ObjectivesThe objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England.MethodsClinical 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.ResultsA 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.ConclusionsThis 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.
HighlightsFirst complete sequence of a floR plasmid from Actinobacillus pleuropneumoniaeExtended similarity to floR plasmids in other Pasteurellaceae speciesConjugal transfer between between species confirmed
A main goal of evolutionary biology is to understand the genetic basis of adaptive evolution. Although the genes that underlie some adaptive phenotypes are now known, the molecular pathways and regulatory mechanisms mediating the phenotypic effects of those genes often remain a black box. Unveiling this black box is necessary to fully understand the genetic basis of adaptive phenotypes, and to understand why particular genes might be used during phenotypic evolution. Here, we investigated which genes and regulatory mechanisms are mediating the phenotypic effects of the Eda haplotype, a locus responsible for the loss of lateral plates and changes in the sensory lateral line of freshwater threespine stickleback (Gasterosteus aculeatus) populations. Using a combination of RNAseq and a cross design that isolated the Eda haplotype on a fixed genomic background, we found that the Eda haplotype affects both gene expression and alternative splicing of genes related to bone development, neuronal development and immunity. These include genes in conserved pathways, like the BMP, netrin and bradykinin signalling pathways, known to play a role in these biological processes. Furthermore, we found that differentially expressed and differentially spliced genes had different levels of connectivity and expression, suggesting that these factors might influence which regulatory mechanisms are used during phenotypic evolution. Taken together, these results provide a better understanding of the mechanisms mediating the effects of an important adaptive locus in stickleback and suggest that alternative splicing could be an important regulatory mechanism mediating adaptive phenotypes.
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