Comparison of PCR-based methods for typing Escherichia coli

Abstract

To establish a library typing system appropriate for studying cross-transmission of Escherichia coli. Eighteen epidemiologically unrelated isolates were genotyped by means of pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), repetitive (rep) PCR, and fluorescent amplified fragment length polymorphism (AFLP). Fingerprints were analyzed either by Pearson correlation or, in the case of AFLP, by Dice coefficients employing the novel 'uncertain band' software tool from GelCompar II. During a nine-month period, 112 isolates taken from 93 patients hospitalized in five intensive care units were analyzed by use of the two most discriminative PCR typing methods. Genotyping by RAPD and rep-PCR revealed insufficient discrimination. Among 18 epidemiologically unrelated strains with 17 different PFGE patterns, IS3 rep-PCR and AFLP distinguished 10 and 18 types, respectively. Comparison of the different methods for analysis of AFLP fingerprints showed that the Dice coefficients, which ignore 'uncertain bands', offered the best concordance with visual interpretation. Consecutive isolates originating from the same patient differed in less than three fragments. AFLP analysis showed the highest discriminative capacity for PCR typing of E. coli isolates. Analysis of fingerprints employing the Dice coefficients proved the most efficient method for an automated software-based retrieval of visually indistinguishable genotypes in an AFLP fingerprint database.