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.

Comparative analysis of amplified fragment length polymorphism and pulsed field gel electrophoresis in a hospital outbreak and subsequent endemicity of ampicillin-resistantEnterococcus faecium

“…D'Agata et al [36] compared PFGE and AFLP and reported that PFGE is more discriminatory for vancomycin-resistant E. faecium, however only 22 isolates were included. Antonishyn et al [19] also compared PFGE and AFLP in a study that included 30 vancomycin-resistant E. faecium isolates and showed similar discriminatory power of the two techniques.…”

Identification and tracing ofEnterococcusspp. by RAPD-PCR in traditional fermented sausages and meat environment

“…Among the methods described, pulsed-field gel electrophoresis is considered to be the gold standard (Descheemaeker et al 1997). Unfortunately, this method is technically demanding and time-consuming and for that reason, other molecular typing techniques such as plasmid profiling (Farber 1996;Jurkovic et al 2007), randomly amplified polymorphic DNA (RAPD)-PCR (Cocconcelli et al 1995;Descheemaeker et al 1997), repetitive element sequence-based PCR (Svec et al 2005b) and amplified fragment length polymorphisms (Antonishyn et al 2000;Vancanneyt et al 2002) have been widely used to characterize clinical and food isolates of enterococci.…”

Genetic diversity and safety aspects of enterococci from slightly fermented sausages

“…Genetic typing techniques, such as plasmid profiling (Farber et al 1996), pulsed-field gel electrophoresis of DNA macro-restriction patterns (Descheemaeker et al 1997;Vancanneyt et al 2002), randomly amplified polymorphic DNA (RAPD)-PCR (Cocconcelli et al 1995;Descheemaeker et al 1997) and amplified fragment length polymorphisms (Antonishyn et al 2000;Vancanneyt et al 2002) have been widely used to characterize clinical and dairy isolates of enterococci.…”