title: Curing vector for IncI1 plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3791 on Klebsiella pneumoniae.
abstract: 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 Klebsiella 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 vector. The IncI1 CTX-M-1 plasmid was reintroduced by conjugation into the cured K. pneumoniae strain and also a naturally IncI1 plasmid free S. enterica serotype 4,5,12:i:-, S348/11. Original, cured and complemented strains were tested for metabolic competence using Biolog 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 and l-proline as 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.
authors.affiliation: Animal and Plant Health Agency, New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Irene
authors.initials: I
authors.lastname: Freire Martín
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Thomas
authors.affiliation: School of Biosciences, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Laing
authors.affiliation: Animal and Plant Health Agency, New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Manal
authors.initials: M
authors.lastname: AbuOun
authors.affiliation: Animal and Plant Health Agency, New Haw, Addlestone, Surrey KT15 3NB, UK.|School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey GU2 7AL, UK.
authors.forename: Roberto M
authors.initials: RM
authors.lastname: La Ragione
authors.affiliation: Department of Food and Nutritional Sciences, University of Reading, Whiteknights Park, Reading RG6 5AP, UK.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Woodward
mesh_terms: D004734:Energy Metabolism; D005827:Genetics, Microbial / Q000379:methods*; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D053858:Metabolic Networks and Pathways; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D001618:beta-Lactamases / Q000096:biosynthesis*
chemical_list: C055184:beta-lactamase TEM-3; D001618:beta-Lactamases