Shiga toxin, cytolethal distending toxin, and hemolysin repertoires in clinical Escherichia coli O91 isolates

Abstract

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains of serogroup O91 are the most common human pathogenic eae-negative STEC strains. To facilitate diagnosis and subtyping of these pathogens, we genotypically and phenotypically characterized 100 clinical STEC O91 isolates. Motile strains expressed flagellar antigens H8 (1 strain), H10 (2 strains), H14 (52 strains), and H21 (20 strains) or were H nontypeable (Hnt) (10 strains); 15 strains were nonmotile. All nonmotile and Hnt strains possessed the fliC gene encoding the flagellin subunit of the H14 antigen (fliC(H14)). Most STEC O91 strains possessed enterohemorrhagic E. coli hlyA and expressed an enterohemolytic phenotype. Among seven stx alleles identified, stx(2dact), encoding mucus- and elastase-activatable Stx2d, was present solely in STEC O91:H21, whereas most strains of the other serotypes possessed stx(1). Moreover, only STEC O91:H21 possessed the cdt-V cluster, encoding cytolethal distending toxin V; the toxin was regularly expressed and was lethal to human microvascular endothelial cells. Infection with STEC O91:H21 was associated with hemolytic-uremic syndrome (P = 0.0015), whereas strains of the other serotypes originated mostly in patients with nonbloody diarrhea. We conclude that STEC O91 clinical isolates belong to at least four lineages that differ by H antigens/fliC types, stx genotypes, and non-stx putative virulence factors, with accumulation of virulence determinants in the O91:H21 lineage. Isolation of STEC O91 from patients' stools on enterohemolysin agar and the rapid initial subtyping of these isolates using fliC genotyping facilitate the identification of these emerging pathogens in clinical and epidemiological studies and enable prediction of the risk of a severe clinical outcome.

FIG. 1.

HhaI fliC RFLP patterns of STEC O91 isolates of different serotypes and of Hnt and NM strains compared with E. coli H type strains expressing H8, H10, H14, and H21. Lane 1, E. coli H8 type strain H 515b (O103:H8), which displays the H8b fliC genotype (3); lane 2, STEC O91:H8 (06-03971); lane 3, E. coli H10 type strain E 77a (O35:H10); lane 4, STEC O91:H10 (05-06323); lane 5, E. coli H14 type strain F 10018-41 (O18ab:H14); lane 6, STEC O91:H14 (00-04243); lane 7, STEC O91:Hnt (01-04459); lane 8, STEC O91:NM (02-03777); lane 9, E. coli H21 type strain U 11a-44 (O8:H21); lane 10, STEC O91:H21 (00-04445-2); lane M, 100-bp ladder (Invitrogen).

FIG. 2.

Photomicrographs of HBMECs after 3 days (A) and 5 days (B) of incubation with supernatant of a CDT-V-producing STEC O91:H21 strain. (C) Control HBMECs incubated 5 days in cell culture medium. Bars, 200 μm.