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

Abstract

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.

Characterisation of a mobilisable plasmid conferring florfenicol and chloramphenicol resistance in Actinobacillus pleuropneumoniae

“…Initially, florfenicol resistance was transferred to E. coli Stellar cells (Clontech) by heat shock with the MIDG3446 plasmid extract, according to the manufacturer's protocol, with selection of transformants on LB agar supplemented with 10 μg/ml florfenicol. Subsequently, mobilisation into selected Pasteurellaceae strains was assessed using a mating protocol previously described ( Bossé et al, 2009; Bossé et al, 2015 ). Recipient strains included nalidixic acid resistant derivatives of M. haemolytica (MIDG1579Nal R ), P. multocida (MIDG1570 Nal R ), and Haemophilus parasuis (MIDG3176Nal R ), as well as the NAD-independent A. pleuropneumoniae isolate, MIDG2331Δ ureC :: nadV , previously described ( Bossé et al, 2009; Bossé et al, 2014 ).…”

“…Transconjugants were selected on Brain Heart Infusion agar (with or without 0.01% NAD or 20 μg/ml nalidixic acid, as appropriate) supplemented with 2 μg/ml florfenicol. Transformants and transconjugants were tested by PCR for the presence of floR , as above, and for the nadV gene, as previously described ( Bossé et al, 2015 ), where appropriate. MICs for florfenicol and chloramphenicol were determined, as above, for recipient strains +/− plasmid.…”

Trimethoprim and other nonclassical antifolates an excellent template for searching modifications of dihydrofolate reductase enzyme inhibitors

“…Tang et al [72] found and reported in K. pneumoniae, in a single putative multireplicon plasmid pHS0091147, the coexistence of blaKPC-2 and dfrA25, which carried five resistance genes (blaKPC-2, blaCTA-M-14, blaTEM-1, sul1, and dfrA25). In turn, Bosse et al [73] reported on the presence of the mobilize plasmids dfrA14 in the Pasteurellaceae spp. In addition, widely reported PCR assays confirmed, that mobilize plasmids conferred TMP resistance in Actinobacillus pleuropneumoniae TMP-insensitive Staphylococci spp.…”

Complete Genome Sequence of MIDG2331, a Genetically Tractable Serovar 8 Clinical Isolate of Actinobacillus pleuropneumoniae

“…Serovar 8, found in many countries around the world ( 1 ), is the predominant serovar in the United Kingdom ( 2 ) and southeastern Brazil ( 3 ). A United Kingdom clinical serovar 8 isolate, MIDG2331, was shown to be highly competent for natural transformation ( 4 ) and amenable to conjugal transfer of plasmids ( 5 ). This isolate is ideally suited for facile construction of gene deletions as well as complementation using shuttle vectors such as pMC-Express and pMK-Express ( 6 ).…”