The sequences of these genes were identical in the parental strains of 18A and PAO1 and their dispersal isolates (data not shown). Mutations may be mediated by other genes, such as the recombinase systems encoded by xerD and sss (Martinez-Granero et al., 2005), or through the action of lytic phage, the appearance of which correlates with the appearance of variants from biofilms of P. aeruginosa (Webb et al.,

2004; Rice et al., 2009). Alternatively, variant formation may be the result of growth phase–dependent expression of DNA repair systems, as is the case for low-level expression of the methyl-directed mismatch repair genes during stationary-phase growth in E. coli (Feng et al., 1996). The mutation frequency of the biofilm population decreased for both strains 18A and PAO1 during

the period when the biofilm biomass Crenolanib manufacturer was increasing the fastest. It would therefore be of particular interest to quantify the expression of repair and recombination genes Gefitinib molecular weight at different stages of biofilm development. Similarly, sequencing of the genes encoding AHL synthetases (lasI and rhlI) and their cognate receptors (lasR and rhlR), as well as regulatory genes such as mvaT and vfr that are known to influence QS, revealed no mutations between the variants and the parent (data not shown). Therefore, changes in the expression of those genes and the subsequent production of AHL signals must be the result of mutations elsewhere in the genome. It has been shown that low protease production in clinical isolates could be complemented by overexpressing regulatory genes, and therefore, it is possible that the mutations lie in regulatory regions rather than in the genes encoding AHL synthesis or elastase production (Tingpej et al., 2007). In summary, the Sinomenine results presented here show that increased diversification occurs in P. aeruginosa when it grows as a biofilm rather than planktonically. This was shown for both a representative CF chronic infection isolate and

the laboratory strain PAO1. Longitudinal studies of CF isolates from chronically colonised individuals have suggested that infecting strains evolve to a chronic infection phenotype characterised by the loss of acute virulence determinants (Smith et al., 2006a; Rau et al., 2010). Acute infection phenotypes are, however, seen during exacerbations of disease. Here, we have shown that some clinical strain variants regain hallmarks of an acute infection isolate when grown as a biofilm in vitro but not when grown as a planktonic culture. We propose that by perpetuating this cycle and leading to diversification in traits that may enhance survival in differing niches, biofilm growth increases in vivo survival and persistence resulting in intractable infection.