Detection of Escherichia coli O157:H7 from Stool by Polymerase Chain Reaction

Kim, Eui Chong; Oh, Jun Ho; Lee, Hoan Jong

Abstract

BACKGROUND: Since a recent large outbreak of E. coli O157:H7 infection in Japan, the surveillance for this infection has been performed by the Ministry of Health and Welfare, Korea. Some of hospital laboratories have tried to isolate E. coli O157:H7 from stools on routine culture base. However, E. coli O157: H7 infections was not confirmed in Korea until this time. E. coli O157 can be detected on sorbitol-MacConkey agar (SMAC), which is used as the primary inoculation media for the stool. However, this method is not sensitive for the detection of E. coli O157 from stool. In this study, polymerase chain reaction (PCR) was performed for the detection of E. coli O157.
METHODS
Stool was inoculated in LB broth with vancomycin and incubated overnight. One milliliter of this culture broth was centrifuged and the pellet was resuspended with one milliliter of distilled water. After boiling for 15 minutes, five microliters of supernatant was used as a DNA template. The primers for multiplex PCR were stx1F, stx1R, stx2F, and stx2R as designed by Paton, et al. (J Clin Microbiol 36:598, 1998). The stool showing positive PCR was diluted with normal saline and inoculated on SMAC, and the colorless colonies were picked up. Agglutination tests of colonies, which were probably E. coli on the basis of the pink colony on MacConkey agar, were performed with O157 and H7 antisera.
RESULTS
A positive PCR was observed in a seven year old boy, who visited the emergency room of the Children's Hospital at Seoul National University on October 13, 1998 complaining of a high fever and abdominal pain. Colorless colonies were observed on SMAC at a ratio of 1:200. Nine of the 12 colorless colonies exhibited a colonial appearance of E. coli on MacConkey agar, and six were agglutinated with O157 and H7 antisera. The titers of stx1 and stx2 by VTEC-RPLA test were >1:256 and 1:128, respectively.
CONCLUSION
E. coli O157:H7 could be detected by multiplex PCR, but not by the direct inoculation of stool on SMAC because this bacteria existed in the very small numbers in the stool. Therefore, E. coli O157:H7 can be sensitively detected by the selective enrichment culture of the stool and PCR. In addition, the several colorless colonies on SMAC should be tested with O157 antisera.