In vivo analysis of knockout mutants of differentially expressed genes in persister strains Cisplatin cost will aid in identifying the factors leading to persistence. Materials and Methods Strains used in this study Ethics approval for this study was given by the University of Sydney Human Research Ethics Committee (Protocol Number: X07-0029, Reference Number 6999). The Australian Epidemic Strain-1 (AES-1, previously known as m16, C3789 or PI), is one of two dominant eastern Australian mainland clonal complexes. AES-1R was isolated from a child aged 14 months at the time as the deaths of five CF-infants infected with AES-1 [53]. AES-1M was isolated from the same patient at 11 years 9 months. AES-1 was not eradicated in the patient in the intervening period (D. Armstrong pers. comm.).

Written informed consent was obtained by the Royal Children’s Hospital Melbourne, and The Southern Health Service, Melbourne. Genotyping of AES-1R and AES-1M AES-1R and AES-1M were genotyped using pulse field gel electrophoresis (PFGE) [54]. Briefly, bacterial cells embedded in agarose plugs were lysed using EC lysis buffer (Sigma-Aldrich Australia), and digested using restriction enzyme SpeI to generate a small number (ca. 15�C40) of large DNA fragments. After electrophoresis of plugs containing digested DNA on 1.2% w/v agar, band pattern analyses were performed using cluster analysis software (GelComparII?, Applied Maths, Belgium) and the criteria developed by Tenover et al [55] (different genotype if more than a two band difference).

AES-1R genome sequencing and construction of the PANarray The AES-1R genome was sequenced using a 454 Genome Sequencer GS20 (Roche Diagnostics, Basel, Switzerland). This produced 598131 reads totalling 58 Mbp, providing approximately nine times coverage of the genome. De novo assembly using the Newbler assembler with default parameters, produced 1968 contigs totalling 6.28 Mbp, which were ordered and oriented to the P. aeruginosa PAO1 genome [56]. Putative genes were predicted using GeneMarkS 4.6b [57]. The whole genome shotgun sequence of the AES-1R genome (ID: 64619) is available at http://www.ncbi.nlm.nih.gov/nuccore/AFNF00000000. Drug_discovery To examine gene homology amongst the eight P. aeruginosa genomes (AES-1R, PAO1, PA7/PSPA7, PA14, PACS2, Pa_2192, Pae/PLES and c3719), all gene protein sequences were clustered into orthologous groups using OrthoMCL version 1.4 [58]. Genes were assigned an origin based on the highest identity by BLAST analysis as described on the web based annotation system for prokaryotes of the Victorian Bioinformatics Consortium at Monash University (WASABI) http://vbc.med.monash.edu.au/wasabi/.