Arbitrarily primed PCR and sequencing of 16S rDNA for epidemiological typing and species identification of Burkholderia cepacia isolates from Swedishpatients with cystic fibrosis reveal genetic heterogeneity

To investigate whether arbitrarily primed (AP)-PCR and/or 16S rDNA sequenci ng could be used as rapid methods for epidemiological typing and species id entification of clinical Burkholderia isolates from patients with cystic fi brosis (CF), a total of 39 clinical B. cypacia isolates, including 33 isola tes from 14 CF patients, were fingerprinted. ERIC-2 primer was used for AP- PCR. The AP-PCR clustering analysis resulted in 14 different clusters at a 70% similarity level. The AP-PRC patterns were individual despite considera ble similarities. To sequence rDNA, a broad-range PCR was applied. The PCR product included four variable loops (V8, V3, V4 and V9) of the 16S ribosom al small subunit RNA gene. The multiple sequence alignment produced 12 diff erent patterns, 5 of them including more than one isolate. Heterogeneity of the bases in the V3 region, indicating the simultaneous presence of at lea st two different types of 16S rRNA genes in the same cell, was revealed in 10 isolates. Most of the CF patients were adults who had advanced disease a t follow-up. Both the sequencing and the AP-PCR patterns revealed genetic h eterogeneity of isolates between patients. According to the results obtaine d, AP-PCR could advantageously be used for epidemiological typing of Burkho lderia, whereas partial species identification could effectively be obtaine d by sequencing of the V3 region of the 16S RNA gene.