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

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.

Rapid Molecular Detection of Escherichia coli O157:H7 / 감염

BACKGROUND: Sorbitol fermenting Escherichia coli O157 were reported. And E. coli O157:H7 produce various Shiga toxin (Stx) such as Stx1, Stx2, or variants of Stx2. In this study, we tried to establish laboratory methods that detect E. coli O157:H7 quickly and precisely by analyzing sensitivity of colony hybridization test and PCR technique. METHODS: Stx1-producing E. coli ATCC 43890, Stx2-producing E. coli ATCC 43889, and Stx2vha- producing E. coli ATCC 51435 were tested. Three strains of E. coli were diluted with 0.1 g of diarrheal stools from 107 CFU to 101 CFU respectively. The stool samples were incubated overnight in MacConkey agar plates. A mean of 63 colonies were hybridized by stx1- and stx2-specific oligonucleotide probes. PCR for stx1 gene and stx2 gene was done after overnight- incubation of stool samples in the LB broth with vancomycin (6 ug/mL). Positive colonies by colony hybridization were confirmed by PCR for stx1 gene and stx2 gene. RESULTS: Colony hybridization test could detect Stx1-producing E. coli at 103 CFU per 0.1 g of stool, Stx2-producing E. coli at 105 CFU per 0.1 g of stool, and Stx2vha-producing E. coli at 104 CFU per 0.1 g of stool. PCR technique after enrichment in LB broth with vancomycin (6 ug/mL) could detect stx1-, stx2-, and stx2vha-containing E. coli at 10 CFU per 0.1 g of stool respectively. CONCLUSOIN: A combination of colony hybridization and PCR after enrichment in broth with vancomycin (6 ug/mL) is useful for the rapid and precise diagnosis of infections of Shiga toxin-producing E. coli O157:H7.

COLONY HYBRIDIZATION WITH BIOTIN LABELED DNA PROBE / 微生物学通报

A simple, rapid method for colony hybridization has been developed. The DNA probes were labeled by digo xigenin. The signal of hybridization was detected by streptavidin and poly (AP) system. The results showed that this method is sensitive, specific and repoducible, it can be used for colony hybridization instead of isotopic.