Fluorescent amplified-fragment length polymorphism analysis of an outbreak of group A streptococcal invasive disease

Abstract

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was carried out for an outbreak of group A streptococcal (GAS) invasive disease. Streptococcal genomic DNAs were digested with endonucleases EcoRI and MseI, site-specific adaptors were ligated, and PCR amplification was carried out with an EcoRI adaptor-specific primer labelled with fluorescent dye. Amplified fragments of up to 600 bp in size were separated on a polyacrylamide sequencing gel which contained internal size markers in each lane. These data were automatically scanned and analyzed, fragments were precisely sized (+/-1 bp), and electropherograms were generated for each genome with GeneScan 2.1 software. All isolates were compared in this way. Among 27 GAS isolates examined, we found 18 FAFLP profiles, compared with 12 macrorestriction profiles by pulsed-field gel electrophoresis. FAFLP readily distinguished genotypes for two clones of GAS serotype M77 which were responsible for outbreaks of invasive disease in a care-of-the-elderly system. It provided an automated analysis of the whole genome of bacterial isolates. It was reproducible, more discriminatory, and capable of higher throughput than other molecular typing methods. Given agreed conditions, FAFLP would be reproducible between laboratories for rapid characterization of outbreak strains.

FIG. 1

GeneScan 2.1 software-derived electropherograms (FAFLP profiles) for EcoRI+0 plus MseI+T amplifications of three GAS genomes. Panel 1, profile A1; the first outbreak strain (Table 1); the two sections represent FAFLP fragments from 90 to 275 bp and from 276 to 600 bp, respectively. Panel 2, profile A2; the second outbreak strain. Panel 3, profile A10; a non-outbreak-related isolate which had the same PFGE profile as that of the second outbreak strain shown in panel 2. The solid arrowheads and peaks indicate a fragment characteristic of that profile (sizes are indicated in base pairs). Open arrowheads indicate the absence of a polymorphic fragment from that profile.