Cn supplement

Longitudinal samples were collected from seven adult CF patients, each chronically infected with the P. aeruginosa LES, as described previously [16 (link)]. Briefly, a sputum sample was collected from each patient at a routine visit to the Regional Adult Cystic Fibrosis Unit in Liverpool, UK, during January 2009. Sputum was treated with an equal volume of Sputasol (Oxoid), incubated at room temperature with shaking at 200 r.p.m. for 15 min, and then cultured on Pseudomonas selective agar under aerobic conditions with CN supplement (Oxoid) and 40 LES colonies were selected to maximize colony morphology diversity and identified as described previously [16–18 (link)]. Details concerning age, sex and clinical status of patients are summarized in Tables S1 and S2 (available in the online version of this article) and in a previous publication [17 (link)]. CF01, CF03, CF05 and CF07 are known to have been LES-positive since at least 1995; CF04 and CF09 since 2004; and CF08 since at least 2008.

On arrival at the laboratory, the fecal samples were inoculated into tryptic soy broth and incubated at 37°C for 24 h. Next, a loopful of previously inoculated broths was cultured on Pseudomonas agar base medium with C-N supplement (Oxoid, Hampshire, UK) and incubated at 37°C for 48 h. Subculture on nutrient agar plates was also done for further identification.