title: Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.
abstract: OBJECTIVE
  The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.
  
  
  METHODS
  Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids.
  
  
  RESULTS
  A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene.
  
  
  CONCLUSIONS
  This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial.
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK j.bosse@imperial.ac.uk.
authors.forename: Janine T
authors.initials: JT
authors.lastname: Bossé
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors.forename: Yanwen
authors.initials: Y
authors.lastname: Li
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Walker
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors.forename: Tom
authors.initials: T
authors.lastname: Atherton
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors.forename: Roberto
authors.initials: R
authors.lastname: Fernandez Crespo
authors.affiliation: Animal and Plant Health Agency (APHA) Bury St Edmunds, Rougham Hill, Bury St Edmunds, Suffolk IP33 2RX, UK.
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Williamson
authors.affiliation: Animal and Plant Health Agency (APHA) Bury St Edmunds, Rougham Hill, Bury St Edmunds, Suffolk IP33 2RX, UK.
authors.forename: Jon
authors.initials: J
authors.lastname: Rogers
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Roy R
authors.initials: RR
authors.lastname: Chaudhuri
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Lucy A
authors.initials: LA
authors.lastname: Weinert
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Olusegun
authors.initials: O
authors.lastname: Oshota
authors.affiliation: The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.
authors.forename: Matt T G
authors.initials: MT
authors.lastname: Holden
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Duncan J
authors.initials: DJ
authors.lastname: Maskell
authors.affiliation: Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK.
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Tucker
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
authors.forename: Brendan W
authors.initials: BW
authors.lastname: Wren
authors.affiliation: Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hawkshead Campus, Hatfield, Hertfordshire AL9 7TA, UK.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Rycroft
authors.affiliation: Section of Paediatrics, Department of Medicine, Imperial College London, St Mary's Campus, London W2 1PG, UK.
authors.forename: Paul R
authors.initials: PR
authors.lastname: Langford
mesh_terms: D000189:Actinobacillus Infections / Q000382:microbiology / Q000662:veterinary*; D016977:Actinobacillus pleuropneumoniae / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification; D000818:Animals; D000890:Anti-Infective Agents / Q000494:pharmacology; D004739:England; D016680:Genome, Bacterial; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010957:Plasmids; D017422:Sequence Analysis, DNA; D013444:Sulfisoxazole / Q000494:pharmacology; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014295:Trimethoprim / Q000494:pharmacology; D014296:Trimethoprim Resistance
chemical_list: D000890:Anti-Infective Agents; D013444:Sulfisoxazole; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase
keywords: animal infections; antibiotic resistance; respiratory tract