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Journal of Clinical Microbiology, June 2000, p. 2440-2442, Vol. 38, No. 6
Department of Health and Epidemiology, Osaka
City Institute of Public Health and Environmental Sciences, 8-34, 543-0026, Osaka, Japan,1 and Laboratory
of Enteric Pathogens, Central Public Health Laboratory, London NW9
5HT, United Kingdom2
Received 12 August 1999/Returned for modification 1 December
1999/Accepted 28 February 2000
One hundred sixty-nine strains of enterohemorrhagic
Escherichia coli serogroup O157 were examined for the
correlation between the genotype of their Shiga toxin genes
(stx) and manifestation of bloody diarrhea (BD). It was
shown that the strains carrying only stx2vha were probably
less virulent and caused BD less frequently.
Enterohemorrhagic Escherichia
coli (EHEC) of serogroup O157 was recognized as the causative
agent following two outbreaks of hemorrhagic colitis (HC) in
1982 (12). The organisms are characterized by their ability
to elaborate Shiga toxin (Stx) (2, 8). The association of
EHEC with HC and hemolytic-uremic syndrome (HUS) implies that Stx is a
major virulence factor in these diseases (4, 11, 15, 17).
However, the role of Stx in the pathogenesis of HC remains to be
elucidated. Tzipori et al. showed that Stx-negative variant strains
still caused diarrhea (19). Attempts to link Stx with
clinical manifestations of HC have been performed with epidemiological,
clinical, and animal model investigations (6, 7, 10).
However, these studies were performed prior to the discovery of Stx2 by
Scotland et al. (14). Hence, it is not clear from reading
the original sources whether single or multiple forms of Stx were being produced.
To assess the relationship between the manifestation of bloody diarrhea
(BD) and Stx, EHEC O157 strains were examined by typing of the Stx
genes (stx) and Stx production. The properties of the strains were correlated with the clinical symptoms of the persons from
whom the strains were isolated.
A total of 357 EHEC O157 strains originally isolated from 1996 to 1998 in Osaka City, Japan, was used. They were assigned to groups on the
basis of phage typing and DNA typing with pulsed-field gel
electrophoresis as reported (Y. Nishikawa, A. Hase, J. Ogasawara, T. Cheasty, G. A. Willshaw, B. Rowe, and A. Yasukawa,
presented at the Third Int. Symp. Workshop
Verocytotoxin-Producing Escherichia coli Infections,
Baltimore, Md., 1997). To provide independent clones for further
analysis, 169 strains were chosen as the representatives of 162 types.
The representative strain was the earliest isolate among strains
belonging to each type. Thirteen strains that represented six types but
were isolated in different years were treated as putative different
clones. Finally, 169 strains of 162 types were examined to assess the
relationship between Stx genotypes and manifestation of BD.
Stx2 genotypes were analyzed by the predicted restriction enzyme
fragment length polymorphisms of PCR products (18). Stx2e and Stx2ev genes were examined using primer pairs reported by Cao
et al. and Johnson et al., respectively (1, 3). The strains
were examined for Stx production within two months after isolation
using the RPLA test (VTEC-RPLA lots 31608, 29809, and 27808; Denka
Seiken Co. Ltd., Tokyo, Japan) (5); it was confirmed that
the sensitivities of the kits used differed by no more than a factor of
2 throughout the study.
The 169 strains were assigned to seven groups according to their type
of stx (Table 1). There were
no O157 strains that were positive for stx2vhb,
stx2e, or stx2ev in this study. This is concordant with the study of Tyler et al., in which stx2vhb
was found only among non-O157 Stx-producing E. coli strains
(18). Symptoms of BD were reported for 50% (48 of 96) of
patients from whom isolates possessing both stx1 and
stx2 were isolated; BD was defined as diarrhea that
contained blood that could be observed macroscopically. BD was reported
for 55% (6 of 11), 39% (9 of 23), 18% (2 of 11), and 8% (2 of 25)
of patients infected by isolates possessing only stx2, both
stx2 and stx2vha, both stx1 and
stx2vha, and only stx2vha, respectively (Table
1). After the strains were assigned to three groups on the basis of
Stx2 genotypes, these results indicated that patients with strains that
possessed stx2 or both stx2 and
stx2vha often presented with BD. On the other hand, the
patients from whom organisms having only stx2vha were isolated manifested BD with lower frequency (Table
2).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Relationship of Genetic Type of Shiga Toxin to Manifestation of
Bloody Diarrhea due to Enterohemorrhagic Escherichia
coli Serogroup O157 Isolates in Osaka City, Japan
ABSTRACT
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TABLE 1.
Relationship among type of Stx genes, anti-Stx2 RPLA
titer of EHEC O157 strains, and manifestation of BD
TABLE 2.
Relationship between type of Stx2 genes of EHEC O157
strains and manifestation of BD
Significant differences were not observed in the ages and sexes of
patient groups from which each stx type of organism was isolated (Table 2); however, the patients with BD were significantly younger than infected persons without BD (Table
3). It was not suggested that particular
phage and/or DNA types were related to manifestation of BD (data not
shown).
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Why the strains that possessed only stx2vha caused BD less
frequently remains to be elucidated. However, it may reflect functional difference between Stx2 and Stx2vha. Stx1 and Stx2 bind preferentially to Gb3 [Gal
(1
4)Gal
(14)Glc-ceramide]
(20), while Stx2vha and Stx2e bind preferentially to
Gb4
[GalNAc(13)Gal(14)Gal-(14)Glc-ceramide] (13). Takeda
et al. observed that Vero cell cytotoxicity and mouse lethality of
Stx2vh were somewhat lower than those of Stx2 (16). Tyler et
al. (18) also reported that the culture supernatants of
strains carrying only Stx2 variant genes gave low cytotoxin titers in HeLa cell assays. Differences in these biological activities can possibly explain why the strains that possessed only stx2vha
caused BD less frequently.
Alternatively, it was possible that the quantity of Stx produced was an
important factor. The amount of Stx that was reactive to the anti-Stx2
latex particles depended on the Stx genotype. Strains possessing
stx2vha tended to have lower titers than strains with
stx2, regardless of the presence or absence of
stx1 (Fig. 1). It is possible
that the anti-Stx2 latex was less reactive to Stx2vha. To address this,
gene sequences in strains possessing only stx2vha were
preliminarily examined. No difference was found in the sequence of
stx2vha between two strains that produced high titers and
two with low titers (data not shown), suggesting that Stx2vha molecules
produced by these strains were identical. Therefore, the low titers
expressed by the majority of strains possessing only stx2vha
seemed to reflect a lower production of Stx2vha. If so, the discrepancy
in manifestation of BD could be due to the Stx levels produced by each
strain.
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It appeared that the strains carrying stx2 tended to cause BD more than strains possessing only stx2vha. Classical HUS develops typically a few days after the onset of an acute diarrheal prodromal illness, which is often bloody. In cases of patients infected with EHEC O157, it may be that HUS is also caused by organisms having stx2 or both stx2 and stx2vha rather than bacteria with only stx2vha, although BD is not an essential symptom for HUS. In fact, all six strains isolated from HUS patients who first contracted BD in this study possessed both stx1 and stx2. Ostroff et al. suggested that EHEC O157 strains that produced Stx2 alone were more likely to be associated with the development of HUS than strains that produced Stx1 alone or Stx1 and Stx2 (9). In the present investigation, however, the possession of stx1 did not have any negative influence on manifestation of BD. BD was found more among patients infected by strains with both stx1 and stx2vha (18%) than among those infected by strains with stx2vha alone (8%) (Table 1). Of 18 strains that did not possess stx1 and gave a low RPLA titer of Stx2 (<80), only one was from a patient with BD. On the other hand, 6 of 22 strains that gave an Stx2 titer of less than 80 but had stx1 were from patients with BD. These differences, however, were not statistically significant, and the role of Stx1 in BD could not be evaluated since there was only a single strain that produced only Stx1 in this study.
In conclusion, the present study indicates that the typing of Stx2 genes of EHEC O157 isolates provides useful information not only for epidemiological analysis but for prognosis, as strains carrying only stx2vha are possibly less virulent and cause BD less frequently.
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ACKNOWLEDGMENTS |
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We thank Sylvia M. Scotland for her valuable comments on the manuscript.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Food and Nutrition, Faculty of Human Life Science, Osaka City University, 558-8585, Japan. Phone: (06) 6605-2883. Fax: (06) 6605-2914. E-mail: nisikawa{at}life.osaka-cu.ac.jp.
Present address: Changchun University of Agriculture and Animal
Sciences, Changchun, China.
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Journal of Clinical Microbiology, June 2000, p. 2440-2442, Vol. 38, No. 6
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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