CAREER: Degradation and Deactivation of Extracellular and Intracellular Antibiotic Resistance Genes during Disinfection Processes

CBET-1254929迈克尔·多德华盛顿大学？西雅图在过去的几十年里，抗生素耐药性特征在卫生保健环境中的细菌种群中的流行率急剧增加。同时，抗生素抗性细菌（ARB）及其相关的抗生素抗性基因（ARG）广泛分布于水生环境系统（例如，城市废水沃茨、农业废水流、甚至处理过的饮用沃茨）。在这种情况下，消毒剂的使用（即，应用于含水基质或无生命表面的化学或物理试剂）和防腐剂（即，应用于活组织的化学或物理试剂）与供水、城市废水或污染表面上的ARB的结合，可为减轻抗生素耐药性的传播提供关键手段。然而，即使ARB在消毒过程中完全失活，其含ARG DNA的完整片段也可能保留在所得细胞碎片中，并通过不需要活DNA供体细胞的水平基因转移过程（如自然转化）将抗生素抗性性状赋予时间或空间分离的细菌群体。正在进行的这一研究项目的目的是：（a）量化完整的、具有生物活性的精氨酸生成物的可能性？生存？甚至在ARG B灭活后，也能用于消毒过程，以及（B）提供必要的数据，以评估和减轻由ARG在环境和临床相关细菌群落中促进抗生素耐药性传播方面造成的风险。一套常规和分子微生物学分析工具将用于检查暴露于水处理和卫生保健环境中最常用的消毒剂和防腐剂期间各种细菌物种的游离（细胞外）和细胞相关（细胞内）染色体和质粒携带ARG的命运。从这些研究中获得的结果将使目前应用于水处理和卫生保健实践的消毒过程中细胞外和细胞内ARG失活的定量建模成为可能，这一工作将提供第一个系统的调查使用的消毒剂和防腐剂明确的降解，去激活ARG。由此产生的数据不仅有利于优化消毒过程，以最大限度地减少天然和工程水生环境和卫生保健环境之间的完整ARG转移，而且还将大大提高对各种消毒剂和防腐剂的DNA反应性的基本理解，以及细菌细胞在消毒过程中失活的机制。由于公众对抗生素耐药性的社会重要性的高度认识，这个主题也代表了一个特殊的机会，让K-12教师和学生参与加强STEM课程。因此，该项目将作为一个平台，与来自地区高中的STEM教师和代表性不足的学生团体合作，以开发教学实验室模块套件，重点是与项目范围相关的概念。这些实验室模块随后将在区域高中课程中实施，并由PI用于帮助培训新的本科生和研究生研究助理。该项目还将支持一名研究生和几名代表性不足的本科生的职业发展。

CBET-1254929Michael DoddUniversity of Washington ? SeattleThe prevalence of antibiotic resistance traits has increased dramatically amongst bacterial populations within health-care settings during the last several decades. Concurrently, it has also become clear that antibiotic resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) are widely distributed within aquatic environmental systems (e.g., municipal wastewaters, agricultural waste streams, and even treated drinking waters). Within this context, the use of disinfectants (i.e., chemical or physical agents applied to aqueous matrixes or inanimate surfaces) and antiseptics (i.e., chemical or physical agents applied to living tissues) to inactivate ARB present in water supplies, in municipal wastewater, or on contaminated surfaces can provide a critical means for mitigating dissemination of antibiotic resistance. However, even if ARB are fully inactivated during disinfection processes, intact segments of their ARG-containing DNA may remain within the resulting cell debris and confer antibiotic resistance traits to temporally- or spatially-separated bacterial populations by means of horizontal gene transfer processes not requiring live DNA donor cells, such as natural transformation. This research project is being undertaken to: (a) quantify the likelihood of intact, biologically-active ARGs ?surviving? disinfection processes even after ARB inactivation, and (b) to provide the data necessary to evaluate and mitigate risks posed by such ARGs in promoting dissemination of antibiotic resistance amongst environmental and clinically-relevant bacterial communities. A suite of conventional and molecular microbiological analytical tools will be utilized to examine the fate of free (extracellular) and cell-associated (intracellular) chromosomal and plasmid-borne ARGs from a variety of bacterial species during exposure to the disinfectant and antiseptic agents most commonly applied in water treatment and health-care settings. The results obtained from these investigations will enable quantitative modeling of extracellular and intracellular ARG deactivation during disinfection processes currently applied in water treatment and health-care practice, in turn making it possible to design disinfectant and antiseptic applications specifically for deactivation of ARGs.This work will provide the first systematic investigation into the use of disinfectant and antiseptic agents expressly for the degradation and deactivation of ARGs. The resulting data will not only facilitate optimization of disinfection processes for minimizing the transfer of intact ARGs amongst natural and engineered aquatic environments and health-care settings, but will also greatly improve fundamental understanding of DNA reactivity toward various disinfectants and antiseptics and the mechanisms by which bacterial cells are inactivated during disinfection processes. Because of high public awareness as to the societal importance of antibiotic resistance, this topic also represents an exceptional opportunity to engage K-12 teachers and students in strengthening STEM curricula. The project will therefore be utilized as a platform from which to partner with groups of STEM teachers and underrepresented students from regional high schools, in order to develop suites of instructional laboratory modules focusing on concepts relevant to the project scope. These laboratory modules will subsequently be implemented within regional high school curricula, as well as utilized by the PI to aid in the training of new undergraduate and graduate research assistants. The project will also support the career development of a graduate student and several underrepresented undergraduate students.