The Effect of Biocides on Antimicrobial Resistance Transmission

Antimicrobial resistance (AMR) poses a significant global threat to human health and the economy both now and in the future. The One Health approach emphasizes the interconnectedness of human, animal, and environmental health in addressing AMR. Certain environments, such as wastewater, are considered to be key drivers of AMR dissemination by acting as hotspots that contain pollutants, diverse plasmids harboring antibiotic resistance genes (ARGs), and a wide variety of bacteria. These conditions facilitate horizontal gene transfer processes like conjugation, where bacteria exchange genetic material, such as plasmid-borne ARGs. Xenobiotic pollutants, such as biocides, may exacerbate the AMR crisis by stimulating conjugation, as observed in prior studies conducted with simple mating assays. However, these studies often rely on pure cultures of donors and recipients and a single RP4 model plasmid, which limit their ability to capture the diversity of environmental hotspots containing various plasmids and bacterial species. It remains unclear how factors such as the plasmid type (Incompatibility (Inc) group/mobilization (MOB) family) or bacterial species influence the modulation of conjugation. To address these gaps, this thesis investigates the impact of various biocides on conjugation, incorporating a broader diversity of plasmids and bacterial strains. Additionally, this Ph.D. project explores the underlying mechanism behind biocide-induced modulation of conjugation, hypothesizing that reactive oxygen species (ROS) generation and subsequent RecA activation play a vital role.

In the first study, we developed a novel liquid mating assay coupled with a liquid quantification method to accurately evaluate the effect of biocides on conjugation by minimizing the bias. Indeed, the conventional plate quantification method often overestimate transfer frequencies due to plasmid transfer on transconjugant-selective plates, especially for plasmids that favor solid mating. Here, we also established a cell density threshold at or below 28 ± 4 CFU/mm² (approximately 10⁵ CFU/plate) to avoid the abovementioned bias, if conventional selective plating is the choice of quantification method. By mitigating such bias, this assay enabled more accurate estimations of transfer frequencies.

In the second study, the liquid mating assay was applied to test biocides from different chemical classes in pure culture systems. The results revealed that conjugation is more often inhibited than stimulated, with three key factors determining the outcome: the biocides, the plasmid Inc group/MOB family, and the bacterial strain involved. Intracellular ROS levels were measured in donor strains exposed to two biocides, CuSO₄ and DDAC, revealing significant differences in ROS generation depending on the biocide and bacterial species. Further experiments using a recA-knockout (KO) E. coli mutant as donor demonstrated that RecA plays a role in biocide-induced modulation of conjugation. These findings highlight how RecA interacts with plasmids from different Inc groups/MOB families, which regulate transfer machinery in distinct ways.

In the final study, we tested if the findings from pure culture experiments could be observed using a complex microbial donor community that contains diverse plasmids and bacterial species. A GFP-tagged E. coli recipient was used to isolate exogenous plasmids from an influent wastewater donor community under CuSO₄ or DDAC exposure. Although no statistical significance was observed in the MOB family composition across treatments, some patterns under CuSO₄ exposure were noted, such as the increase of MOB_P and decrease of MOB_F conjugative plasmids. This suggests that additional ecological interactions obscure the results identified in simpler pure culture systems.

Overall, this thesis provides new insights into the complex interplay between biocides, plasmids, and bacterial species, as well as the mechanisms underlying the modulation of conjugation. From a biotechnological and societal perspective, these findings may assist in the development of novel mitigation strategies, guide policymaking, and improve environmental risk assessment efforts to address the growing AMR crisis.