Detection and genetic analysis of the enteroaggregative Escherichia coli heat-stable enterotoxin (EAST1) gene in clinical isolates of enteropathogenic Escherichia coli (EPEC) strains

Abstract

Background: The enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) encoded by astA gene has been found in enteropathogenic E. coli (EPEC) strains. However, it is not sufficient to simply probe strains with an astA gene probe due to the existence of astA mutants (type 1 and type 2 SHEAST) and EAST1 variants (EAST1 v1-4). In this study, 222 EPEC (70 typical and 152 atypical) isolates were tested for the presence of the astA gene sequence by PCR and sequencing.

Results: The astA gene was amplified from 54 strains, 11 typical and 43 atypical. Sequence analysis of the PCR products showed that 25 strains, 7 typical and 18 atypical, had an intact astA gene. A subgroup of 7 atypical strains had a variant type of the astA gene sequence, with four non-synonymous nucleotide substitutions. The remaining 22 strains had mutated astA gene with nucleotide deletions or substitutions in the first 8 codons. The RT-PCR results showed that the astA gene was transcribed only by the strains carrying either the intact or the variant type of the astA gene sequence. Southern blot analysis indicated that astA is located in EAF plasmid in typical strains, and in plasmids of similar size in atypical strains. Strains carrying intact astA genes were more frequently found in diarrheic children than in non-diarrheic children (p < 0.05).

Conclusion: In conclusion, our data suggest that the presence of an intact astA gene may represent an additional virulence determinant in both EPEC groups.

Figure 1

Nucleotide sequences of the PCR products from tEPEC (T) and aEPEC (A) strains. The nucleotide sequences of the EAST1, SHEAST1 and SHEAST2 genes are shown for comparison. Identical nucleotides are shown as dots. Asterisks indicate the positions of nucleotide deletion.

Figure 2

Agarose gel electrophoresis of the RT-PCR products of representative strains of tEPEC (T) and aEPEC (A). EAEC 042 strain (C+) was used as positive control. Molecular size standard bands are at left.

Figure 3

Southern blot hybridization of the plasmids of tEPEC (T) and aEPEC (A) strains. (A) and (C) Hybridization results with the astA probe. (B) Hybridization results with the EAF probe. EAEC 042 and EPEC E2348/69 were used as positive controls (C+) for astA and EAF probes, respectively. The arrows in panels A and C indicate the pAA2 plasmid (65-MDa) for EAEC 042 strain and the arrow in panel B indicate the EAF plasmid (60-MDa) for EPEC E2348/69 strain. Molecular size standard bands are at left.

Figure 4

Nucleotide sequence of the EAST1 gene and its variants, including the new one described in this study. Identical nucleotides are shown as dots.