Kinetic and Mechanistic Understanding of Degradation and Deactivation of Antibiotic Resistance Genes by Conventional and Alternative Disinfectants

Abstract

Antibiotic resistance is an increasingly critical global public health issue. The overuse and misuse of antibiotics in healthcare and agriculture promote the selection of antibiotic-resistant bacteria (ARB) and lead to the proliferation of antibiotic resistance genes (ARGs) in various environments, including soil, water, and wastewater systems. These ARGs can be transferred among bacterial populations through horizontal gene transfer (HGT), further accelerating the spread of antibiotic resistance. Wastewater treatment plants (WWTPs) can be significant contributors to ARG dissemination, serving as hotspots for the accumulation of antibiotics, bacteria, viruses, and ARGs. Therefore, effective water treatment processes are essential as crucial barriers to mitigating ARG spread and controlling antibiotic resistance. Addressing this issue requires a comprehensive understanding of how different water disinfection processes can be optimized to effectively target ARGs and minimize associated risks. The aim of this doctoral dissertation was to assess the degradation and deactivation efficiency of ARGs by various conventional and alternative water disinfection processes, focusing on understanding the role of different oxidants and water conditions in enhancing ARG removal. The study was divided into three main topics: i) evaluating the degradation kinetics of ARGs during bromination and chlorination, with a focus on bromide's (Br-) impact on chlorination efficiency, ii) exploring the influence of bromide on the performance of peracetic acid (PAA) treatments for ARG degradation, and iii) comparing the degradation and deactivation rates of ARGs during different disinfection treatments (UV, O3, chlorine, bromine, PAA, and hydroxyl radicals (•OH)). These topics were investigated to provide a comprehensive understanding of how different disinfection strategies can be optimized for effective ARG removal, contributing to enhanced water quality and reduced risk of antibiotic resistance proliferation. In the first part, this study examines the degradation kinetics of ARGs, specifically tetA and blaTEM-1, during Br--containing water chlorination. During chlorination, the presence of Br- led to the formation of bromine, which exhibited higher reactivity than chlorine and significantly enhanced ARG degradation. Bromine reactions followed second-order kinetics, with rate constants ranging from 4.0×102 to 1.6×103 M⁻¹ s⁻¹ for blaTEM-1_216 to tetA_1136 amplicons. A kinetic model was developed to simulate ARG degradation during chlorination in the presence of Br-, providing insights into how Br- can enhance chlorination processes for more efficient ARG degradation. In the second part, the effect of Br- on PAA treatment was explored for its effectiveness in degrading ARGs. Low concentrations of Br- enhanced ARG degradation due to the in situ formation of bromine, while higher concentrations led to decreased degradation rates because bromine was consumed by hydrogen peroxide (H2O2), a component of PAA solutions. Removing H2O2 resulted in enhanced degradation, underscoring the importance of managing H2O2 concentration to optimize PAA-based disinfection. These results demonstrate that while PAA alone may have limited efficiency in degrading ARGs, its efficacy can be significantly improved by carefully controlling Br- and H2O2 levels In the third part, the study compared the degradation and deactivation rates of ARGs across various disinfection treatments-UV, O3, chlorine, bromine, PAA, and •OH. •OH were the most effective due to their ability to induce significant DNA fragmentation, which disrupts ARG integrity and prevents transformation. In contrast, UV, O3, chlorine, bromine, and PAA primarily modified DNA bases without causing extensive fragmentation, resulting in moderate deactivation. Transformation assays using different bacterial recipients highlighted the biological factors influencing ARG deactivation. A. baylyi demonstrated higher deactivation rates compared to E. coli DH5α, likely due to its natural transformation competence, which integrates ARGs into chromosomal DNA less effectively. The structural form of plasmid DNA, whether circular or fragmented, played a key role in its use by recipient cells. Circular plasmids were less readily utilized because they require an exact matching region with chromosomal DNA during the Campbell-like mode of insertion, whereas fragmented plasmids were more readily integrated and utilized with chromosomal DNA via (non)-homologous recombination during replacement insertion. Based on these findings, deactivation rates were inferred from gene degradation rates and compared to empirical measurements, supporting the applicability of this comparison method. These results emphasize that understanding these biological interactions is crucial for accurately evaluating disinfection efficacy. These findings underscore that optimized disinfection strategies targeting both ARG degradation and deactivation are essential to improving wastewater treatment efficacy and mitigating antibiotic resistance. Future research should focus on refining these strategies to accommodate diverse water matrices and environmental conditions, thereby contributing to more robust barriers against the spread of antibiotic resistance.