Molecular epidemiology of antimicrobial resistance plasmids in bacteria from animals

这项建议是我们利用分子方法更好地了解动物细菌抗菌素耐药性(AMR)流行病学的扩展努力的一部分。提出的独特方法是将重点放在地理有限的区域(安大略省)，但同时关注比过去传统检查更广泛的细菌群(即不仅是大肠杆菌和沙门氏菌，而且是肠杆菌科细菌)。基于更有针对性的研究结果，我想测试的假设是，AMR决定因素不会从一个生态间隔平等地流动到另一个生态间隔，导致1)在细菌物种水平，2)在动物宿主物种水平，以及3)在共生/病原体水平。我的第四个假设是，后一种现象可以部分解释为AMR和毒力决定因素在可移动的遗传元件(质粒)上的联系，这些联系稳定了AMR决定因素，从而部分解释了AMR在病原体中的较高患病率。为了验证这些假设，我将集中精力研究头孢菌素和氟喹诺酮类药物的耐药性，并比较四个农场和同伴动物物种的共生体和病原体中这些临床关键抗菌素的耐药性决定因素的分布。这些决定因素在细菌物种、动物物种和共生/病原体中的分布将允许检验分隔假说，并确定AMR决定因素扩散的主要共性和瓶颈。观察到的分布将允许选择具有代表性的共生体和病原体，并对它们的基因组进行排序，以便准确地了解AMR基因之间以及AMR与毒力决定因素之间的关联/联系。最后，为了验证最后一个假设，将使用野生型和基因工程大肠杆菌菌株进行体内实验，以评估具有连锁和非连锁AMR和毒力决定因素的耐药性和病原体的持久性。这项研究的预期结果将为在细菌物种、宿主物种和水库-病原体相互作用的复杂界面上的AMR流行病学提供新的和更广泛的见解，而不是过去对动物细菌中AMR的不协调研究所提供的。拟议的工作将构成培训一名硕士、一名博士和至少五名本科生的基础。

This proposal is part of our extended efforts to understand better the epidemiology of antimicrobial resistance (AMR) in bacteria from animals using molecular methods. The unique approach proposed is to focus on a geographically limited region (Ontario), but to look simultaneously at a broader group of bacteria (i.e. not only E. coli and Salmonella, but Enterobacteriaceae in general) than has traditionally been examined in the past. Based on results from more focused studies, the hypotheses I want to test are that AMR determinants do not flow equally from one ecological compartment to another, resulting in compartmentalization 1) at the bacterial species level, 2) at the animal host species level, and 3) at the commensal/pathogen levels. My fourth hypothesis is that the latter phenomenon can be partially explained by the linkage of AMR and virulence determinants on mobile genetic elements (plasmids) and that these linkages stabilize AMR determinants, thus partially explaining the higher prevalence of AMR in pathogens. To test these hypotheses, I will concentrate efforts on resistance to cephalosporins and fluoroquinolones, and compare the distribution of resistance determinants to these clinically critically important antimicrobials in commensals and pathogens from four farm and companion animal species. The distribution of these determinants among bacterial species, animal species, and commensal/pathogens will allow to test the compartmentalization hypothesis and to identify the main commonalities and bottlenecks in the diffusion of AMR determinants. The observed distribution will allow to select representative commensals and pathogens and to sequence their genomes in order to understand precisely the associations/linkages between AMR genes and between AMR and virulence determinants in the population under study. Finally, to test the last hypothesis, in vivo experiments will be conducted to assess the persistence of resistance and pathogens with linked and non-linked AMR and virulence determinants using wild type and genetically engineered E. coli strains. The expected results of this study will provide new and broader insights into AMR epidemiology at the complex interface of bacterial species, host species, and reservoir-pathogen interactions than has been provided by the past uncoordinated studies on AMR in bacterial from animals. The proposed work will form the basis for the training of one MSc, one PhD, and at least five undergraduate students.