Tetracyclines 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.
