The plates were incubated at 37°C overnight and the clear zone at the agar/Petri dish interface was measured as per Harunur-Rashid and Kornberg [30] followed by staining with coomassie brilliant blue G250 (0.5% (w/v) in 25% (v/v) isopropanol/10% (v/v)

acetic acid) for 30 Selumetinib price min to increase contrast. All motility assays were performed in triplicate. Detection of pilA and fliC genes was confirmed as described by Kus et al. [31] with modifications in the primers as shown in Table 1. PilA genes of isolates 1, 40 and 48 were amplified with the primer set pilB2 and tRNAThr, and for isolate 72, the primer set pilA and tRNAThr. FilC genes of isolates 1 and 72 were amplified with primers fliCFor3 and fliCRev2 [32], and for isolates 40, 41 and 48 the primer set fliCFor2 and fliCRev2. The resultant amplicons were ligated into a pT7Blue-2 cloning vector and transformed into NovaBlue Singles using a Perfectly Blunt Cloning Kit (Novagen). Plasmid DNA was extracted from broth cultures using a Rapid Plasmid Miniprep Kit (Qiagen) and the inserts sequenced. Primers SeqU19, SeqT7 and pre-pilA were used in the sequencing of all cloned KPT330 pilA genes. In addition, clones from isolates 1, 40 and 48 required use of primer pilB2 while isolate 72 required the primer pilA. Primers SeqT7 and SeqU19 were used to sequence the cloned fliC genes from all four isolates.

The sequences for isolates 1, 40, 41 and 48 have been deposited in GenBank. For the fliC gene the accession numbers are EF418192, EF418193, EF418194, and EF418195 respectively while for the pilA gene EF418188, EF418189, EF418190 and EF418191, respectively). Gfp tagging of P. aeruginosa isolates was carried out by mobilising the pBK-miniTn7-gfp3 and pUX-BF13 plasmids (Table 2) as per Koch et al. [13]. Insertion was confirmed by PCR using transrev/transfor primers (Table 1) giving a 150 bp amplicon.

Table 2 Strains and plasmids used in this study. Strain/plasmids Genotype/phenotype Source/reference E. coli E coli JM109 End1 recA1 gyrA96 this hsdR17(rk -mk +) relA1 supE44 Δlac-proAB (F’ traD36 proAB DNA ligase lacIqZΔAM15) Promega P. aeruginosa ATCC 15442   Centre for Biofilm Engineering, Montana Plasmids     pRK2013 ColE1-Tra(RK2)+Kmr Figurski & Helinski, (1979) [47] pUX-BF13 R6 K replicon -based helper plasmid providing the Tn7 transposition function in trans. Apr, mob+ Bao et al. (1991) [48] pBK-miniTn7-gfp3 pUC19 based delivery plasmid or miniTn7-gfp3. Kmr, Apr, Cmr, Smr, mob+ Koch et al. (2001) Microtitre plate assay for assessment of biofilm formation P. aeruginosa strains were grown to an attenuance (D600 nm) of 0.5 and diluted 100-fold with LB broth following which 100 μl aliquots were dispensed into triplicate microtitre plates which were incubated at 37°C.