ELIMINATION OF ANTIBIOTIC RESISTANCE GENES FROM WATER MATRICES USING CONVENTIONAL AND ADVANCED TREATMENT PROCESSES

Abstract

The overuse and misuse of antibiotics to treat bacterial infections, the release of unmetabolized residuals into the sewer system, and the incomplete removal antibiotic residues by wastewater treatment plants (WWTPs) pose a severe threat to human health. The accumulation of antibiotic residue induces selective pressure on the bacterial population, resulting in the spread of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) in water. This study investigated the degradation of different types of ARGs in water matrices using a wide variety of treatment technologies. Real wastewater samples were collected from a WWTP in urban Philadelphia and the presence of single and multidrug-resistant bacteria and resistance genes were investigated using molecular-based techniques. Subsequently, an analytical method was developed and validated for the detection and quantification of the ARGs against a range of antibiotics, such as tetracycline (TCN), ciprofloxacin (CIP), and levofloxacin (LVX). Finally, to remove the ARGs from water matrices, different conventional and advanced oxidation processes were applied. At the very onset, conventional treatment processes such as chlorine treatment was used to inactivate the E.coli resistant strains. It was observed that chlorination can potentially deactivate the ARBs by applying a lower dose and contact time. However, the effectiveness of chlorine treatment in removing all types of ARGs from water matrices was limited. For instance, no significant degradation of extracellular ARGs (e-ARGs) was observed in DI water during chlorine treatment. Subsequently, a peracetic acid (PAA) based treatment process was used to degrade the genomic and plasmid-encoded ARGs from the water matrices. Similar to chlorine treatment, no significant changes were observed in the degradation of extracellular ARGs (e-ARGs) in DI and WW. Then, the degradation kinetics of ARGs across different types (gyrAR, tetAR, qnrSR) and forms (chromosomal, plasmids) were evaluated using the Ultraviolet (UV) disinfection process. Compared to chlorination and PAA, UV treatment showed better removal efficiencies for the degradation of different types of e-ARGs in DI water. The degradation profile of e-ARGs showed 1-4 log reductions at a UV fluence of 900 mj/cm2. The i-ARGs showed similar degradation rates as compared to e-ARGs in phosphate buffer saline (PBS) at the same UV dosage. On the other hand, the regrowth potential of ARBs at low UV dosage (60–180 mJ/cm2) showed the evidence of damage repairment after several hours of exposure to light (photoreactivation) and dark conditions, making it susceptible again to the resistance spread. To resolve this issue, process parameters were optimized, and no regrowth of the ARBs were found from the higher fluence from 300 to 600 mJ/ cm2. Later, UV/ H2O2 based AOP was applied to evaluate the degradation and deactivation of the same resistant genes. The addition of H2O2 during the UV treatment produces strongly reactive •OH radicals during the treatment and showed considerable improvements in e-ARGs degradation (1.2-5 logs) compared to UV treatment alone. However, this AOP showed minimal contribution to i-ARG degradation (1-2.4 logs), possibly due to the scavenging of •OH radicals by the cellular components in PBS. In contrast to PBS, the wastewater matrix moderately enhanced the gene degradation during the treatment. In terms of plasmid degradation, the conformational differences of the supercoiled structures showed 1.2-2.8 times slower degradation rates than chromosomal ARGs. In addition, the degradation kinetics of the free residual ARGs (f-ARGs) were assessed during the treatment to reduce the AMR dissemination risk from the treated sample. This study also examined the potential of ozone (O3) based oxidation process to degrade and deactivate the extracellular and intracellular ARGs, and MGE (plasmid, intl-1) from E.coli ARBs. The degradation kinetics of the ARGs across different sizes (118-454 bps) and types were evaluated in different water matrices (DI water, PBS, and WW), and showed a significantly higher removal for chromosomal, and plasmid encoded ARGs than other treatment technologies. For the e-ARGs in DI water, 3.8-5.2 logs removal was observed at ozone dosage of 2.0 × 10-2 M.s. i-ARGs in PBS and wastewater showed nearly similar degradation (3.8-5 logs) during O3, indicating the elimination of i-ARGs was not dependent on the cellular components and effluent organic matter. Moreover, an analysis of environmental DNA (eDNA) from wastewater was conducted to examine the degradation of DNA and ARGs for different storage periods and temperatures (-20°C, 0°C, 4°C, 22±0.87°C). Result indicated that water samples kept at -20°C and 0°C showed the best performance in preventing the DNA concentration and gene degradation over time. Additionally, the effectiveness of different preservatives (Longmire buffers: LB1 and LB2, benzalkonium chloride at 0.1%, 0.01%) were investigated in preserving the DNA integrity and the gene degradation at an ambient temperature. It was found that the Longmire buffer (LB1) exhibited lowest gene degradation during the three-week storage period. In summary, this research provided a comprehensive assessment on the degradation of e-ARGs, i-ARGs, and free ARGs from water using different treatment technologies (i.e., UV, UV/H2O2, O3, PAA, chlorine). Additionally, this study suggested valuable information on optimizing the process parameters of the selected methods and developed a comparative assessment of removing the ARGs from the water matrix (DI/PBS, WW). The estimation of Electrical Energy per Order (EEO, kWh/m3) during UV and ozone treatments provided a comparison of the energy consumption for ARGs degradation in the water. Overall, the findings of this study can be useful for evaluating different types and forms (chromosomal, plasmid) of ARG degradation from water matrices and can help to reduce the risk of AMR dissemination in the environment.