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

大环内酯类是治疗牛和猪呼吸道感染的常用抗菌药物，其发病机制与溶血性曼氏菌和多杀性巴氏杆菌密切相关。细菌对大环内酯类的耐药性是由多种机制介导的，包括酶失活、核糖体靶位点的化学修饰、主动外排以及23 S rRNA或核糖体蛋白基因的突变。大环内酯类耐药基因通常位于移动的遗传元件（MGE）上，如质粒、转座子或整合和接合元件（ICE），这些元件支持它们跨菌株、种和属边界的水平转移。在这些MGE上，大环内酯类耐药基因通常与其他抗菌药物耐药基因共定位，这进一步促进了它们在其他类别的抗菌药物也施加的选择压力下的共选择和持久性。在德国从牛和猪分离的溶血性和多杀性巴氏杆菌。然而，大环内酯类耐药菌株（M.溶血性巴氏杆菌n=20;多杀性巴氏杆菌n=102）在2008 - 2020年期间检测到的频率越来越高。此外，牛大环内酯耐药M.最近在德国鉴定了溶血性巴氏杆菌和多杀性巴氏杆菌分离株，其中大环内酯抗性的遗传基础是由于作为新ICE（M.溶血性巴氏杆菌）或所有六个23 S rRNA操纵子中的新突变（多杀性巴氏杆菌）。此外，还有4株大环内酯类耐药M.在本研究中，我们将一方面阐明122株M中大环内酯类药物耐药的遗传基础，另一方面，我们还将研究122株M中大环内酯类药物耐药的遗传基础。为此，计划进行全基因组分析，作为所有进一步实验方法的基础，包括检测大环内酯类耐药基因和大环内酯类耐药介导突变。此外，进一步的关键行动是分析可能与相应抗性基因相关的MGE、其结构和组织、其他抗性基因的内容及其转移模式和宿主谱。另一方面，我们将对4 M进行深入分析。大环内酯类耐药机制未知的溶血性分离株耐药的遗传基础。这也包括比较密切相关的大环内酯类药物敏感的分离株的基础上的全基因组序列，外膜蛋白作为大环内酯类药物的渗透性屏障的分析，和转移研究。此外，我们将建立高度特异性的分子鉴定系统，为新的大环内酯类耐药基因在本研究中确定，以快速，可靠地检测这些基因。

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.