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Shiga Toxin Activatable by Intestinal Mucus in Escherichia coli Isolated from Humans: Predictor for a Severe Clinical Outcome

  1. Martina Bielaszewska1,2,
  2. Alexander W. Friedrich1,2,
  3. Thomas Aldick1,
  4. Robin Schürk-Bulgrin1, and
  5. Helge Karch1,2
  1. 1National Consulting Laboratory on Hemolytic Uremic Syndrome, Institute for Hygiene, University of Münster, and Interdisciplinary Center for Clinical Research (IZKF) Münster, Münster, Germany
  2. 2Interdisciplinary Center for Clinical Research (IZKF) Münster, Münster, Germany
  1. Reprints or correspondence: Dr. Martina Bielaszewska, Institut für Hygiene, Universität Münster, Robert Koch Str. 41, 48149 Münster, Germany (mbiela{at}uni-muenster.de).
 
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Abstract

Background. Some Escherichia coli produce Shiga toxin (Stx) in which cytotoxicity is increased (activated) by intestinal mucus and elastase (Stx2dactivatable). These strains are highly virulent in mice, but their association with human disease is poorly understood. We investigated the prevalence of Stx2dactivatable among Stx-producing E. coli (STEC) isolated from humans and the association between production of this Stx and the clinical outcome of infection.

Methods. A total of 922 STEC isolates obtained from patients with hemolytic uremic syndrome or bloody or nonbloody diarrhea or from asymptomatic carriers were tested for the gene encoding Stx2dactivatable by PCR and PstI restriction analysis. The toxin activatibility by human and mouse intestinal mucus and by an elastase was determined by quantifying the cytotoxicity using the Vero cell assay.

Results. The stx2d-activatable gene was identified in 60 (6.5%) of 922 STEC strains; in 31 of these strains, it was the sole stx gene. Thirty of these 31 strains produced Stx2dactivatable. All of them lacked the intimin-encoding eae gene. Among eae-negative STEC, which typically cause mild diarrhea or asymptomatic infection, production of Stx2dactivatable was significantly associated with the ability to cause severe disease, including bloody diarrhea (P < .001), and with systemic complications, such as hemolytic uremic syndrome (P < .001).

Conclusions. Production of Stx2dactivatable by the infecting STEC may predict a severe clinical outcome of the infection, with progression to hemolytic uremic syndrome. A prompt and comprehensive subtyping of stx genes in STEC isolates is necessary to alert the treating physician that a patient is at risk of developing hemolytic uremic syndrome, even though the infecting STEC lacks eae.

Infection with Shiga toxin (Stx)–producing Escherichia coli (STEC) can result in a spectrum of outcomes, ranging from asymptomatic carriage to uncomplicated diarrhea, bloody diarrhea, and hemolytic uremic syndrome (HUS) [1, 2]. HUS consists of hemolytic anemia, thrombocytopenia, and acute renal failure [3, 4], and it is the leading cause of acute renal failure in children [5]. The mortality rate associated with HUS during the acute phase is 2%–5% [1, 6], and there is a high rate of renal or nonrenal sequelae in survivors [7]. STEC O157:H7 is the predominant cause of HUS [1, 2, 4, 6, 8], but several non-O157:H7 serotypes have been emerging [2, 6, 8–12].

Stxs are the major virulence factors implicated in the pathogenesis of HUS [4, 13]. These AB5 toxins have been subdivided, on the basis of toxin neutralization assays and sequence analysis of stx genes, into 2 families, Stx1 and Stx2 [14]. Each family consists of the major Stx type and variants. The members of the Stx1 family are Stx1, Stx1c [15], and Stx1d [16]. The more heterogeneous Stx2 family includes Stx2, Stx2c [17], Stx2c2 [18], Stx2d [19, 20], Stx2e [21], and Stx2f [22]. The Stx2d group comprises 2 independently described [19, 20] and distinct toxins, Stx2dEH250 [20] and Stx2dactivatable [19]. The designation of the latter toxin has been derived from its capacity to be activated in its biological activity (demonstrated as a significant increase in its cytotoxicity for cultured cells) by mouse or human intestinal mucus [19]. This mucus activatibility differentiates Stx2dactivatable from the other presently known Stxs [19, 23].

The activator of Stx2dactivatable in mouse mucus is an elastase that is highly homologous to human elastase IIIB [24]. The activation involves cleavage by the elastase of the last 2 C-terminal amino acids from an enzymatically active A subunit of Stx2dactivatable [23]. As a result of this activation, STEC that produce Stx2dactivatable are considerably more virulent in a streptomycin-treated mouse model of STEC infection (oral 50% lethal dose [LD50] of <10 colony-forming units [cfu]) than STEC producing Stx2 or Stx2c (LD50 of 1010 cfu) [19, 25]. The augmented virulence of Stx2dactivatable-producing STEC in this model led to the hypothesis that the presence of an activatable Stx in STEC infecting humans and the activation of this Stx by human intestinal mucus during infection might result in increased virulence of such strains for humans [19]. However, the association of STEC that produce Stx2dactivatable with human disease is poorly understood. Therefore, we investigated the prevalence of E. coli that produce Stx2dactivatable in a large collection of STEC from clinically well-defined subjects, and we determined the association between production of Stx2dactivatable by the infecting STEC and clinical outcome of the infection.

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Patients and Methods

Patients and strains. A total of 922 STEC strains were isolated from 1994 to 2005 from stool samples obtained from individual, epidemiologically unrelated patients with HUS (339 strains), bloody diarrhea (51 strains), or nonbloody diarrhea (414 strains) or from asymptomatic carriers (118 strains), as described elsewhere [26, 27]. The strains belonged to 109 serotypes, as determined by a microtiter serotyping method [28]. Six hundred and eighty-three strains belonging to 30 serotypes contained the eae gene encoding the STEC adherence factor intimin [29], and 239 strains belonging to 79 serotypes were eae negative (table 1). Reference Stx2dactivatable-producing E. coli strain B2F1 [19], which was isolated from a patient with HUS [30], was kindly provided by M. A. Karmali (Public Health Agency of Canada, Guelph, Canada).

Table 1
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Table 1

Serotypes, eae genes, and clinical associations of Shiga toxin–producing Escherichia coli strains investigated for stx2d-activatable.

Case definition. Patients with nonbloody diarrhea were defined as patients with ⩾3 watery stools without visible blood per day. Patients with bloody diarrhea were defined as patients with ⩾3 watery stools per day in which visible blood was noted in the stool. HUS was defined as a case of microangiopathic hemolytic anemia (hematocrit of <30% with peripheral evidence of intravascular hemolysis), thrombocytopenia (platelet count, <150,000 platelets/mm3), and renal insufficiency (serum creatinine concentration greater than the upper limit of the normal range for age) [3].

Detection of stx genes. The presence of the gene encoding Stx2dactivatable in the 922 STEC strains was tested for using PCR with primers SLT-II-vc and CKS2 [18] and restriction analysis of the resulting 890–base pair (bp) amplicon with PstI [18]. PstI does not cleave the amplicon derived from stx2d-activatable but cleaves this amplicon into 504-bp and 386-bp fragments if the target is stx2 or stx2c. The presence of stx1, stx1c, stx1d, stx2, stx2c, stx2dEH250, and stx2e was sought, as described elsewhere [15, 16, 21, 26].

Isolation of mouse and human intestinal mucus. Mucus was isolated from the intestines (small and large) of 5 BALB mice and from pooled effluents obtained during colonoscopy from 3 humans [19]. The mucus preparations were adjusted to a protein concentration of 5 mg/mL with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES; 2.4 g/L) buffer (pH, 7.4) and were frozen in aliquots at -70°C.

Mucus and elastase activation assay. In the mucus activation assay [19], sterile-filtered supernatants were preincubated (for 2 h at 37°C) with mouse or human intestinal mucus (1 mg/mL) or HEPES buffer and tested for cytotoxicity using Vero cells [31]. The level of activation was determined as the ratio between the cytotoxicity titer of the mucus-treated supernatant and that of the HEPES-treated supernatant. The toxin was considered to be activatable when the cytotoxicity titer increased at least 8-fold after incubation with the mouse intestinal mucus and at least 4-fold after incubation with the human colonic mucus. These cut-off values were based on the lowest levels of activation produced by mouse (8-fold) and human (4-fold) mucus in the supernatant of the control Stx2dactivatable-producing strain B2F1. None of the mucus preparations were toxic to Vero cells at the concentration used.

In the elastase activation assay, supernatants were incubated (for 2 h at 37°C) with 1 unit of porcine pancreatic elastase (EC 3.4.21.36; Calbiochem) (used in lieu of mouse intestinal elastase, which is not commercially available) or with phospate elastase resuspension buffer (50 mM KH2PO4 and 50 mM Na2HPO4; pH, 7.6). The activation was determined as described above. The toxin was considered to be activatable when the cytotoxicity titer increased at least 4-fold after incubation with 1 unit of the elastase. This elastase concentration was chosen, because it is not toxic to Vero cells. In contrast, after 72 h, 5 and 10 units of the elastase caused cytotoxicities in titers of 1 : 8 and 1 : 32, respectively.

Detection of other enteric bacterial pathogens in stools. Salmonella species, Shigella species, Yersinia enterocolitica, and Campylobacter jejuni were sought using standard procedures.

Serological investigation. IgM antibodies against lipopolysaccharide O157 in serum samples obtained from patients in the acute phase of HUS were detected using an immunoblot [32].

Statistical analysis. Statistical analysis was performed using the χ2 test, Yates's corrected χ2 test, and Epi-Info, version 2002 (Centers for Disease Control and Prevention and the World Health Organization). P values <.05 were considered to be significant.

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Results

Prevalence of stx2d-activatable among STEC and characterization of strains harboring the gene and the associated patients. The gene encoding Stx2dactivatable was identified in 60 (6.5%) of 922 STEC isolates investigated. Thirty-one of these 60 strains contained stx2d-activatable as the sole stx gene; the other 29 strains possessed stx2d-activatable in combination with stx1 and/or stx2 (table 2). STEC harboring stx2d-activatable belonged to 26 serotypes, the most common of which were O91:H21 (16 isolates), O22:H8 (8 isolates), and O113:H21 (7 isolates) (table 2). All 60 STEC strains harboring stx2d-activatable were eae negative. This resulted in a significantly higher prevalence of stx2d-activatable among eae-negative STEC (60 [25.1%] of 239 strains) than among eae-positive STEC (0 of 683 strains) (χ2, 140.26; P < .001; 95% CI, 19.33–997.78). Thirteen of 16 cases of severe disease (HUS or bloody diarrhea) associated with the stx2d-activatable-harboring STEC occurred in patients who were infected with strains containing stx2d-activatable as the only stx (table 2). These patients were substantially older (as indicated by median age) than those infected with strains harboring stx2d-activatable in combination with other stx genes (table 2).

Table 2
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Table 2

The stx genotypes, serotypes, and clinical associations of 60 Shiga toxin–producing Escherichia coli strains harboring stx2d-activatable.

Vero cell cytotoxicity and activation of Stx produced by STEC harboring stx2d-activatable by the intestinal mucus and elastase. Supernatants of all 31 STEC strains that harbored stx2d-activatable as the sole stx gene were cytotoxic to Vero cells (titer range, 6–256; median titer, 64) (table 3). Preincubation with the mouse intestinal mucus resulted in ⩾8-fold increases in cytotoxicity titers in all but 1 strain (range, 8–21.3-fold; median increase, 10.7-fold) (table 3). In these 30 strains, cytotoxicity also significantly increased after preincubation with the human colonic mucus (range, 4–16-fold; median increase, 6.7-fold) and elastase (range, 4 –10.7-fold; median increase, 6.7-fold) (table 3). A similar extent of activation was observed after incubation with the mucus preparations and elastase in the control Stx2dactivatable-producing strain B2F1 (table 3).

Table 3
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Table 3

Vero cell cytotoxicity and Shiga toxin (Stx) activatibility by mouse and human intestinal mucus and elastase for Stx-producing Escherichia coli strains harboring stx2d-activatable as the sole stx gene.

Activation of other Stx types by the intestinal mucus and elastase. Eight hundred and sixty-two STEC strains harbored genes encoding other Stx types, including Stx1, Stx1c, Stx1d, Stx2, Stx2c, Stx2dEH250, and Stx2e. Some of these Stxs have been shown to be nonactivatable by intestinal mucus [19, 23]. To investigate the mucus and elastase activatibility of all of these Stx types, 3–5 randomly selected strains that produced each of these Stxs as a sole Stx (31 strains altogether) were subjected to the activation assay. None displayed an increase in the cytotoxicity that fulfilled the definition of activation after incubation with either mouse or human intestinal mucus or elastase. Thus, all of these Stxs are very probably nonactivatable.

Association between production of Stx2dactivatable and clinical outcome of infection. Because all STEC strains that produced Stx2dactivatable were eae negative, we analyzed the association between production of Stx2dactivatable and clinical outcome of infection among individuals infected with eae-negative STEC. A stratified analysis including the 30 STEC strains that produced Stx2dactivatable as the sole Stx was performed to avoid confounding by coproduction of nonactivatable Stx types (Stx1 and/or Stx2) by the other 29 strains and possible contribution of these Stxs to the clinical outcome.

The eae-negative STEC represent only small proportions of the total numbers of STEC isolated from patients with HUS (3.3%) and bloody diarrhea (12.0%) (table 4), but STEC that produce Stx2dactivatable account for most of the eae-negative STEC strains isolated from patients with HUS (7 [63.6%] of 11 strains) and all of the eae-negative STEC strains isolated from patients with bloody diarrhea (table 4). Among STEC isolates obtained from patients with nonbloody diarrhea and from asymptomatic carriers, for whom the proportions of eae-negative strains are higher (28.5% and 73.1%, respectively), the relative proportions of Stx2dactivatable-producing strains among the eae-negative STEC isolates are considerably lower (15.0% and 0%, respectively) (table 4). All 30 STEC strains producing Stx2dactivatable were isolated from subjects who were ill (tables 3 and 4), demonstrating that the production of Stx2dactivatable by the infecting eae-negative STEC is significantly associated with its ability to cause disease, compared with asymptomatic infection (χ2, 15.13; P < .001; 95% CI, 2.15–111.53) (table 4). Interestingly, the only isolate obtained from an asymptomatic individual, which harbored stx2d-activatable as the sole stx, did not express the activatable phenotype (table 3). Among eae-negative STEC isolated from symptomatic individuals, production of Stx2dactivatable by the infecting strain was significantly associated with the ability to cause severe disease (HUS and bloody diarrhea), compared with milder disease (nonbloody diarrhea) (χ2, 14.65; P < .001; 95% CI, 1.81–6.06) (table 4). Among eae-negative isolates obtained from patients who developed diarrhea but not HUS, production of Stx2dactivatable was significantly associated with bloody diarrhea, compared with nonbloody diarrhea (χ2, 11.3; P < .001; 95% CI, 1.55–27.59) (table 4), although the number of isolates obtained from patients with bloody diarrhea was low. Moreover, production of Stx2dactivatable by the infecting eae-negative STEC was significantly associated with the ability to cause HUS, compared with other outcomes (χ2, 22.94; P < .001; 95% CI, 3.16–59.61) (table 4). Taken together, this analysis demonstrated that, in patients infected with eae-negative STEC, production of Stx2dactivatable by the infecting strain is a risk factor for a severe clinical outcome and for systemic progression of the infection to HUS.

Table 4
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Table 4

Association between clinical outcome of infection due to eae-negative Shiga toxin (Stx)–producing Escherichia coli (STEC) strains and production of Stx2dactivatable.

Characterization of patients infected with Stx2dactivatable-producing STEC. The median age of 24 of 30 patients from whom the STEC strains that produced Stx2dactivatable were isolated was 21 years (range, 1–89.5 years). Ages of 6 patients were unknown. Ages were available for 5 of the 7 patients with HUS; 4 were adults (aged 21, 45, 57, and 64 years), and 1 was a 3-year-old child. One adult patient (a 64-year-old woman) died during the acute phase of HUS (tables 2 and 3). None of the 4 surviving patients with HUS for whom ages were known developed anti-O157 lipopolysaccharide IgM, demonstrating that E. coli O157:H7, which is the most common cause of STEC-associated HUS, was not missed by the microbiological evaluation. Serum samples from the remaining 3 patients with HUS were not available. Stool samples from all 30 patients infected with Stx2dactivatable-producing STEC were negative for Salmonella species, Shigella species, Y. enterocolitica, and C. jejuni.

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Discussion

The clinical outcome of an infection due to STEC depends, in large part, on the Stx type produced by the infecting strain and on the presence of the eae gene, which encodes the adherence factor intimin. STEC that produce Stx2 or Stx2c, most of which possess eae [26], cause severe disease, such as hemorrhagic colitis or HUS [10, 26, 33, 34], whereas eae-negative STEC that produce Stx2dEH250 or Stx2e have been associated with uncomplicated diarrhea or asymptomatic infection [20, 21, 26].

A subset of eae-negative STEC strains (i.e., STEC that produce Stx2dactivatable) differ from other eae-negative STEC strains by their strong association with severe disease. Our systematic analysis of the association between production of Stx2dactivatable by the infecting STEC strain and the outcome of infection suggests that production of the activatable Stx may be a risk factor for the development of HUS after infection with eae-negative STEC, which otherwise only rarely causes HUS (table 4) [26, 33, 34]. By its association with severe outcome of the infection caused by eae-negative STEC, Stx2dactivatable parallels the pathogenetic significance of Stx2 among eae-positive STEC [26, 33, 34]. However, in contrast to Stx2, which is associated with HUS development in children <5 years old [26], most of the patients with HUS from whom we isolated Stx2dactivatable-producing STEC were adults.

From a practical standpoint, our data demonstrate a need for a rapid and comprehensive subtyping of stx genes in STEC isolates by microbiologists and the immediate interpretation and reporting of results to clinicians. The information about the presence of the gene encoding Stx2dactivatable in the infecting STEC could alert the treating physician that a patient is at risk for developing HUS, even though this strain lacks eae. Such a patient, if symptomatic, should be monitored for HUS development and given intravenous volume expansion [35], which has been associated with a reduced risk of anuric renal failure in patients with HUS due to STEC O157:H7. Although the precise risk of developing HUS for a patient with diarrhea who is infected with an Stx2dactivatable-producing STEC is unknown, these organisms might have consequences for infected humans similar to those associated with STEC O157; we believe that it would be appropriate to treat such patients similarly, at least until more data emerge. Because the activatable Stx was expressed by the majority of STEC isolates that contained stx2d-activatable as the sole stx (30 of 31 isolates), we propose that the identification of the gene itself, without a need to perform a labor-intensive mucus or elastase activation assay, might be a sufficient predictor for the highly pathogenic potential of the isolated STEC. These data also demonstrate the need for more extensive, rapid, and accurate characterization of infecting strains, beyond the mere assessment of their ability to produce Stx.

The increased pathogenicity of STEC harboring stx2d-activatable for humans, as demonstrated in our study, has been suspected on the basis of previous occasional isolations of such strains from severely ill persons, including patients with bloody diarrhea or HUS [30, 34, 36]. In a study from the United States [18], STEC that produced Stx2dactivatable were associated with a more severe course of diarrhea, as demonstrated by a higher rate of diagnostic or therapeutic procedures in patients infected by such strains; however, the association with bloody diarrhea did not reach statistical significance. The apparent disproportionate virulence of Stx2dactivatable-producing STEC for humans is also suggested by their ability to cause outbreaks, which are exceptionally associated with eae-negative STEC [37]. Of note, the eae-negative STEC strain O104:H21, which caused a large milkborne outbreak of hemorrhagic colitis in the United States [38], produces Stx2dactivatable [23].

Reasons for the excess virulence of strains producing Stx2dactivatable are presently not understood, but they are probably complex. It has been suggested [19] that the production of an activatable toxin might compensate for the lack of intimin, which mediates the intimate intestinal adhesion of eae-positive STEC [29] and, thus, enables an efficient delivery of Stx from the intestine to the bloodstream. Another plausible explanation might be a high rate of the expression of Stx2dactivatable in strains that contain this gene, as observed in our study. In contrast, a subset of eae-negative STEC that harbor structurally intact genes encoding other Stx2 variants (e.g., Stx2dEH250 and Stx2e) [39] do not secrete Stxs [34, 39]. Alternatively, STEC that produce Stx2dactivatable might express additional, yet unidentified, virulence factors that could contribute to their high human virulence. It should be noted that production of the activatable Stx is not essential for the ability of eae-negative STEC to cause HUS. Four eae-negative HUS isolates in this study (table 4) and an eae-negative STEC O113:H21 that caused an HUS cluster in Australia [37] possessed nonactivatable Stxs. Recently, a plant-based oral vaccine for protection against systemic intoxication caused by Stx2 has been developed [40]. The ability of this vaccine to completely protect mice from the renal pathology and resulting death caused by Stx2dactivatable-producing strain B2F1 [40] is promising and might be useful in preventing human diseases caused by such strains.

In summary, we identified Stx2dactivatable as an important cause of severe human disease caused by STEC. Diagnostic strategies should be modified to identify allelic variants in STEC, and symptomatic patients should be considered to be at increased risk of microangiopathic sequelae and treated accordingly.

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Acknowledgments

We thank Philip I. Tarr (Washington University School of Medicine, St. Louis, MO), for fruitful discussions, and Dagmar Mense (Institute for Hygiene, University of Münster, Münster, Germany), for technical assistance.

Financial support. Interdisciplinary Center of Clinical Research (IZKF), Münster (grant Ka2/061/04 to M.B.).

Potential conflicts of interest. All authors: no conflicts.

  • Received May 18, 2006.
  • Accepted July 5, 2006.
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  1. Clin Infect Dis. (2006) 43 (9): 1160-1167. doi: 10.1086/508195
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