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Genomic context of antimicrobial resistance genes in bovine Pasteurella multocida isolates from Germany

Elias Dix, Henrike Krüger-Haker

In this project, genome sequences of 166 Pasteurella multocida from cattle in Germany were analyzed. The main goal was to assess the presence of antimicrobial resistance (AMR) genes (i) to determine the frequency of their co-occurrence, and (ii) to characterize the specific surrounding contexts within the bacterial genome. Of particular interest was their potential location on certain mobile genetic elements, the integrative and conjugative elements (ICEs). A local comparison of the genome sequences against the AMRFinder database was conducted using the AMRFinderPlus software to localize AMR genes. ICEs within the genomes were identified using the ICEfinder tool to compare them against the ICEberg 3.0 database. ICEfinder provides information not only about AMR genes found on the ICE, but also about genes mediating ICE mobility, provided they are included in the database. The genomic sequences of the ICEs were clustered using CD-HIT (90% identity). A Python-based pipeline incorporating EMBOSS stretcher allowed graphical comparison of ICE sequences and identification of structural patterns. Among the 166 genomes, regions in which several AMR genes were located close to each other could be determined. In some isolates, multiple regions of this nature were identified. Overall, AMR genes were found in 152/166 genomes, most of which were part of a region comprising several AMR genes. Furthermore, 111 genomes contained an ICE, that could be categorized within one of two main clusters of distinct ICE types, as well as several sub-clusters. Within these ICEs, the AMR genes appeared in a specific region, which was mostly conserved but could vary. Occasionally, ICEs lacked AMR genes but shared similar structures with AMR-carrying ICEs. In addition to the AMR regions within ICEs, some isolates contained other regions harboring AMR genes within their genome. Furthermore, the genomic data also revealed the presence of four extrachromosomal plasmids, another type of mobile genetic element, which also carried AMR genes. However, as the main focus was on ICEs, these were not investigated further yet. In conclusion, a general trend for AMR genes to be located close to each other within certain packages of associated genes, particularly as part of ICEs although not exclusively, was observed among P. multocida from cattle in Germany. As ICEs can be transferred horizontally between individual bacteria, this promotes the rapid spread of AMR within the bacterial population.