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

大环内酯类药物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海姆病和多杀性巴氏杆菌密切相关。细菌对大环内酯类药物的耐药性是由多种机制介导的，包括酶失活、核糖体靶位的化学修饰、主动外排以及23S 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遗传元件（MGEs）上，如质粒、转座子或整合和共轭元件（ICEs），这支持它们在菌株、物种和属边界之间的水平转移。在这些MGEs上，大环内酯类耐药基因通常与其他抗微生物药物耐药基因共定位，这进一步促进了它们在其他类别抗微生物药物的选择压力下的共选择和持久性。在国家耐药性监测规划GERM-Vet中，德国从牛和猪分离的溶血性支原体和多杀性支原体的耐药性情况普遍有利。然而，在2008 - 2020年期间，大环内酯耐药分离株（溶血支原体n=20；多杀性支原体n=102）的检测频率越来越高。此外，最近在德国鉴定出牛大环内酯耐药的溶血支原体和多杀性支原体分离株，其中大环内酯耐药的遗传基础是由于耐药基因mef(C)和mph(G)作为新型ICE（溶血支原体）的一部分或所有6个23S rRNA操纵子的新突变（多杀性支原体）。此外，从以往的研究中发现了4株对大环内酯类耐药的溶血支原体，其中没有确定已知的大环内酯类耐药机制。在该项目中，我们将一方面阐明从德国菌种-兽医分离的122株溶血分枝杆菌和多杀假单胞菌对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，分析与各自抗性基因相关的MGEs、它们的结构和组织、附加抗性基因的含量、它们的转移方式和宿主谱是进一步的关键工作。另一方面，我们将对4株具有未知大环内酯耐药机制的溶血分枝杆菌分离株进行深入分析，以了解耐药的遗传基础。这还包括在全基因组序列的基础上与密切相关的大环内酯敏感分离株的比较，分析外膜蛋白作为大环内酯的渗透屏障，以及转移研究。此外，我们将为本研究鉴定的新型大环内酯类耐药基因建立高度特异性的分子鉴定系统，以便快速可靠地检测出这些基因。

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.