Professional Documents
Culture Documents
Method
INTRODUCTION
Escherichia coli that causes diarrhea in humans is referred
to as the diarrheagenic E. coli (DEC) and has been categorized
into the following six groups: Shiga toxin-producing E. coli
(STEC), enterotoxigenic E. coli (ETEC), enteropathogenic E.
coli (EPEC), enteroinvasive E. coli (EIEC), enteroaggregative
E. coli (EAggEC), and diffusely adhering E. coli (DAEC)
(1). These groups of DEC are differentiated on the basis of
their virulence mechanisms, such as their specific toxin productions, ability to attach to or invade target cells, etc., and
thus different immunological, infectious, and molecular assays have been developed for the identification of different
groups of DEC (2). However, identification of the six groups
of DEC is very laborious, time consuming, and costly, and
therefore many clinical laboratories routinely perform only
serotyping assays for the identification, which is sometimes
very useful for the detection of STEC of the serotype O157,
O26, and so on (3).
In recent years, the genes related to the pathogenicity of
the DEC, except for DAEC, have been identified (1), which
helped in the development of different molecular detection
assays like DNA hybridization (4), real-time polymerase chain
reaction (PCR) (5,6), loop-mediated isothermal amplification
(LAMP) (7), the DNA microarray method (8), and multiplex
PCR (mPCR) (9). Among these techniques, the mPCR assay
is one of the most useful routine diagnostic methods, and
some mPCR systems have already been reported for the identification of several categories of DEC (10-13), or for all the
five categories for which it was intended (14-17). Aranda et
al. (15) reported two mPCR assays to identify eight target
genes (eae, bfpA, CVD432, LT gene, ST gene, ipaH, stx1,
and stx2) except aggR and astA, coding for the transcriptional
activator of adherence fimbriae and the enterotoxin, respectively. The one-shot mPCR assay reported by Kimata et al.
Inc., Shiga, Japan and Nippon Gene Co., Tokyo, Japan), 25 50 M of each primer, and 2.5 l of the DNA template. These
samples, which had been preheated at 94C for 1 min, were
amplified for 25 cycles using a thermal cycler (TaKaRa PCR
Thermal cycler MP [TaKaRa], and BioRAD i-cycler [BioRad, Hercules, Calif., USA]). Each cycle consisted of denaturation at 94C for 1 min, annealing at 55C for 1 min, and
extension at 72C for 1 min. A final extension step was performed at 72C for 10 min. The PCR products were separated
by electrophoresis (100 V, 40 min; Mupid-21; Cosmo Bio
Ltd., Tokyo, Japan) in 3% agarose gels (Wako Pure Chemical Industries Ltd., Osaka, Japan) and stained with ethidium
bromide.
Applying mPCR assays to clinical isolates of E. coli:
To demonstrate the diagnostic usefulness of the two mPCR
assay systems, we examined stool specimens collected from
outpatients attending different private hospitals around
Hirosaki, Aomori, and Sendai cities in Japan, between February 2005 and November 2006. These stool specimens were
examined for the presence of Salmonella enterica, Shigella,
Yersinia, Vibrio, Campylobacter, and E. coli following standard identification methods. A total of 683 E. coli-like isolates
were pure cultured on 5% sheep-blood agar plates (Becton
Dickinson Co., Sparks, Md., USA) and serotyped with O antisera purchased from Denka Seiken. They were then identified
by the Enterotube II (Becton Dickinson). All these strains
were grown on Kajiton agars that were preserved at 4C
before the mPCR assays were performed, as described above.
Description
Target gene
E76
E32
E31
E-O169
E1231
E33
E1407
STEC
ETEC
ETEC
ETEC
EPEC
EIEC
EAggEC
RESULTS
The control strains were analyzed by each single primer
set of PCR to detect the virulence genes (stx1, stx2, eaeA,
invE, aggR, LT gene, ST gene, and astA). However, the ST
gene was detected as a weak band (data not shown). At first,
we tried simultaneously the eight primer sets of PCR, but
only a few genes were detected. Next we devided these primer
sets into a group comprising primers for stx1, eaeA, LT gene,
and astA and a group comprising primers for stx2, invE, aggR,
DEC, diarrheagenic Escherichia coli; STEC, shiga toxinproducing E. coli; ETEC, enterotoxigenic E. coli; EPEC,
enteropathogenic E. coli; EIEC, enteroinvasive E. coli;
EAggEC, enteroaggregative E. coli.
Sequence (5 to 3)
AGTTAATGTGGTGGCGAA
GACTCTTCCATCTGCCG
TTCGGTATCCTATTCCCG
TCTCTGGTCATTGTATTA
AAACAGGTGAAACTGTTGCC
CTCTGCAGATTAACCTCTGC
ATATCTCTATTTCCAATCGCGT
GATGGCGAGAAATTATATCCCG
GTATACACAAAAGAAGGAAGC
ACAGAATCGTCAGCATCAGC
TTTATTTCTGTATTGTCTTT
ATTACAACACAGTTCACAG
TCTGTATTATCTTTCCCCTC
ATAACATCCAGCACAGGC
CCCTCAGGATGCTAAACCAG
TTAATAGCACCCGGTACAAGC
AGCAGGTTTCCCACCGGATCACCA
GTGCTCAGATTCTGGGTCTC
GCCATCAACACAGTATATCC
GAGTGACGGCTTTGTAGTCC
477
Amplicon
size (bp)
Concentration of
primers (M)
Reference
817
25
18
474
25
18
454
25
19
382
25
10
254
25
20
171
50
10
186
50
21
166
25
21
130
25
10
106
25
19
and the ST gene. As a result, the target genes (stx1, stx2, eaeA,
invE, aggR, LT gene, and astA) were detected as clear bands.
However, the ST gene was either not detected or was detected only with the single PCR assay, but not with mPCR.
Therefore, the ST gene primer was divided into the STp gene
and STh gene. The completed mPCR was comprised of two
combinations: one group with stx1, eaeA, invE, STp gene,
and astA, and the other group with stx2, aggR, STh gene, and
LT gene (Fig. 1).
All the 683 clinical E. coli-like isolates were examined by
mPCR, and 51 DEC and 38 astA gene-positive E. coli strains
(Table 3) were detected (EPEC, 21; EAggEC, 15; ETEC, 9;
STEC, 6; and astA positive strains, 38). Five (eaeA, three;
aggR, four) of these DEC strains were O antigen untypable
(OUT) strains. In addition, we analyzed all these 683 E. coli
strains for confirmation tests with individual nine-kind PCR
with each primer set, and the results were identical to those
Fig. 1. The results of control DEC by mPCR. Lane 1, E76 + E33 + EO169; 2, E76; 3, E33; 4, E-O169; 5, DNA molecular size markers
(100-bp ladder); 6, E76 + E1407 + E31; 7, E76; 8, E1407; 9, E31.
stx1 + stx2
+ eaeA
stx1 + eaeA
STp gene
+ astA
LT gene
astA
5
3
1
1
1
1
4
3
2
5
3
1
4
1
1
1
1
3
1
1
2
2
4
2
1
3
1
1
1
3
2
2
1
1
1
1
2
1
4
3
2
20
478
11
38
REFERENCES
1. Beinke, C., Laarmann, S., Wachter, C., et al. (1998): Diffusely adhering
Escherichia coli strains induce attaching and effacing phenotypes and
secrete homologs of Esp proteins. Infect. Immun., 66, 528-539.
2. Nataro, J.P. and Kaper, J.B. (1998): Diarrheagenic Escherichia coli.
Clin. Microbiol. Rev., 11, 142-201.
3. Takeda, Y., Igarashi, A. and Kojima, S. (1999): Emerging and Re-emerging
Infectious Diseases. p. 16-29. Kindai Shuppan, Tokyo (in Japanese).
4. Scaletsky, I.C.A., Fabbricotti, S.H., Aranda, K.R., et.al. (2002): Comparison of DNA hybridization and PCR assays for detection of putative
pathogenic enteroadherent Escherichia coli. J. Clin. Microbiol., 40,
1254-1258.
5. Mller, J., Eis-Hbinger, A.M., Dumer, M., et al. (2007): A novel
internally controlled real-time reverse transcription-PCR assay for HIV1 RNA targeting the pol integrase genomic region. J. Viral. Methods,
142, 127-135.
6. Fortin, N.Y., Mulchandani, A. and Chen, W. (2001): Use of real-time
polymerase chain reaction and molecular beacons for the detection of
Escherichia coli O157. Anal. Biochem., 289, 281-288.
7. Notomi, T., Okayama, H., Masubuchi, H., et al. (2000): Loop-mediated
isothermal amplification of DNA. Nucleic Acids Res., 28, e63, 1-7.
8. Richmond, C.S., Glasner, J.D., Mau, R., et al. (1999): Genome-wide
expression profiling in Escherichia coli K-12. Nucleic Acids Res., 27,
3821-3835.
9. Bej, A.K., McCarty, S.C. and Atlas, R.M. (1991): Detection of coliform
bacteria and Escherichia coli by multiplex polymerase chain reaction:
comparison with defined substrate and plating methods for water quality monitoring. Appl. Environ. Microbiol., 57, 2429-2432.
10. Itoh, F., Ogino, T., Itoh, K., et al. (1992): Differentiation and detection
of pathogenic determinants among diarrheagenic Escherichia coli by
polymerase chain reaction using mixed primers. Jpn. J. Clin. Med., 50,
343-347 (in Japanese).
11. Cebula, T.A., Payne, W.L. and Feng, P. (1995): Simultaneous identification of strains of Escherichia coli serotype O157:H7 and their Shigalike toxin type by mismatch amplification mutation assay-multiplex
PCR. J. Clin. Microbiol., 33, 248-250.
12. Wang, G., Clark, G.C. and Rodgers, F.G. (2002): Detection in Escherichia
coli of genes encoding the major virulence factors, the defining the
O157:H7 serotype, and components of the type 2 Shiga-toxin family by
multiplex PCR. J. Clin. Microbiol., 40, 3613-3619.
13. Pass, M.A., Odedra, R. and Batt, R.M. (2000): Multiplex PCRs for
identification of Escherichia coli virulence gene. J. Clin. Microbiol.,
38, 2001-2004.
14. Toma, C., Lu, Y., Nakasone, N., et al. (2003): Multiplex PCR assay for
identification of human diarrheagenic Escherichia coli. J. Clin.
Microbiol., 41, 2669-2671.
15. Aranda, K.R.S., Fagundes-Neto, U. and Scaletsky, C.A. (2004): Evaluation of multiplex PCRs for diagnosis of infection with diarrheagenic
Escherichia coli and Shigella spp. J. Clin. Microbiol., 42, 5849-5853.
16. Kimata, K., Shima, T., Shimizu, M., et al. (2005): Rapid categorization
of pathogenic Escherichia coli by multiplex PCR. Microbiol. Immunol.,
49, 485-492.
17. Aranda, K.R.S., Fabbricotti, S.H., Fagundes-Nto, U., et al. (2007):
Single multiplex assay to identify simultaneously enteropathogenic,
enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxinproducing Escherichia coli strains in Brazilian children. FEMS Microbiol.
Lett., 267, 145-150.
18. Kobayashi, K. (1991): Detection of enterohemorrhagic Escherichia coli
using PCR. Clin. Microbiol., 18, 507-513 (in Japanese).
19. Yatsuyanagi, J., Saito, S., Sato, H., et al. (2002): Characterization of
enteropathogenic Escherichia coli isolated from diarrheal outbreaks. J.
Clin. Microbiol., 40, 294-297.
20. Ratchtrachenchai, O.A., Subpasu, S. and Ito, K. (1997): Investigation
on enteroaggregative Escherichia coli infection by multiplex PCR. Bull.
Dept. Med. Sci., 39, 211-220.
21. Schultsz, C., Pool, G., Ketel, R.V., et al. (1994): Detection of enterotoxigenic
Escherichia coli in stool samples by using nonradioactively labeled
oligonucleotide DNA probes and PCR. J. Clin. Microbiol., 32, 23932397.
22. Brk, C., Dietrich, R., Aar, G., et al. (2003): Identification and characterization of a new variant of Shiga toxin 1 in Escherichia coli ONT:H19
of bovine origin. J. Clin. Microbiol., 41, 2106-2112.
23. Suzuki, M., Kondo, F., Ito, Y., et al. (2004): Identification of a Shigatoxin type I variant containing an IS1203-like element, from Shiga-toxin
ACKNOWLEDGMENTS
We thank K. Abe, Aomori Prefectural Institute of Public Health and Environment, and J. Ono, Public Health Department, Aomori Health Welfare
479
24.
25.
26.
27.
28.
29.
30. Zhang, W.L., Khler, B., Oswald, E., et al. (2002): Genetic diversity of
intimin genes of attaching and effacing Escherichia coli strains. J. Clin.
Microbiol., 40, 4486-4492.
31. Yamamoto, D., Hernandes, R.T., Blanco, M., et al. (2009): Invasiveness
as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia coli strains with different uncommon intimin
types. BMC Microbiol., 9:146. doi:10.1186/1471-2180-9-146.
32. Blanco, M., Blanco, J.E., Dahbi, G., et al. (2006): Typing of intimin
(eae) genes from enteropathogenic Escherichia coli (EPEC) isolated
from children with diarrhea in Montevideo, Uruguay: identification of
two novel intimin variants (B and R/2B). J. Med. Microbiol., 55,
1165-1174.
33. Savarino, S.J., Fasano, A., Watson, J., et al. (1993): Enteroaggregative
Escherichia coli heat-stable enterotoxin 1 represents another subfamily
of E. coli heat-stable toxin. Proc. Natl. Acad. Sci. USA, 90, 3093-3097.
34. Yatsuyanagi, J., Saito, S., Miyajima, Y., et al. (2003): Characterization
of atypical enteropathogenic Escherichia coli strains harboring the astA
gene that were associated with a waterborne outbreak of diarrhea in
Japan. J. Clin. Microbiol., 41, 2033-2039.
35. Eklund, M., Scheutz, F. and Siitonen, A. (2001): Clinical isolates of
non-O157 Shiga toxin-producing Escherichia coli: serotypes, virulence
characteristics, and molecular profiles of strains of the same serotype.
J. Clin. Microbiol., 39, 2829-2834.
480