, 2010; Workentine et al., 2010). Phenotypic variants such as mucoid variants (Govan & Deretic, 1996), small colony variants (SCVs) (Häußler et al., 2003; Häußler, 2004) and quorum-sensing (QS) variants (D’Argenio

et al., 2007; Hoffman et al., 2009) are commonly CT99021 clinical trial isolated from chronic infections. It has therefore been suggested that long-term adaptation to the lung of the host results in reduced expression of acute virulence factors to develop a chronic infection type which may facilitate long-term, chronic infection (Proctor et al., 2006; Bragonzi et al., 2009; Hogardt & Heesemann, 2010). We have demonstrated that mucoid clinical strains of P. aeruginosa isolated from the sputa of chronically infected patients with CF exhibit seeding dispersal during in vitro biofilm growth, as do model laboratory strains. Intriguingly,

dispersal of clinical strains resulted in a higher frequency and colony diversity of dispersal variants than the dispersal population of the laboratory strain PAO1 (Kirov et al., 2005, 2007). Colony morphotype is only one indicator of variation, and therefore, this study characterised the biofilm dispersal population of a mucoid, chronic infection CF isolate of P. aeruginosa and the laboratory strain, PAO1, for a variety of functional traits to determine the extent of diversification that occurs during biofilm development. The traits examined included those that are likely to enhance the capacity of P. aeruginosa to establish chronic airway infection, such as the capacity to utilise different carbon sources, as this has been shown to be important FK506 mw for the growth of P. aeruginosa in artificial sputum

and is of functional significance for bacterial survival in the CF lung mucus (Sriramulu et al., 2005; Palmer et al., 2007; Starkey et al., 2009). Additionally, the dispersal population was tested for properties likely to contribute to niche colonisation and persistence in the CF airway, including attachment and biofilm formation, QS signal production and virulence factor production such as general protease and elastase production. The mutation frequencies of the parental strains were quantified under planktonic and biofilm growth to correlate mutation frequency with variant formation. Pseudomonas aeruginosa PAO1 (Holloway, 1955) and the clinical strain 18A, isolated from the sputum of a chronically infected patient Aurora Kinase with CF in Tasmania, Australia, and characterised as previously described (O’May et al., 2006), were used here. The latter strain was selected for further study as it was representative of a number of clinical isolates that showed seeding dispersal and marked heterogeneity in the morphotypes of its dispersal cells (Kirov et al., 2005, 2007). Cultures were stored at −80 °C, and all strains and biofilm-derived isolates were routinely grown on LB10 agar [Luria–Bertani agar: 10 g L−1 of tryptone, 5 g L−1 of yeast extract, 10 g L−1 of NaCl and 15 g L−1 of agar (Research Organics Inc.)] at 37 °C.