Poster Presentation International Pasteurellaceae Conference 2014

Ampicillin resistance in UK isolates of Actinobacillus pleuropneumoniae is mediated by a 4.7 kb plasmid carrying blaRob1 (#41)

Janine T. Bosse 1 , Yanwen Li 1 , Stephanie Walker 1 , Tom Atherton 1 , Susanna M. Williamson 2 , Jon Rogers 2 , Roy R. Chaudhuri 3 4 , Lucy A. Weinert 3 , Matt T. Holden 5 , Duncan J. Maskell 3 , Alexander W. Tucker 3 , Brendan W. Wren 6 , Andrew N. Rycroft 7 , Paul R. Langford 1 , on behalf of the BRaDP1T Consortium 1 3 6 7
  1. Department of Medicine, Imperial College London, London, UK
  2. Animal Health and Veterinary Laboratories Agency (AHVLA), Bury St Edmunds, UK
  3. Department of Veterinary Medicine, University of Cambridge, Cambridge, UK
  4. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, UK
  5. The Pathogen Sequencing Unit, The Wellcome Trust Sanger Institute, Cambridge, UK
  6. Department of Pathogen Molecular Biology, London School of Hygiene & Tropical Medicine, London, UK
  7. Department of Pathology and Pathogen Biology, The Royal Veterinary College, Hatfield, UK

As part of a screen to determine antimicrobial resistance profiles of UK isolates of Actinobacillus pleuropneumoniae (APP) collected between 1995-2011, we determined that 21% of 107 strains were resistant to ampicillin (MIC>8 mg/L). This frequency was much higher than anticipated, as AmpR strains have rarely been identified in the UK. In order to determine the genetic basis of the resistance, we analysed shotgun genome sequence data (Illumina HiSeq 2000) generated from the AmpR strains, and identified the presence of the blaRob1 gene in all cases. Plasmids were conjugated from selected AmpR APP clinical isolates into an NAD-independent recipient APP strain (MIDG2331 ureC::nadV), with counterselection on BHI containing 25 µg/ml ampicillin. Isolated plasmids (4747 bp) were fully sequenced and were found to be very similar to the pB1000 plasmid (4613 bp), previously described in Haemophilus parasuis,  Haemophilus influenzae, and Pasteurella multocida1 2 3 . BLAST searches against the genomes of the other AmpR APP isolates identified almost identical plasmids in all of them. The sequenced APP plasmid has 134 bp downstream of the blaRob1 gene that are not present in pB1000. Interestingly, in a related P. multocida plasmid, pB10022  (5685 bp; virtually identical to the APP7_A plasmid from APP serovar 7 strain AP76), there is evidence of this 134 bp sequence downstream of the blaRob1 gene, though it is disrupted by ISApl14 . All of these related Pasteurellaceae plasmids carry mobilization genes (mobCAB), which may be responsible for horizontal dissemination within and between the species.

Acknowledgements

This work was supported by a Longer and Larger (LoLa) grant from the Biotechnology and Biological Sciences Research Council (grant numbers BB/G020744/1, BB/G019177/1, BB/G019274/1 and BB/G003203/1), the UK Department for Environment, Food and Rural Affairs and Zoetis awarded to the Bacterial Respiratory Diseases of Pigs-1 Technology (BRaDP1T) consortium.

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