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Polymorphic Amplified Typing Sequences Provide a Novel Approach to Escherichia coli O157:H7 Strain Typing

Indira T. Kudva,¹ Peter S. Evans,^2, Nicole T. Perna,³ Timothy J. Barrett,⁴ G. Joel DeCastro,^1, Frederick M. Ausubel,^5,6 Frederick R. Blattner,² and Stephen B. Calderwood^1,7*

Division of Infectious Diseases,¹ Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114,⁵ Department of Microbiology and Molecular Genetics,⁷ Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,⁶ Laboratory of Genetics,² Department of Animal Health and Biomedical Sciences, University of Wisconsin—Madison, Madison, Wisconsin 53706,³ Foodborne and Diarrheal Diseases Branch, Centers for Disease Control and Prevention, Atlanta, Georgia 30333⁴

Received 23 July 2001/ Returned for modification 17 October 2001/ Accepted 4 January 2002

Escherichia coli O157:H7 (O157) strains are commonly typed by pulsed-field gel electrophoresis (PFGE) following digestion of genomic DNA with the restriction enzyme XbaI. We have shown that O157 strains differ from each other by a series of discrete insertions or deletions, some of which contain recognition sites for XbaI, suggesting that these insertions and deletions are responsible for the differences in PFGE patterns. We have devised a new O157 strain typing protocol, polymorphic amplified typing sequences (PATS), based on this information. We designed PCR primer pairs to amplify genomic DNA flanking each of 40 individual XbaI sites in the genomes of two O157 reference strains. These primer pairs were tested with 44 O157 isolates, 2 each from 22 different outbreaks of infection. Thirty-two primer pairs amplified identical fragments from all 44 isolates, while eight primer pairs amplified regions that were polymorphic between isolates. The isolates could be differentiated solely on the basis of which of the eight polymorphic amplicons was detected. PATS correctly identified 21 of 22 outbreak pairs as being identical or highly related, whereas PFGE correctly identified 14 of the 22 outbreak pairs as being identical or highly related; PATS was also able to type isolates from three outbreaks that were untypeable by PFGE. However, PATS was less sensitive than PFGE in discriminating between outbreaks. These data suggest that typing by PATS may provide a simple procedure for strain typing of O157 and other bacteria and that further evaluation of the utility of this method for epidemiologic investigations is warranted.

Present address: Institute of Human Virology, Baltimore, MD 21201.

Present address: College of Physicians and Surgeons, Columbia University, New York, NY 10032.

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