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

对大环内酯类抗生素耐药巴氏杆菌的研究目的multocida und Mannheimia（M.）溶血性弧菌分离株的耐药机制主要包括：（1）鉴定相关耐药机制，（2）表征存在的耐药基因或耐药介导突变，（3）分析耐药决定簇的调控和可转移性，以及研究由相应耐药决定簇介导的潜在交叉耐药。在SCHW 382/10-1项目的第一阶段，在多杀性巴氏杆菌和分枝杆菌中发现了3个新的大环内酯类药物耐药基因，分别为p53（42）、msr（E）和mph（E）。产自北美的溶血性弧菌。基因位于一个新的移动的遗传元件，整合和接合元件ICEPmu 1。该ICE的大小为82 kb，在大小约为15.7和9.8 kb的两个抗性基因区域中总共含有12个抗菌素抗性基因。ICEPmu 1以2.9 × 10-6至1 × 10-4的高频率从原始多杀性巴氏杆菌分离株转移到多杀性巴氏杆菌、M.溶血性和大肠杆菌受体菌株，其中它赋予多重抗性表型。PCR被开发用于快速和可靠地鉴定这些新的抗性基因。进一步的研究表明，这些基因也赋予了对替地罗新或加米霉素的抗性。泰地罗新和加米霉素是大环内酯类的最新抗菌药物，已于2011年获批用于治疗牛和猪的呼吸道感染。12个耐药基因共定位在一个移动的元件上，能够在属间进行高频自我转移，这是一个严重的威胁，极大地限制了食品动物呼吸道感染有效抗菌治疗的选择。不携带任何新的大环内酯类耐药基因的溶血性分离株，（2）分析巴斯德菌科中ICE在其耐药基因区域的结构变异性，并研究ICE作为耐药基因的复制者和分布者的潜力，而且（3）分析第一大环内酯-第一阶段的研究结果提供了巴氏杆菌科细菌对大环内酯类抗生素耐药性的重要基础信息。后续研究旨在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斯德菌科中鉴定出赋予多重耐药性的ICE，但后续研究将深入了解这些新型移动的元素的结构变异性，并预测无法再用抗菌剂治疗的泛耐药性巴斯德菌科的进一步发展。

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.