Assessing an Algae-Based Treatment for Mitigation of Antibiotic Resistance

The widespread prevalence and growing dissemination of antibiotic resistant bacteria (ARB) via environmental pathways is becoming an issue of global proportions. Wastewater treatment plants are a source of constituents that may contribute to antibiotic resistance in the environment; namely, antibiotics, antibiotic resistance genes (ARGs), and ARB. The exposure of bacteria to antibiotics, ARGs, and ARB during bacteria-based wastewater treatment creates a selective pressure for the development of more ARB. Resistance may then be transmitted “vertically” to future offspring of those organisms or “horizontally” to other organisms in the same generation, resulting in proliferation of ARB within wastewater treatment plants and downstream receiving waters. Although ARB are theoretically deactivated during wastewater disinfection processes, many antibiotic drug compounds and their corresponding ARGs are present in the effluent and discharged to the environment. Thus, there is strong interest in developing wastewater treatment technologies that can deactivate these entities efficiently, inexpensively, and sustainably.

This dissertation assesses the capacity of a novel algae-mediated biological treatment using the freshwater alga, Scenedesmus dimorphus, to remove wastewater constituents that can stimulate antibiotic resistance in downstream environmental bacteria. Interdisciplinary techniques are brought together to measure the removal of the antibiotic ciprofloxacin (CIP), the residual potency of treated CIP effluents during short- and long-term exposures to model bacteria, and the deactivation of plasmid pEX18Tc, which carries the tet ARG. Results show significant CIP removal in light control samples without algae and algae treated samples: 53% and 93%, respectively, over 6 days. A residual antibiotic potency assay reveals that untreated CIP is significantly more growth-inhibiting to a model bacterium (Escherichia coli) than the algae-treated and light control samples during short exposures (6 hours). Adaptive laboratory evolution, again using E. coli, reveals that treated samples exhibit reduced capacity to stimulate CIP resistance during sustained exposures compared to untreated CIP. Finally, observed CIP resistance in the CIP-exposed bacterial lineages is corroborated via genotype characterization, which reveals the presence of resistance-associated mutations in gyrase subunit A (gyrA) that are not present in bacterial lineages exposed to algae treated or light control samples.

Preliminary qPCR and transformation assay experiments reveal that the algae background matrix suppresses plasmid pEX18Tc amplification and transformation relative to controls in pure matrices. Final results for deactivation of the model plasmid pEX18Tc show 100% reduction in plasmid transformation efficiency in less than 3 days for both the algae treatment and light control. As such, algae-mediated tertiary treatment could be effective in deactivating wastewater constituents that stimulate antibiotic resistance in bacterial communities downstream from wastewater treatment plants. In addition, adaptive laboratory evolution and transformation assays are useful for assessing the potential of antibiotics and ARGs to stimulate antibiotic resistance downstream.

The results of this dissertation will not only inform decision-making about treatment technology options and facilitate meaningful risk analysis related to the dissemination of antibiotic resistance via environmental pathways, but also constitute methodological advancements in the environmental engineering field with a particular emphasis on effects-based treatment evaluations.