Novel diagnostic PCR assay for Burkholderia cenocepacia epidemic strain ST32 and its utility in monitoring infection in cystic fibrosis patients

BackgroundA highly transmissible Burkholderia cenocepacia sequence type (ST) 32 strain caused a major outbreak at the Prague Cystic Fibrosis (CF) Centre in the late 1990s and early 2000s. Because a large number of CF patients were affected by ST32, a rapid and easy-to-use diagnostic tool for ST32 infection was urgently needed for the detection of new cases as well as for long-term surveillance. The present study sought to identify unique DNA sequences within the ST32 genome to develop an ST32 strain-specific PCR assay.MethodsGenomic subtractive hybridisation between B. cenocepacia ST32 and the closely related genome-sequenced strain B. cenocepacia ST28 identified a 325 bp long region that was absent in all but one Burkholderia strain, as demonstrated by our newly designed PCR.ResultsOut of 57 strains, only B. cenocepacia ST33 cross-reacted with ST32, resulting in a PCR specificity of 98.2%. This specificity was further tested by various genotyping methods, which revealed the practical indistinguishibility of ST32 and ST33. The PCR sensitivity, checked on a panel of 50 ST32 clinical isolates, was 100%. A closer examination of the ST32-specific sequence revealed no significant homology apart from a fragment of the ISBmu3 transposase.ConclusionsThis novel ST32-specific PCR assay allows the rapid and reliable detection of a globally distributed B. cenocepacia epidemic strain. Its routine use is especially well suited to infection surveillance programs for CF populations with a high rate of ST32 infection. This PCR method can also be used to detect ST33, a clonal variant of ST32.