Molecular basis of macrolide resistance among Pasteurella multocida and Mannheimia haemolytica isolates

研究耐大环内酯巴氏杆菌（P.）和溶血曼海氏菌（M.）分离株的目的是：(1)鉴定所涉及的耐药机制；(2)鉴定存在的耐药基因或耐药介导突变；(3)分析耐药决定因素的调控和可转移性，并调查各自耐药决定因素介导的潜在交叉耐药。在第一期SCHW382/10-1项目中，从北美多杀假单胞菌和溶血假单胞菌分离株中鉴定出3个新的大环内酯类耐药基因erm（42）、msr(E)和mph(E)。这些基因被定位在一个新的可移动遗传元件——整合共轭元件ICEPmu1上。该ICE的大小为82 kb，在两个大小分别为15.7和9.8 kb的耐药基因区中共含有12个耐药基因。ICEPmu1以2.9 × 10-6至1 × 10-4的高频率从原始的多杀假单胞菌分离物转移到多杀假单胞菌、溶血假单胞菌和大肠杆菌受体菌株中，在那里它赋予了多重抗性表型。为了快速、可靠地鉴定这些新的抗性基因，开发了pcr。进一步的研究表明，这些基因也赋予了对替地匹罗辛或加红霉素的抗性。替地匹罗辛和加红霉素是2011年批准用于治疗牛和猪呼吸道感染的大环内酯类的最新抗菌剂。12个耐药基因的共定位在一个能够跨属边界高频自转移的移动元件上，这是一个严重的威胁，并极大地限制了食用动物呼吸道感染的有效抗菌治疗的选择。后续研究的目的是：(1)确定不含任何新的大环内酯类耐药基因的溶血分枝杆菌分离株的大环内酯类耐药机制，(2)分析巴氏杆菌中ice耐药基因区域的结构变异性，并研究ice作为耐药基因积累和分布者的潜力，以及(3)分析来自德国的第一个对大环内酯类耐药的巴氏杆菌分离株。第一期研究结果为巴氏杆菌对大环内酯类抗生素的耐药性研究提供了重要的基础资料。后续研究的目的是鉴定其他迄今未知的耐药基因和/或机制。虽然在第一项研究中首次在巴氏杆菌中发现了多重耐药ice，但后续研究将深入了解这些新型移动元件的结构变动性，并允许预测进一步发展到无法再使用抗菌药物治疗的泛耐药巴氏杆菌。

Aims of the study on macrolide-resistant Pasteurella (P.) multocida und Mannheimia (M.) haemolytica isolates were (1) the identification of the resistance mechanisms involved, (2) the characterization of the resistance genes or resistance-mediating mutations present, and (3) the analysis of the regulation and transferability of the resistance determinants, as well as the investigation of potential cross-resistances mediated by the respective resistance determinants. In the first period of the project SCHW382/10-1, three novel macrolide resistance genes, erm(42), msr(E) and mph(E), could be identified in P. multocida und M. haemolytica isolates from North America. The genes were located on a novel mobile genetic element, the integrative and conjugative element ICEPmu1. This ICE had a size of 82 kb and harbored in total twelve antimicrobial resistance genes in two resistance gene regions of approximately 15.7 and 9.8 kb in size. ICEPmu1 transferred with a high frequency of 2.9 x 10-6 to 1 x 10-4 from the original P. multocida isolate into P. multocida, M. haemolytica and Escherichia coli recipient strains where it conferred a multiresistance phenotype. PCRs were developed for a fast and reliable identification of these the novel resistance genes. Further studies showed that the genes also conferred resistance to tildipirosin or gamithromycin. Tildipirosin and gamithromycin represent the latest antimicrobial agents of the macrolide class which have been approved in 2011 for the treatment of respiratory tract infections in cattle and pigs. The co-localization of twelve resistance genes on a mobile element capable of high frequency self-transfer across genus boundaries represents a serious threat and drastically limits the options of an efficient antimicrobial therapy of respiratory tract infections in food-producing animals.The aims of the follow-up study are (1) to identify the macrolide resistance mechanism(s) in M. haemolytica isolates that do not harbor any of the new macrolide resistance genes, (2) to analyze ICEs in Pasteurellaceae for structural variabilities in their resistance gene regions and to investigate the potential of ICEs as accumulators and distributers of resistance genes, but also (3) to analyze the first macrolide-resistant Pasteurellaceae isolates from Germany.The results of the first study period provided important basic information on resistance to macrolide antibiotics in Pasteurellaceae. The follow-up study aims at the identification of other so far unknown resistance genes and/or mechanisms. While multiresistance-conferring ICEs were identified for the first time in Pasteurellaceae during the first study, the follow-up study will provide in-depth insight into the structural variability of these novel mobile elements as well as allow for a prediction of further developments towards pan-resistant Pasteurellaceae which cannot be treated anymore with antimicrobial agents.