Escherichia coli O8:H8 Carrying a Novel Variant of the Heat-Labile Enterotoxin LT2 Gene Caused Outbreaks of Diarrhea

Abstract

No outbreaks caused by Escherichia coli-producing heat-labile enterotoxin LT2 have been reported to date. Here, we revealed that the E. coli O8:H8 strains isolated from patients in 2 independent diarrhea outbreaks were negative for any known virulence determinants in routine microbiological tests, were very closely related, and carried a prophage-encoded gene for a novel LT2 variant (LT2d) and the genes for colonization factor antigen III. We also showed that LT2d has a cytotonic activity similar to LT1. These data indicate the importance of E. coli strains producing LT2d as a human pathogen.

Keywords: colonization factor antigen; diarrhea outbreak; heat-labile enterotoxin; phage; scherichia coli.

Figure 1.

The phylogenetic analyses, genetic structure of the LT2d-encoding phage, and CHO cell elongation assay. A, A core gene–based maximum likelihood (ML) tree of Escherichia coli O8:H8 strain 16F5M1D1 and an E. coli reference strain set. A cryptic Escherichia clade I strain TW15838 was included as an outgroup. The tree was constructed based on 225 254 SNP sites located on 2569 core genes. Phylogroups, pathotypes, and serotypes are indicated. B, Neighbor-joining (NJ) trees based on the nucleotide sequences of the A and B subunit genes of elt2d and other known elt1 and elt2 variants. The cholera toxin genes (ctx) were also included in this analysis. Accession numbers of each gene are indicated in parentheses. C, The genetic structure of the LT2d-encoding phage is shown. In panel, the genome sequence of the LT2d phage was compared with that of 2 Stx2 phages, to which the LT2d phage genome showed the highest similarity in the early and late regions, respectively. Sequence identities are indicated by different colors. D, The results of the CHO cell elongation assay. CHO cells (2×10⁵ cells/well/500 µL) in a 24-well plate were treated with 100-fold diluted bacterial cell lysates for 48 hours at 37°C and visualized under a light microscope (×100). Cell lysates were prepared by sonicating bacterial cultures incubated with the presence or absence of 500 ng/mL of mitomycin C or 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 5 hours at 37°C. Enterotoxigenic E. coli O6:HNT (LT1-positive) and E. coli K-12 MG1655 were used as positive and negative controls, respectively. Untreated: CHO cells untreated with bacterial lysate.