Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. 2003 Feb; 69(2): 1059–1066.
PMCID: PMC143677

Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing Escherichia coli Isolates


There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.

Shiga toxin-producing Escherichia coli (STEC) are emerging human pathogens that can cause diarrheal disease as well as more-severe clinical manifestations, including hemorrhagic colitis and hemolytic-uremic syndrome (HUS) (19, 46). The main environmental reservoirs of STEC are cattle and other ruminants, which often carry STEC among their intestinal floras (25); however, infected animals do not exhibit symptoms. Humans become infected with STEC primarily through ingestion of contaminated meat or other food products or by fecal-oral transmission of STEC from infected humans or animals (46). Only a subgroup of STEC within the bovine reservoir are thought to be capable of causing infection and disease (3). Evidence for this hypothesis includes differences in the prevalence of STEC serotypes isolated from cattle versus that of STEC serotypes isolated from infected humans (3, 25, 42). It is presently unclear whether serotype per se contributes directly to STEC pathogenesis (16) or whether other genetic differences account for the range of STEC pathogenicity (2).

STEC are mucosal pathogens that produce several virulence factors thought to contribute to their pathogenicity. Among these, the principal virulence factor is Shiga toxin (Stx), an A-B-type toxin that inhibits protein synthesis in target cells (reviewed in reference 48). Stx produced by STEC within the intestine is thought to enter the systemic circulation and result in damage to distal organs. Several observations suggest that the risk of serious complications of STEC infection (e.g., HUS) is correlated with the presence and amount of Stx produced during infection. First, intravenous administration of Stx to baboons resulted in microangiopathic hemolytic anemia (47), the key pathological feature seen in HUS (33). Second, according to the results of investigations with a mouse model, antibiotics that increase Stx production in the intestine can also increase mortality in mice infected with STEC (58). Similarly, administration of antibiotics to patients with STEC infection has been epidemiologically associated with more-severe clinical outcomes (55). The mechanism by which this occurs has yet to be elucidated; however, a strong possibility is that the antibiotics induced toxin production (17, 29, 58) and exacerbated the Stx-linked symptoms of STEC infection.

In addition to stx, several other genes are thought to contribute to STEC virulence. For example, genes within the locus of enterocyte effacement pathogenicity island are thought to mediate the adhesion of STEC to epithelial cells. In particular, the locus of enterocyte effacement gene eae, which encodes intimin, enables intimate attachment of bacteria to the gut epithelium (49; reviewed in reference 13). However, this gene is apparently not required for virulence, as eae-negative STEC have been isolated from patients with HUS (39). Additional potential virulence genes include an enterohemolysin, ehxA (also known as enterohemorrhagic E. coli hlyA) (4), a catalase-peroxidase (katP) (6), an extracellular serine protease (espP) (7), and a secreted metalloprotease, stcE (26). Of these, most is known about ehxA, the presence of which is associated with severe disease in humans (2, 5, 27, 45). The roles of these additional factors in STEC pathogenicity await additional confirmation.

Two main types of Stx are found in STEC, Stx1 and Stx2, each of which has one or more closely associated variant(s), namely Stx1c (57) and Stx2c, Stx2d, Stx2e, and Stx2f (14). The genes for these toxins are encoded in the genomes of bacteriophages related to phage lambda (20, 36, 38, 50, 54). Within these phage genomes, the stx genes are located between orthologues for the lambda genes Q and S, which are important for late gene transcription and lysis, respectively (23, 37, 41). Based on studies of relatively few disease-linked STEC isolates, it is thought that Stx production can be regulated by a number of mechanisms. Study of lysogens of the Stx1-encoding phage H-19B showed that low environmental iron concentrations can augment stx1 transcription by relieving iron-dependent Fur-mediated repression at an stx1 promoter (8). Stx2 production has not been found to be iron regulated; however, stx2 expression can be induced by prophage-inducing stimuli, including the DNA-damaging agent mitomycin C (35, 56), H2O2 (52), and members of certain classes of antibiotics (17, 29). These stimuli induce the bacterial SOS response, which results in cleavage of the phage repressor, CI (43). Following cleavage of CI, the late phage genes, including stx, are efficiently transcribed and phage particles are produced. Recently, Wagner et al. demonstrated that expression of stx2 under these circumstances is primarily dependent on initiation of transcription at the phage promoter pR′ and upon antitermination by the antiterminator Q (54). stx1 expression by H-19B is also induced by mitomycin C; however, pR′ and Q appear to be less important in this strain (53).

Most previous comparative studies of human disease and bovine-linked STEC have focused on the distribution of virulence-associated genes rather than on production of the factors they encode. Since Stx production appears to be a critical determinant in the development of severe disease, we examined whether HUS- and bovine-associated isolates produced differing amounts of Stx. We compared levels of Stx production by these isolates in response to a variety of stimuli. The human isolates used for this study all came from nonepidemic HUS patients. We found that mitomycin C induced Stx2 production to higher levels in disease-associated isolates than in bovine-associated isolates. Similarly, low iron concentrations induced Stx1 production to a greater extent in disease-associated isolates. These findings suggest that environmental control of Stx production differs among disease-associated STEC isolates.


Sample collection, isolation, and characterization of STEC.

To obtain human disease-associated STEC isolates, stool samples were collected from 31 patients diagnosed with HUS during an ongoing study of the epidemiology of STEC in the United States. Each patient represented a sporadic case and was the source of only one STEC strain. In addition, six Stx1-producing STEC isolates from patients with HUS were kindly provided by H. Schmidt (Institut fur Hygiene und Mikrobiologie der Universitat Wurzburg, Wurzburg, Germany). STEC isolates with no known human disease association were isolated from bovine fecal samples obtained from cattle pastures in Nebraska; some fecal samples were taken from different pastures on the same farm, while other samples were obtained from geographically distant farms. Fresh fecal samples were collected and stored frozen until arrival at the laboratory.

Homogenized stool (or 25 ml of water in those situations in which we were testing water from the cattle drinking supply) was added to MacConkey broth, and the culture was incubated overnight with shaking at 37°C. The overnight culture was then screened for Stx production by enzyme immunoassay (Premier EHEC; Meridian Diagnostics Inc., Cincinnati, Ohio). Stx-producing single colonies were subsequently identified using a colony immunoblot assay (21). The presence of stx1 and stx2 genes was determined by PCR analysis using primers specific for the A subunit coding region as described by Paton and Paton (40). To simplify the interpretation of the results, only those isolates that contained either stx1 or stx2 (which represented 43% of the total number of isolates obtained), but not both, were used in subsequent experiments. Using PCR followed by digestion of the amplification product with HaeIII and FokI as described by Friedrich et al. (14), variants of the stx2 gene were identified. The stx1c variant was identified by PCR as described by Zhang et al. (57). Genes encoding intimin (eae) and enterohemolysin (ehxA) were detected by multiplex PCR essentially as described by Paton and Paton (40). In this assay, the primers used to detect eae are specific for the conserved region at the start of the gene; hence, both STEC and enteropathogenic E. coli intimin types are amplified. Typing for the E. coli O antigen was performed using standard methods (Mike Davis, E. coli Reference Laboratory, University Park, Pa.).

Mitomycin C induction experiments.

Overnight cultures of each isolate were subcultured (5% inoculum) into two tubes containing 5 ml of fresh Luria broth and grown with shaking at 37°C to an optical density at 600 nm of 0.6. Mitomycin C (200 ng ml−1 final concentration) was added to one tube, while the second received an equivalent volume of distilled water. At 3 h after mitomycin C addition, 1 ml of culture was removed and sonicated (Sonifier 450; Branson Sonic Power, Danbury, Conn.) until cell lysis was evident (based on assessment of the extent of visual turbidity and confirmed by microscopic examination of selected samples). The total Stx concentration in each sample was then determined by an enzyme-linked immunosorbent assay (ELISA) as described previously (11). All the Stx1 and Stx2 variants identified in the STEC isolates studied here were detectable by the ELISA used in this study.

Influence of iron concentration on Stx1 production.

A single colony from each isolate was inoculated into 2 ml of syncase broth (low-iron medium) (12) and grown overnight at 37°C with shaking. The overnight cultures were diluted to an optical density at 600 nm of 0.1 in fresh syncase medium or syncase medium supplemented with iron (10 μg of Fe [as FeCl3] ml−1) (iron-replete medium) and grown with shaking at 37°C for 3 h. The cultures were treated with polymyxin B (2 mg ml−1) to release periplasmic Stx1 (10), and the total (periplasmic and extracellular) Stx1 concentrations were determined by ELISA. Polymyxin B was used instead of sonication in this experiment to facilitate rapid sample processing.

Statistical analysis.

The median and the interquartile range (IR) of the Stx concentration obtained from each group are presented below. The IR describes the distribution of the central 50% of observations (i.e., those which are located between the 25th and 75th percentiles). The Wilcoxon rank-sum test was used to determine whether there were statistically significant differences between mitomycin C-treated and/or low-iron-induced total Stx1 or Stx2 concentrations produced by HUS- and bovine-associated STEC (9). All experiments were carried out in duplicate.


Characterization of Stx2-producing STEC isolates.

A total of 13 Stx2-producing STEC isolates that contained amplifiable stx2 but not stx1 were obtained from stools of patients diagnosed with HUS. The majority of these isolates contained only stx2, and two isolates contained both stx2 and stx2c. PCR assays revealed that all of these isolates contained amplifiable eae and ehxA sequences. The majority of these isolates (11 of 13) belonged to serogroup O157, while the remaining 2 isolates belonged to serogroup O121 (Table (Table1).1). This association of the O157 serogroup with HUS following infection with Stx2-producing STEC corroborates previous observations (18).

Characteristics of Stx2-producing HUS- and bovine-associated STEC isolates

Twenty-five isolates of STEC producing only Stx2 were obtained from cattle pastures. One of these isolates was obtained from a water supply, while all of the others were isolated directly from bovine feces. Of these 25 isolates, 11 contained stx2, 9 contained only stx2c, and 5 contained both stx2 and stx2c (Table (Table1).1). In contrast to the disease-associated STEC isolates, only 6 of the 25 bovine-associated STEC isolates contained amplifiable eae sequences and only 13 of the 25 contained amplifiable ehxA sequences. Also in contrast to the disease-associated isolates, there was a large range of O serogroups present in the bovine isolates (Table (Table1).1). Some isolates were classified as serogroup OX, which refers to a provisional designation for as-yet-unclassified or nonstandardized O-antigen groups. In addition, 5 of the 25 bovine-associated STEC isolates did not react to any of the 181 O antisera used in testing. The bovine-associated O157 isolates (three of three) were all ehxA negative, and two were eae negative. Since eae and ehxA are generally present in human O157 isolates (as noted above), additional tests were performed to confirm the species and serogroup classification of these three isolates.

Characterization of Stx1-producing STEC isolates.

No HUS-associated STEC isolates producing Stx1 only were identified in the ongoing epidemiological study of STEC in the United States; consequently, Stx1-producing isolates from Germany were obtained. All these isolates contained stx1 and not a recently identified variant, stx1c (Table (Table2).2). The six Stx1-producing HUS-associated STEC isolates all contained amplifiable eae and ehxA sequences. However, unlike the Stx2-producing isolates, none belonged to serogroup O157; instead, they belonged to a range of serogroups including O103 and O111 (Table (Table2),2), which are the most common serogroups of Stx1-producing STEC isolates obtained from HUS patients (28).

Characteristics of Stx1-producing HUS- and bovine-associated STEC isolates

A total of 20 Stx1-producing STEC isolates from cattle pastures were obtained from feces and water supplies and, in one instance, from coyote feces found in a pasture enclosure. Of the 20 isolates, 7 contained the stx1c gene. Similar to the bovine-associated Stx2-producing STEC, only 8 of the 20 Stx1-producing STEC isolates contained eae sequences and 13 of 20 contained ehxA sequences. In all 26 of the Stx1-producing STEC isolates investigated in this study, eae and ehxA status appeared to be serogroup specific. For example, all O103 and O145 isolates contained amplifiable eae and ehxA sequences whereas all O109 isolates contained amplifiable ehxA sequences only. The bovine-associated Stx1-producing isolates belonged to a diverse range of O-antigen groups, including O103, O121, and O142 (Table (Table22).

Mitomycin C-induced Stx production.

There were marked differences in the effects of mitomycin C, an agent known to induce prophages, on Stx2 production by HUS- and bovine-associated isolates. Stx2 production by HUS-associated isolates increased dramatically during growth in mitomycin C; typically, at least 100-fold increases in Stx2 production were observed for these HUS-associated isolates (median fold increase [median] = 134; IR = 117 to 195) (Fig. (Fig.1A).1A). In contrast, mitomycin C treatment of bovine-associated Stx2-producing isolates resulted in variable, but significantly lower (P < 0.001), induction of Stx2 production (median = 41; IR = 6 to 78). The median concentration of Stx2 after mitomycin C treatment was also significantly greater (P < 0.001) for HUS-associated STEC than for bovine-associated STEC (Table (Table1).1). Stx2 concentrations in non-mitomycin C-treated cultures were also significantly greater (P < 0.01) for HUS-associated STEC (median = 77; IR = 49 to 102) than for bovine-associated STEC (median = 14; IR = 2 to 46). Some bovine isolates were highly induced (>100-fold for 4 of 25 isolates) and/or produced high concentrations (>10,000 ng ml−1 for 4 of 25 isolates) of Stx2 following mitomycin C treatment. There was no correlation between highly mitomycin C-inducible Stx2 production by bovine-associated isolates and serogroup or eae or ehxA status. In contrast to the marked increases in Stx2 production by all HUS-associated O157 isolates, Stx2 production by each of the three bovine-associated O157 isolates was induced <50-fold by mitomycin C and two of the three isolates showed hardly any induction of toxin production. Thus, mitomycin C inducibility of Stx2 production is not an intrinsic feature of all O157 STEC.

FIG. 1.
Increase in Stx production following mitomycin C treatment of Stx2-producing (A) and Stx1-producing (B) HUS- and bovine-associated STEC. Each assay was performed on duplicate cultures; the mean and the standard deviation are presented for each isolate. ...

In contrast to the dramatic induction of Stx2 production by mitomycin C, this prophage-inducing agent had minimal effect on Stx1 production by HUS-associated isolates (Fig. (Fig.1B).1B). Following mitomycin C treatment, Stx1 concentrations ranged from 300 to 2,400 ng of Stx1 ml−1 in cultures of these isolates (Table (Table2);2); in the majority, no increase (median = 1; IR = 1 to 1.5) in Stx1 concentration was observed. In the majority of bovine-associated Stx1-producing isolates (16 of 20), increases were less than 10-fold, although mitomycin C treatment of 4 isolates resulted in 13- to 60-fold increases in Stx1 production (Fig. (Fig.1B).1B). Although the median fold increase in Stx1 production in response to mitomycin C treatment did not significantly differ between Stx1-producing HUS- and bovine-associated STEC isolates (Fig. (Fig.1B),1B), there was a statistically significant difference (P < 0.01) in the total amounts of Stx1 produced following mitomycin C treatment of these two groups of STEC isolates (production of Stx by HUS isolates [median = 915; IR = 510 to 1,965] was greater than that by bovine isolates [median = 210; IR = 58 to 416]). This discrepancy reflects the fact that in the absence of mitomycin C, the HUS-associated isolates produced significantly greater (P < 0.001) amounts of Stx1 than bovine isolates; without addition of mitomycin C, the median Stx1 concentrations were 860 (IR = 660 to 1,110) and 120 (IR = 20 to 170) ng of Stx1 ml−1, respectively, for these two groups.

Association of stx and bacteriophage genes.

Since we found that mitomycin C treatment did not increase Stx production in all STEC isolates and previous experiments had shown that mitomycin C can induce toxin production by lysogens of both Stx1- and Stx2-encoding phages (51), we used PCR to assess whether the stx genes studied here were located in phage genomes. Primers were designed based on the sequences of the Stx1-encoding phage H-19B or the Stx2-encoding phage 933W, as described previously (50). These primers amplify the segments between Q and stx and between stx and S. While the stx genes of all 13 HUS-associated Stx2-producing STEC were found adjacent to 933W-like Q sequences, linkage to a 933W-like S sequence was observed in only 3 (Table (Table1).1). Conversely, the stx genes in only 7 of 25 bovine-associated Stx2-producing STEC isolates were adjacent to the 933W-like Q, whereas the stx genes were linked to the 933W-like S sequence in a greater proportion of these isolates (20 of 25). These results reflect the diversity and likely mosaicism present in these stx2-encoding phages. Overall, of the eight Stx2-encoding STEC isolates in which Stx2 production did not increase (defined as <10-fold) in response to mitomycin C treatment, seven contained stx2 genes demonstrated by PCR to be linked to phage sequences. The stx genes of only three of six Stx1-producing HUS-associated STEC isolates were linked with H-19B-like sequences (Q or S; Table Table2).2). Of 20 Stx1-producing bovine-associated isolates, linkage between the stx genes and H-19B-like Q or S sequences occurred in only 7 or 5, respectively. However, failure to detect the presence of neighboring phage genes did not always correspond with a lack of mitomycin C inducibility nor was detection of phage genes always accompanied by mitomycin C inducibility. Overall, the primary conclusion that can be drawn from these PCR assays is that although the majority of STEC isolates studied had stx genes flanked by phage sequences, the sequences surrounding some stx genes in STEC isolates were variable and appeared to contain sequences that differ from those of H-19B or 933W.

Low-iron-induced Stx1 production.

Since Stx1 production had been shown previously to be induced when lysogens of H-19B were grown in low-iron medium, we compared levels of Stx1 production by HUS- and bovine-associated isolates to determine whether regulation of Stx1 production by iron concentration differs in these groups. The median fold increase in Stx1 production in low-iron medium was significantly greater (P < 0.01) in HUS-associated isolates (median = 6.5; IR = 6 to 9) than in bovine-associated STEC isolates (median = 3.5; IR = 1 to 5) (Fig. (Fig.2).2). However, note that Stx1 production by bovine-associated isolates was highly variable in both low- and high-iron media; some isolates produced high levels of Stx1 (>10,000 ng of Stx1 ml−1). In contrast, there was less variability in Stx1 production by HUS-associated isolates grown in low- and high-iron media. Overall, levels of total Stx1 production following growth in low-iron medium did not differ significantly between the two groups. No correlation was observed between isolates in which Stx1 production was highly induced by growth in low-iron medium and those in which Stx1 production was induced by mitomycin C (Table (Table22).

FIG. 2.
Increase in Stx1 production following growth in low-iron medium of Stx1-producing HUS- and bovine-associated STEC. Each assay was performed on duplicate cultures; the mean and the standard deviation are presented for each isolate. The bars represent the ...


Regulation of virulence factor production in response to environmental stimuli is thought to be a critical feature of microbial pathogenicity (32). Previous studies have documented how prophage-inducing stimuli (e.g., mitomycin C) and low-iron conditions can greatly augment Stx production (1, 15, 51, 53). However, these studies analyzed a limited number of human STEC isolates, and until now, little information existed regarding how bovine-associated STEC isolates respond to these stimuli. In this study, we found that not all STEC respond similarly, as marked differences in Stx production were found between HUS- and bovine-associated STEC isolates. The augmentation of Stx production by the presence of either mitomycin C or low iron concentration was, in general, significantly greater in HUS-associated STEC isolates than that observed for bovine-associated STEC isolates. That a subgroup of STEC can be more virulent for or more readily transmitted to humans was previously suggested by detection of genotypic variations in bovine and human O157 isolates (24, 31). Our results suggest that response to prophage-inducing stimuli or low iron contributes to the ability of a subgroup of STEC to cause severe disease in humans.

Among the HUS isolates, mitomycin C-inducible toxin production was detected in all the Stx2-producing isolates tested but, unexpectedly, this prophage-inducing agent did not augment toxin production by Stx1-producing isolates. Previous work (for examples, see references 51, 53, and 54) has shown that this stimulus induces lysogens carrying either stx1 or stx2 to increase toxin synthesis, as it promotes phage transcription and replication. Our PCR analyses revealed that stx1 was flanked by Q and/or S sequences in three of six of the HUS-associated isolates we studied; thus, these isolates might have been expected to be inducible with mitomycin C. Presumably, in these cases the stx1 genes lie within defective prophages. In this regard, they may be like the stx genes in Shigella dysenteriae type 1, which have been shown to be adjacent to phage-like sequences (30) but are not transmissible.

Our results extend previous observations that there is a considerable diversity of stx-containing prophages (22, 50, 51). Both the range of augmentation of Stx production in response to mitomycin C treatment and the differing sizes of the PCR products amplified from the toxin-linked phage sequences are reflections of this heterogeneity. Another indication of this variability was the finding that Stx2 production in bovine-associated STEC was augmented by provision of the H-19B-like Q in trans in only a subset of the isolates tested (data not shown).

Our observations suggest that prophage induction is not as important for Stx1 production as it is for Stx2 production, since, as mentioned above, the HUS-associated Stx1-producing isolates we tested did not respond to mitomycin C treatment. The nature of the process by which Stx1 is released from bacteria in the absence of prophage induction is unclear, since phage-induced bacterial lysis is the only cellular program shown (at least in vitro) to allow Stx to escape from the periplasm (53). Our present data raise the possibility that additional processes also enable toxin release (at least from HUS-associated Stx1-producing strains). However, the apparent absence of prophage regulation of Stx1 production might account for the fact that Stx2-producing STEC isolates, either alone or in combination with Stx1, are more frequently associated with HUS than isolates that produce Stx1 alone (18). Alternatively, it is possible that some Stx-encoding phages are induced in vivo via pathways distinct from the SOS response (44). Overall, further study of prophage induction in vivo is warranted to determine whether host responses (e.g., H2O2 production by neutrophils) (52) have a significant impact or whether exogenous stimuli (e.g., antibiotic treatment) are the dominant modulators of toxin production.

The median levels of Stx production following growth in Luria broth also differed between the HUS-associated and bovine-associated STEC isolates we studied. This basal Stx production by both Stx1- and Stx2-producing HUS-associated isolates was significantly greater than that by bovine isolates. In particular, basal Stx production was high in Stx1-producing HUS isolates. This finding, coupled with the fact that prophage-inducing stimuli had a less marked effect on Stx1-producing isolates than on the Stx2-producing HUS isolates, suggests that Stx1 inducibility is not critical for the pathogenicity of these strains.

Factors other than basal and inducible Stx production are also important in STEC pathogenesis. As previous analyses have suggested (2), we found that ehx and eae are likely to be important contributors to STEC pathogenicity. Both genes were present in all HUS-associated STEC isolates analyzed. In contrast, only 13 of 45 of the bovine-associated STEC isolates contained both genes and 18 of 45 contained neither gene. However, neither ehxA nor eae appears to be essential for STEC pathogenicity, since clinical isolates lacking either ehxA or eae have been reported (34, 39). Still, these virulence factors appear to be more critical than serogroup for STEC pathogenicity; we found that HUS-linked isolates were from a variety of serogroups. Future studies of these factors should include quantitation of virulence gene expression, since we found that quantifying Stx production revealed a previously unrecognized distinction between disease- and bovine-associated STEC.


We thank Brigid Davis, Cheleste Thorpe, and Anne Kane for critical reading of the manuscript and Herbert Schmidt for the Stx1-producing HUS isolates.

Financial support was provided by Food and Drug Administration grant FD-U-001574, Center for Gastroenterology Research on Absorptive and Secretory Processes grant P30 DK-34928, National Institutes of Health grants AI 39067 (all D.W.K.A.) and AI 42347 (M.K.W.), and the HHMI (M.K.W. and P.L.W.).


1. Al-Jumaili, I., D. A. Burke, S. M. Scotland, H. Al-Mardini, and C. O. Record. 1992. A method of enhancing verocytotoxin production by Escherichia coli. FEMS Microbiol. Lett. 93:121-126.
2. Barrett, T. J., J. B. Kaper, A. E. Jerse, and I. K. Wachsmuth. 1992. Virulence factors in Shiga-like toxin-producing Escherichia coli isolated from humans and cattle. J. Infect. Dis. 165:979-980. [PubMed]
3. Beutin, L., D. Geier, H. Steinruck, S. Zimmermann, and F. Scheutz. 1993. Prevalence and some properties of verotoxin (Shiga-like toxin)-producing Escherichia coli in seven different species of healthy domestic animals. J. Clin. Microbiol. 31:2483-2488. [PMC free article] [PubMed]
4. Beutin, L., M. A. Montenegro, I. Orskov, F. Orskov, J. Prada, S. Zimmermann, and R. Stephan. 1989. Close association of verotoxin (Shiga-like toxin) production with enterohemolysin production in strains of Escherichia coli. J. Clin. Microbiol. 27:2559-2564. [PMC free article] [PubMed]
5. Boerlin, P., S. A. McEwan, F. Boerlin-Perzold, J. B. Wilson, R. P. Johnson, and C. L. Gyles. 1999. Associations between virulence factors of Shiga toxin-producing Escherichia coli and disease in humans. J. Clin. Microbiol. 37:497-503. [PMC free article] [PubMed]
6. Brunder, W., H. Schmidt, and H. Karch. 1996. KatP, a novel catalase-peroxidase encoded by the large plasmid of enterohaemorrhagic Escherichia coli O157:H7. Microbiology 142:3305-3315. [PubMed]
7. Brunder, W., H. Schmidt, and H. Karch. 1997. EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol. Microbiol. 24:767-778. [PubMed]
8. Calderwood, S. B., and J. J. Mekalanos. 1987. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J. Bacteriol. 169:4759-4764. [PMC free article] [PubMed]
9. Dawson-Saunders, B., and R. G. Trapp. 1990. Basic and clinical biostatistics. Appleton and Lange, East Norwalk, Conn.
10. Donohue-Rolfe, A., and G. T. Keusch. 1983. Shigella dysenteriae 1 cytotoxin: periplasmic protein releasable by polymyxin B and osmotic shock. Infect. Immun. 39:270-274. [PMC free article] [PubMed]
11. Donohue-Rolfe, A., D. W. K. Acheson, A. V. Kane, and G. T. Keusch. 1989. Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies. Infect. Immun. 57:3888-3893. [PMC free article] [PubMed]
12. Donohue-Rolfe, A., G. T. Keusch, C. Edson, D. Thorley-Lawson, and M. Jacewicz. 1984. Pathogenesis of Shigella diarrhea. IX. Simplified high yield purification of Shigella toxin and characterisation of subunit composition and function by the use of subunit-specific monoclonal and polyclonal antibodies. J. Exp. Med. 160:1767-1781. [PMC free article] [PubMed]
13. Frankel, G., A. D. Phillips, I. Rosenshine, G. Dougan, J. B. Kaper, and S. Knutton. 1998. Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol. Microbiol. 30:911-921. [PubMed]
14. Friedrich, A. W., M. Bielaszewska, W. L. Zhang, M. Pulz, T. Kuczius, A. Ammon, and H. Karch. 2002. Escherichia coli harboring shiga toxin 2 gene variants: frequency and association with clinical symptoms. J. Infect. Dis. 185:74-84. [PubMed]
15. Fuchs, S., I. Muhldorfer, A. Donohue-Rolfe, M. Kerenyi, L. Emody, A. Rossen, R. Alexiev, P. Nenkov, and J. Hacker. 1999. Influence of RecA on in vivo virulence and Shiga toxin 2 production in Escherichia coli pathogens. Microb. Pathog. 27:13-23. [PubMed]
16. Gerber, A., H. Karch, F. Allerberger, H. M. Verweyen, and L. B. Zimmerhackl. 2002. Clinical course and the role of Shiga toxin-producing Escherichia coli infection in the hemolytic uremic syndrome in pediatric patients, 1997-2000, in Germany and Austria: a prospective study. J. Infect. Dis. 186:493-500. [PubMed]
17. Grif, K., M. P. Dierich, H. Karch, and F. Allerberger. 1998. Strain-specific differences in the amount of Shiga toxin released from enterohemorrhagic Escherichia coli O157 following exposure to subinhibitory concentrations of antimicrobial agents. Eur. J. Clin. Microbiol. Infect. Dis. 17:761-766. [PubMed]
18. Griffin, P. M., and R. V. Tauxe. 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. [PubMed]
19. Griffin, P. M., P. S. Mead, and S. Sivapalasingam. 2002. Escherichia coli O157:H7 and other enterohemorrhagic E. coli, p. 627-642. In M. J. Blaser, P. D. Smith, J. I. Ravdin, et al. (ed.), Infections of the gastrointestinal tract, 2nd ed. Lippincott Williams & Wilkins, Philadelphia, Pa.
20. Huang, H., J. Friesen, and J. L. Bruton. 1987. Characterization of a bacteriophage that carries the genes for production of Shiga toxin 1 in Escherichia coli. J. Bacteriol. 169:4308-4312. [PMC free article] [PubMed]
21. Hull, A. E., D. W. K. Acheson, P. Echeverria, A. Donohue-Rolfe, and G. T. Keusch. 1993. Mitomycin immunoblot colony assay for detection of Shiga-like toxin-producing Escherichia coli in fecal samples: comparison with DNA probes. J. Clin. Microbiol. 31:1167-1172. [PMC free article] [PubMed]
22. Johansen, B. K., Y. Wasteson, P. E. Granum, and S. Brynestad. 2001. Mosaic structure of Shiga-toxin-2-encoding phages isolated from Escherichia coli O157:H7 indicates frequent gene exchange between lambdoid phage genomes. Microbiology 147:1929-1936. [PubMed]
23. Karch, H., H. Schmidt, C. Janetzki-Mittmann, J. Scheef, and M. Kroger. 1999. Shiga toxins even when different are encoded at identical positions in the genomes of related temperate bacteriophages. Mol. Gen. Genet. 262:600-607. [PubMed]
24. Kim, J., J. Nietfeldt, and A. K. Benson. 1999. Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle. Proc. Natl. Acad. Sci. USA 96:13288-13293. [PMC free article] [PubMed]
25. Kobayashi, H., J. Shimada, M. Nakazawa, T. Morozumi, T. Pohjanvirta, S. Pelkonen, and K. Yamamoto. 2001. Prevalence and characteristics of Shiga toxin-producing Escherichia coli from healthy cattle in Japan. Appl. Environ. Microbiol. 67:484-489. [PMC free article] [PubMed]
26. Lathem, W. W., T. E. Grys, S. E. Witowski, A. G. Torres, J. B. Kaper, P. I. Tarr, and R. A. Welch. 2002. StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol. Microbiol. 45:277-288. [PubMed]
27. Levine, M. M., J. Xu, J. B. Kaper, H. Lior, V. Prado, B. Tall, J. Nataro, H. Karch, and K. Wachsmuth. 1987. A DNA probe to identify enterohemorrhagic Escherichia coli of O157:H7 and other serotypes that cause hemorrhagic colitis and hemolytic uremic syndrome. J. Infect. Dis. 156:175-182. [PubMed]
28. Mariani-Kurkdjian, P., E. Denamur, A. Milon, B. Picard, H. Cave, N. Lambert-Zechovsky, C. Loirat, P. Goullet, P. J. Sansonetti, and J. Elion. 1993. Identification of a clone of Escherichia coli O103:H2 as a potential agent of hemolytic-uremic syndrome in France. J. Clin. Microbiol. 31:296-301. [PMC free article] [PubMed]
29. Matsushiro, A., K. Sato, H. Miyamoto, T. Yamamura, and T. Honda. 1999. Induction of prophages of enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. J. Bacteriol. 181:2257-2260. [PMC free article] [PubMed]
30. McDonough, M. A., and J. R. Butterton. 1999. Spontaneous tandem amplification and deletion of the Shiga toxin operon in Shigella dysenteriae I. Mol. Microbiol. 34:1058-1069. [PubMed]
31. McNally, A., A. J. Roe, S. Simpson, F. M. Thomson-Carter, D. E. E. Hoey, C. Currie, T. Chakraborty, D. G. E. Smith, and D. L. Gally. 2001. Differences in levels of secreted locus of enterocyte effacement proteins between human disease-associated and bovine Escherichia coli O157. Infect. Immun. 69:5107-5114. [PMC free article] [PubMed]
32. Mekalanos, J. J. 1992. Environmental signals controlling expression of virulence determinants in bacteria. J. Bacteriol. 174:1-7. [PMC free article] [PubMed]
33. Moake, J. L. 2002. Thrombotic microangiopathies. N. Engl. J. Med. 347:589-600. [PubMed]
34. Morabito, S., H. Karch, P. Mariani-Kurkdjian, H. Schmidt, F. Minelli, E. Bingen, and A. Caprioli. 1998. Enteroaggregative, Shiga toxin-producing Escherichia coli O111:H2 associated with an outbreak of hemolytic-uremic syndrome. J. Clin. Microbiol. 36:840-842. [PMC free article] [PubMed]
35. Muhldorfer, I., J. Hacker, G. T. Keusch, D. W. K. Acheson, H. Tschape, A. V. Kane, A. Ritter, T. Olschlager, and A. Donohue-Rolfe. 1996. Regulation of the Shiga-like toxin II operon in Escherichia coli. Infect. Immun. 64:495-502. [PMC free article] [PubMed]
36. Neely, M. N., and D. I. Friedman. 1998. Arrangement and functional identification of genes in the regulatory region of lambdoid phage H-19B, a carrier of a Shiga-like toxin. Gene 223:105-113. [PubMed]
37. Neely, M. N., and D. I. Friedman. 1998. Functional and genetic analysis of regulatory regions of coliphage H-19B: location of Shiga-like toxin and lysis genes suggest a role for phage functions in toxin release. Mol. Microbiol. 28:1255-1267. [PubMed]
38. O'Brien, A. D., J. W. Newland, S. F. Miller, R. K. Holmes, H. W. Smith, and S. B. Formal. 1984. Shiga-like toxin-converting phages from Escherichia coli strains that cause hemorrhagic colitis or infantile diarrhea. Science 226:694-696. [PubMed]
39. Paton, A. W., M. C. Woodrow, R. M. Doyle, J. A. Lanser, and J. C. Paton. 1999. Molecular characterization of a Shiga toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremic syndrome. J. Clin. Microbiol. 37:3357-3361. [PMC free article] [PubMed]
40. Paton, A. W., and J. C. Paton. 1998. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157. J. Clin. Microbiol. 36:598-602. [PMC free article] [PubMed]
41. Plunkett, G., III, D. J. Rose, T. J. Durfee, and F. R. Blattner. 1999. Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181:1767-1778. [PMC free article] [PubMed]
42. Pradel, N., V. Livrelli, C. de Champs, J.-B. Palcoux, A. Reynaud, F. Scheutz, J. Sirot, B. Joly, and C. Forestier. 2000. Prevalence and characterization of Shiga toxin-producing Escherichia coli isolated from cattle, food, and children during a one-year prospective study in France. J. Clin. Microbiol. 38:1023-1031. [PMC free article] [PubMed]
43. Ptashne, M. 1992. A genetic switch: phage lambda and higher organisms, 2nd ed. Cell Press & Blackwell Science, Cambridge, Mass.
44. Rozanov, D. V., R. D'Ari, and S. P. Sineoky. 1998. RecA-independent pathways of lambdoid prophage induction in Escherichia coli. J. Bacteriol. 180:6306-6315. [PMC free article] [PubMed]
45. Schmidt, H., and H. Karch. 1996. Enterohemolytic phenotypes and genotypes of Shiga toxin-producing Escherichia coli O111 strains from patients with diarrhea and hemolytic-uremic syndrome. J. Clin. Microbiol. 34:2364-2367. [PMC free article] [PubMed]
46. Su, C., and L. J. Brandt. 1995. Escherichia coli O157:H7 infection in humans. Ann. Intern. Med. 123:698-714. [PubMed]
47. Taylor, F. B., Jr., V. L. Tesh, L. DeBault, A. Li, A. C. K. Chang, S. D. Kosanke, T. J. Pysher, and R. L. Siegler. 1999. Characterization of the baboon responses to Shiga-like toxin: descriptive study of a new primate model of toxic response to Stx-1. Am. J. Pathol. 154:1285-1298. [PMC free article] [PubMed]
48. Thorpe, C. M., J. M. Ritchie, and D. W. K. Acheson. 2002. Enterohemorrhagic and other Shiga toxin-producing Escherichia coli. In M. Donnenberg (ed.), Escherichia coli: virulence mechanisms of a versatile pathogen. Academic Press, San Diego, Calif.
49. Tzipori, S., I. K. Wachsmuth, C. Chapham, R. Birner, J. Brittingham, C. Jackson, and J. Hogg. 1986. The pathogenesis of hemorrhagic colitis caused by Escherichia coli O157:H7 in gnotobiotic piglets. J. Infect. Dis. 154:712-716. [PubMed]
50. Unkmeir, A., and H. Schmidt. 2000. Structural analysis of phage-borne stx genes and their flanking sequences in Shiga toxin-producing Escherichia coli and Shigella dysenteriae type 1 strains. Infect. Immun. 68:4856-4864. [PMC free article] [PubMed]
51. Wagner, P. L., D. W. K. Acheson, and M. K. Waldor. 1999. Isogenic lysogens of diverse Shiga toxin 2-encoding bacteriophages produce markedly different amounts of Shiga toxin. Infect. Immun. 67:6710-6714. [PMC free article] [PubMed]
52. Wagner, P. L., D. W. K. Acheson, and M. K. Waldor. 2001. Human neutrophils and their products induce Shiga toxin production by enterohemorrhagic Escherichia coli. Infect. Immun. 69:1934-1937. [PMC free article] [PubMed]
53. Wagner, P. L., J. Livny, M. N. Neely, D. W. K. Acheson, D. I. Friedman, and M. K. Waldor. 2002. Bacteriophage control of Shiga toxin 1 production and release by Escherichia coli. Mol. Microbiol. 44:957-970. [PubMed]
54. Wagner, P. L., M. N. Neely, X. Zhang, D. W. K. Acheson, M. K. Waldor, and D. I. Friedman. 2001. Role for a phage promoter in Shiga toxin 2 expression from a pathogenic Escherichia coli strain. J. Bacteriol. 183:2081-2085. [PMC free article] [PubMed]
55. Wong, C. S., S. Jelacic, R. L. Habeeb, S. L. Watkins, and P. I. Tarr. 2000. The risk of the hemolytic-uremic syndrome after antibiotic treatment of Escherichia coli O157:H7 infections. N. Engl. J. Med. 342:1930-1936. [PMC free article] [PubMed]
56. Yee, A. J., S. de Grandis, and C. L. Gyles. 1993. Mitomycin-induced synthesis of a Shiga-like toxin from enteropathogenic Escherichia coli H.I.8. Infect. Immun. 61:4510-4513. [PMC free article] [PubMed]
57. Zhang, W., M. Bielaszewska, T. Kuczius, and H. Karch. 2002. Identification, characterization, and distribution of a Shiga toxin 1 gene variant (stx1c) in Escherichia coli strains isolated from humans. J. Clin. Microbiol. 40:1441-1446. [PMC free article] [PubMed]
58. Zhang, X., A. D. McDaniel, L. E. Wolf, G. T. Keusch, M. K. Waldor, and D. W. K. Acheson. 2000. Quinolone antibiotic induces Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J. Infect. Dis. 181:664-670. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try


Save items

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Compound
    PubChem Compound links
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...