Background/Objectives: Actinobacillus pleuropneumoniae is an important respiratory tract pathogen of swine worldwide. Insertion sequences (ISs) play a major role in the transfer of antimicrobial resistance (AMR) among various porcine respiratory tract pathogens. In this study, three A. pleuropneumoniae genomes were investigated for the presence of a novel IS. Methods: Analysis of the draft genomes of three A. pleuropneumoniae serovar 8 isolates (AP_1, AP_120, AP_123) suggested the presence of a novel IS. A closed whole-genome sequence was generated for strain AP_123 by hybrid assembly of Oxford Nanopore MinION long-reads and Illumina MiSeq short-reads, followed by sequence analysis using standard online tools. Transfer was tested by natural transformation. Antimicrobial susceptibility testing was conducted by broth microdilution following Clinical and Laboratory Standards Institute standards. Results: A novel IS, designated ISApl4, was detected in all three genomes. ISApl4 is 712 bp in size and has a transposase gene (tnp) of 654 bp. Moreover, it has perfect terminal 14-bp inverted repeats and produces 8-bp direct repeats at its integration site. This IS was found in 39 copies in the AP_123 genome, two of which formed the 5,765-bp pseudo-compound transposon Tn7560. This transposon carries four AMR genes: sul2 (sulfonamide resistance), strA-strB (streptomycin resistance), and tet(Y) (tetracycline resistance). RT-PCR confirmed tnp gene expression and horizontal transfer of Tn7560 into A. pleuropneumoniae MIDG2331. Conclusions: This study identified the novel ISApl4 in porcine A. pleuropneumoniae and its association with the novel pseudo-compound transposon Tn7560, which proved to be an active element capable of disseminating multidrug resistance amongst A. pleuropneumoniae.
