epidermidis >100 cfu 47 22 12 mixed coagulase-negative Staphylococci 90 39 61 69 0.81 3.20 0.07 13 S. epidermidis >100 cfu 24 16 14 P. aeruginosa >100 cfu 48 19 Total 239 51 Ethics approval for this study was granted by the Royal Brisbane and Women’s Hospital Human Ethics Board (Protocol 2008/059) and Griffith University Human Ethics Board. Semi-quantitative method The removal ACs were
examined using the semi-quantitative method [12]. This method is based on rolling a segment, usually the tip, of the removed catheter back and forth on 5% sheep blood agar plates (Oxoid, Australia) after removal. The plates were incubated at 35°C under aerobic conditions for 2-4 days. Microorganisms were then isolated LXH254 chemical structure and identified according to standard hospital protocol. Semi-quantitative tip culture was considered colonised if the HM781-36B purchase plate grew ≥15 colony forming unit (cfu). If <15 cfu were grown, the catheter tip was considered to be uncolonised. Detailed molecular methods DNA extraction and PCR amplification Catheter tips were suspended in 200 μl of lysis buffer, which contained 20 mg/ml lysozyme, 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 1.2% Triton, and Proteinase K at 37°C overnight. After that, catheter
tips were taken out and bacterial DNA was extracted using the Evofosfamide mouse QIAamp DNA mini kit (Qiagen, Australia). For each catheter, a control (unused) AC was taken from the original packaging and rolled back and forth on blood many agar plates, with bacterial DNA extracted as above. Sixteen S rRNA genes were amplified from purified genomic DNA using the primers 8F and 1490R [20]. For each 25
μl reaction, conditions were as follows: 3 μl of DNA template (concentration ranged from neat to 1:103), 2.5 μl of 10 × reaction buffer containing 20 mM MgCl2, 2 μl of 25 mM dNTPs, 1 μl of each primer (10 μM), 0.1 U of Taq DNA polymerase (Qiagen, Australia), 5 μl of 5 × BSA and 10.4 μl of sterile deionised water (sdH2O). Each PCR run contained a negative control (sdH2O instead of template DNA) and a positive control (E. coli instead of template DNA). For each DNA sample, three replicate PCRs were performed. Thermocycling was as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of a 1-min denaturation, 1-min annealing at 55°C and 2-min elongation at 72°C, all followed by a final extension of 10 min at 72°C. Cloning and sequencing of 16S rDNA PCR products After purification using the Qiaquick PCR Purification kit (Qiagen, Australia), the PCR amplified 16S rRNA gene fragment were ligated into TOPO TA vector Cloning® system (Invitrogen, Ausralia) according to the manufacturer’s instructions. Two microliters of the ligation mixture was transferred to 1.5 ml sterile tube which was with competent Escherichia coli TOP10 cells provided by the manufacturer. The mixture was chilled on ice for 20 min before heat shocking for 45 seconds at 42°C.