Molecular analysis of H antigens reveals that human diarrheagenic Escherichia coli O26 strains that carry the eae gene belong to the H11 clonal complex

Abstract

Fifty-seven Escherichia coli O26 strains isolated from patients in six countries were investigated by PCR restriction fragment length polymorphism (RFLP) analysis of the flagellin-encoding (fliC) gene (fliC RFLP analysis). The strains were determined by serotyping to belong to five different H types or were nonmotile. The fliC RFLP analysis revealed only two different patterns among the 57 strains. One fliC RFLP pattern was displayed by 54 strains and was identical to that of E. coli H11 reference strain Su4321-41. The other fliC RFLP pattern was observed for three strains and was identical to that for E. coli H32 reference strain K10. The 54 strains with the H11 fliC RFLP pattern included 22 strains of serotype O26:H11, 23 nonmotile strains, and 9 strains that were initially serotyped as H2, H8, H21, and H32 but that were confirmed to express H11 by repeat serotyping. All 54 strains with the H11 fliC RFLP pattern contained the attaching-and-effacing (eae) gene. The three strains with the H32 fliC RFLP pattern belonged to serotype O26:H32, and all were eae negative. The fliC genes of 14 selected E. coli O26:H11 strains isolated between 1964 and 1999 had identical nucleotide sequences. Our results demonstrate that E. coli O26 strains that carry the eae gene belong exclusively to the H11 clonal complex. Since there were no H11 fliC allelic variations among the O26 strains tested, E. coli O26:H11 may have emerged recently. The fliC PCR-RFLP test is a reliable, easy-to-perform, and rapid method for determination of the H types of E. coli O26 isolates.

FIG. 1

fliC PCR products and fliC RFLP patterns of E. coli H reference strains. Lanes M, molecular mass markers (1-kb DNA ladder; Gibco, BRL). In lanes 1 to 5 and 6 to 10, the fliC PCR products and fliC RFLP patterns, respectively, of the following strains (serotypes in parentheses) are shown: Bi7455-41 (O43:K⁻:H2) (lanes 1 and 6), Ap32oc (O2:K⁻:H8) lanes 2 and 7), Su4321-41 (O13:K11:H11) (lanes 3 and 8), U11a-44 (O8:K49:H21) (lanes 4 and 9), and K10 (O114:K⁻:H32) (lanes 5 and 10).

FIG. 2

fliC RFLP patterns of E. coli O26 isolates with different H antigens determined by serotyping or determined to be nonmotile (H⁻). Lanes M, molecular mass marker (1-kb DNA ladder; Gibco, BRL). Lanes 1 to 16, E. coli O26 strains (H antigens in parentheses) 1616/96 (H11) (lane 1), 8590/64 (H11) (lane 2), 6422/94 (H⁻) (lane 3), 4393/66 (H⁻) (lane 4), TB 285A (H2) (lane 5), 3820/99 (H8) (lane 6), 4118/99 (H8) (lane 7), C1137-76 (H21) (lane 8), C1138-76 (H21) (lane 9), C1218-96 (H21) (lane 10), C1540-99 (H21) (lane 11), C1215-99 (H21) (lane 12), 15/77 (H32) (lane 13), 98/78 (H32) (lane 14), 1897/88 (H32) (lane 15), and 1976/88 (H32) (lane 16). On the basis of the data derived from nucleotide sequence analysis of the fliC gene in E. coli O26:H11 strain 6061/96 (accession number AJ243796), bands of 280 and 150 bp in the H11 fliC RFLP pattern (lanes 1 to 13) each consist of two fragments of similar sizes (269 and 276 bp and 141 and 142 bp, respectively) that could not be separated on the gel. Three additional fragments of 41, 31, and 14 bp could not be detected on the gel.