Genetic basis, transferability and co-selection of macrolide resistance among Mannheimia haemolytica and Pasteurella multocida from respiratory tract infections of cattle and swine

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Macrolides are commonly used antimicrobial agents for the therapy of respiratory tract infections in cattle and swine, in the pathogenesis of which Mannheimia haemolytica and Pasteurella multocida are significantly involved. Bacterial resistance to macrolides is mediated by various mechanisms, including enzymatic inactivation, chemical modification of the ribosomal target sites, active efflux as well as mutations in 23S rRNA or genes for ribosomal proteins. Macrolide resistance genes are often located on mobile genetic elements (MGEs), such as plasmids, transposons or integrative and conjugative elements (ICEs), which support their horizontal transfer across strain, species and genus boundaries. On these MGEs, macrolide resistance genes are commonly co-located with other antimicrobial resistance genes, which furthers their co-selection and persistence under the selective pressure also imposed by antimicrobial agents of other classes.In the national resistance monitoring programme GERM-Vet, a generally favourable resistance situation has been seen for M. haemolytica and P. multocida isolates from cattle and swine in Germany. However, macrolide-resistant isolates (M. haemolytica n=20; P. multocida n=102) have been detected at increasing frequencies during 2008 – 2020. In addition, bovine macrolide-resistant M. haemolytica and P. multocida isolates in Germany have recently been identified, in which the genetic basis of macrolide resistance was due to the resistance genes mef(C) and mph(G) as part of a novel ICE (M. haemolytica) or novel mutations in all six 23S rRNA operons (P. multocida). In addition, there are 4 macrolide-resistant M. haemolytica isolates from previous studies available, in which no known macrolide resistance mechanism could be identified.In the proposed project, we will on the one hand elucidate the genetic basis of macrolide resistance among the 122 M. haemolytica and P. multocida isolates from GERM-Vet. For this, whole genome analyses are planned which serve as a basis for all further experimental approaches, including the detection of macrolide resistance genes and macrolide resistance-mediating mutations. In addition, the analysis of MGEs potentially associated with the respective resistance genes, their structure and organization, content of additional resistance genes and their modes of transfer and host spectra are further key actions. On the other hand, we will perform in-depth analysis of the 4 M. haemolytica isolates with unknown macrolide resistance mechanisms for the genetic basis of resistance. This also includes the comparison with closely related macrolide-susceptible isolates on the basis of whole genome sequences, the analysis of outer membrane proteins as permeability barriers for macrolides, and transfer studies. Moreover, we will establish highly specific molecular identification systems for the novel macrolide resistance genes identified in this study in order to detect these genes quickly and reliably.