Identification of Enterohemorrhagic Escherichia coli by Colony Hybridization with stx-specific Oligonucleotide

Shin, Hyoung Shik

Abstract

BACKGROUND: Shiga toxin (Stx)-producing Enterohemorrhagic Escherichia coli (EHEC) infections are increasing in incidence. Sorbitol fermenting E. coli O157 has been reported and many serotypes of E. coli produce various Stx such as Stx1, Stx2, or variants of Stx2. The aim of the present study is to examine the validity of colony hybridization using stx-specific oligonucleotide to identify EHEC.
METHODS
Stx1-producing E. coli ATCC 43890, Stx2-producing E. coli ATCC 43889, and Stx2c- producing E. coli ATCC 51435 were used as reference strains. Multiplex PCR was conducted on diarrheal stools and colony hybridization with stx1- and stx2- specific oligonucleotide was tested on PCR positive and PCR negative diarrheal stools. Biochemical tests and O serotyping were performed for the colonies isolated by colony hybridization. Vero cell cytotoxicity was determined to confirm whether the isolated E. coli produced Stx.
RESULTS
Colony hybridization of 3 reference strains could detect Stx-producing E. coli at 10(3) CFU per 0.1 g of stool. Among 131 stools, 2 stx1 gene positive stools and 5 stx2 gene positive stools were detected by PCR. 124 PCR negative stools were also negative in colony hybridization. Colony hybridization detected 1 stx1-positive E. coli and 4 stx2-positive E. coli of 7 PCR positive stools. The serotypes of positive E. coli were O146, O8, O153, O26, but 1 strain was non-typable. All of the isolated E. coli fermented sorbitol, and 3 strains had EHEC-hlyA. All of the isolated E. coli showed characteristic cytotoxicity in Vero cell monolayer culture.
CONCLUSION
Colony hybridization with stx-specific oligonucleotide is a sensitive and highly specific diagnostic test to identify EHEC. However to improve the sensitivity, stool culture in a selective enrichment media before colony hybridization should be considered.