Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae

Abstract

Objectives: 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.

Keywords: animal infections; antibiotic resistance; respiratory tract.

Figure 1.

Isolation and characterization of newly identified dfrA14-containing A. pleuropneumoniae plasmids. (a) Comparison of plasmid extracts from MIDG2331ΔureC::nadV (Lane 1), conjugal donor strains (Lane 2 = MIDG3224 and Lane 4 = MIDG3389) and respective trimethoprim-resistant transconjugants, showing the transfer of plasmids (Lane 3 = pM3224T and Lane 5 = pM3389T) into MIDG2331ΔureC::nadV. (b) PCR amplification of dfrA14 (343 bp amplicon; Lane 1 in each section), sul2 (220 bp amplicon; Lane 2 in each section) and nadV (1.5 kb amplicon; Lane 3 in each section) from MIDG2331ΔureC::nadV, MIDG3224, MIDG2331ΔureC::nadV+pM3224T, MIDG3389 and MIDG2331ΔureC::nadV+pM3389T, as indicated for each section of the gel. (c) Schematic comparison of pM3224T with the most closely related Pasteurellaceae plasmid, pB1003, and pCERC1, a dfrA14-containing plasmid found in Enterobacteriaceae. (d) Schematic comparison of pM3389T with the most closely related Pasteurellaceae plasmid, pIG1, and pCERC1. Reading frames are indicated by arrows, with arrowheads showing the direction of transcription; only relevant genes have been annotated (sul2: sulphonamide resistance; strA, strB: streptomycin resistance; dfrA14: trimethoprim resistance; mobA, mobB, mobC: plasmid mobilization; strB′: partial strB; strA′: partial strA). Dark grey blocks between sequences indicate ≥99% nucleotide sequence identity.