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.
Many of the bacteria that cause infections in humans have become resistant to the antibiotics we use to combat them. Frequently, the genes that make them resistant are encoded on pieces of DNA called plasmids that have the ability to spread from one species to another, introducing the resistance genes to previously susceptible bacteria. In this study, we are capturing and characterizing such plasmids from urban wetlands, which we have shown act as reservoirs of plasmid-encoded resistance genes. This approach will allow us to identify resistance plasmids before they emerge in the clinic, aiding future effors to prevent their dissemination among human pathogens.