Fig. 16).
The structural genes that encode SLT-I were cloned on a 2.5-kb DNA fragment
from a phage from strain H19, on a 3.1-kb DNA fragment from phage 933J, and
on a 1.7-kb DNA fragment from phage H19B (Huang et al., 1986; Newland et
al., 1985).
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The recombinant plasmid containing the 1.7-kb fragment from phage H19B
confers the ability to produce high levels of SLTs on transformed E. coli cells.
By an in vitro transcription/translation system that the cloned fragment specifies
the two subunit peptides which have masses of 31 and 5.5 kDa which different
from 70,000 estimated (Huang et al., 1986).
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A 0.75 kb probe of the SLT gene was used to hybridize clinical strains. Some
SLT-positive strains only hybridized at low stringency and these results indicate
that there are differences in the SLT genes of EEC (Willshaw et al., 1985).
Genes controlling production of SLT-II were cloned from the phage 933W.
Subcloning analysis identified a region within the 4.9-kb EcoRI fragment was
associated with SLT-II production by minicell experiment. These experiments
showed the genetic organization of the SLT-II genes to be the same as that of the
SLT-I genes, with the coding sequence for the large A subunit adjacent to that
for the smaller B subunit. The mobilities of the SLT-II subunits in SDS-PAGE
gels were slightly greater than those determined for the SLT-I subunits.
Although apparent processing of the SLT-I subunits was observed with
polymyxin B treatment of the labeled minicells, no processing of the SLT-II
subunits was detected. Southern blot hybridization studies suggested that the
DNA fragment carrying the SLT-II structural genes shares approximately 50 to
60% homology with the DNA of the SLT-I structural genes (Newland et al.,
1987). The molecular weight of the A and B subunits of SLT-II, deduced from
the translated nucleotide sequences, were 33,135 and 7187, respectively (Smith
and Scotland, 1988).
The structural gene of a SLT-II variant (SLT-IIv) produced by a strain of E.

coli was cloned and analysed. The A subunit genes for this SLT-IIv and SLT-II
were highly homologous (94%), whereas the B subunit were less homologous
(79%). The A subunit of SLT-IIv, like those of other members of this toxin
family, had regions of homology with the plant lectin ricin. SLT-IIv did not bind
to galactose-α-1-4-galactose conjugated to bovine serum albumin, which is an
analog of the eucaryotic cell receptor for shiga-toxin and SLTs. SLT-IIv may
bind to a different cellular receptor than do other members of the shiga toxin
family (Weinstein et al., 1988b).
5.5. Role in Disease
Epidemiological Evidence
As is the case with Shiga toxin, there is no direct proof that E. coli SLTs plays
a role in disease. Some of the strongest circumstantial evidence comes from
epidemiological studies of E. coli strains isolated from humans and animals.
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Most of the high level cytotoxin producers were associated with diarrhea,
hemorrhagic colitis, or hemolytic uremic syndrome (HUS) (Table 4) (Smith and
Scotland, 1988).
It appears that food is the primary source of infection in man. E. coli O157
has been isolated from hamburger meat and unpasteurised milk which was also
associated with hemorrhagic colitis and HUS. The O157:H7 were isolated from
1.5-3.7% of samples of beef, pork, poultry and lamb (Smith and Scotland,
1988).
Animal Models
A number of animal models, including chicks, mice, rabbits, calves, and pigs,
has been used to study the pathogenesis of SLT producing E. coli.
6. HEMOLYSINS
E. coli produce cell-free and cell-bound hemolysins, designated as the α- (AH)
23
and β-hemolysin, respectively. This is an unfortunate designation and has
caused some confusion since both the α- and β-hemolysins cause β-hemolysis
(clear zone of lysis) around colonies on blood agar plates. An γ-hemolysin was
also produced by mutants resistant to nalidixic acid and this hemolysin does not
hemolyze human or rabbit RBC but does hemolyze RBC of other species
(Cavalieri et al., 1984). The AH has been studied intensively and discussed as
follows.
6.1. Production and Purification
Hemolysin-producing E. coli isolates are found primarily but not exclusively

in serogroups O4, O6, O18, and O75.
Ah is produced by growing hemolytic isolates in an alkaline meat extract

broth, casein hydrolysate, or a chemical defined medium at 37C, aerobic,
anaerobic condition and also in CO2. Aerobic growth enhances AH production.
Both AH and β-hemolysin are produced during the log phase of growth
(Cavalieri et al., 1984).
Iron concentration above 100 μM represses hemolysin production. It is

suggested that a major function of AH in vivo may be to provide iron for growth
under iron-limiting conditions.
AH has been purified by various biochemical methods. However,

electrophoresis of AH preparations in PAGE gels under nondenaturing
conditions was unsuccessful, probably due to the high molecular weight of the
molecule. Electrophoresis after denaturing with SDS and 2-mercaptoethanol
and boiling revealed one well-defined protein band. AH was also purified by
affinity chromatography. An immunoadsorbent was prepared by coupling
Affi-Gel and the monoclonal antibody (Bohach and Snyder, 1986).
6.2. Characteristics
AH, purified by affinity column chromatography, contains several proteins

and lipopolysaccharides. Thus, AH exists as a macromolecular complex and
may be exported from E. coli cells by outer membrane fragmentation (Bohach
and Snyder, 1986). Complexing of AH proteins to LPS could account for
discrepancies between measurements of the size of active AH (150,000 to
24
300,000 daltons) and the 106,000 to 110,000-dal predicted by the size of its
structural gene (Pollard et al., 1990). Treatment of AH with DNase, RNase,
lecithinase, or lysozyme has no effect on AH activity, indicating that nucleotides,
lecithin, or peptidoglycan do not comprise the active site. However, enzymatic
treatment with lipases destroys hemolytic activity, suggesting that a lipid
component may be necessary for AH activity (Cavalieri et al., 1984).
Divalent cation calcium, strontium, or barium was required to demonstrate

hemolytic activity in cultures of E. coli. The requirement of Calcium is not
completely understood. Boehm et al. (Boehm et al., 1990) showed that calcium
is required for binding of AH to erythrocyte membranes. Calcium
autoradiography of the recombinant hemolysins separated by SDS-PAGE and
transferred to nitrocellulose showed that full-length, active hemolysin bound
calcium (Fig.17).
AH is not a heat-stable protein, and it is inactivated by heating at 56C for as

little as 10 min. However, some species of AH are relatively more stable to heat.
Stability to heat is depending on the medium of treatment. Also, AH is
inactivated by formalin and urea.
25
6.3. Genetics
The AH gene locates on various incompatible plasmids. It was shown that

Insertion element (IS) occur in these plasmids that is the possible explanation
for the finding of hemolysin determinants on various types of plasmids
(Cavalieri et al., 1984).
Strains harboring these plasmids produce a cell-free extracellular pool of

hemolysin as well as an intracellular pool. Two types of nonhemolytic mutants
were obtained by chemical and transposon mutagenesis of the plasmids. Some
mutants produce intracellular AH but cannot export it into the medium.
Cloning and the following analysis of AH gene have been done. At least three
cistrons, designated as hlyA, hlyB, and hlyC, clustered in the AH determinant
were found to be involved in synthesis and secretion of AH (Fig. 18) (Cavalieri
et al., 1984). hlyA is responsible for synthesis of precursor, hlyC is responsible
for the processing, and hlyB is responsible for export of AH. The gene product
of hlyA was found to be a 106,000- to 107,000-dal nonsecreted cytoplasmic
protein which is probably the inactive hemolysin precursor. hlyC codes for a
18,000-dal protein that appears to be involved in the conversion of the precursor
hemolysin to active hemolysin with a proposed molecular weight of 58,000.
The hlyC gene product is believed to have dual functions of (i) activation and (ii)
transport of hemolysin through the cytoplasmic membrane to the periplasm. The
hemolysin not treated with HlyC could not bind to RBC (Boehm et al., 1990).
hlyB is not involved in synthesis of hemolysin but is required for transport of
hemolysin from the periplam to the exterior of the cell. The hlyBa cistron codes
for a 46,000-dal protein located in the outer membrane that binds the hemolysin
and transports it through the outer membrane. hlyBb codes for a protein of
62,000-dal, most of which is found in the outer membrane, and presumably
functions in release of hemolysin from the outer membrane (Cavalieri et al.,
1984).
26
Hemolysin determinant on chromosome has also been cloned and studied. As
with AH plasmid, at least three cistrons (A, B, and C) are present on the
chromosomal AH determinant. Cistron hlyA seems to be most variable, whereas
hlyB and hlyC are highly conserved.
The primary structure of E. coli hemolysin (HlyA) contains a 9-amino-acid

sequence which is tandemly repeated 13 times near the C terminus and which is
essential for hemolytic activity. The domain involved in binding calcium was
identified as the tandemly repeated sequences, since the deletion derivative
missing 11 of the 13 repeats did not bind calcium (Boehm et al., 1990).
6.4. Role in Virulence and Mode of Action
AH is toxic and lethal when intravenously injected to animal. It also shows

cytotoxicity and the toxicity can be neutralized by antiserum treatment.
The AH had a rather low activity in membranes formed of pure lipids, such as
phosphatidylcholine or phosphatidylserine. In membranes from asolectin, a
crude lipid mixture from soybean, hemolysin was able to increase the
conductance by many order of magnitude in a steep concentration-dependent
fashion. The asolectin may contain a receptor needed for membrane activity of
27
the toxin. The results of single-channel records showed that the membrane
activity of hemolysin is due to the formation of ion-permeable channels with a
single-channel conductance of about 500 pS in 0.15 M KCl (Benz et al., 1989).
7. ADHERENCE
7.1. In Enterotoxigenic E. coli
Adhesion of ETEC to the small intestinal mucosa is now recognized as an

important early event in colonization and the development of diarrheal disease.
Special classes of protein fimbriae which promote mucosal adhesion of ETEC
have been identified in some ETEC strains. The colonization factor antigens
(CFAs) now include CFA/I, CFA/II, CFA/III, and CFA/IV (formerly PCF8775).
A putative human ETEC colonization factor (PCF0159:H4) has been described
in ETEC serotype O159:H4. The CFA/I, CFA/III, and PCF0159 are probably
homogenous rodlike fimbrial antigens (Fig. 19). CFA/II is composed of three
surface-associated antigenic components termed coli surface antigens (CS), CS1,
CS2, and CS3. Strains of serotype O6:H16 produce either CS1 or CS2 in
associated with CS3, while CS3 alone is found in most other CFA/II serotypes.
CFA/IV also exhibits heterogeneity and currently consists of three distinct CS
antigens, CS4, CS5, and CS6; CS4 and CS5 are rodlike fimbriae, where a
structure has not been reported for CS6. The adhesion mediated by the fimbriae
is shown in Fig. 20 (Knutton et al., 1989b).
28
A new nonfimbrial adhesive factor (antigen 8786), with mol.Wt. 16,300 Da,
was found on the bacterial surface of enterotoxigenic E. coli O117:H4 (Aubel et
al., 1991).
A plasmid was also demonstrated coding for CS5, CS6, heat-stable

enterotoxin, and colicin in O167 (Thomas et al., 1987). Infact, the structural
genes of colonization factors are located on high-molecular-weight plasmids,
except CS1 and CS2, which are chromosomal (Aubel et al., 1991).
Synthesis and Action
The synthesis of fimbriae by E. coli 469-3 (O21:H-) which was isolated from
an infantile enteritis was studied at different temperature. It synthesized
fimbriae at 37C, but not at 18C. No transcripts were detected, indicating that
environmental temperature affects expression by regulating transcription
(Williams and Hinson, 1987).
Colonizing factor occurs in ETEC, however, its role in causing diarrhea

remains unclear. Small bowel colonization by colonizing, nontoxigenic E. coli
impairs water and electrolyte absorption and sucrase activity in the absence or
recognized enterotoxin, cytotoxin, invasion, or effacement traits (Schlager et al.,
1990).
29
Toxicity of heat-labile enterotoxin secreted by an ETEC was 40-fold
enhanced in mixtures containing organisms capable of adhering (Streptococcus
pyogenes) to the cell (Y-1 adrenal mouse cells) compared with monolayers
exposed to organisms whose adherence was inhibited by mannoside (Ofek et al.,
1990). Toxin produced by bacteria adherent to cells are targeted more efficiently
and become relatively inaccessible to neutralization by toxin inhibitors (Table 5)
(VL645 abd VL647 are isogenic strains Fim+ and Fim- strains of E. coli, each
harboring LT+ plasmid, H-10407-p is an enterogenic strain lacking CFA/I but
expressing type 1 fimbriae) (Ofek et al., 1990).
The function of the coli surface antigens of CFA/IV (PCF8775) was studied.
Mutants of PCF8775-positive enterotoxin producing E. coli were obtained.
Mutants carrying CS6 alone colonized the intestine equally as well as strains
carrying CS4-CS6 or CS5-CS6 did, whereas CS-negative mutants were
excreted in the stool for a significantly shorter period. Rabbits previously
infected with mutants carrying CS6 alone or CS6 in combination with CS4 or
CS5 developed diarrhea with a significantly lower frequency after reinfection
with a normally highly diaarheagenic dose. These results suggest that the CS6
component is a colonization factor in rabbits and that it is also capable of
inducing protective immunity (Svennerholm et al., 1988).
Molecular Biology of CFA
The CFA/I gene has been isolated and sequenced. The amino acid sequence
deduced from the nucleotide sequence is composed of 170 amino acids. The
first 23 amino acids are considered to be the signal peptide of the CFA/I protein
since they are not present in the protein sequence. Among the remaining amino
acids, only two are different from the protein sequence. The CFA/I gene has a
typical Shine-Dalgarno sequence located 10 bp upstream from the initiation
30
codon. The sequence TACAAT located 48 bp upstream from the initiation
codon was tentatively designated the -10 sequence of the CFA/I gene promoter.
No sequence homologous to the consensus -35 promoter sequence was found. A
pair of inverted repeat sequences followed by a stretch of eight A's are located
45 bp downstream from the termination codon of the CFA/I gene; this region
may be a p-independent transcriptional terminator (Karjalainen et al., 1989).
The genetic determinant encoding the biosynthesis of 987P fimbriae in the E.

coli strains isolated from neonatal pigs has been cloned into cosmid vector
pHC79, and also cloned into pBR322. Analysis revealed that the 987P gene
cluster contains a transposon that encodes the synthesis of ST and is flanked by
inverted repeats of IS1. Hybridization experiments with the ST- and
987P-specific probes demonstrated that a variety of ST+ 987P+ wild-type E.
coli strains contained contiguous ST-987P DNA, most likely on their
chromosome (Klaasen et al., 1990).
7.2. In Enteropathogenic E. coli
It has been shown that many EPEC strains adhere to cells (e.g. HEp-2, HeLa)
in characteristic patterns termed localized adherence (LA) and diffuse
adherence (DA) (Benz and Schmidt, 1989).
It was demonstrated that hemagglutination (pattern termed HAIII) factor of

EPEC is very similar to the type 1-fimbriae antigenically. Type 1-fimbriae have
been shown to mediate adherence to intestinal mucosa. But not all the EPEC
strains carry type 1-fimbriae, so other structures are likely to be involved in the
adhesion process. Electron microscopy failed to show fimbriae or pilus-like
structures on the bacteria which exhibited adherence to HEp-2 and HeLa cells
(Pal and Ghose, 1990).
Adherence Factor on Plasmids
EPEC are capable of adhering to tissue culture cells. EPEC of classic

serotypes usually form distinctive microcolonies on the surface of epithelial
tissue culture cells. This so called localized adherence (LA) is associated with
the presence of a plasmid of 50 to 70 MDa. Such plasmids encode the so-called
EPEC adherence factor (EAF) (Fletcher et al., 1990). The fragment A from
31
pMAR2 was used as probe in Southern blot analysis, and the result showed high
degree of sequence conservation among these plasmids. Adherence genes from
pMAR2 were cloned as two distinct plasmid regions which confer the
adherence phenotype only when complementing each other in trans (Nataro et
al., 1987).
A DNA probe has been constructed from one of the adherence plasmids
(pMAR-2) and has been used in field trials to detect EPEC (Fletcher et al.,
1990). By comparing the restriction maps, other plasmids associated with cell
adhesion are not similar to pMAR-2 (Fletcher et al., 1990).
Another plasmid, pYR111 from serotype O111:NM, was also associated with
localized adherence (LA) with HeLa cells. Curing of this plasmid yielded
strains which lost the ability to exhibited LA, resistance to the antibiotics, and
expression of lipopolysaccharide (LPS) O-antigenic polysaccharide (Riley et al.,
1987). By conjugation experiment, both the mannose-resistant hemagglutinin
and cell adherence factors were shown to be encoded by the same plasmid in
two diarrheagenic E. coli strains belonging to an EPEC (O86) (Table 6) (Pal and
Ghose, 1990).
The diffuse adherence (DA) was also demonstrated to be associated with a
32
large plasmid in EPEC. EPEC strain 2787 (O127:H27), isolated from a case of
infantile diarrhea, exhibited three major properties: (i) DA to HeLa cells, (ii)
carried two large (about 100-kb) plasmids and one small plasmid (3-kb), and (iii)
no fimbriae. A 6.0-kb fragment from the largest plasmid was cloned to E. coli
and expressed DA phenotype. This insert encoded the production of a
100,000-dal protein mediating adhesion (Benz and Schmidt, 1989).
The cellular adherence factors were associated with cell surface structures of
bacteria that were proteinaceous in nature. So, cellular adherence properties
could be substantially reduced by pronase treatment and by heat treatment
(100C for 5 min) of bacteria (Pal and Ghose, 1990).
Adherence Factor on Chromosome
However, adherence factor may also exist in chromosome. TnphoA insertion

mutants of EPEC with various adherence and pathogenic activity were obtained.
By Southern hybridization of plasmid and total DNA of each strain was
performed to determine the location of each TnphoA insert, and each TnphoA
insert along with flanking EPEC sequences was also cloned. These studies
resulted in the grouping of the mutants into five main categories: (A) strains
with plasmid and chromosomal insertions that alter adherence, (B)
chromosomal insertions that alter the ability to induce actin polymerization, (C)
chromosomal insertions that do not affect adherence or actin polymerization
(Table 7). It indicates that genes affecting EPEC adherence may be located on
both the plasmid and chromosome, that several genes are involved in the
induction of actin polymerization in epithelial cells, and that EPEC invasion is a
complex process involving multiple genetic loci (Donnenberg et al., 1990). The
chromosomally encoded gene products are required for full expression of
enteropathogenicity (Knutton et al., 1987b; Knutton et al., 1987a).
33
Studying the adhesion by using electron microscope, a two-stage model of
EPEC adhesion is proposed (Fig. 21, 22) (Knutton et al., 1987b): (i) initial
attachment of bacteria promoted by plasmid-encoded adhesion, and (ii)
effacement of brush border microvilli and intimate EPEC attachment. The
second stage can occur in the absence of the first atage, but the presence of
plasmid-encoded adhesions appears to greatly enhance the ability of EPEC to
colonize the mucosa.
34
35
Adherence and Pathogenesis
Although EPEC strains possess toxic and adhesive capabilities which are
likely to be involved in the disease process, it has been proposed that the
intimate attachment of EPEC strains to intestinal mucosa could disturb the
function of the microvillous border and bring about diarrhea. Evidence that
adhesive non-toxigenic organisms can cause disease has been demonstrated in a
distinct class of strains of E. coli. These strains of E. coli do not belong to the
EPEC serotypes and are designated enteroadherent E. coli (EAEC) (Knutton et
al., 1987b).
Methodology
Fluorescence Actin Staining (FAS) Method
When bacteria are attached, microvilli are lost. The underlying

cytoskeleton of the epithelial cell is disorganized, with a proliferation of
filamentous actin. The polymerization of actin at the sites of the attaching
and effacing lesion forms the basis of a recently described diagnostic test for
EPEC. Fluorescein isothiocyanate (FITC)-phalloidin, the fluorescein conjugate
of a phallotoxin, binds specifically to polymerized actin (Knutton et al., 1989a;
Knutton et al., 1991).
7.3. In Enterohemorrhagic E. coli
A plasmid of 60 MDa was also found in EHEC (e.g. O157:H7) (Toth et al.,
1990; Tzipori et al., 1987). But experiment in gnotobiotic piglets showed that
the presence of such plasmid is not essential for expression of virulence (Tzipori
et al., 1987). Such plasmid appears to modify the eukaryotic cell adherence of E.
coli O157:H7 and to confer that adherence on E. coli HB101 through surface
structures other than pili. By electron microscopy, the wildtype strain and the
plasmid cured strain which showed reduced adherence had pili (Fig. 23) (Toth
et al., 1990).
36
8. INVASIVENESS
Enteroinvasive E. coli (EIEC) are strains with pathogenicity close to Shigella.

Epithelial-cell invasiveness as detected by the ability of the organism to cause
keratoconjunctivitis in the guinea-pig eye (Sereny test) is absent in
enteropathogenic E. coli. Strains of Salmonella and Shigella were internalized
after attachment to animal cells. Such process also occurs after the adherence of
the E. coli to HEp-2 cells or Henle 407 cells and multiplication has been seen to
take place. The E. coli invasion of human intestinal tissue in vivo has not been
demonstrated (Law, 1988; Miliotis et al., 1989).
9. DETECTION
9.1. Using glucuronidase assay
It was reported in 1976 that β-glucuronidase is limited to E. coli, Shigella

species, and Salmonella species in the family Enterobacteriaceae. Then,
β-glucuronidase substrates have been incorporated in diverse media to detect E.
coli in samples from a variety of sources, such as environmental, food, seawater,
and clinical sources. Constitutive enzyme test demonstrate that 87 to 97% of E.
37
coli isolates are positive, and inducible procedures show 91 to 100% positivity.
A method designated as Colilert system is described as follows. The sample is
plated on MacConkey agar, and the suspected colonies are picked and
resuspended in Colilert tube (Access Analytical Systems, Branford, Conn.,
U.S.A.) (medium containing 4-methylumbelliferyl-β-glucuronide, MUG, as the
fluorogenic indicator) rehydrated with 10 ml of destilled water. Tubes are read
for fluorescence after 24, 28, and 120 h of incubation at 35C. The tube becomes
yellow if total coliforms were present and fluorescent (under long UV light
source) if E. coli is present (Rice et al., 1990).
Using the Colilert system, 95.5% of E. coli isolates from human and animals
were β-glucuronidase-positive in 24 h and 99.5% positive after 28 h of
incubation (Rice et al., 1990). However, in another report 34% of E. coli from
volunteers was β-glucuronidase-negative assayed in lauryl sulfate tryptose broth
containing MUG (LST-MUG, Difco) (Chang et al., 1989).
9.2. Animal Tissue Culture
Some of the bioassays for detecting EEC are listed in Table 8 (O'Brien and
Holmes, 1987).
Some cell lines such as Chinese Hamster Ovary (CHO) and Y1 Adrenal cells
response to the LT. Cells could be cultivated in petri dishes or microplates.
Bacterial cultures plus animal tissue culture medium are added to cells and
incubate for 1 to several hours and replaced with fresh culture medium. The
rounding of cells could be observed at 18 to 24 h (Sack and Sack, 1975).
The EPEC and EHEC adhere to the intestinal mucosa and produce and
38
attaching and effacing lesion in the brush border microvillous membrane, this
phenomenon also appears in tissue culture cells exposed to these bacteria or
toxins. Dense concentrations of microfilaments are present in the apical
cytoplasm beneath attached bacteria. Such polymerization of actin can by
detected by Fluorescein-labeled phallotoxin (FAS). So FAS can be a simple,
highly sensitive diagnostic test for EPEC and EHEC (Knutton et al., 1989a).
9.3. Animal Assays
Rabbit ligated ileal loop assay (RIL) is usually employed. Test using infant
mice is a convenient assay for STa. Supernatants of cultures (0.1 ml) could be
injected with a no. 30 hypodermic needle into the milk-filled stomachs of infant
mice (1-4 day old) and fluid accumulation in the intestine was measured after 4
h by determining the ration of intestine to whole body weight. Usually two
drops of a 2% solution of pontamine sky blue 6BX (DuPont) are added to each
1 ml of inoculum. Results from mice with no dye in the intestine tract at
autopsy are discarded. CT did not dilate infant-mouse intestine significantly,
even in high concentrations.
The younger mice and/or mice held at lower temperatures (e.g. 25C) tended
to accumulate intestinal fluid, higher gut weight/body weight ratios (Moon et al.,
1978).
9.4. Immunological Methods
Membrane Filter-Enzyme-Labeled Antibody Procedure
A hydrophobic grid membrane filter (HGMF)-enzyme-labeled antibody

method was developed for the rapid detection of hemorrhagic O157 in food. An
O-antigen-specific monoclonal antibody was labeled by horseradish
peroxidase-protein A. The blended sample (e.g. meat) was pipetted through
disposable prefilters (pore size 100-æm), dispensed in 10 ml of peptone water,
and filtered through the HGMFs. The membranes were incubated at 43C for 16
to 20 h on HC agar. Antiserum, horseradish peroxidase-protein A conjugate
were added in 1 ml of Tris-buffered saline (TBS, 20 mM Tris-500 mM NaCl,
pH7.5) containing 1% gelatin. Filtered blocked in 3% gelatin-TBS and colored
developed in coloring reagent containing 4-chloro-1-naphthol, hydrogen
peroxide, etc. This method yielded presumptive identification within 24 h and
39
recovered, on average, 95% of the E. coli O157:H7 artifically inoculated into
the meats (Todd et al., 1988).
Enzyme-linked Immunosorbent Assay (ELISA)
A competitive ELISA for STa is commercially available (COLI ST EIA,

Denka Seiken). This use a peroxidase-conjugated monoclonal antibody to STa
together with STa prepared by peptide synthesis (Scotland et al., 1989).
Monoclonal anti-LT and anti-ST antibodies were also used in GM1-ganglioside
ELISA which does not require advanced equipments(Scotland et al., 1989).
Monoclonal antibodies against different epitopes on colonization factors of
ETEC were produced (Lopez-Vidal et al., 1988) and these antibodies can be
used to detect these virulence factors.
Shiga-like toxin (Stx) can be detected by enzyme immunoassay (EIA)

(ProSpecT STEC; Remel, Lenexa, KS) and by culturing on chromogenic agar
(Chromagar O157; BD BBL, Sparks, MD) (Grys et al., 2009).
Antibody array constructed on solid supports using nitrocellulose membrane

and poly-l-lysine (PLL) glass slide was developed for the detection of E. coli
(Karoonuthaisiri et al., 2009). Different types of immunosensors (biosensors)
proposed for rapid identification of E. coli O157:H7 were evaluated (Tokarskyy
and Marshall, 2008).
Latex Agglutination Test
A simple latex agglutination test (E. coli O157 latex test, DR620, Oxoid) was
developed for rapid presumptive detection of E. coli serotype O157:H7 during
outbreak of hemorrhagic colitis. It is highly efficient and reliable test with 100%
sensitivity and specificity. The latex test includes two reagents: test latex,
consisting of latex particles sensitized with specific rabbit antibody reactive
with the E. coli O157 antigen, and control latex, consisting of latex particles
sensitized with preimmune rabbit globulins. It is a slide agglutination format,
best used in conjunction with Sorbitol-MacConkey medium (SMAC). The
SMAC medium is not very suitable for screening food samples (March and
Ratnam, 1989). A V. cholerae enterotoxin Reverse Phase Latex Agglutination
kit is commercialized by Seiken and this kit is also can be used to detect LT of E.
coli.
40
Commercial latex agglutination kit for the detection of LT-producing E. coli
is also available (Oxoid, TD920) (Notermans et al., 1991).
VTEC can be detected by VTEC-Screen 'Seiken'. In a modified method, only

parts of the commercial kit were used, including the Polymyxin B sulution,
diluent, sensitized latex, control latex and the positive control. The basis for the
test was the coagglutination test for strains of E. coli which produce the
heat-labile enterotoxin (LT). Growth from each culture was emulsified in 50 μl
polymyxin solution in the well of a U-bottomed microtitre tray. For each strain,
25 μl diluent were dispensed into one well of a V-bottomed microtitre tray.
Using a multichannel pipette, 25 μl of supernantant fluid were carefully
removed from the U-bottomed tray, avoiding contact with the precipitated
bacterial cells, and added to the diluent. Following thorough mixing, 25 μl were
placed in an adjacent well. To one of this pair of wells was added 25 μl
sensitized latex and to the second well, 25 μl control latex. In addition, 25 μl
positive control were placed into two wells and treated with the two latex
preparations. The tray was sealed and left in the dark at room temperature for 2
h. It was then centrifuged 500 g for 15 min at 22°C and set at an angle of about
45° for up to 1 h at room temperature. The tray was regularly examined for
bleeding of the control latex while the positive control with the sensitized latex
remained intact. Where bleeding occurred in the test wells, these were
considered negative; where they remained intact, these were considered positive
(Bettelheim, 2001)
E. coli in water samples were assayed by traditional and immunomagnetic

separation/adenosine triphosphate (IMS/ATP) methods. Pearson's correlation
analysis showed strong, significant, linear relations between IMS/ATP and
traditional methods for all sample treatments; strongest linear correlations were
with the direct analysis (r = 0.62 for E. coli) (Fig. 24). Simple linear regression
was used to estimate bacteria concentrations as a function of IMS/ATP results
(Bushon et al., 2009). In the ATP method, ATP of E. coli was released into
solution to which luciferin-luciferase was added to produce light.
41
9.5. Enzymatic bio-nanotransduction
Enzymatic bio-nanotransduction is based on the production and measurement

of biological nano-signals (nucleic acid sequences) in response to the biological
recognition of the targeted organism or toxin. Specifically, biological
recognition molecules (such as antibodies) are linked to DNA templates that
code for a T7 RNA polymerase promoter and a given nucleotide sequence. The
specific capture and concentration of a target organism or toxin that is bound to
a recognition molecule is followed by an in vitro transcription reaction of the
bound DNA template. Detection of the RNA nano-signals on a detection
platform is correlated with the presence or absence of the target in the original
sample. By this approach, it is possible to detect multiple targets and target
types (e.g., DNA, RNA, protein, whole cells) in a single sample by changing the
recognition element (e.g., antibody, nucleic acid probe, aptamer,
peptidomimetic) linked to the DNA template. In addition, it is possible to link
this flexible detection system to nucleic acid detection platforms such as
biosensors (Branen et al., 2007).
9.6. Nucleic Acid Probes
42
There are a number of reports on the detection of various toxin producers of
EEC using various nucleic acid probes in different formats. Nucleic acid probes
based on different virulence factors have been developed for the detection of
pathogenic E. coli.
Detection of Heat-labile and Heat-stable Enterotoxins
LT probe isolated from pEWD299 after cleavage with HindIII and EcoRI
(Sommerfelt et al., 1988a), or a 850-bp HincII digestion were used (Echeverria
et al., 1989). Oligonucleotide probes were used used for the detection of LT,
e.g. 5'-A CGT TCC GGA GGT CTT ATG CCC AGA GGG CAT AAT-3'
(Sommerfelt et al., 1988a). PCR were used to amplify LT. A conservative
sequence of LT subunit A was amplified by PCR and detected by radio- or
enzyme-labeled oligonucleotide probes. 10 μl of saline suspension of bacteria
was used and combined in a total volume of 10 μl with a 10X PCR buffer; a 10
μl of 8 mM deoxynucleoside triphosphate stock (2 mM each of dATP, dGTP,
dTTP, and dCTP); and 4 μl of each primer (10 μM each), and distilled water to
final volume of 99 μl. The reaction mixtures were overlaied with mineral oil
and heated initially to 95C for 10 min to lyse the bacteria and denature the DNA
(Olive, 1989; Victor et al., 1991).
215-bp HapII fragment from the STa-II gene of pSLM004 into the AccI site
of pUC8 and later cleaved with BamHI and PstI and used as probe (Sommerfelt
et al., 1988a). Aslo, a 157-bp HinfI fragment from the STa-I gene cloned into
the SmaI site of pUC8 and cleaved by EcoRI and BamHI and used as probe.
Oligonucleotide probes were also used, e.g. STa-I, 5'-GAA CTT TGT AAT CCT
GCC TGT GCT GGA TGT-3'; STa-II, 5'-GAATTG TGT AAT CCT GCT TGT
ACC GGG TGC-3' (Sommerfelt et al., 1988b; Sommerfelt et al., 1988a). A
number of gene probes for various types of ST were used to detect ST genes in
E. coli strains isolated in Brazil (Maas et al., 1985).
One to four oligonucleotide probes labeled by alkaline phosphatase are used

to detect each of the enterotoxin gene types and these probes are provided by
Bresatec Ltd., Adelaide, South Australia, Australia (Medon et al., 1988).
A single RNA probe was synthesized and used to detect simultaneously the
LT and ST in E. coli strains. 911-bp of DNA where the regulatory elements and
the signal peptide of eltB (for LT) were replaced by estA2 DNA (for ST) was
43
cloned into pSP plasmids containing SP6 promoter. Plasmid pYK159 (estA-eltB)
was first linearized with HindIII and then transcribed with SP6 RNA
polymerase with biotin-labeled synthetic UTP. The results with the biotinulated
or radioactive probe correlated 100% with the biological assay results for both
toxins. This probe could facilitate large epidemiological studies (S:aez-Llorens
et al., 1989).
Detection of Invasion
An EIEC probe, 17-kb EcoRI digestion fragment of pRM17, was studied

(Echeverria et al., 1989).
Detection Shiga-like Toxin
Toxin gene probes consisting of the 1,142-bp TaqI-HincII fragment of parts

of the SLT-IA and SLT-IB subunits and the 842-bp SmaI-PstI fragment of the
SLT-IIA subunit were excised, purified, and labeled with 32P. Colony blot from
cultures on Modified Trypticase soy agar (BBL Microbiol.) and dot-blot with
cultures from enrichment broth (Modified Trypticase soy broth, BBL) were
hybridize with these probes. The dot-blot technique yielded results within 48 h
and can be used as a fast and sensitive method of detection for SLT-producers
(Samadpour et al., 1990). In Thailand, these probes were used in determination
of SLT-producers in children with diarrhea (Brown et al., 1989a; Seriwatana et
al., 1988a), also in meats (Suthienkul et al., 1990).
Synthetic oligonucleotide probes labeled with 32P were also used for detecting
E. coli producing SLT-I, SLT-II, and a variant of SLT-II (Brown et al., 1989b;
Karch and Meyer, 1989a; Meyer et al., 1989). The results were affected by the
hybridization temperatures (Brown et al., 1989b). Oligonucleotide probes for
the detection of LTI, ST-Ia, ST-Ib, inv, EAF, SLT-I, and SLT-II were described
(Olsvik et al., 1991).
Unique restriction fragments present in plasmids of serogroup O157 E. coli

strains were used as DNA probe for the detection of enterohemorrhagic E. coli
(Huck et al., 1995).
Detection of adherence factor
44
A 21-base oligonucleotide probe was constructed on the basis of a sequence
from within the 1-kb E. coli adherence factor (EAF) probe and was shown to
have greater sensitivity and specificity than the EAF fragment probe in
detecting localized adherent E. coli. These isolates of enteropathogenic E. coli
that exhibit localized adherence to HEp-2 cells (Jerse et al., 1990).
9.7. Using polymerase chain reaction
Polymerase chain reaction (PCR) methods have been developed.

Nonradioactive reporting material, digoxigenin-11-dUTP, was incorporated
(Jackson, 1991). PCR has been used for amplifying segment of distinct SLT
genes (Karch and Meyer, 1989b). The primers deduced from a conserved region
among SLT genes are so-called degenerate-sequence primers; e.g., they contain
intentionally introduced sequence ambiguities to overcome minor sequence
variations within different SLT genes. In direct gel hybridization with genomic
DNA, both primers recognized SLT-I and SLT-II sequences
The PCR was used as a tool for the detection of heat-labile enterotoxin (LT)
gene. The nucleotide sequences of the left- and right-hand amplimers were
5'-CCTCTCTATATGCACACGGAGCTCCCCAG-3' and 5'-CTATATGTTGAC
TGCCCGGACTTCGACC-3', respectively. For identification of the amplified
PCR product, an internal 20-mer probe with the sequence 5'-ATACGGAATC
GATGGCAGGC-3' was used. When 3 CFU of E. coli LT was added to the 25-g
samples of minced meat prior to enrichment culturing, the PCR assay yielded
positive results (Wernars et al., 1991).
Inosine-containing oligonucleotide primers were used for enzymatic

amplification of different alleles of the heat-stable toxin type I gene. Inosine
residues have no destabilizing effect on DNA duplexes regardless of the
opposing bases (Candrian et al., 1991).
PCR and gene probe detection of target lacZ (β-galactosidase) and uidA
(β-glucuronidase) genes were used to detect total coliform bacteria and E. coli,
respectively, for determining water quality (Bej et al., 1991a; Bej et al., 1991b).
On the basis of virulence markers and clinical data, test strains could be split
into different pathogroups, such as uropathogenic E. coli, enteropathogenic E.
coli, Shiga toxin-producing E. coli, and enteroaggregative E. coli. H serotyping
45
and genotyping of the flagellin (fliC) gene revealed 11 different H types and a
close association between certain H types, virulence markers, and pathogroups
was found. Nucleotide sequence analysis of the O-antigen gene cluster revealed
putative genes for biosynthesis of O15 antigen. PCR assays were developed for
sensitive and specific detection of the O15-antigen-specific genes wzx and wzy
(Fig. 25) (Beutin et al., 2005).
A novel AB5 subtilase cytotoxin is produced by certain Shiga toxigenic E.

coli (STEC) strains. This potentially lethal toxin may contribute to severe
gastrointestinal and systemic disease in humans. A multiplex PCR assay for the
detection of the novel toxin A subunit gene subA, as well as stx1 and stx2. The
three primer pairs used in the assay do not interfere with each other and
generate amplification products of 556, 180, and 255 bp, respectively (Fig. 26)
(Paton and Paton, 2005).
46
Real-time PCR methods were also developed. PCR protocol for rapid
identification of enterohemorrhagic E. coli on a LightCycler instrument. In a
multiplex assay, the genes encoding Shiga toxin 1 and Shiga toxin 2 are
detected in a single reaction capillary. A complete analysis of up to 32 samples
takes about 45 min (Bellin et al., 2001).
Real-time PCR method targeting on the fliC gene was developed. The fliC
allele of STEC O91:H21 strain B2F1 was amplified and sequenced. The
nucleotide sequence obtained was compared with fliC genes of E. coli
O157:H21, O8:H21 and O113:H21 strains. A pair of oligonucleotide primers
and a TaqMan minor groove binder probe specific for fliC-H21 were designed
and used in a 5'-nuclease PCR assay. The primers fliCH21-F (5’-ACAGGAT
AAAGATGGCAAACAAGTT) and fliCH21-R (5’-GCAGCCACTGCAAGC
TTAGTT) and the TaqMan minor groove binder (MGB) probe (5’-FAM-
ACCAGCAGTGATACTCMGB) were selected using Primer Express software
(Applied Biosystems). This method was evaluated using a panel of 138 diverse
bacterial strains and was shown to be 100% specific for H21. PCR amplification
of fliC-H21 from one cell per reaction mixture was possible, and an initial
47
inoculum of 10 STEC H21 colony-forming units per 25 g of ground beef was
detected after overnight enrichment (Fig. 27) (Auvray et al., 2008).
Samples from Australia were also tested using real-time PCR (with SYBR
Green I dye) for the presence of potential pathogenic microorganisms. Results
showed that in the 27 rainwater samples tested, 17 (63%), 21 (78%), 13 (48%),
and 24 (89%) were positive for E. coli, enterococci, C. perfringens, and
48
Bacteroides spp., respectively. 11 (41%), 7 (26%), 4 (15%), 3 (11%), and 1 (4%)
were PCR positive for the Campylobacter coli ceuE gene, the Legionella
pneumophila mip gene, the Aeromonas hydrophila lip gene, the Salmonella
invA gene, and the Campylobacter jejuni mapA gene (Ahmed et al., 2008).
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ENTEROPATHOGENIC ESCHERICHIA COLI

Escherichia coli is part of the normal large-bowel flora of humans and

E. coli is usually considered to be an opportunistic pathogen which

E. coli was first incriminated as an enteropathogen in 1945, responsible for an

There are several subgroups of EEC: (A) enterotoxigenic (ETEC), (B)

In 1995, the enteropathogenic E. coli (EPEC) pathotype is divided into two

2.1. General Characteristics

Two partially cross-reacting antigenic variants of plasmid-coded LT,

Another heat-labile enterotoxin was discovered in extracts of E. coli SA53, a

2.2. Gene and Regulation

The gene of LT-IIa was studied. It is organized in a transcriptional unit

The carboxy-terminal domain of EtxB (encodes B subunit) mediates A

2.3. Mode of Action

LT from E. coli is a protein of approximately 86,000 daltons that consists of

The LT-II, similar to CT and LT-I, increases cAMP by activating adenylate

Separation of ETEC bacteria from target intestinal epithelial monolayers by

Enterotoxigenic E. coli LT-induced diarrhea is the leading cause of infant death

3.1. General Characteristics

The heat-stable enterotoxins are low-molecular-weight, heat-stable,

Generally STs are known to be small peptide toxins consisting of 18 (STb) or

STb is a heat-stable enterotoxin which does not cause intestinal fluid

In swine, the most common and severe enterotoxigenic E. coli (ETEC)

3.2. Mode of Action and Regulation

The enteropathogenic strains (O8:KX105) which colonized the small

Enterotoxin plasmids from classical strains (frequently associated with

Enteropathogenic E. coli (EPEC, e.g. O26:H11, O128:B12, O138:K81) and

Iron is known to depress Shiga toxin production by Shigella dysenteriae 1,

5.3. Production and Regulation

Production of SLT is activated in iron-limited media. By a gene fusion

Phage conversion is responsible for controlling the production of several

The structural gene of a SLT-II variant (SLT-IIv) produced by a strain of E.

5.5. Role in Disease

E. coli produce cell-free and cell-bound hemolysins, designated as the α- (AH)

6.1. Production and Purification

Hemolysin-producing E. coli isolates are found primarily but not exclusively

Ah is produced by growing hemolytic isolates in an alkaline meat extract

Iron concentration above 100 μM represses hemolysin production. It is

AH has been purified by various biochemical methods. However,

AH, purified by affinity column chromatography, contains several proteins

Divalent cation calcium, strontium, or barium was required to demonstrate

AH is not a heat-stable protein, and it is inactivated by heating at 56C for as

The AH gene locates on various incompatible plasmids. It was shown that

Strains harboring these plasmids produce a cell-free extracellular pool of

The primary structure of E. coli hemolysin (HlyA) contains a 9-amino-acid

6.4. Role in Virulence and Mode of Action

AH is toxic and lethal when intravenously injected to animal. It also shows

7.1. In Enterotoxigenic E. coli

Adhesion of ETEC to the small intestinal mucosa is now recognized as an

A plasmid was also demonstrated coding for CS5, CS6, heat-stable

Synthesis and Action

Colonizing factor occurs in ETEC, however, its role in causing diarrhea

Molecular Biology of CFA

The genetic determinant encoding the biosynthesis of 987P fimbriae in the E.

7.2. In Enteropathogenic E. coli

It was demonstrated that hemagglutination (pattern termed HAIII) factor of

Adherence Factor on Plasmids

EPEC are capable of adhering to tissue culture cells. EPEC of classic

The diffuse adherence (DA) was also demonstrated to be associated with a

Adherence Factor on Chromosome

However, adherence factor may also exist in chromosome. TnphoA insertion

Fluorescence Actin Staining (FAS) Method

When bacteria are attached, microvilli are lost. The underlying

7.3. In Enterohemorrhagic E. coli