Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is an important zoonotic pathogen, often multi-resistant to antimicrobial agents. Among swine, LA-MRSA of clonal complex (CC) 398 dominates in Europe, Australia and the Americas, while LA-MRSA-CC9 is the main epidemic lineage in Asia. Here, we comparatively investigated the metabolic properties of rare and widespread porcine LA-MRSA isolates from Germany and China using Biolog Phenotype MicroArray technology to evaluate if metabolic variations could have played a role in the development of two different epidemic LA-MRSA clones in swine. Overall, we were able to characterize the isolates’ metabolic profiles and show their tolerance to varying environmental conditions. Sparse partial least squares discriminant analysis (sPLS-DA) supported the detection of the most informative substrates and/or conditions that revealed metabolic differences between the LA-MRSA lineages. The Chinese LA-MRSA-CC9 isolates displayed unique characteristics, such as a consistently delayed onset of cellular respiration, and increased, reduced or absent usage of several nutrients. These possibly unfavorable metabolic properties might promote the ongoing gradual replacement of the current epidemic LA-MRSA-CC9 clone in China with the emerging LA-MRSA-CC398 lineage through livestock trade and occupational exposure. Due to the enhanced pathogenicity of the LA-MRSA-CC398 clone, the public health risk posed by LA-MRSA from swine might increase further.

Background/Objectives: Pasteurella multocida commonly colonizes the bovine respiratory tract and can occasionally cause intramammary infections. Here, eight P. multocida isolates from clinical cases of bovine mastitis were investigated for their molecular characteristics as well as phenotypic and genotypic antimicrobial resistance (AMR) properties. Methods: The isolates originated from quarter milk samples obtained in Germany for diagnostic purposes. Antimicrobial susceptibility testing (AST) by broth microdilution was performed according to the Clinical and Laboratory Standards Institute. Closed whole-genome sequences were generated by hybrid assembly of Illumina MiSeq short-reads and Oxford Nanopore MinION long-reads, followed by consecutive sequence analysis. Results: The P. multocida isolates belonged either to capsular:lipopolysaccharide type A:3 (n = 7) or A:6 (n = 1), and multi-locus sequence types 1 (n = 7) or 7 (n = 1). Seven isolates carried AMR genes, such as mef(C), mph(G), strA, strB, aphA1, aadA31, tet(H), tet(Y), floR, catA3, and sul2, as part of an integrative and conjugative element (ICE). These mobile genetic elements, 58,382–78,401 bp in size, were highly similar to the ICEs Tn7406 or Tn7407 that have been previously described in bovine Mannheimia haemolytica and P. multocida, respectively. Moreover, the isolates showed elevated minimal inhibitory concentrations corresponding to the identified AMR determinants. Conclusions: Molecular typing and ICE organization suggest the bovine respiratory tract as reservoir of the investigated mastitis-associated P. multocida. Horizontal cross-genus transfer of multidrug-resistance-mediating ICEs seems to occur under in vivo conditions among different pathogens from cattle in Germany, which underlines the importance of pathogen identification followed by AST for successful bovine mastitis therapy.

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.