For competition between 345-2RifC(RP1) and P1 or P2 agar contained ampicillin at 25 μg/ml. For competition between wild-type plasmids and see more their respective host strains it contained ampicillin for RP1 carrying strains, and tetracycline for the pUB307 and N3 carrying strains. Six replicates of each competition experiment were performed. Average per generation fitness (W) was calculated as W = 1 – b, where b is equal to t he gradient of the graph

of ln(strain x count/strain y count) per transfer, divided by the number of generations per transfer (T). T was calculated as ln(dilution factor)/ln(2). The students t-test was used to estimate the statistical significance of results. Investigation of in vitro reversion to resistance The recovery of resistance by isolates with intact but silent RP1 encoded resistance genes was investigated by spreading undiluted and serially diluted overnight nutrient broth cultures onto IsoSensitest agar containing the appropriate antibiotic (ampicillin, 25 μg/ml; kanamycin 30 μg/ml; tetracycline, 25 μg/ml). To calculate reversion frequencies, total cell counts were obtained following plating serial dilutions of the same culture onto antibiotic-free medium. Animal experiments Animal experiments were carried out using a modified method of that described previously [24]. For each experiment, six organic piglets

from two litters of Saddleback-Duroc cross, weaned at five weeks of age, were housed as a single group for two weeks, to allow the animals to acclimatize to their CAL-101 solubility dmso surroundings. They were then randomly separated into two groups of three into pens with individual HEPA filtration and fed a standard organic feed (Organic feed company, grower/finisher pellets, UK) ad libitum. All procedures complied with the Animals (Scientific Procedures) Act 1986 and were performed under Home Office License. Briefly, bacterial strains (E. Morin Hydrate coli 345-2RifC(pVE46), 345-2RifC(RP1), L5 and P1) were inoculated separately into six piglets as a single dose of 1010 cfu per animal by oral gavage. Faecal samples were collected from

each animal by digital manipulation on day 3, 5, 7, 10, 12, 14, 17, 19 and 21 post-inoculation and analysed within 24 hours. One gram of faeces was suspended in nine millilitres of saline and plated at appropriate dilutions onto six MacConkey agar plates containing 50 μg/ml rifampicin (detection limit 2 cfu/g). They were incubated overnight at 37°C and colonies obtained replica plated onto MacConkey agar containing 50 μg/ml rifampicin with ampicillin (25 μg/ml), tetracycline (25 μg/ml), sulfamethoxazole (500 μg/ml) or streptomycin (25 μg/ml) for L5, and rifampicin with ampicillin, tetracycline or kanamycin (30 μg/ml) for P1, followed by replica plating onto MacConkey agar with rifampicin only. Nucleotide sequence accession number The N3 DNA sequence has been submitted to EMBL under the accession number FR850039.