A diagnostic PCR assay for the detection of an Australian epidemic strain of Pseudomonas aeruginosa

Abstract

Background: Chronic lung infection with the bacterium Pseudomonas aeruginosa is one of the hallmarks of cystic fibrosis (CF) and is associated with worsening lung function, increased hospitalisation and reduced life expectancy. A virulent clonal strain of P. aeruginosa (Australian epidemic strain I; AES-I) has been found to be widespread in CF patients in eastern Australia.

Methods: Suppression subtractive hybridization (SSH) was employed to identify genetic sequences that are present in the AES-I strain but absent from the sequenced reference strain PAO1. We used PCR to evaluate the distribution of several of the AES-I loci amongst a collection of 188 P. aeruginosa isolates which was comprised of 35 AES-I isolates (as determined by PFGE), 78 non-AES-I CF isolates including other epidemic CF strains as well as 69 P. aeruginosa isolates from other clinical and environmental sources.

Results: We have identified a unique AES-I genetic locus that is present in all 35 AES-I isolates tested and not present in any of the other 153 P. aeruginosa strains examined. We have used this unique AES-I locus to develop a diagnostic PCR and a real-time PCR assay to detect the presence of P. aeruginosa and AES-I in patient sputum samples.

Conclusions: We have developed diagnostic PCR assays that are 100% sensitive and 100% specific for the P. aeruginosa strain AES-I. We have also shown that Whatman FTA Elute cards may be used with PCR-based assays to rapidly detect the presence of P. aeruginosa strains in CF sputum.

Figure 1

PCR detection of oprL and the AES-I HW2 locus from CF sputum. Sputum from 2 patients that had been shown by PFGE screening to contain either AES-I or a unique strain of P. aeruginosa (UCF) was swabbed onto Whatman FTA^® Elute cards. The oprL and HW2 loci were amplified by PCR from these sputum samples (UCF-FTA, AES-I-FTA) or chromosomal DNA (UCF-DNA, AES-I-DNA) purified from the P. aeruginosa strains isolated from the same sputum sample. These strains were also confirmed by PFGE to be AES-I or a unique strain of P. aeruginosa (data not shown).