Widespread pyocyanin over-production among isolates of a cystic fibrosis epidemic strain

Abstract

Background: Some isolates of the Liverpool cystic fibrosis epidemic strain of Pseudomonas aeruginosa exhibit an unusual virulence-related phenotype, characterized by over-production of quorum sensing-regulated exoproducts such as pyocyanin and LasA protease. Our aim was to determine the prevalence of this unusual phenotype amongst isolates of the epidemic strain, and to study other intraclonal phenotypic and genotypic variations.

Results: The unusual phenotype was detected in at least one epidemic strain isolate from the majority of cystic fibrosis patients tested, and can be retained for up to seven years during chronic infection. Multiple sequential isolates of the epidemic strain taken from six patients over a period of up to nine years exhibited a wide range of phenotypes, including different antimicrobial susceptibilities. Our data suggest that each sputum sample contains a mixture of phenotypes and genotypes within the epidemic strain population, including within colony morphotypes. Many isolates exhibit premature (during early rather than late exponential growth) and over-production of pyocyanin, which has a number of toxic effects directly relevant to cystic fibrosis.

Conclusion: The widespread occurrence of this unusual phenotype suggests that it may play an important role in the success of the epidemic strain.

Figure 1

Pyocyanin production in culture supernatants after overnight growth in Luria broth. Mean absorbance values are shown for isolates of P. aeruginosa falling into the following categories: (a) water isolates (n = 10); (b) keratitis isolates (n = 10); (c) non-LES CF isolates (n = 29); (d) all LES isolates (n = 48); (e) LES isolates omitting known lasR mutants (n = 38); (f) LES known lasR mutants (n = 10); (g) strain PA01.

Figure 2

Pyocyanin assays on LES isolates. Production of pyocyanin in culture supernatants during growth of LES isolates in L-broth is shown for all the LES isolates tested in this study. Each combination of colour and shape plotted represents a different LES isolate. The only non-LES isolate included is strain PA01 (blue squares, indicated by the arrow). LES isolates could be sub-divided on the basis of pyocyanin assays into various groups: (i) OP (early- and over-production of pyocyanin) [circles]. (ii) Intermediate [diamonds]. (iii) Normal or negative [squares]

Figure 3

Pyocyanin production by sequential isolates from CF patients A-C. Production of pyocyanin in culture supernatants during growth of LES isolates in L-broth is shown for isolates from three CF patients (patient A-F). Chronological order of isolation is indicated by colour from the earliest to the most recent in the following order : yellow (1995), red (1996), blue (1998), black (2000), green (2003) and orange (2004). Different isolates from the same year are indicated by different shapes.

Figure 4

Pyocyanin production by sequential isolates from CF patients D-F. Production of pyocyanin in culture supernatants during growth of LES isolates in L-broth is shown for isolates from three CF patients (patient D-F). Chronological order of isolation is indicated by colour from the earliest to the most recent in the following order : yellow (1995), red (1996), blue (1998), black (2000) and pink (2002). Different isolates from the same year are indicated by different shapes.