these observations, further work should now concentrate on understanding the molecular mechanisms responsible so that the underlying process are understood and used to help develop better treatment and prevention and Gamma-secretase inhibitor control strategies. Methods Bacterial strains and plasmids E. coli 345-2RifC, E. coli 345-8 and 343-9 are all commensal isolates of porcine origin. E. coli 345-2RifC is marked with a no-cost Blasticidin S ic50 rifampicin-resistance mutation in RpoB (H526Y). Strains 99-24 and 99-40 are human urinary isolates, whilst E. coli K12 JM109 is a laboratory strain. Study strains were chosen on the basis that they did not carry acquired antibiotic resistance genes and that they exhibited good growth characteristics in laboratory media, with doubling ranging between 21 and 27 minutes in nutrient broth. Their phylogenetic group was determined as described previously . The relatedness of the isolates was investigated by randomly amplified polymorphic DNA (RAPD) PCR . The broad-host range plasmids
RP1, pUB307, Proteases inhibitor R46, pVE46 and N3 were introduced into host strains by conjugation using the agar mating method . The 345-2RifC(pVE46) strain used was a variant passaged in the laboratory, the same from which silent isolates arose . Derivatives of 345-2RifC(pVE46) and 345-2RifC(RP1), carrying silent antibiotic resistance genes were as described previously . The characteristics of strains and plasmids used in this study are listed in Table 3. DNA sequencing and analysis DNA of IncN plasmid N3 was prepared
by alkaline SDS maxiprep and CsCl/EtBr density gradient centrifugation . The E. coli N3 plasmid was sequenced to approximately learn more 37-fold shotgun sequence, totalling 1711 end sequences, from pUC19 (with insert sizes of 2-4 kb; 4-6 kb) genomic shotgun libraries that were sequenced using big-dye terminator chemistry on ABI3730 automated sequencers. The assembly was generated using phrap2gap. All repeat regions and gaps were bridged by read-pairs or end-sequenced polymerase chain reaction (PCR) products again sequenced with big dye terminator chemistry on ABI3730 capillary sequencers. The sequence was manipulated to the ‘Finished’ standard . Competition experiments to assay in vitro fitness To assess the fitness impact of the plasmids upon E. coli host strains growth competition between plasmid-carrying and plasmid-free isogenic strain pairs was carried out as described previously in Davis minimal medium with 25 mg/ml glucose (DM25) . To estimate bacterial counts, competition cultures were diluted as appropriate and spread in triplicate onto IsoSensitest agar (Oxoid) and onto IsoSensitest agar containing the relevant antibiotic.