Trimethoprim resistance in a porcine Pasteurella aerogenes isolate is based on a dfrA1 gene cassette located in a partially truncated class 2 integron

Sir, Folate pathway inhibitors, such as sulphonamides and trimethoprim, are used for the control of various bacterial pathogens in veterinary medicine. In 2005, they represented the third most frequently used class of antimicrobial agents, accounting for 12.9% of all antimicrobial agents sold in veterinary medicine in Germany (http://www.p-e-g.org/econtext/germap2008). A recent comparison of the sales of veterinary antimicrobial agents between 10 European countries identified folate pathway inhibitors among the four most frequently sold classes of antimicrobial agents.1 Resistance to folate pathway inhibitors is common in a wide range of bacteria and numerous resistance genes, whose products confer resistance to either sulphonamides or trimethoprim, have been identified. In members of the genus Pasteurella, sulphonamide resistance is mainly due to the gene sul2, which has been identified—most frequently in combination with the streptomycin resistance gene strA—on plasmids or in the chromosomal DNA in various Pasteurella and Mannheimia species.2 However, the genetics of trimethoprim resistance in Pasteurellaceae of animal origin are largely unknown. So far, only a single trimethoprim resistance gene, dfrA20, has been described in a single Pasteurella multocida isolate.3 During PCR screening of the Pasteurella collection of the Institute of Farm Animal Genetics for trimethoprim resistance genes, the porcine Pasteurella aerogenes isolate 343 was identified to carry a dfrA1 gene. This isolate originated from the faecal sample of a pig suffering from diarrhoea and was identified to species level by standard biochemical procedures, as previously described.4,5 Hybridization with a dfrA1 probe identified this gene on a small plasmid. The plasmid, designated pCCK343, was transformed into the pan-susceptible P. multocida B130,3 where it mediated resistance not only to trimethoprim (MIC >256 mg/L), but also to streptomycin and spectinomycin (MICs of >256 mg/L each). In addition, a high MIC of >256 mg/L was seen for the streptothricin antibiotic nourseothricin. Plasmid pCCK343 was linearized by XbaI digestion, cloned into the vector pBluescript II SK+ and sequenced completely on both strands by primer walking, starting with the M13 universal and reverse primers. The organization of the 5415 bp plasmid pCCK343 is shown in Figure 1(a). Plasmid pCCK343 consisted mainly of two segments, which showed homology to either the tetracycline resistance plasmid pHS-Tet from Haemophilus parasuis6 or to the integrative and conjugative element AGI-5 from Escherichia coli strain BEN374 (GenBank accession no. GU725392). The initial 485 bp of pCCK343 corresponded exactly to the mobC gene region and the 5′ terminus of the mobA gene of pHS-Tet. A 57 bp sequence comprising positions 481–537 in the pCCK343 sequence was identical to an internal part (positions 30 376–30 432) of the Tn7-associated transposase gene tnsD in AGI-5 of E. coli BEN374. The 2975 bp segment immediately downstream differed by only 2 bp from the corresponding E. coli BEN374 sequence. This part comprised a truncated integrase gene intI2 as well as three complete gene cassettes for the dihydrofolate reductase gene dfrA1, the streptothricin acetyltransferase gene sat2 and the aminoglycoside adenyltransferase gene aadA1 for combined resistance to streptomycin and spectinomycin. With regard to the length of the coding sequence as well as to the size and structure of the 59-base element, these gene cassettes were indistinguishable from what has been described previously.7–9 Recombination sites 1 and 2 (Figure 1b) show the junctions of the tnsD segment in comparison with the mobA and ΔintI2 sequences. Adjacent to this E. coli BEN374-homologous part, two smaller segments of homology to pHS-Tet were found. The 125 bp segment corresponded to an internal part in the 5′ terminus of the mobA gene, whereas the 175 bp segment corresponded to the 3′ terminus of mobA, including the non-coding region downstream of the translational stop codon of mobA. Recombination sites 3 and 4 show the respective junctions between the sequences of E. coli BEN374 and mobA from pHS-Tet in relation to the pCCK343 sequence (Figure 1b). The remaining 1606 bp of pCCK343 differed by 2 bp from the corresponding pHS-Tet sequence. Recombination site 5 (Figure 1b) shows the pHS-Tet sequences downstream of mobA and downstream of tet(B) in relation to the respective pCCK343 sequence. A detailed analysis of the sequences of the junctions between the different homologous parts suggested that plasmid pCCK343 has developed from a pHS-Tet-like ancestor plasmid by the integration of a partly truncated class 2 integron as well as the deletion of large parts of the mobA gene (including the mobB gene) and the tetracycline resistance gene tet(B). (a) Comparison of the maps of the plasmids pCCK343 (FR687372) and pHS-Tet (AY862435) as well as part of the integrative and conjugative element AGI-5 from E. coli strain BEN374 (GU725392). A distance scale in kb is given below each map. The arrows indicate the extents and directions of transcription of the genes mobA, mobB and mobC (plasmid mobilization), tet(B) (tetracycline resistance), dfrA1 (trimethoprim resistance), sat2 (streptothricin resistance), aadA1 (streptomycin/spectinomycin resistance) and ΔintI2 (truncated integrase gene). Regions of >97.5% nucleotide sequence identity are marked by grey shading. The map of pHS-Tet was redrawn from the original database entry to better show the structural homology with pCCK343. Recombination sites 1–5 are displayed as filled black circles with white numbers. (b) Detailed analysis of recombination sites 1–5. Vertical bars indicate identical nucleotides in comparison with the pCCK343 sequence. The numbers of the recombination sites refer to their positions in (a). The sequence positions refer to the original database entries of pCCK343 (FR687372), pHS-Tet (AY862435) and AGI-5 from E. coli strain BEN374 (GU725392). To the best of our knowledge, this is the first description of class 2-associated resistance gene cassettes in members of the genus Pasteurella. In contrast to P. multocida, which is found in the respiratory tract of various animal species, P. aerogenes is commonly found in the intestinal tract of swine, and may be associated with diarrhoea and other infections.5 As such, it is in contact with Enterobacteriaceae, which frequently carry class 1 and class 2 integrons on plasmids.9 Since plasmids from E. coli and other Enterobacteriaceae usually cannot replicate in Pasteurella hosts, recombination of a transferred enterobacterial plasmid with plasmids indigenous to Pasteurella ensures a stable maintenance of the newly acquired resistance genes in the Pasteurella host. The in-depth analysis of plasmid pCCK343 from P. aerogenes provides further support for such inter- and intra-plasmid recombination events. Moreover, the data presented in this study substantiate the assumption of a gene flux between Enterobacteriaceae and P. aerogenes, which has previously been suggested based on the finding that the tetracycline resistance gene tet(B)—the predominant tet gene among Enterobacteriaceae—is most frequently found in P. aerogenes.10 This is a self-funded project of the Friedrich-Loeffler-Institut. None to declare. We thank Vera Nöding for excellent technical assistance.