title: Polymorphic amplified typing sequences provide a novel approach to Escherichia coli O157:H7 strain typing.
abstract: 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.
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.
authors.forename: Indira T
authors.initials: IT
authors.lastname: Kudva
authors.forename: Peter S
authors.initials: PS
authors.lastname: Evans
authors.forename: Nicole T
authors.initials: NT
authors.lastname: Perna
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Barrett
authors.forename: G Joel
authors.initials: GJ
authors.lastname: DeCastro
authors.forename: Frederick M
authors.initials: FM
authors.lastname: Ausubel
authors.forename: Frederick R
authors.initials: FR
authors.lastname: Blattner
authors.forename: Stephen B
authors.initials: SB
authors.lastname: Calderwood
mesh_terms: D015373:Bacterial Typing Techniques; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016521:Electrophoresis, Gel, Pulsed-Field; D004927:Escherichia coli Infections / Q000382:microbiology; D019453:Escherichia coli O157 / Q000145:classification* / Q000235:genetics* / Q000472:pathogenicity; D006801:Humans; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D014774:Virulence / Q000235:genetics
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; C025580:endodeoxyribonuclease XBAI; D015252:Deoxyribonucleases, Type II Site-Specific