Bacteriology
The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome

https://doi.org/10.1016/j.diagmicrobio.2007.04.013Get rights and content

Abstract

Shiga toxins (Stx) are believed to play a key role in the pathogenesis of diseases caused by Stx-producing Escherichia coli (STEC), including the potentially life-threatening hemolytic uremic syndrome (HUS). In this study, 201 STEC strains collected from patients and environmental sources were investigated with regard to the stx genotypes and pathogenicity. The stx2 and stx2c alleles were associated with high virulence and the ability to cause HUS, whereas stx2d, stx2e, stx1, and stx1c occurred in milder or asymptomatic infections. Quantification of Stx using an enzyme immunoassay and the Vero cell cytotoxicity assay showed no significant differences between the strains associated with HUS and those causing milder diseases. We hypothesize that the stx genotype and perhaps other yet unknown virulence factors rather than the amount of Stx or the in vitro cytotoxicity correlate with the development of HUS.

Introduction

Shiga toxin (Stx)-producing Escherichia coli (STEC) have emerged worldwide as a cause of severe human diseases including bloody diarrhea and hemolytic uremic syndrome (HUS) (Sonntag et al., 2005a, Tarr et al., 2005). HUS is characterized by the appearance of thrombocytopenia, hemolytic anemia, and acute nephropathy, which are the systemic complications after an intestinal infection with STEC. Approximately 15% of patients develop HUS, in particular, children younger than 10 years infected with E. coli O157:H7, the most pathogenic STEC. Several non-O157 STEC serotypes have also been identified as significant causes of bloody diarrhea and HUS (Brooks et al., 2005, Eklund et al., 2001, Friedrich et al., 2002, Gerber et al., 2002, Jelacic et al., 2003, Johnson et al., 2006, Karch et al., 2005, Mellmann et al., 2005, Misselwitz et al., 2003, Sonntag et al., 2004, Zhang et al., 2006), among which STEC O26 is the most common (Brooks et al., 2005, Friedrich et al., 2002, Gerber et al., 2002, Jelacic et al., 2003, Johnson et al., 2006, Karch et al., 2005).

Stxs are thought to play a major role in the pathogenesis of diseases involving STEC. Stxs are a family of bipartite protein toxins consisting of 2 toxin types, Stx1 and Stx2 (O'Brien et al., 1992). Variants have been identified within each of these types, including Stx1c (Friedrich et al., 2003, Zhang et al., 2002), Stx1d (Kuczius et al., 2004), Stx2c (Schmitt et al., 1991), Stx2c2 (Jelacic et al., 2003), Stx2d (Melton-Celsa et al., 1996, Pierard et al., 1998), Stx2e (Sonntag et al., 2005a, Weinstein et al., 1988), Stx2f (Schmidt et al., 2000, Sonntag et al., 2005b), and Stx2g (Leung et al., 2003). Within the Stx2d group, 2 independently described toxins exist, which differ by their nucleotide sequence and by the activatability of one by intestinal mucus (Melton-Celsa et al., 1996, Pierard et al., 1998), specifically due to its component elastase (Kokai-Kun et al., 2000).

In the study described here, for clarity, these toxins are referred to as Stx2dactivatable (Melton-Celsa et al., 1996) and Stx2d (Pierard et al., 1998). According to results from nucleotide sequence analyses and restriction fragment length polymorphism analyses, additional variants may also exist (De Baets et al., 2004), but the terminology is not consistent with the proposed nomenclature (Calderwood et al., 1996).

STEC strains isolated from patients with HUS produce mostly Stx2 and/or Stx2c (Friedrich et al., 2002), and in some pathogenic serotypes such as O26:H11/NM (nonmotile) and O145:H28/NM, there is evidence that the occurrence of strains harboring Stx2 is increasing (Bielaszewska et al., 2005a, Sonntag et al., 2004). Nucleotide sequence analyses of stx genes demonstrated that stx1 is essentially identical to stx of Shigella dysenteriae type 1, but that it shares only 57% and 60% nucleotide sequence identity in its A and B subunit, respectively, with the corresponding subunits of stx2 (Jackson et al., 1987). Variants of stx2 show 63% to 99.7% nucleotide sequence identity in their A subunits and 57.4% to 95.2% nucleotide sequence identity in their B subunits with the corresponding subunits of classical stx2 (Leung et al., 2003, Pierard et al., 1998, Schmidt et al., 2000, Schmitt et al., 1991, Weinstein et al., 1988). The highest divergency within the stx family is present in stx2e, stx2f, and stx2g.

In addition to Stxs, STEC also contains several other putative virulence factors. One of the best characterized is intimin, encoded by the eae gene, which is located on EE, the locus of enterocyte effacement. STEC containing eae are considered to be highly pathogenic to humans (Werber et al., 2003).

The aim of this study was to examine STEC strains, isolated from patients and environmental sources, for stx genotypes and Stx production. The association between the various stx subtypes, the source of the isolates, other virulence factors, and the disease manifestations was used to create a risk profile for the different STEC strains. We also compared the amount of Stx and the Vero cell cytotoxic activity of the strains causing HUS with those causing milder forms of the disease.

Section snippets

Bacterial isolates

A total of 201 STEC strains (132 eae positive and 69 eae negative) from humans (n = 150), animals (n = 37), and food (n = 14) were investigated (Table 1). The strains were isolated in the Austrian Reference Centre for Enterohaemorrhagic E. coli (EHEC), Innsbruck Medical University, Innsbruck, Austria, during routine diagnostic procedures and epidemiologic investigations between 2003 and 2005. The isolation of STEC strains from stools was performed as described previously (Friedrich et al., 2002

stx genotypes

A total of 16 different stx genotypes were identified among the 201 STEC investigated. The distribution of stx genotypes in isolates originating from humans, animals, and food is shown in Table 1. Fifty-eight strains harbored stx1 or stx1c as the only stx allele; 75 strains contained stx2 and/or stx2 variants, but not stx1 or stx1c; and 68 isolates contained stx1 or stx1c in combination with stx2 and/or stx2 variants.

stx1c was found in 19 of 126 isolates harboring genes of the stx1 group; 12 of

Discussion

There is evidence that subtyping stx genes is useful for predicting the severity of clinical symptoms in patients with STEC infections (Bielaszewska et al., 2006a, Eklund et al., 2001, Friedrich et al., 2002, Jelacic et al., 2003). This investigation in Austrian STEC isolates collected from patients and the environment over a 3-year period has demonstrated that stx2 and stx2c are associated with high virulence and the ability to cause HUS, whereas stx2d, stx2e, stx1, and stx1c are associated

Acknowledgment

The authors thank C. Ortner and A. Rief for excellent technical assistance. This study was supported by the Network of Excellence EuroPathoGenomics (LSHB-CT-2005-512061).

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