Identification of Burkholderia species and genomovars from cystic fibrosis patients by AFLP fingerprinting

Abstract

AFLP is a genomic fingerprinting technique based on the selective amplification of restriction fragments from a total double-digest of genomic DNA. The applicability of this method to differentiate between species and genomovars of the genus Burkholderia was tested, with particular emphasis on taxa occurring in cystic fibrosis patients. In this study, 78 well-characterized strains and field isolates were investigated by two methods of AFLP fingerprinting. In the manual procedure, a radioactively labelled primer was used, amplified fragments were separated by conventional PAGE and the patterns were revealed by autoradiography. In the automated procedure, a fluorescently labelled primer was used in combination with electrophoresis and on-line data collection by means of an automated DNA sequencer. Overall, there was good agreement between the two AFLP procedures and the data were mostly consistent with results obtained from SDS-PAGE of whole-cell proteins and DNA-DNA hybridization experiments. The automated AFLP procedure has considerable technical advantages compared with the manual AFLP procedure, but a thorough visual analysis of the DNA profiles was required to avoid misidentification of some Burkholderia cepacia genomovar III strains.