Bioavailability of Tetracycline in Water and Soil to Bacteria for Expressing Antibiotic Resistance Response

1438105水和土壤中四环素对细菌表达耐药性的生物利用度四环素是牲畜和人类最常见的抗生素，四环素耐药细菌的扩散已成为日益严重的全球人类健康威胁。四环素广泛分布于水和土壤中，对当地微生物群落产生选择性压力。抗生素耐药性产生的先决条件是细菌对四环素的摄取，但对许多可能的水溶和吸附四环素形式的细菌的相对生物利用度知之甚少。环境四环素和抗生素耐药基因的同时增加表明，微量四环素对本地微生物群落施加了选择压力。然而，在四环素和细菌的主要联系点，如废水处理厂或动物饲养设施，没有证明两者之间的关系。拟议中的项目针对的是一个紧迫的社会问题，即抗生素功效受到的威胁。在这个项目中，生物利用度的统一概念将环境和分析化学与微生物生态学相结合。所有的学生和博士后发展多种互补的实验方法和学科观点。各级鼓励妇女和代表性不足的群体，包括勤工俭学的本科生和实习生。结果和方法将在多个研究生和本科课程中呈现，并通过pi参与国际和多州工作组、多个部门、中心和资助而更广泛地传播。这些研究所有两个协同研究项目，一个是关于抗生素通过径流从土壤转移到地表水，另一个是关于被土壤强烈隔离的二恶英的生物利用度。由于项目具有广泛的社会意义，环境教育和推广的机会对于公众和K-12，监管机构和从业者来说是有希望的，并且将由PIs与MSU Extension一起追求。本提案旨在发展四环素物种形成（在其吸附和含水物种之间）对自然和工程系统中抗生素耐药性发展的机制理解。pi假设；A)某些水四环素物种将优先被细菌利用，b)四环素在土壤吸附剂中的封存将限制其生物利用度。本研究将利用四环素诱导启动子与绿色荧光蛋白转录融合构建大肠杆菌生物报告基因。在每次实验中，项目团队将测量生物报告蛋白中四环素的细胞内浓度，并通过定量生物报告蛋白荧光来估计抗生素耐药性的表达。具体目标是：(1)鉴定在水溶液中优化生物利用度和抗生素抗性基因激活的四环素物种；(2)评估四环素吸附在整个土壤和土壤组分上的生物利用度；(3)确定吸附四环素引起的抗生素抗性的表达与土壤含水量、土壤吸附表面生物膜的形成以及细菌细胞对吸附颗粒的潜在摄取有关。PIs假设细菌四环素耐药性不是四环素总浓度的简单函数，而是取决于抗生素的详细环境物种。pi建议发现这样的物种形成联系。本项目将推进生物地球化学因素（pH、金属阳离子和天然有机配体）、四环素对各种地吸附剂的吸附、土壤水分含量和生物膜形成等作用下细菌吸收四环素的机理知识。数据将用于化学物种形成模型，以确定优先生物可利用的四环素物种，最大限度地在大肠杆菌中发展抗生素耐药性。这种对诱导抗生素耐药性的生物地球化学控制机制的改进理解应允许合理制定改进的环境废物管理方案。例如，在废物处理前进行石灰处理会增加水中的Ca2+和pH值，这两者都可能抑制四环素对细菌的生物利用度。此外，原位吸附改性剂如生物炭可能会隔离四环素，降低生物利用度，从而减少抗生素耐药性的自然选择。

1438105LiBioavailability of Tetracycline in Water and Soil to Bacteria for Expressing Antibiotic Resistance ResponseTetracyclines are the most common group of antibiotics for livestock and people, and the proliferation of tetracycline-resistant bacteria has become an ever-increasing global threat to human health. Tetracyclines are widely distributed in water and soils, where they exert selective pressure on indigenous microbial communities. A prerequisite for development of antibiotic resistance is bacterial uptake of tetracycline, but little is known about the relative bioavailabilities to bacteria of the many possible aqueous and sorbed tetracycline forms. The concurrent increases in environmental tetracyclines and antibiotic resistance genes suggest that trace amounts of tetracycline exert selective pressure on indigenous microbial communities. However, no relationships have been demonstrated at the primary nexuses of tetracyclines and bacteria, such as wastewater treatment plants or animal feeding facilities. The proposed project attacks a pressing social problem, the threats to the efficacy of antibiotics. The unifying concept of bioavailability in this project integrates environmental and analytical chemistry with microbial ecology. All students and postdocs develop multiple complementary experimental approaches and disciplinary perspectives. Women and underrepresented groups are encouraged at all levels, including work-study undergraduates and interns. Results and approaches will be presented in multiple graduate and undergraduate courses, and disseminated more widely through PIs involvement in international and multi-state working groups, multiple departments, centers, and grants. The PIs have two synergistic research projects, one on transport of antibiotics from soils to surface water by runoff, and one on bioavailability of dioxins which are strongly sequestered by soils. Due to the projects broad social relevance, opportunities for environmental education and outreach are promising for the public and K-12, regulators, and practitioners, and will be pursued by the PIs in conjunction with MSU Extension.This proposal seeks to develop a mechanistic understanding of the effect of tetracycline speciation (among its sorbed and aqueous species) on the development of antibiotic resistance in natural and engineered systems. The PIs hypothesize that; a) certain aqueous tetracycline species will be preferentially bioavailable to bacteria, and, b) tetracycline sequestration in soil geosorbents will limit its bioavailability. An E. coli bioreporter constructed by transcriptional fusions between a tetR-regulated tetracycline-inducible promoter and green fluorescent protein will be used in this study. In each experiment, the project team will measure intracellular concentrations of tetracycline in the bioreporter and also estimate the expression of antibiotic resistance by quantifying bioreporter fluorescence. Specific aims are: (1) to identify the tetracycline species that optimizes bioavailability and activation of antibiotic resistance genes in aqueous solutions, (2) to evaluate the bioavailabilities of tetracycline sorbed to whole soils and soil components, and, (3) to determine the expression of antibiotic resistance evoked by sorbed tetracycline as functions of soil water content, biofilm formation on geosorbent surfaces, and potential uptake of sorbent particles by bacterial cells. The PIs hypothesize that bacterial tetracycline resistance is not a simple function of total tetracycline concentration, but instead depends on the detailed environmental speciation of the antibiotic. The PIs propose to discover such speciation linkages. This project will advance the mechanistic knowledge of tetracycline uptake by bacteria as functions of biogeochemical factors (pH, metal cations, and natural organic ligands), tetracycline sorption to various geosorbents, soil moisture content, and biofilm formation. Data will be used in chemical speciation modeling to identify the preferentially bioavailable tetracycline species that maximize the development of antibiotic resistance in E. coli. This improved mechanistic understanding of biogeochemical controls on the induction of antibiotic resistance should allow the rational development of improved environmental waste management schemes. For example, liming before waste application would increase aqueous Ca2+ and pH, which both may suppress the bioavailability of tetracyclines to bacteria. Furthermore, in-situ sorbent amendments such as biochar may sequester tetracycline, decrease bioavailability, and thereby diminish natural selection for antibiotic resistance.