FEMS Microbiology Letters 178 (1999) 177^182

Comparison of eae, tir, espA and espB genes of bovine

and human attaching and eacing Escherichia coli
by multiplex polymerase chain reaction
Bernard China *, Frederic Goaux, Vinciane Pirson, Jacques Mainil
Laboratory of Bacteriology, Faculty of Veterinary Medicine, University of Liege, Sart Tilman B43a, B-4000 Liege, Belgium
Received 21 May 1999; received in revised form 13 July 1999; accepted 14 July 1999

Abstract
Attaching and effacing Escherichia coli (AEEC) virulence genes include the eae, the tir, the espA and the espB genes. These
genes have been sequenced from several AEEC strains. The sequences alignments revealed the presence of constant and
variable regions. Multiplex polymerase chain reactions were developed, in order to determine the subtype of each gene present
in a particular isolate. AEEC strains isolated from calves dead of diarrhea, from healthy calves and from infected humans were
compared. The same pathotypes were found in sick and healthy calves but in inverted proportion. These pathotypes were also
found in human AEEC. Although, the human EHEC strains from serotype O157 possessed their own pathotype. 1999
Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Virulence gene; Polymerase chain reaction ; Attaching and eacing Escherichia coli

1. Introduction
Attaching and eacing Escherichia coli (AEEC)
have been implicated in diarrhea and dysentery in
man and various animals including 2^8-week old
calves [1,2]. Attaching and eacing is the term used
to describe an intestinal lesion produced by these E.
coli: attaching indicates the intimate attachment of
bacteria to the enterocyte, eacing relates to the localized eacement of brush border microvilli. AEEC
groups together enteropathogenic E. coli (EPEC)
and enterohemorrhagic E. coli (EHEC) [3]. Humans

* Corresponding author. Tel.: +32 (4) 366 40 52;

Fax: +32 (4) 366 40 55; E-mail: bchina@ulg.ac.be

infections by EHEC can be of bovine origin and

bovine meat is considered as a major source of contamination [3].
The genes responsible for the development of this
lesion are clustered on the chromosome forming a
pathogenicity island called LEE for locus of enterocyte eacement [4,5]. The LEE human EPEC strain
E2348/69 O127H6 was the rst one to be cloned and
sequenced [6]. This 35-kb long DNA fragment includes genes encoding a type III secretion system,
genes encoding type III secreted proteins (espA,
espB, espD and tir) and the eae gene encoding intimin, an outer membrane protein involved in the intimate attachment. A human EHEC strain from serotype O157H7 [4,7] and a rabbit EPEC strain RDEC1 from serotype O15 [8] and several bovine AEEC

0378-1097 / 99 / $20.00 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 3 5 2 - 3

FEMSLE 8942 11-8-99

178

B. China et al. / FEMS Microbiology Letters 178 (1999) 177^182

strains [9] all possess a LEE related to theone of

the human EPEC strain E2348/69 although dierences in length, insertion site and gene sequences
have been reported. Variants of the eae, tir and esp
genes have been cloned and sequenced from human
EPEC and EHEC and from rabbit EPEC strains
[6,7,10^12].
In addition, eae from a dog EPEC [13] and a bovine EHEC [14] have likewise been cloned and sequenced. The eae gene variants of human strains
have been recently classied as K, L, Q and O ([15],
Oswald, personal communication). They can be distinguished by PCR or gene probes [15]. The eae
genes of rabbit and bovine origin are of the L type
and the canine eae gene is of the N type. The aim of
this study was to develop a multiplex PCR specic
for variants of the eae, tir, espA and espB genes already described to compare bovine and human
AEEC strains with respect to those virulence-associated genes.

2. Materials and methods

2.1. Strains
The AEEC strains belonged to three populations.
37 AEEC isolated from the intestinal contents of 24
dierent 2^8-week old calves which died of diarrhea,
34 AEEC isolated from the intestinal contents of
four dierent 24^48-week old cows from the slaughterhouse and 26 AEEC strains isolated independently from stools of humans with diarrhea, dysentery
or hemolytic uremic syndrome. The strains were classied as AEEC because of a positive hybridization
with the probe for the eae gene [16]. Several of these
strains have already been tested for their capacity to
induce an AE lesion in the rabbit ileal loop assay [9].
The 97 strains were serotyped using the following
antisera: O5, O8, O20, O26, O111, O118 and O157
[17]. The human EPEC strain E2348/69 of serotype
O127H6 representing the K group of genes, the rab-

Table 1
Primers used in this study
Genes and
subtypes

Strain
(accession number)

Sequences of the deduced primers

Optimala annealing
temperature (C)

Amplicon size
(bp)

eae
Type K
eae
Type Q
eae
Type L
tir
Type K
tir
Yype Q
tir
Type L
espA
Yype K
espA
Type Q
espA
Type L
espB
Type K
espB
Type Q
espB
Type L

E2348/69
(EMBL X60439)
EDL933
(EMBL Z11541)
193
(GenBank AF043226)
E2348/69
(GenBank AF022236)
EDL933
(GenBank AF07134)
95ZG1
(GenBank AF070068)
E2348/69
(GenBank Z54352)
EDL933
(GenBank Y13068)
RDEC-1
(GenBank U80908)
E2348/69
(GenBank AF022236)
EDL933
(GenBank Y13068)
RDEC-1
(GenBank U80796)

B73: TACTGAGATTAAGGCTGATAA
B138: GACCAGAAGAAGATCCA
B73: TACTGAGATTAAGGCTGATAA
B74: AGGAAGAGGGTTTTGTGTT
B73: TACTGAGATTAAGGCTGATAA
B137: TGTATGTCGCACTCTGATT
B139: CRCCKCCAYTACCTTCACA
B152: CGCTAACCTCCAAACCATT
B139: CRCCKCCAYTACCTTCACA
B141: GTCGGCAGTTTCAGTTTCAC
B139: CRCCKCCAYTACCTTCACA
B140: GATTTTTCCCTCGCCACTA
B163: TGAGGCATCTAARGMGTC
B165: GCTGGCTATTATTGACCG
B163: TGAGGCATCTAARGMGTC
B164: ATCACGAATACCAGTTACCA
B163: TGAGGCATCTAARGMGTC
B166: TGCCTTTCTTATTCTTGTCA
B148: GCCGTTTTTGAGAGCCA
B151: TCCCCAGGACAGATGAGAT
B148: GCCGTTTTTGAGAGCCA
B150: GCACCAGCAGCCTTTGA
B148: GCCGTTTTTGAGAGCCA
B149: CTTTCCGTTGCCTTAGT

50.4

452

50.2

778

50.8

520

54.2

342

54.7

781

53.4

560

48.9

269

47.9

172

46.4

101

50.6

94

53.1

188

50.9

233

R = A+G, K = T+G, Y = C+T, M = A+C.

a
As calculated by the oligo software.

FEMSLE 8942 11-8-99

B. China et al. / FEMS Microbiology Letters 178 (1999) 177^182

179

Table 2
Restriction analysis of amplicons
Gene

Primers

Strain

Amplicon size (bp)

Enzyme

Restriction fragment length (bp)

eaeK
eaeL
eaeQ
tirK
tirL
tirQ
espAK
espAL
espAQ
espBK
espBL
espBQ

B73-B138
B73-B137
B73-B74
B139-B152
B139-B140
B139-141
B163-B165
B163-B166
B163-B164
B148-B151
B148-B149
B148-B150

E2348/69
RDEC-1
ATCC43888
E2348/69
RDEC-1
ATCC43888
E2348/69
RDEC-1
ATCC43888
E2348/69
RDEC-1
ATCC43888

452
520
778
342
560
781
269
101
172
94
233
188

SspI
RsaI
SspI
PstI
XhoI
EcoRV
HaeII
FokI
MnlI
HpaII
Sau3A
Sau3A

298^154
220^200^80
435^343
215^127
320^240
460^321
189^80
61^50
108^64
50^44
133^100
94^94

bit EPEC strain RDEC-1 of serotype O15 representing the L group of genes and the human EHEC
strain ATCC43888 of serotype O157H7 representing
the Q group of genes have been used as positive controls.
2.2. Primers and amplications
The primers used in this study are listed in Table
1. The DNA was prepared as previously described
[18]. The PCR mixture was: 5 Wl of template DNA,
5 Wl of 2 mM dNTP, 5 Wl of 10Ubuer (100 mM
Tris-HCl, 15 mM MgCl2 , 500 mM KCl, pH 8.3),
0.5 Wl of each primer, 1 U of Taq DNA polymerase
(Boehringer Mannheim, Germany) and sterile distilled water till 50 Wl. The amplication was performed in a Gene Cycler (Bio-Rad). For espA, the
following cycles were used: 1U94C for 5 min and
30U(94C for 30 s, 48C for 30 s, 72C for 30 s). For
espB, eae and tir, the following cycles were used:
94C for 5 min and 30U(94C for 30 s, 50C for
30 s, 72C for 30 s). The PCR products were analyzed by gel electrophoresis in 2^2.5% agarose. In
order to be sure that the amplicon was obtained
from the appropriate genes, amplicons were digested
with restriction endonucleases (Table 2).

3. Results
3.1. Selection and specicity of primers
The available sequences of the eae, tir, espA and

espB genes of the K, L and Q groups were aligned and

from each sequence alignment, an upper primer was
selected into a constant part and a lower primer was
selected into a variable region of each gene subtype
to obtain one cocktail of four primers for each gene.
The primers corresponding to one gene were selected
in order to have the same annealing temperature for
each couple and to obtain an amplicon of dierent
size. First, the specicity of the primers was tested on
control strains (Fig. 1). For the eae gene, an amplicon of 778 bp for the amplication of the eaeQ gene
by primers B73 and B74, an amplicon of 452 bp for
the amplication of the eaeK gene by primers B73
and B138 and an amplicon of 520 bp for the amplication of the eaeL gene by primers B73 and B137
were expected. For the tir gene, an amplicon of 342
bp for the amplication of the tirK gene by primers
B139 and B152, an amplicon of 781 bp for the amplication of the tirQ gene by primers B139 and B141
and an amplicon of 560 bp for the amplication of
the tirL gene by primers B139 and B140 were expected.
For the espB gene, we expected an amplicon of 94
bp for the amplication of the espBK gene by primers
B148 and B151, an amplicon of 188 bp for the amplication of the espBQ gene by primers B148 and
B150 and an amplicon of 233 bp for the amplication of the espBL gene by primers B148 and B149
were expected. For the espA gene, 269-, 172- and
101-bp amplicons for espAK , espAQ and espAL amplications were expected, respectively. In each case, an
amplicon of the expected size was observed (Fig. 1
and Table 1). Moreover, all the amplicons gave re-

FEMSLE 8942 11-8-99

180

B. China et al. / FEMS Microbiology Letters 178 (1999) 177^182

Fig. 1. Analysis of PCR amplications. The eae, tir, espA and espB genes were amplied by multiplex PCR. Amplication products were
analyzed by electrophoresis on a 3% agarose gel. Lane 1: eae gene from O127 human EPEC strain E2348/69 amplied with primers B73,
B74, B137 and B138; lane 2: eae gene from O15 rabbit EPEC strain RDEC-1 amplied with primers B73, B74, B137 and B138; lane 3:
eae gene from O157H7 human EPEC strain ATCC43888 amplied with primers B73, B74, B137 and B138; lane 4: DNA from E. coli
strain JM109 amplied with primers B73, B74, B137 and B138 ; lane 5: tir gene from strain E2348/69 amplied with primers B139, B140,
B141 and B152; lane 6: tir gene from strain RDEC-1 amplied with primers B139, B140, B141 and B152; lane 7: tir gene from strain
ATCC43888 amplied with primers B139, B140, B141 and B152 ; lane 8: DNA from E. coli strain JM109 amplied with primers B139,
B140, B141 and B152; lane 9: espA gene from strain E2348/69 amplied with primers B163, B164, B165 and B166; lane 10: espA gene
from strain RDEC-1 amplied with primers B163, B164, B165 and B166; lane 11: espA gene from ATCC43888 amplied with primers
B163, B164, B165 and B166; lane 12: DNA from E. coli strain JM109 amplied with primers B163, B164, B165 and B166; lane 13 : espB
gene from strain E2348/69 amplied with primers B148, B149, B150 and B151; lane 14: espB gene from strain RDEC-1 amplied with
primers B148, B149, B150 and B151; lane 15: espB gene from strain ATCC43888 amplied with primers B148, B149, B150 and B151;
lane 16 : DNA from E. coli strain JM109 amplied with primers B148, B149, B150 and B151. Molecular mass ladder is in bp.

striction fragments of the expected size (Table 2,

data not shown). No amplication product was obtained from E. coli K12 JM109.
3.2. Typing of bovine AEEC strains
The four primer cocktails were used to amplify
eae, tir, espA and espB genes from bovine AEEC
isolated either from the intestinal content of 2^8week old calves dead of diarrhea or from the intestinal content of 24^48-week old healthy calves at the
slaughterhouse (Table 3). In calves with diarrhea, the
eae gene mainly (67.5%) observed is the eaeL gene
while the other strains possessed an eaeQ gene. In
healthy calves, the image was inverted since 82% of
the strains possessed an eaeQ gene and only 18% an
eaeL gene. Interestingly, the eaeK gene was not recovered from either sick or healthy calves. For the tir

gene, in calves with diarrhea, the tirL was dominant

and in healthy calves, the tirK was dominant. The tirQ
was never amplied from either sick or healthy
calves. For the espB gene, in calves with diarrhea,
the espBL was dominant (67.5%) and in healthy
calves, the espBK was dominant (82%). The espBQ
was not amplied from both calf populations.
The same results were obtained for the espA gene.
In general, only two gene combinations were found
in both calf populations: eaeL tirL espAL espBL and
eaeQ tirK espAK espBK . Moreover, in AEEC strains
isolated from calves with diarrhea, the pathotype
eaeL tirL espAL espBL was dominant (67.5%) and
the pathotype eaeQ tirK espAK espBK was present as
a minority. In contrast, in AEEC isolated from
healthy calves, the pathotype eaeQ tirK espAK espBK
was dominant (82%) and the pathotype eaeL tirL espAL espBL was present as a minority (18%). The per-

FEMSLE 8942 11-8-99

B. China et al. / FEMS Microbiology Letters 178 (1999) 177^182

centage was extremely dierent (P 6 0.001) from one

population to the other as calculated by the Fisher
exact test.
Pathotype eaeL tirL espAL espBL was associated
with serogroups O5 (1/31), O20 (1/31) and O26 (5/
31) and pathotype eaeQ tirK espAK espBK was associated with serogroups O5 (3/36), O20 (1/36), O111 (6/
36) and O118 (4/36) but the majority of bovine
AEEC strains were untypable using our antisera
(49/71).
3.3. Typing of human AEEC strains
Since bovine meat is considered as a major source
of human contamination, we investigated if the combination eaeQ tirK espAK espBK could be recovered
from human AEE. Seven O111 human EHEC
strains, eight human O26 EHEC and 11 human
O157 EHEC strains were isolated from patients
with diarrhea, dysentery or haemolytic uremic syndrome. The results indicated that the seven O111
EHEC were eaeQ tirK espAK espBK , the eight O26
EHEC were eaeL tirL espAL espBL and the 11 O157
EHEC were eaeQ tirQ espAQ espBQ (Table 3).
4. Discussion
AEEC cause diarrhea and dysentery in 2^8-week
old calves and are recovered from healthy adults
[2,19] but also in humans where bovine product is
the major contamination source [3]. The subdivision
based on the serotype is less and less clearly dened
as the range of serotypes isolated from both cattle
and humans widens continuously. The multiplex

181

PCR seems to be a good alternative. We constructed

multiplex PCR for variants of the eae, tir, espA and
espB genes. The nomenclature used by Adu-Bobie et
al. [15] for the eae gene was extended to the other
genes. First, we studied bovine AEEC isolated either
from calves dead of diarrhea or from healthy calves.
Only two pathotypes (out of 81 combinations) were
found with dierent frequencies in bacteria from two
calf populations. Although both pathotypes (eaeL
tirL espAL espBL and eaeQ tirK espAK espBK ) were
present in healthy and diseased calves, the pathotype
eaeL tirL espAL espBL seems to be more frequently
associated with virulence. Second, we addressed the
question of the correlation between pathotype and
serogroup and of the comparison with human
AEEC belonging to three serogroups also found in
cattle: O26 and O111 causing diarrhea in calves and
isolated from healthy cattle and O157 isolated from
healthy cattle [3,20]. All the tested O26 strains
possessed the pathotype eaeL tirL espAL espBL and
all the tested O111 strains possessed the pathotype
eaeQ tirK espAK espBK . For O157 strains, a new
pathotype appeared (eaeQ tirQ espAQ espBQ ). On the
other hand, the pathotype detected was not correlated to other serogroups, such as O5, O20 or
O118 (Table 3).

Acknowledgements
This work was supported by Grant 5740A from
the `Ministere des Classes moyennes et de l'Agriculture, DGVI, Recherche et Developpement'. We
thank Dr Denis Pierard of the `AZ-VUB' for providing the human EHEC strains.

Table 3
Pathotype of tested AEEC
Source

Number of
strains

Pathotype

Calves with diarrhea

37

Healthy calves

34

Human

26

eaeL
eaeQ
eaeL
eaeQ
eaeL
eaeQ
eaeQ

tirL
tirK
tirL
tirK
tirL
tirK
tirQ

espAL espBL
espAK espBK
espAL espBL
espAK espBK
espAL espBL
espAK espBK
espAQ espBQ

Number of
positive

Serogroup (number of strains)

25
12
6
28
8
7
11

O5 (1), 026 (2), O118 (1), NT (21)

O111 (1), NT (11)
O20 (1), O26 (3), NT (2)
O5 (3), O20 (1), O111 (5), O118 (4), NT (15).
O26 (8)
O111 (8)
0157 (11)

NT: non-typable using our antisera.

FEMSLE 8942 11-8-99

182

B. China et al. / FEMS Microbiology Letters 178 (1999) 177^182

References
[1] Mainil, J.G., Jacquemin, E., Kaeckenbeeck, A. and Pohl, P.
(1993) Association between the eacing gene (eae) and the
Shiga-like toxin-encoding genes in Escherichia coli isolates
from cattle. Am. J. Vet. Res. 54, 1064^1068.
[2] China, B., Pirson, V. and Mainil, J. (1998) Prevalence and
molecular typing of attaching and eacing Escherichia coli
among calf populations in Belgium. Vet. Microbiol. 63, 249^
259.
[3] Nataro, J.P. and Kaper, J.B. (1998) Diarrheagenic Escherichia
coli. Clin. Microbiol. Rev. 11, 142^201.
[4] McDaniel, T.K., Jarvis, G., Donnenberg, M.S. and Kaper,
J.B. (1995) A genetic locus of enterocyte eacement conserved
among diverse enterobacterial pathogens. Proc. Natl. Acad.
Sci. USA 92, 1664^1668.
[5] McDaniel, T.K. and Kaper, J.B. (1997) A cloned pathogenesis
island from enteropathogenic Escherichia coli confers the attaching and eacing phenotype on E. coli K12. Mol. Microbiol. 23, 399^407.
[6] Elliott, S.J., Wainwright, L.A., McDaniel, T.K., Jarvis, K.G.,
Deng, Y.K., Lai, L.C., McNamara, B.P., Donnenberg, M.S.
and Kaper, J.B. (1998) The complete sequence of the locus of
enteroycte eacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol. Microbiol. 28, 1^4.
[7] Perna, N., Mayhew, G.F., Posfai, G., Elliot, S., Donnenberg,
M.S., Kaper, J.B. and Blattner, F.R. (1998) Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 66, 3810^3817.
[8] Karaolis, D.K.R., McDaniel, T.K., Kaper, J.B. and Boedeker,
E.C. (1997) Cloning of the RDEC-1 locus of enterocyte eacement (LEE) and functional analysis of the phenotype on Hep2 cells. In: Mechanisms in the Pathogenesis of Enteric Diseases (Paul, P.S., Francis, D.H. and Beneld, D.A., Eds.),
Adv. Exp. Med. Biol. 412, pp. 241^245. Plenum, New York.
[9] Goaux, F., Mainil, J., Pirson, V., Charlier, G., Pohl, P.,
Jacquemin, E. and China, B. (1997) Bovine attaching and
eacing Escherichia coli possess a pathogenesis island related
to the LEE of the human enteropathogenic E. coli strain
E2348/69. FEMS Microbiol. Lett. 154, 415^421.
[10] Agin, T.S., Cantey, J.R., Boedeker, E.C. and Wolf, M.K.
(1996) Characterization of the eaeA gene from rabbit enter-

[11]

[12]

[13]

[14]

[15]

[16]

[17]
[18]

[19]

opathogenic Escherichia coli RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-afacing lesions. FEMS Microbiol. Lett. 14, 249^258.
Abe, A., Kenny, B., Stein, M. and Finlay, B.B. (1997) Characterization of two virulence proteins secreted by rabbit enteropathogenic Escherichia coli, EspA and EspB, whose maximal
expression is sensitive to host body temperature. Infect. Immun. 65, 3547^3555.
Paton, A.W., Mannig, P.A., Woodrow, M.C. and Paton, J.C.
(1998) Translocated intimin receptor (Tir) of Shiga-toxigenic
Escherichia coli isolates belonging to serogroups, O26, O111,
and O157 react with sera from patients with hemolytic-Uremic
syndrome and exhibit marked sequence heterogeneity. Infect.
Immun. 66, 5580^5586.
An, H., Fairbrother, J.M., Dubreuil, D. and Harel, J. (1997)
Cloning and characterization of the eae gene from a dog attaching and eacing Escherichia coli strain 4221. FEMS Microbiol. Lett. 148, 239^245.
China, B., Devrin, A.C., Jacquemin, E., Pirson, V. and Mainil, J. (1999) Heterogeneity of the eae genes in attaching/eacing Escherichia coli from cattle : comparison with human
strains. Res. Microbiol. 150, 323^332.
Adu-Bobie, J., Frankel, G., Bain, C., Gonzales, A.G., Trabulsi, L.R., Douce, G., Knutton, S. and Dougan, G. (1998) Detection of intimins K, L, Q, and N four intimin derivates expressed by attaching and eacing microbial pathogens. J. Clin.
Microbiol. 36, 662^668.
Jerse, A.E., Yu, J., Tall, B.D. and Kaper, J.B. (1990) A genetic locus of enteropathogenic Escherichia coli necessary for
the production of attaching and eacing lesions on tissue culture cells. Proc. Natl. Acad. Sci. USA 87, 7839^7843.
Orskov, F. and Orskov, I. (1984) Serotyping of Escherichia
coli. Method. Microbiol. 14, 43^112.
China, B., Pirson, V. and Mainil, J. (1996) Typing of bovine
attaching and eacing Escherichia coli by multiplex in vitro
amplication of virulence-associated genes. Appl. Environ.
Microbiol. 62, 3462^3465.
Blanco, M., Blanco, J.E., Blanco, J., Moro, A., Prado, C.,
Alonso, M.P., Mourino, M., Madrid, C., Balsalobre, C. and
Juarez, A. (1997) Distribution and characterization of faecal
verotoxin-producing Escherichia coli (VTEC) isolated from
healthy cattle. Vet. Microbiol. 54, 309^319.

FEMSLE 8942 11-8-99