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

Abstract

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

FIG. 1

Phylogram from distance-based phylogenetic analysis of fbAFLP data. The bootstrap values from 100 replicates are shown at each respective node. Bootstrap values of >75% are displayed and represent the amount of support contained in the fbAFLP data matrix for the clustering arrangements depicted in the tree. Branch lengths graphically depict the number of band differences between isolates. The scale correlates branch length to percent genetic difference. The fbAFLP ID assignment is annotated to the right of the phylogram.

FIG. 2

Screen capture of electropherogram analysis using Genescan software. Panel A displays the 233- to 243-bp portion of the profiles from isolate 5 (fbAFLP ID 3-1). Panel B contains the same size range for isolate 22 (fbAFLP ID 1-1). Each panel contains all three fbAFLP fingerprints from the same isolates superimposed. The FAM-labeled primer set is indicated with the blue electropherogram. The HEX-labeled primer set is indicated in green. The fbAFLP band sizing data for the peak indicated with the arrow is circled in red.

FIG. 3

Graph of random tree length distribution analysis on fbAFLP data.