Evaluating a gas biosensor for the measurement of conjugative transfer in wastewater communities
Rice, Eric W
Stadler, Lauren B.
Master of Science
Antibiotic resistance poses a threat to global public health causing simple infections to become untreatable and lead to potential fatality. Antibiotic resistance genes (ARGs) can be exchanged between microorganisms via horizontal gene transfer (HGT) enabling the rapid dissemination of resistance. HGT of ARGs present on broad host range plasmids (BHRP) can be transferred among phylogenetically distant bacteria. There is a need to characterize the bacteria that transfer BHRPs, as well as the transfer rates of BHRPs to inform antibiotic resistance mitigation strategies. This work is focused on the development and application of an engineered BHRP that reports on HGT by producing a volatile gas after HGT occurs. The project focused on characterizing the performance of this bioreporter in Escherichia coli. Specifically, the Escherichia coli was engineered as a donor of the BHRP reporter and its performance was measured. In addition, the reporter signal was transferred intraspecies to receiver Pseudomonas putida and gas output was characterized across several strains. Gas detection in complex matrices (e.g. wastewater) was also demonstrated. Results indicated that background donor signal, variability of gas production in transconjugant hosts, and gas degradation in activated sludge (AS) are important factors that must be accounted for to deploy the gas reporter in real wastewaters. Future research using targeted single-cell fusion PCR methods can be applied to characterize the hosts of the BHRP in a mixed community. Knowledge of BHRP hosts is critical to evaluate the risk of ARG spread between environmental and clinically-relevant pathogenic bacteria.