Eur J Clin Microbiol Infect Dis (2012) 31:899–904
DOI 10.1007/s10096-011-1395-7
REVIEW
Differentiation between Shigella, enteroinvasive Escherichia
coli (EIEC) and noninvasive Escherichia coli
M. J. C. van den Beld & F. A. G. Reubsaet
Received: 26 April 2011 / Accepted: 18 August 2011 / Published online: 7 September 2011
# Springer-Verlag 2011
Abstract Shigella causes bacillary dysentery and is classi-
fied into four species based on their antigen characteristics.
This classification does not reflect genetic relatedness; in
fact, Shigella species are so related to Escherichia coli ,
they should be classified as one distinctive species in the
genus Escherichia. The differentiation of Shigella and E.
coli is even more complicated with the description of
enteroinvasive E. coli (EIEC). EIEC are strains that possess
some of the biochemical characteristics of E. coli and have
the ability to cause dysentery using the same method of
invasion as Shigella does. Sequencing of multiple house-
keeping genes indicates that EIEC is more related to
Shigella than to non-invasive E. coli. Shigella and EIEC
evolved from the same ancestor and form a single pathovar
within E. coli. Shigella and EIEC could be separated from
other E. coli by a PCR targeting the ipaH-gene; this is a
multicopy gene exclusively found in all Shigella and EIEC.
It is possible to differentiate Shigella and all E. coli,
including EIEC, by using multiple tests, including ipaH-
gene PCR, physiological and biochemical typing and
serological typing. Based on literature study, a key is
designed for daily use in diagnostic laboratories to identify
Shigella and all E. coli.
In the Netherlands, physicians and heads of laboratories
have a notification obligation towards health authorities for
certain infectious diseases. Shigellosis is one of these
infectious diseases, while an infection with Escherichia
M. J. C. van den Beld (*) : F. A. G. Reubsaet
Bacteriology, Laboratory for Infectious Diseases and Perinatal
Screening, National Institute for Public Health
and the Environment,
PO Box 1, Postvak 22, 3720 BA Bilthoven, The Netherlands
e-mail: maaike.van.den.beld@rivm.nl
coli, other than enterohemorrhagic E. coli, is not. The aim
of this study was to develop a key to differentiate Shigella
from all E. coli based on literature study.
Shigella is a human-specific causative agent of bacillary
dysentery, a serious illness characterized by abdominal
cramps, nausea, fever and a bloody and mucus diarrhea.
Shiga first described Shigella as Bacillus dysenteriae in
1898. He named it Bacillus because it seemed to be related
to Bacillus coli, which is now referred to as Escherichia
coli [1, 2]. In the 1940s, Ewing proposed to classify four
species in the new genus Shigella, namely S. dysenteriae, S.
flexneri, S. boydii and S. sonnei, based on the antigen
characteristics of those species. Later research by Lan et al.
showed that the relatedness of serotypes is not parallel to
genetic relatedness [3].
The invasiveness of Shigella can be tested by the Serény
test, which demonstrates the virulent nature of Shigella by
causing a keratoconjunctivitis in guinea pigs or rabbits [4,
5]. This test involves testing animals and is not applicable
on a daily basis in the laboratory [6].
As Shiga already noticed in 1898, Shigella and E. coli
are related. In fact they are so closely related that they
should be classified as one distinctive species within the
genus Escherichia [7, 8]. In fatty acid analysis, Shigella and
E. coli form one cluster with other genera of the family
Enterobacteriaceae (study of author, data not shown).
Brenner et al. determined the nucleotide similarity of
Shigella and E. coli at 80–90%, while other Escherichia
species have a much lower degree of similarity and are
genetically distant. Because the gene encoding for 16S
rRNA is highly conserved, analysis of 16S rDNA have
been used all over the world to determine bacterial
relationships and to support or reject taxonomical decisions.
Based on their sequence of the 16S rRNA-gene Shigella
and E. coli are highly related [9, 10]. The sequencing of
900
numerous other housekeeping genes, virulence genes and
even whole genomes also indicates a high degree of
relatedness between Shigella and E. coli [2, 3, 11–14].
However, because of the severity of dysentery it is a
taxonomical decision to maintain the disposition of Shigella
and E. coli into two different genera.
Despite their relatedness, Shigella could always be
separated from E. coli based on their physiological and
biochemical characteristics. Most of the E. coli strains
(>80%) are motile, lysine decarboxylase (LDC) positive,
form gas from D-glucose and are indol positive. In contrast,
Shigella strains are always non-motile, always LDC
negative and never form gas from D-glucose, except
Shigella flexneri serotype 6, which is indol negative. Acid
formation from salicine and esculine hydrolysis are never
found in Shigella [15–19]. However, separation was
hindered when strains were found that turned out to be
some sort of intermediate form between Shigella and E.
coli. While these strains possess some of the E. coli
biochemical characteristics, they demonstrate the pathogen-
ic behavior of Shigella. Those dysentery causing hybrid
strains that could cross-react with Shigella anti-sera were
first described in 1944, and were later called enteroinvasive
E. coli (EIEC) [2, 3]. EIEC is associated with specific E.
coli O-serotypes. However, because the genetic relations of
Shigella and EIEC are more in agreement with physiolog-
ical and biochemical characteristics than with serotyping,
the first mentioned are still the recommended techniques to
distinguish EIEC from Shigella [3].
The whole-genome sequencing of E. coli and all four
Shigella species revealed that they share a common
backbone of approximately 3,9 kb. E. coli-specific DNA
and Shigella-specific DNA interrupt the common back-
bone. The most striking difference between the genomes of
E. coli and Shigella is the presence of many IS-elements in
the Shigella chromosome which contributes to a very
dynamic genome [11, 20]. The easy acquisition and loss
of genes promote the success of Shigella as a pathogen,
because fast genetic adaptation is necessary to survive in
different circumstances in the host [11, 14, 21]. The four
Shigella species all have very diverse genomes, with a
different mapping of genes presumably caused by transloca-
tion through IS-elements [14, 20]. While the whole genome
of EIEC has not yet been compared to whole genomes of
Shigella, the sequencing of multiple housekeeping genes
indicates that EIEC is genetically more related to Shigella
than to noninvasive E. coli [3]. The above described
characteristics suggests that Shigella could be separated from
non-invasive E. coli genetically, but until now it has not been
possible to distinguish Shigella species from each other and
from EIEC by molecular DNA techniques.
Shigella and EIEC have both evolved from nonpatho-
genic E. coli. The sequencing of multiple housekeeping
Eur J Clin Microbiol Infect Dis (2012) 31:899–904
genes indicates that Shigella has risen on seven different
occasions from seven different ancestors within the group
of nonpathogenic E. coli. All Shigellae group into three
major clusters and four single-strain groups [2]. EIEC
seems to have five origins within the noninvasive E. coli
based on their housekeeping genes. All EIEC strains, but
one, group into four major clusters; the one remaining
strain was probably misclassified (Fig. 1). Shigella-EIEC
forms one single pathovar of E. coli, in which the same
species or serotypes are not necessary phylogenetically
related to each other [3].
Pupo et al. have estimated the time since the descent of
the three above mentioned clusters of Shigella from a
common ancestor, based on the molecular clock rates that
Whittam, Gutman and Dykhuizen described [2]. In their
estimation, clusters 1 and 2 derived from their ancestors
50,000–270,000 years ago, and the derivation of cluster 3 is
estimated at 35,000–170,000 years ago (Fig. 1). Both times
of descent are relatively recent when one takes in account
that a major nonpathogenic E. coli cluster diverged from
other bacteria 8–22 million years ago. The estimated time
of derivation of Shigella coincides with the paleolithic
expansion and the rise of early man. These data are
probably no coincidence because pathogenesis of Shigella
is based on surviving in the intestinal epithelial cells of
humans only—a perfect host-adaptation [2, 12, 22].
Sequence variations in the clusters of Shigella and EIEC
indicate that EIEC evolved from E. coli ancestors later than
Shigella did. Based on this later derivation of EIEC, two
hypotheses about EIEC in relation to Shigella are stated.
First, EIEC is an ancestral form that in time will develop
into ‘real’ Shigella. Second, EIEC is a different group of
organisms that is adapted to human hosts like Shigella is,
but is better equipped to survive outside the host [3].
The major event that probably gave rise to Shigella and
EIEC is the acquisition of the invasion plasmid (pINV)
(Fig. 1) [1]. All virulent Shigellae and EIEC harbor this
pINV, which is a non self-transferable large single-copy
plasmid of 180–230 [23–26]. Early molecular techniques
have revealed that pINVs of different species form a closely
related family and share a common origin outside E. coli.
The pINVs evolved independently; however, the genes that
code for intracellular invasion and survival are highly
conserved [23, 27]. The independently evolved pINVs also
proved to be functionally interchangeable between different
serotypes [26]. The virulence associated genes on the pINV
are probably obtained from another unrelated genus,
because the A + T content of the nucleotides of these genes
is 75%, while the A + T content of all Shigella and E. coli
genomes is 50% [1, 28].
With the acquisition of the pINV, Shigella and EIEC
were able to colonize the human intestinal epithelial cells as
a new niche. For the invasion and maintaining in the host,
Eur J Clin Microbiol Infect Dis (2012) 31:899–904
Fig. 1 Shigella and enteroinva-
sive E. coli (EIEC) evolved
multiple times from
Origin major Shigella cluster 1
non-pathogenic E. coli
Origin major Shigella cluster 2
Origin major Shigella cluster 3
Escherichia coli
Years -320,000 -280,000 -240,000 -200,000 -160,000 -120,000
rise of early man
P = Invasion plasmid
A = Pathoadaptation
Shigella and EIEC need a combined expression of genes
located both on the pINV as well as on the chromosome
[21, 26]. The chromosome of Shigella has adapted to the
acquisition of the invasion plasmid by pathoadaptation.
Pathoadaptation can occur by multiple different events,
such as bringing newly acquired virulence under control of
an already present regulator, or by point mutations within
genes, or by the suppression or expression of certain genes,
or by deletion of anti-virulence genes. The deletions of
these anti-virulence genes are generating so-called ‘black
holes’ [21]. For example, the loss of anti-virulence gene
cadA is a black hole in EIEC and Shigella. CadA encodes
for lysine decarboxylase activity (LDC), which is
present in virtually all non-enteroinvasive E. coli. The
product of decarboxylation of lysine is cadaverin, which is
an inhibitor of Shigella enterotoxins and blocks trans-
epithelial migration. Because of the inhibiting influence of
cadaverin on the virulence of Shigella, natural selection
pressures deletion of the cadA-gene by different genetic
arrangements [21]. Consequently, LDC is a trait which is
very useful to differentiate between non-enteroinvasive E.
coli versus Shigella and EIEC, but not between EIEC and
Shigella.
The pINV, unique to Shigella and EIEC, contains many
virulence genes that in cooperation with virulence-
associated genes on the chromosome evoke a cascade of
events that lead to infection. Shigella causes infection by
901
P A
P A
P A
Origin EIEC cluster 4 P A
Origin EIEC cluster 5 P A
Origin EIEC cluster 6 P A
Origin EIEC cluster 7 P A
-80,000 -40,000 0
invading the intestinal epithelial cells followed by intracel-
lular multiplication and spread to adjacent cells [1, 29].
Recombination techniques and the sequencing of the
invasion plasmid and chromosomal genes associated with
virulence, gave insight into the precise mechanism of
infection by Shigella. First, the bacteria in the intestinal
lumen invade the submucosal side of the colon by trans-
cytosis through microfold cells (M-cells) [30]. Once they
access the submucosa, Shigella is taken up by macro-
phages. After cell death, the bacteria are released in the
submucosa from where they invade epithelial cells by
endocytosis. During the invasion of the epithelial cells,
ipaBCD and mxiAB genes are brought to expression. These
genes are part of the ipa-mxi-spa island on the invasion
plasmid, and the loss of this island results in avirulence [1,
20, 23, 25, 26, 31, 32]. IpaD is believed to play a role in
attaching to host cell membranes, and subsequently ipaB
plays a role in the endocytic uptake of the bacteria. The
roles of the other known virulence genes associated with
invasion of the cell have yet to be discovered. Once inside
the endocytic vacuoles, the bacteria escape rapidly into the
cytoplasm of the host cell [1, 33]. Little is known about the
exact escaping mechanism. When the bacteria have escaped
from the endocytic vacuoles, they are able to deposit F-actin,
which enables intra- and intercellular spreading. The genes
necessary for this feature are virA and virG, located on the
invasion plasmid [1, 19].
902 Eur J Clin Microbiol Infect Dis (2012) 31:899–904

The IpaH-gene is a multicopy gene on the pINV as well copies on the invasion plasmid were acquired from a
as on the chromosome, which is exclusively found in different source [14]. The arrangements of the ipaH copies
Shigella and EIEC [1, 4, 34]. Venkatesan et al. discovered on the invasion plasmids are different, and are bordered by
that the copies of ipaH-genes in Shigella and EIEC are not IS629. It is possible that these IS-elements function as
equivalent but nevertheless all contain a conserved core recombination elements, as a result of which the genes can
region (bp720-1557 of ipaH7.8) and a variable region ‘jump’ through the pINV that could determine which copy
outside this core [35]. The different copies were named is expressed [36].
after the size of the HindIII fragments in which they were Little is known about the exact function in terms of
discovered, for example, ipaH1.4, ipaH2.5, ipaH4.5, ipaH7.8 virulence of Shigella and EIEC of the proteins encoded by
and ipaH9.8 [20, 36]. Most of the copies of ipaH-genes on the ipaH-genes. The discovery that this protein could evoke
the chromosome are present in large ipaH- islands. The an anti-body response indicates that the protein is probably
other genes on this ipaH-island are genetically homologous presented at the surface of the bacteria. However, the
with Escherichia coli phage P27 genes. This homology protein does not contain a signal peptide and it is not
indicates that the ipaH-genes on the chromosome were known which transport-system provides for transport
acquired through transduction of P27 genes [11, 14, 21]. In throughout the cell [31, 34]. All products of ipaH-gene
contrast, the ipaH-gene copies on the pINV are not linked copies have a leucine-rich repeat at the amino terminal end,
with phage genes, which strongly suggests that the ipaH similar to the yopM protein of Yersinia pestis, but are never

Agglutination with EIEC associated antisera d

Agglutination with Shigella antisera

Gas from D-Glucose and indol a
Esculin hydrolysis a
ipaH-gene PCR b

Salicin, acid a
Motility c
LDC a

+ E. coli non-enteroinvasive
- - E. coli non-enteroinvasive
+ + EIEC
- + EIEC
- + EIEC
- + EIEC
- + EIEC
- + Shigella
- Provisional Shigella

a. Based on Edwards and Ewing’s identification of Enterobacteriaceae, 4th edition,

1986 and/or Cowan and Steel’s manual for the identification of medical bacteria,
3rd edition, 1993.
b. Performed with a standard PCR protocol, with primers designed to amplify a part
of the conserved region of ipaH7.8, as described by Buysse et al., Microb. Pathog.
19(5):335-349.
c. Incubated for 24 h in BHI-medium at 37°C.
d. Known O:H serotypes of EIEC according to Bergey’s manual of Systematic
Bacteriology, 2nd edition, volume 2, The Proteobacteria, Part B The
Gammaproteobacteria.

Fig. 2 Key for differentiation

Eur J Clin Microbiol Infect Dis (2012) 31:899–904
found in any other bacteria [34, 35]. However, this leucine-
rich repeat is common in eukaryotic cells, which indicates
that the ipaH protein could play a role in manipulating gene
expression of the host [11]. This hypothesis is supported
with the fact that ipaH-gene mutants cause an over-reactive
response in the Serény test.
The multicopy nature of ipaH (4–10 copies in total) and
the fact that multiple copies were also present on the
chromosome of Shigella and EIEC made it a useful tool for
identification even in plasmid-cured strains or in case of
major deletions [6, 22, 34, 37–39].
In conclusion, Shigella and EIEC can be differentiated
from non-enteroinvasive E. coli by testing for presence of
the ipaH-gene and absence of LDC activity. The presence
or absence of these features is of more taxonomic
importance than the phylogenetical weight of a point
mutation in one of the involved genes. Since EIEC can
have physiological and biochemical characteristics that
separate them from Shigella, identification systems will
identify members of the pathovar Shigella-EIEC as either
E. coli or Shigella. Motility and the biochemical character-
istics fermentation of salicin, esculin hydrolysis and the
combined features gas from D-glucose and indol production
can differentiate between EIEC and Shigella. Supplementary
to the physiological and biochemical characteristics,
serotyping can be necessary for differentiation. In this
case, EIEC-associated antisera should be used only in
combination with Shigella antisera, because of the display
of cross reactivity in antibody response. Shigella strains
that do not react with a known antiserum should be
classified to Shigella, but are until now described as
‘provisional Shigella’.
We have designed a key for daily use in diagnostic
laboratories to identify Shigella, EIEC and non-
enteroinvasive E. coli (Fig. 2). This key is applicable to
strains that have a biochemical pattern similar to that of E.
coli and Shigella. If specific O-antisera are not available,
one can use only the first part of the key or send the strain
to the local reference laboratory.
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