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

BACKGROUND

A 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.

METHODS

Genomic 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.

RESULTS

Out 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.

CONCLUSIONS

This 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.