Shiga Toxin Gene Loss and Transfer In Vitro and In Vivo during Enterohemorrhagic Escherichia coli O26 Infection in Humans

Abstract

Escherichia coli serogroup O26 consists of enterohemorrhagic E. coli (EHEC) and atypical enteropathogenic E. coli (aEPEC). The former produces Shiga toxins (Stx), major determinants of EHEC pathogenicity, encoded by bacteriophages; the latter is Stx negative. We have isolated EHEC O26 from patient stools early in illness and aEPEC O26 from stools later in illness, and vice versa. Intrapatient EHEC and aEPEC isolates had quite similar pulsed-field gel electrophoresis (PFGE) patterns, suggesting that they might have arisen by conversion between the EHEC and aEPEC pathotypes during infection. To test this hypothesis, we asked whether EHEC O26 can lose stx genes and whether aEPEC O26 can be lysogenized with Stx-encoding phages from EHEC O26 in vitro. The stx 2 loss associated with the loss of Stx2-encoding phages occurred in 10% to 14% of colonies tested. Conversely, Stx2-and, to a lesser extent, Stx1-encoding bacteriophages from EHEC O26 lysogenized aEPEC O26 isolates, converting them to EHEC strains. In the lysogens and EHEC O26 donors, Stx2-converting bacteriophages integrated in yecE or wrbA. The loss and gain of Stx-converting bacteriophages diversifies PFGE patterns; this parallels findings of similar but not identical PFGE patterns in the intrapatient EHEC and aEPEC O26 isolates. EHEC O26 and aEPEC O26 thus exist as a dynamic system whose members undergo ephemeral interconversions via loss and gain of Stx-encoding phages to yield different pathotypes. The suggested occurrence of this process in the human intestine has diagnostic, clinical, epidemiological, and evolutionary implications.Escherichia coli serogroup O26 has members classified as enterohemorrhagic E. coli (EHEC) or atypical enteropathogenic E. coli (aEPEC). EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and hemolytic uremic syndrome (HUS) in Europe (16,18,19,25,48,51). EHEC O26 is also the most common non-O157 EHEC serogroup in the United States, where, between 1983 and 2002, it accounted for 22% of non-O157 EHEC clinical isolates (10). In a recent prospective study from Montana, half of EHEC O26 isolates originated from patients with bloody diarrhea (23). Moreover, EHEC O26 has spread globally (24).EHEC O26 strains produce Shiga toxin 1 (Stx1) and Stx2, either singly or together (10, 54). Indeed, phage H19B from a clinical EHEC O26 isolate that carries stx 1 was one of the first Stx-converting phages described (45). Moreover, these strains contain the intimin-encoding eae gene (6, 54), an important characteristic of EHEC (33). EHEC O26 represents a highly dynamic group of organisms that rapidly engender new pathogenic clones (54). This is exemplified by emergence of a novel EHEC O26:H11 clonal subgroup in Germany in the 1990s that possessed stx 2 as the sole stx gene, in contrast to stx 1 , exclusively identified in EHEC O26 previously. The pathogenicity of this clone was demonstrated by its strong association with HUS (29, 54) and its ability to spread rapidly (2, 54).aEPEC O26 strains do not harbor stx ...

Cumulative effect of prophage burden on Shiga toxin production in Escherichia coli

“…Theoretically, this ability has clear implications for the severity of Stx-mediated disease symptoms, commensurate with an increased number of toxin gene copies and increased toxin load, but previously this had not been directly and unequivocally demonstrated. Different stx genes, associated with different prophages or prophage remnants, are regularly found in combination within a single host (Allison et al, 2003;Bielaszewska et al, 2007;Eklund et al, 2002;Muniesa et al, 2003;Zheng et al, 2008), and virulence profiles comprising two stx2 genes are frequently associated with HUS and bloody diarrhoea (Banatvala et al, 1996;Eklund et al, 2002;Elliott et al, 2001;Tozzi et al, 2003;Woodward et al, 2002). Furthermore, the presence of two stx operons can lead to increased in vitro toxin expression (Bielaszewska et al, 2006;Cornick et al, 2002;Eklund et al, 2002;Muniesa et al, 2003), though this observation is not without exceptions (Serra-Moreno et al, 2008), nor does it necessarily equate to more severe clinical disease (Bielaszewska et al, 2006;Cornick et al, 2002).…”

Escherichia coli virulence factors

“…Strains producing VTx2a, VTx2c and/or VTx2d are more often associated with the haemolytic-uremic syndrome (HUS) in humans while VTx2e is associated with oedema disease in swine (Mainil and Daube, 2005;Gyles and Fairbrother, 2010;Johannes and Romer, 2010;Karmali et al, 2010;Pennington, 2010;Sandvig et al, 2010;Bolton, 2011). The majority of VTx are encoded by phage-located genes that can be lost or acquired by other E. coli not only in vitro, but also in vivo (Karch et al, 1992;Toth et al, 2003;Bielaszewska et al, 2007).…”

Analysis of Collection of Hemolytic Uremic Syndrome–associated EnterohemorrhagicEscherichia coli

“…In all nonmotile isolates from serogroups O26, O103, O111, O145, and O157, fl iC genes were genotyped (9,10). MLST was performed as described previously (6) with small modifi cations (11). Phylogenetic analyses were based on allelic data that used the BURST algorithm (12) to achieve a more robust interpretation of the clustering and to reduce the infl uences by the effects of the recombination, which are widespread in E. coli (6).…”