Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: Comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a nov el assay based on the fluorescent analysis of an amplified subset of restri ction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA, The ligation o f adapters with primer-specific sites coupled with primers containing selec tive nucleotides allowed the full potential of PCR to be realized while mai ntaining the advantages of restriction endonuclease analysis. Fluorescence- based fragment analysis with polyacrylamide gel electrophoresis provides th e accurate band sizing required for homology assessment. The large number o f phylogenetically informative characters obtained by tbAFLP is well suited for cluster analysis and database development. The method demonstrated exc ellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant e nterococci from an outbreak in a university hospital by mAFLP. Clustering o f fbAFLP data matched epidemiological, microbiological, and pulsed-field ge l electrophoresis data. This study demonstrates the unprecedented utility o f fbAFLP for epidemiological investigation. Future developments in standard ization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.