Employing Metagenomic and Chromatin Capture Techniques to Map the Evolution of Antibiotic Resistance in Coastal Microbiomes

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Project Summary The spread of microbial antibiotic resistance (AR) is a global healthcare problem driven by the extensive clinical and agricultural use of antibiotics. Natural environments serve as massive harbors of antibiotic resistance reservoirs, known as the “resistome”. Genes that encode AR exist on mobile genetic elements and are transferred between microbes through horizontal gene transfer (HGT); this transfer can be triggered in response to antibiotic exposure. Coastal environments have increased exposure to antibiotic waste from anthropogenic inputs such as waste- and stormwater runoff, which influence the spread of AR genes from non-pathogenic microbes to human pathogens. Humans exposed to coastal waters are at increased risk of drug-resistant infections after storms or near wastewater outlets, as well as when eating seafood with antibiotic-resistant microbiomes. Elucidating the sources and patterns of AR gene transfer in environmental reservoirs in response to these runoffs is therefore of great importance. While increases in AR in response to runoffs have been established, mapping the transfer of AR genes to particular community members has yet to be established in coastal microbiomes. In this proposal, we seek to explore the changes in resistome distribution in coastal microbial populations, specifically in response to antibiotics and storm runoff, through a combination of metagenomic approaches. 1) We will employ a metagenomic chromatin conformation capture (MetaHiC) to trace the movement of mobile genetic elements and AR genes between microbial community members in response to treatment with tetracycline, an antibiotic commonly in runoff and known to induce HGT. MetaHiC can map mobile elements to particular microbial hosts, providing a detailed look at how antibiotic contamination affects resistome distribution in complex microbial communities. 2) We will combine MetaHiC data with long- read metagenomic sequencing to define the ratio in which integrative conjugative elements (ICEs, one of the main mobile genetic elements that carry AR genes) are excised or integrated in microbial genomes in response to tetracycline treatment. 3) Using MetaHiC on coastal microbiomes throughout rainstorms, we will build on preliminary data (which revealed taxonomic and resistome composition changes in response to storm runoff) to develop a profile of coastal microbes containing AR genes following rainstorms. We anticipate that a) tetracycline will induce excision and transfer of ICEs in coastal microbiomes in a non-AR gene-specific manner, b) increases in the abundance of known pathogens that are antibiotic-resistant will appear within 24-72 hours of storm runoff, c) increases in the abundance of antibiotic-resistant commensal bacteria will be observed after rainstorms, creating a resistome reservoir for clinically relevant human pathogens to use as a genetic source of AR. This research will be performed by undergraduates and will elucidate the spread of antibiotic resistance in coastal microbiomes.