Irene Freire Martín, Christopher M Thomas, Emma Laing, Manal AbuOun, Roberto M La Ragione, Martin J Woodward

Using a sequence based approach we previously identified an IncI1 CTX-M-1 plasmid, pIFM3791, on a single pig farm in the UK that was harboured by K. pneumoniae, Escherichia coli and Salmonella enterica serotype 4,5,12,i:-. To test the hypothesis that the plasmid had spread rapidly into these differing host bacteria we wished to assess whether the plasmid conferred a fitness advantage. To do this an IncI1 curing vector was constructed and used to displace the IncI1 CTX-M-1 plasmids from K. pneumoniae strain B3791 and several other unrelated IncI1 harbouring strains indicating the potential wider application of the curing plasmid. The IncI1 CTX-M-1 plasmid was re-introduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12,i:-, S348/1. Original, cured and complemented strains were tested for metabolic competence using BiologTM technology and in competitive growth, association to mammalian cells and biofilm formation experiments. The plasmid-cured K. pneumoniae strain grew more rapidly than either the original plasmid-carrying strain or plasmid-complemented strains in competition experiments. Additionally, the plasmid-cured strain was significantly better at respiring with L-sorbose as a carbon source and putrescine, γ-amino-n-butyric acid, L-alanine, L-proline as a nitrogen sources. By contrast, no differences in phenotype were found when comparing plasmid harbouring and plasmid free S. enterica S348/11. In conclusion, the IncI1 curing vector successfully displaced multiple IncI plasmids. The IncI1 CTX-M1 plasmid conferred a growth disadvantage upon K. pneumoniae, possibly by imposing a metabolic burden the mechanism of which remains to be determined.