A group of Shiga-toxin-producing Escherichia coli
(STEC) strains, collectively termed enterohemorrhagic
E. coli (EHEC), causes a wide spectrum of illnesses
ranging from mild diarrhea to severe diseases, such as
hemorrhagic colitis and hemolytic uremic syndrome
(21). Shiga toxin (Stx), which is broadly assigned to
two types (Stx1 and Stx2 or its variants) for STEC, is a
cytotoxin causing lethal damage to the host kidney cells
and is thus considered a major virulence factor respon-
sible for these disease symptoms (15, 22). Meanwhile,
the production of attaching and effacing (A/E) lesions
also contributes to the pathogenicity of EHEC strains
(3, 16). A/E was initially described for enteropathogen-
ic E. coli (EPEC) and characterized by the localized
destruction of microvilli and intimate attachment of
bacteria to the host epithelial cells (28). EPEC is thus
defined at a molecular level by the presence of the A/E
gene (eae) and the absence of the genes for Stxs (stx1
and stx2) (14). EHEC strains share the characteristics of
STEC and EPEC, in which a majority of EHEC strains
harbor both stx and eae as described by Caprioli et al.
(5). Although infections with EHEC of other
serogroups (i.e. O26, O103, and O111) have been docu-
mented (9, 28), strains belonging to serogroup O157 are
by far the most important serotype associated with
severe human diseases including acute diarrhea, haem-
orrhagic colitis, and haemolytic uremic syndrome that
can be fatal (4), and cause large outbreaks almost
worldwide (6, 10). Therefore, development of rapid
and sensitive PCR-based methods for E. coli O157:H7
detection is of paramount importance for clinical diag-
nosis and epidemiological study. Until recently, a num-
ber of PCR-based assays to detect EHEC O157 in foods
and feces have been developed. Some of these assays
have targeted a highly conserved O157 specific O-anti-
gen synthesis gene (7, 17) while others have targeted
the gene of flagellar H7 (8, 20). These genes may be
present in other E. coli strains (i.e. eae and stx negative
E. coli O157:H7 strains), and thus not unique for EHEC
O157. Amplified fragment length polymorphism
(AFLP) is based on selective PCR amplification of
restriction fragments (29). AFLP was applied to several
studies on EHEC strains and shown to be useful for
genetic subtyping of the strains (11, 13). In the course
of AFLP analysis, we observed a fragment common to
all EHEC O157 strains tested but not those of other
serotypes on electrophoresed gels. We here report the
genetic identity of the fragment, which might be a DNA
sequence specific to EHEC O157, developed to a novel
PCR-based method for its early detection in foods and
Identification of Enterohemorrhagic Escherichia coli
O157-Specific DNA Sequence Obtained from
Amplified Fragment Length Polymorphism Analysis
Akihiko Tokunaga1, Masanori Kawano1, Masatoshi Okura1, Sunao Iyoda2, Haruo Watanabe2,
and Ro Osawa*, 1
1Department of Bioresource Science, Graduate School of Agricultural Science, Kobe University, Kobe, Hyogo 657–8501,
Japan, and 2Department of Bacteriology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo 162–8640, Japan
Received April 4, 2007; in revised form, May 22, 2007. Accepted June 1, 2007
Abstract: An approximately 1.1 kbp fragment that was commonly observed only in the enterohemorrhag-
ic Escherichia coli (EHEC) O157 strains in an analysis of amplified fragment length polymorphism was
found to be a partial gene sequence encoding the locus of toxB and a useful molecular marker for the iden-
tification of EHEC O157.
Key words: Enterohemorrhagic Escherichia coli, Identification, AFLP, toxB
Microbiol. Immunol., 51(9), 883–888, 2007
Abbreviations: A/E, attaching and effacing; AFLP, amplified
fragment length polymorphism; DNA, deoxyribonucleic acid;
EHEC, enterohemorrhagic Escherichia coli; EPEC, enteropatho-
Escherichia coli; STEC,
Escherichia coli; Stx, Shiga toxin; PCR, polymerase chain reac-
*Address correspondence to Dr. Ro Osawa, Department of
Bioresource Science, Graduate School of Agricultural Science,
Kobe University, Rokko-dai 1–1, Nada-ku, Kobe, Hyogo
657–8501, Japan. Fax:
10) Griffin, P.M., and Tauxe. R.V. 1991. The epidemiology of
infections caused by Escherichia coli O157:H7, other
enterohemorrhagic E. coli, and the associated hemolytic
uremic syndrome. Epidemiol. Rev. 13: 60–98.
11) Hahm, B.K., Maldonado, Y., Schreiber, E., Bhunia, A.K.,
and Nakatsu, C.H. 2003. Subtyping of foodborne and envi-
ronmental isolates of Escherichia coli by multiplex-PCR,
rep-PCR, PFGE, ribotyping and AFLP. J. Microbiol. Meth-
ods 53: 387–399.
12) Hanahan, D. 1983. Studies on transformation of
Escherichia coli with plasmids. J. Mol. Biol. 166: 557–580.
13) Iyoda, S., Wada, A., Weller, J., Flood, S.J., Schreiber, E.,
Tucker, B., and Watanabe, H. 1999. Evaluation of AFLP, a
high-resolution DNA fingerprinting method, as a tool for
molecular subtyping of enterohemorrhagic Escherichia coli
O157:H7 isolates. Microbiol. Immunol. 43: 803–806.
14) Kaper, J.B. 1996. Defining EPEC. Revista de Microbiolo-
gia, Sao Paulo 27: 130–133.
15) Karch, H., Tarr, P.I., and Bielaszewska, M. 2005. Entero-
haemorrhagic Escherichia coli in human medicine. Int. J.
Med. Microbiol. 295: 405–418.
16) Louie, M., De-Azavedo, J.C.S., Handelsman, M.Y.C.,
Clark, C.G., Ally, B., Dytoc, M., Sherman, P., and Brunton,
J. 1993. Expression and characterization of the eaeA gene
product of Escherichia coli serotype O157:H7. Infect.
Immun. 61: 4085–4092.
17) Maurer, J.J., Schmidt, D., Petrosko, P., Sanchez, S., Bolton,
L., and Lee, M.D. 1999. Development of primers to O-anti-
gen biosynthesis genes for specific detection of Escherichia
coli O157 by PCR. Appl. Environ. Microbiol. 65:
18) McGraw, E.A., Li, J., Selander, R.K., and Whittam, T.S.
1999. Molecular evolution and mosaic structure of α, β,
and γ intimins of pathogenic Escherichia coli. Mol. Biol.
Evol. 16: 12–22.
19) Morabito, S., Tozzoli, R., Oswald, E., and Caprioli, A.
2003. A mosaic pathogenicity island made up of the locus
of enterocyte effacement and a pathogenicity island of
Escherichia coli O157:H7 is frequently present in attaching
and effacing E. coli. Infect. Immun. 71: 3343–3348.
20) Mukhopadhyay, A., and Mukhopadhyay, U.K. 2007. Novel
multiplex PCR approaches for the simultaneous detection of
human pathogens: Escherichia coli 0157:H7 and Listeria
monocytogenes. J. Microbiol. Methods 68:193–200.
21) Nataro, J.P., and Kaper, J.B. 1998. Diarrheagenic
Escherichia coli. Clin. Microbiol. Rev. 11: 142–201.
22) Ray, P.E., and Liu, X.H. 2001. Pathogenesis of Shiga toxin-
induced hemolytic uremic syndrome. Pediatr. Nephrol. 16:
23) Reid, S.D., Selander, R.K., and Whittam, T.S. 1999.
Sequence diversity of flagellin (fliC) alleles in pathogenic
Escherichia coli. J. Bacteriol. 181: 153–160.
24) Tarr, P.I., Schoening, L.M., Lea, Y., Ward, T.R., Jelacic. S.,
and Whittam, T.A. 2000. Acquisition of the rfb-gnd cluster
in evolution of Escherichia coli O55 and O157. J. Bacteriol.
25) Tatsuno, I., Horie, M., Abe, H., Miki, T., Makino, K., Shi-
nagawa, H., Taguchi, H., Kamiya, S., Hayashi, T., and
Sasakawa, C. 2001. toxB gene on pO157 of enterohemor-
rhagic Escherichia coli O157:H7 is required for full epithe-
lial cell adherence phenotype. Infect. Immun. 69:
26) Toma, C., Martinez, E.E., Song, T., Miliwebsky, E., Chi-
nen, I., Iyoda, S., Iwanaga, M., and Rivas, M. 2004. Distri-
bution of putative adhesins in different seropathotypes of
Shiga toxin-producing Escherichia coli. J. Clin. Microbiol.
27) Tozzoli, R., Caprioli, A., and Morabito, S. 2005. Detection
of toxB, a plasmid virulence gene of Escherichia coli
O157, in enterohemorrhagic and enteropathogenic E. coli. J.
Clin. Microbiol. 43: 4052–4056.
28) Vallance, B.A., and Finlay, B.B. 2000. Exploitation of host
cells by enteropathogenic Escherichia coli. Proc. Natl.
Acad. Sci. U.S.A. 97: 8799–8806.
29) Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee,
T., Hornes, M., Frijters, A., Pot, J., Peleman, J., and Kuiper,
M. 1995. AFLP: a new technique for DNA fingerprinting.
Nucleic Acids Res. 23: 4407–4414.
30) Wang, G., Clark, C., and Rodgers, F. 2002. Detection in
Escherichia coli of the genes encoding the major virulence
factors, the genes defining the O157:H7 serotype, and com-
ponents of the type 2 Shiga toxin family by multiplex PCR.
J. Clin. Microbiol. 40: 3613–3619.
31) Wick, L.M., Qi, W., Lacher, D.W., and Whittam, T.S. 2005.
Evolution of genomic content in the stepwise emergence of
Escherichia coli O157:H7. J. Bacteriol. 187: 1783–1791.
A. TOKUNAGA ET AL