Molecular characterization of a Shiga toxigenic Escherichia coli O113:H21 strain lacking eae responsible for a cluster of cases of hemolytic-uremic syndrome

Abstract

Shiga toxigenic Escherichia coli (STEC) strains are a diverse group of organisms capable of causing severe gastrointestinal disease in humans. Within the STEC family, certain strains appear to have greater virulence for humans. STEC strains carrying eae and belonging to serogroup O157 or O111 have been responsible for the vast majority of outbreaks of STEC disease reported to date. Here we describe a STEC O113:H21 strain lacking eae that was responsible for a cluster of three cases of hemolytic-uremic syndrome. This strain produces a single Stx2-related toxin and adheres efficiently to Henle 407 cells.

FIG. 1

Multiplex PCR analysis of fecal cultures from HUS patients and STEC isolates. Crude DNA extracts of broth cultures from the indicated samples or strains were analyzed by multiplex PCR assay 1, as previously described (19). PCR products were electrophoresed on 2% agarose gels and stained with ethidium bromide. Lanes: M, DNA size markers (pUC19 DNA digested with HpaII; fragment sizes visible are 501/489, 404, 331, 242, 190, and 147 bp); 1, negative control; 2, positive control (O111:H⁻ STEC strain 95NR1, which is positive for stx₁, stx₂, eae, and EHEC hlyA); 3, fecal culture from HUS patient 1; 4, fecal culture from HUS patient 2; 5, fecal culture from HUS patient 3; 6, STEC isolate 98NK2; 7, STEC isolate 98BN1. The expected mobilities for the various specific PCR products are also indicated.

FIG. 2

Western immunoblot detection of anti-O113 LPS. Aliquots of LPS purified from E. coli serogroups O113 (lanes 1), O111 (lanes 2), and O157 (lanes 3) were separated by SDS-PAGE, transferred to nitrocellulose filters, and reacted with convalescent-phase sera from the three HUS patients or serum from a healthy control.

FIG. 3

RFLP analysis of STEC O113 isolates. Genomic DNA purified from the indicated STEC strains was digested with EcoRI (A) or SphI (B), electrophoresed, and subjected to Southern hybridization analysis with an stx₂-specific DNA probe, as described previously (24). Lanes: 1, 98NK2; 2, 98BN1; 3, 97MW1; 4, MW10; 5, 1183; 6, 3848. Lane M contains DNA size markers of 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb.

FIG. 4

Pulsed-field gel electrophoresis of XbaI-digested genomic DNA of STEC O113. Lanes 1 to 6 are labelled as for Fig. 3. The lanes marked M contain marker DNA (SmaI-digested genomic DNA of Staphylococcus aureus NCTC8325).

FIG. 5

Deduced amino acid sequences of the A and B subunits of Stx2_O113 aligned with published sequences for Stx2 (10), Stx2c (27), and Stx2d (9). Dots denote residues identical to Stx2_O113; the dash denotes an absent residue. Arrows indicate the first residue of the mature polypeptide for each subunit.