Capture and Characterization of Self-Transmissible Plasmids from Urban Wetlands E

描述(申请人提供)：城市湿地接收流域内所有活动产生的受污染的径流，并对成为各种人为污染物的蓄水池构成潜在威胁。最近的研究表明，在径流事件期间，抗药性细菌和抗药性基因会冲进城市湿地，在径流消退后积聚并持续很长时间。该项目的首要目标是了解城市湿地对临床抗生素耐药性构成的威胁。将从湿地捕获抗生素耐药性质粒，并对其进行特征分析，以确定它们传播到其他物种的可能性以及它们产生的耐药性。这将通过接合将来自本土细菌群落中的细菌的质粒转移到没有质粒的受体菌株中，使用双亲或三亲交配方法，这不需要培养本地质粒宿主。分离的质粒所产生的抗生素耐药性将使用标准纸片扩散法进行测定。将从复制子类型(通过聚合酶链式反应确定)以及使用不同的潜在受体菌株集合的实验宿主范围分析来推断该质粒传播到其他物种的可能性。将使用454焦磷酸测序技术确定对氟喹诺酮类和/或内酰胺类抗生素耐药的广泛宿主范围质粒的完整DNA序列，并对其进行彻底注释和绘制，以确定它们的遗传组织。该项目将揭示城市湿地质粒组的许多内容，包括(1)集体抗生素耐药性，(2)新的耐药性基因、等位基因和组合，以及(3)动员进入临床病原菌的威胁。

DESCRIPTION (provided by applicant): Urban wetlands receive contaminated runoff from all activities within the watershed and pose a potential threat to act as reservoirs of various anthropogenic pollutants. Recent work indicates that antibiotic-resistant bacteria and resistance genes wash into urban wetlands during runoff events where they accumulate and persist long after runoff subsides. The overarching goal of this project is to understand the threat posed by urban wetlands on antibiotic resistance in the clinic. Antibiotic resistance plasmids will be captured from the wetlands, and characterized to determine how likely they are spread to other species and what resistances they confer. This will be done by transferring plasmids from bacteria in the indigenous bacterial communities into a plasmid-free recipient strain by conjugation, using either a bi-parental or tri-parental mating approach, which does not require cultivation of the native plasmid hosts. Antibiotic resistances conferred by the isolated plasmids will be determined using standard disk diffusion methods. The potential for the plasmids to be transmitted to other species will be inferred from the replicon type (determined by PCR) as well as experimental host range assays using a diverse collection of potential recipient strains. The complete DNA sequences of broad host-range plasmids conferring resistance to fluoroquinolone and/or ¿-lactam antibiotics will be determined using 454 pyrosequencing technology and thoroughly annotated and mapped to determine their genetic organization. This project will reveal many things about the urban wetland plasmid metagenome including (1) collective antibiotic resistance, (2) novel resistance genes, alleles, and combinations, and (3) threat of mobilization into clinical pathogens.