Genomic typing of Escherichia coli O157 : H7 by semi-automated fluorescentAFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFL P). Total genomic DNA was digested with two restriction endonucleases (EcoR I and MseI), and compatible oligonucleotide adapters were ligated to the en ds of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction si te sequences. One of the primers from each set was labeled with a fluoresce nt dye, which enabled amplified fragments to be detected and sized automati cally on an automated DNA sequencer. Three AI;LP primer sets generated a to tal of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from t hese same isolates by pulsed-field gel electrophoresis (PFGE) resulted in o nly 21 unique DNA profiles. Also, AFLP fingerprinting was successful for on e DNA sample that was not typable by PFGE, presumably because of template d egradation. AFLP analysis, therefore, provided greater genetic resolution a nd was less sensitive to DNA quality than PFGE. Consequently, this DNA typi ng technology should be very useful for genetic subtyping of bacterial path ogens in epidemiologic studies. (C) 2000 Editions scientifiques et medicale s Elsevier SAS.