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Phenotypic Characterization of Clonal and Nonclonal Pseudomonas aeruginosa Strains Isolated from Lungs of Adults with Cystic Fibrosis

Pholawat Tingpej,^1, Lucas Smith,^1, Barbara Rose,^1* Hua Zhu,^2,3 Tim Conibear,³ Khaled Al Nassafi,¹ Jim Manos,¹ Mark Elkins,⁴ Peter Bye,^4,5 Mark Willcox,^2,3 Scott Bell,^6,7 Claire Wainwright,^8,9 and Colin Harbour¹

Department of Infectious Diseases and Immunology, University of Sydney, Sydney, Australia,¹ Institute for Eye Research, Sydney, Australia,² School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia,³ Department of Respiratory Medicine, Royal Prince Alfred Hospital, Sydney, Australia,⁴ Department of Medicine, University of Sydney, Sydney, Australia,⁵ Adult Cystic Fibrosis Centre, The Prince Charles Hospital, Brisbane, Australia,⁶ Department of Medicine, University of Queensland, Brisbane, Australia,⁷ Department of Respiratory Medicine, Royal Children's Hospital, Brisbane, Australia,⁸ Department of Paediatrics and Child Health, University of Queensland, Brisbane, Australia⁹

Received 22 November 2006/ Returned for modification 21 December 2006/ Accepted 20 March 2007

The emergence of virulent Pseudomonas aeruginosa clones is a threat to cystic fibrosis (CF) patients globally. Characterization of clonal P. aeruginosa strains is critical for an understanding of its clinical impact and developing strategies to meet this problem. Two clonal strains (AES-1 and AES-2) are circulating within CF centers in eastern Australia. In this study, phenotypic characteristics of 43 (14 AES-1, 5 AES-2, and 24 nonclonal) P. aeruginosa isolates were compared to gain insight into the properties of clonal strains. All 43 isolates produced bands of the predicted size in PCRs for vfr, rhlI, rhlR, lasA, lasB, aprA, rhlAB, and exoS genes; 42 were positive for lasI and lasR, and none had exoU. Thirty-seven (86%) isolates were positive in total protease assays; on zymography, 24 (56%) produced elastase/staphylolysin and 22 (51%) produced alkaline protease. Clonal isolates were more likely than nonclonal isolates to be positive for total proteases (P = 0.02), to show elastase and alkaline protease activity by zymography (P = 0.04 and P = 0.01, respectively), and to show elastase activity by the elastin-Congo red assay (P = 0.04). There were no other associations with genotype. Overall, increasing patient age was associated with decreasing elastase activity (P = 0.03). Thirty-two (74%) isolates had at least one N-acylhomoserine lactone (AHL) by thin-layer chromatography. rhl-associated AHL detection was associated with the production and level of total protease and elastase activity (all P < 0.01). Thirty-three (77%) isolates were positive for ExoS by Western blot analysis, 35 (81%) produced rhamnolipids, and 34 (79%) showed chitinase activity. Findings suggest that protease activity during chronic infection may contribute to the transmissibility or virulence of these clonal strains.

Published ahead of print on 28 March 2007.

P.T. and L.S. contributed equally.