Virulence factor expression patterns in Pseudomonas aeruginosa strains from infants with cystic fibrosis.

Pseudomonas aeruginosa is the leading cause of morbidity and mortality in cystic fibrosis (CF). This study examines the role of organism-specific factors in the pathogenesis of very early P. aeruginosa infection in the CF airway. A total of 168 longitudinally collected P. aeruginosa isolates from children diagnosed with CF following newborn screening were genotyped by pulsed-field gel electrophoresis (PFGE) and phenotyped for 13 virulence factors. Ninety-two strains were identified. Associations between virulence factors and gender, exacerbation, persistence, timing of infection and infection site were assessed using multivariate regression analysis. Persistent strains showed significantly lower pyoverdine, rhamnolipid, haemolysin, total protease, and swimming and twitching motility than strains eradicated by aggressive antibiotic treatments. Initial strains had higher levels of virulence factors, and significantly higher phospholipase C, than subsequent genotypically different strains at initial isolation. Strains from males had significantly lower pyoverdine and swimming motility than females. Colony size was significantly smaller in strains isolated during exacerbation than those isolated during non-exacerbation periods. All virulence factors were higher and swimming motility significantly higher in strains from bronchoalveolar lavage (BAL) and oropharyngeal sites than BAL alone. Using unadjusted regression modelling, age at initial infection and age at isolation of a strain showed U-shaped profiles for most virulence factors. Among subsequent strains, longer time since initial infection meant lower levels of most virulence factors. This study provides new insight into virulence factors underpinning impaired airway clearance seen in CF infants, despite aggressive antibiotic therapy. This information will be important in the development of new strategies to reduce the impact of P. aeruginosa in CF.

Rapid single-nucleotide polymorphism-based identification of clonal Pseudomonas aeruginosa isolates from patients with cystic fibrosis by the use of real-time PCR and high-resolution melting curve analysis.

Pseudomonas aeruginosa genotyping relies mainly upon DNA fingerprinting methods, which can be subjective, expensive and time-consuming. The detection of at least three different clonal P. aeruginosa strains in patients attending two cystic fibrosis (CF) centres in a single Australian city prompted the design of a non-gel-based PCR method to enable clinical microbiology laboratories to readily identify these clonal strains. We designed a detection method utilizing heat-denatured P. aeruginosa isolates and a ten-single-nucleotide polymorphism (SNP) profile. Strain differences were detected by SYBR Green-based real-time PCR and high-resolution melting curve analysis (HRM10SNP assay). Overall, 106 P. aeruginosa sputum isolates collected from 74 patients with CF, as well as five reference strains, were analysed with the HRM10SNP assay, and the results were compared with those obtained by pulsed-field gel electrophoresis (PFGE). The HRM10SNP assay accurately identified all 45 isolates as members of one of the three major clonal strains characterized by PFGE in two Brisbane CF centres (Australian epidemic strain-1, Australian epidemic strain-2 and P42) from 61 other P. aeruginosa strains from Australian CF patients and two representative overseas epidemic strain isolates. The HRM10SNP method is simple, is relatively inexpensive and can be completed in <3 h. In our setting, it could be made easily available for clinical microbiology laboratories to screen for local P. aeruginosa strains and to guide infection control policies. Further studies are needed to determine whether the HRM10SNP assay can also be modified to detect additional clonal strains that are prevalent in other CF centres.