aeruginosa as can be found after recent infection, in the sputum or nasopharyngeal samples of CF patients not yet colonized by P. aeruginosa. Methods Culture and identification of bacteria All 8 sputum samples used for this study were collected from cystic fibrosis patients and were cultured on McConkey Agar (MCA) (Becton Dickinson, Cockeysville, MD) and Cetrimide Agar (Cetrimide Broth (Fluka Biochemika, Buchs, Switzerland) + 4% Bacto Agar (Becton Dickinson))(CA) to check for the presence of Pseudomonas aeruginosa. The two sputum samples from the chronically infected CF patients
yielded only P. aeruginosa, as identified by tDNA-PCR and confirmed by OprL PCR [13, 34–37], whereas the six sputum samples from the not chronically infected CF patients were culture and PCR negative for P. aeruginosa, as tested EGFR inhibitor in the routine laboratory and confirmed by our laboratory. Dilution series of P. aeruginosa positive sputum in P. aeruginosa negative sputum All 8 sputa were liquefied by adding v/v Sputasol (Oxoid Ltd, Poole, UK) and incubated during 1 hour at 37°C. The two liquefied sputa
from the CF patients positive for P. aeruginosa were pooled and subsequently diluted tenfold (for dilutions nr 1 and 2) and fivefold (for dilutions nr 3-9) in a pool of liquefied sputa from the six CF patients negative for P. aeruginosa. Written informed consent was obtained from the patients for publication of this report. Copies Selleckchem GSK2126458 of the written consent are available for review by the Editor-in-Chief
of this journal. Culture techniques Fifty μl of each dilution was inoculated onto plates (MCA or CA) or into cetrimide broth and incubated for 24 h at 37°C at ambient atmosphere. Cetrimide Broth was subcultured Olopatadine by inoculating 50 μl onto a Blood Agar plate (Becton Dickinson), which was incubated for 24 h at 37°C (CB). All dilution OSI906 cultures were done in triplicate and P. aeruginosa colonies were counted. DNA-extraction protocols A total of five different DNA-extraction protocols were carried out on each sputum dilution. Two protocols, i.e. Generic 2.0.1. and Specific B, whereby in the latter a double concentration of silica is used and additional washing steps are included, aiming at DNA-extraction from more difficult samples, using the bioMérieux easyMAG Nuclisense extractor (bioMérieux, Marcy-l’Etoile, France), with and without prior proteinase K treatment, were compared with each other and with the manual High Pure PCR Template Preparation Kit (Roche Applied Science, Basel, Switzerland), carried out according to the manufacturer’s recommendations. Proteinase K pretreatment consisted of incubation of 200 μl of each sputum dilution during 1 h at 55°C in 200 μl proteinase K buffer (1 mg/ml proteinase K, 0.5% SDS, 20 mM Tris-HCl, pH 8.3) with vortexing every 15 min. For each extraction the start volume was 200 μl of liquefied sputum and the elution volume was 50 μl. Extracted DNA was stored at -20°C prior to PCR.