Evaluation of a fluorescent amplified fragment length polymorphism (FAFLP) methodology for the genotypic discrimination of Aeromonas taxa

Abstract

A fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting assay is evaluated for its ability to differentiate DNA hybridization groups in the genus Aeromonas. After empirical determination of optimal assay conditions using a limited set of strains, 98 well-characterized type and reference strains encompassing all known Aeromonas taxa were subjected to FAFLP fingerprinting using the standardized protocol. The present study clearly indicates that the use of fluorescent dye-labeled primers does not significantly affect the high capacity of this technique to differentiate among genotypically closely related Aeromonas taxa. Compared to the original AFLP protocol involving the application of radioisotopes, the new FAFLP technology offers a better performance when considering speed of analysis and user safety. On the other hand, FAFLP fingerprints exhibited a significant reduction in the relative number of bands compared to the corresponding autoradiographic patterns. In our hands, the omission of the preselective amplification step and the use of a size standard mix enhanced the cost effectiveness and the reproducibility of the technique. Cluster analysis of FAFLP band patterns generated from Aeromonas type and reference strains demonstrated once more the high correlation of AFLPgenerated data with DNA-DNA homology data. ß

Aeromonas hydrophila subsp. dhakensis subsp. nov., isolated from children with diarrhoea in Bangladesh, and extended description of Aeromonas hydrophila subsp. hydrophila (Chester 1901) Stanier 1943 (approved lists 1980).

“…Strains of type BD-2 displayed high haemolytic and cytotoxic activities and were identified by fatty acid analysis as members of Aeromonas hydrophila DNA hybridization group (HG) 1. Comparison with a genotypic database generated from amplified fragment length polymorphism (AFLP) fingerprinting patterns also allocated the BD-2 isolates to Aeromonas (Huys & Swings, 1999). Clustering was obtained following numerical analysis of digitized FAFLP band patterns using Pearson's product-moment correlation coefficient (expressed as percentages) and the unweighted paired group method using arithmetic means.…”

“…All strains were cultured aerobically on Trypticase soy agar (TSA), containing 3 % (w\v) Trypticase soy broth (BBL) and 1n5% (w\v) bacteriological agar no. 1 (Oxoid), at 28 mC for 24 h. All BD-2 isolates were subjected to FAFLP fingerprinting and the resulting FAFLP profiles were compared with the laboratory-based AEROLIB database (Huys & Swings, 1999). Microscale DNA extraction, AFLP template preparation, selective PCR amplification, electrophoresis on an automated DNA sequencer and data processing were performed as described previously (Huys & Swings, 1999).…”

“…1 (Oxoid), at 28 mC for 24 h. All BD-2 isolates were subjected to FAFLP fingerprinting and the resulting FAFLP profiles were compared with the laboratory-based AEROLIB database (Huys & Swings, 1999). Microscale DNA extraction, AFLP template preparation, selective PCR amplification, electrophoresis on an automated DNA sequencer and data processing were performed as described previously (Huys & Swings, 1999). Microscale DNA extracts were also used for repetitive sequence-based PCR fingerprinting using the ERIC1R and ERIC2 oligonucleotide primers (ERIC-PCR) (Versalovic et al, 1991).…”

“…In order to validate previously reported AFLP identification data obtained with the radioactive methodology (Ku$ hn et al, 1997) and to update the laboratory database generated from FAFLP fingerprints (Huys & Swings, 1999), a subset of BD-2 strains was analysed in a new FAFLP experiment. Comparison of the digitized FAFLP banding patterns with the AEROLIB database (Huys & Swings, 1999) revealed that the BD-2 isolates represented a homogeneous cluster that was clearly separated from all other Aeromonas taxa.…”

The Plesiomonas shigelloides wbO1 gene cluster and the role of O1-antigen LPS in pathogenicity

“…The bacterium has remained within the family Vibrionaceae until molecular studies carried out by Martinez-Murcia et al [8] indicated that P. shigelloides is phylogenetically related to the genus Proteus. Furthermore, Huys and Sings [9] in an evaluation of the amplified fragment length polymorphism technique for genotyping Aeromonas spp. found that P. shigelloides clearly falls out of the major Aeromonas cluster.…”