Elimination of transforming activity of plasmid-encoded antibiotic resistance genes in water oxidation and disinfection processes: kinetics and mechanisms

Abstract

The worldwide increase in propagation and dissemination of ARB (antibiotic resistant bacteria) and ARGs (antibiotic resistance genes) in the aquatic environment has been a threat to public health. To prevent the spread of ARB and ARGs, a proper disinfection process of wastewater prior to its discharge into the aquatic environment is necessary. This dissertation discusses various water treatment technologies adopted for combating the spread of ARB and ARGs, respectively in the matrix of model system and real wastewater. In addition, systematic evaluation of ARG inactivation and deactivation with water disinfection is discussed. As a conclusion of this analysis, the antibiotic resistance expression of disinfected ARG through bacterial strains of varying repair abilities was studied. In the first section of this research, kinetics determination demonstrated that the model ARB and ARG are disinfected by chlorine, UV and UV/H2O2 (•OH). Although many research articles have been reported on ARB & ARG disinfection, as the results are limited by specific experimental conditions, there are few opportunities for inter-comparison and generalization. This chapter assessed the inactivation efficiency of plasmid-encoded ARGs both in extracellular form (e-ARG) and intracellular form (i-ARG; present within Escherichia coli) during water treatment with chlorine, UV (254 nm), and UV/H2O2. A quantitative real-time PCR (qPCR) method was used to quantify the ARG damage to ampR (850 bp) and kanR (806 bp) amplicons, both of which are located in the pUC4K plasmid. Furthermore, plate count and flow cytometry methods were applied to determine the bacterial inactivation parameters, such as culturability and membrane damage, respectively. To address a limitation of this study, additional experimental analysis such as qPCR method with spanning amplicons and ARG’s biological functions were recommended. In the second section of this research, kinetic relationships between transforming activity loss and gene degradation during UV and UV /H2O2 (•OH) were studied. This study extended to investigate the ARG degradation with varying amplicon sizes (192, 400, 603, 851bp) and measured its transforming activity loss. The results can be summarized under two points. Firstly, the gene damage rate of ampR is directly proportional to qPCR amplicon sizes, while interestingly, the enhanced DNA damage by UV/H2O2 for the extracellular plasmids did not result in faster elimination of the transforming activity. Secondly, the resulting fluence-based rate constant (k) of ∼6.2 × 10−2 cm2 mJ−1 was comparable to the k previously determined for plasmids using host cells that were capable of DNA repair, but it was much lower (∼10-fold) for DNA repair deficient cells. These newfound results contributed to the orchestration of the subsequent research topics. In the third section of this research, it was identified that OH radical does not enhance the transforming activity loss despite significant gene degradations. To confirm results and apply the DNA damage by another type of radical species, an efficiency comparison of degradation and deactivation of ARG by UV/H2O2 (OH radical) and UV/PDS (sulfate radical) was conducted. The reaction kinetics of ARG with radicals were assessed. According to the competition kinetics with radical probe compounds, the second-order rate constants of •OH with ampR segments were in the range of 1010−1011 M-1 s-1, which were 2−3 orders of magnitude higher than that of SO4•− (i.e., 107−109 M∙s). Gene transformation assays indicated that the transforming activity loss of ampR during UV/H2O2 and UV/PDS were mainly attributed to UV direct irradiation and the gene damage induced by •OH and SO4•− could be repaired by recipient cells. Finally, in the fourth section of this research, the deactivation of transforming activity loss with differing genotype bacterial strains was assessed. Escherichia coli (E.coli) K12 bacteria strains AB1157 (wild-type, uvrA+ recA+), AB1886 (uvrA-), AB2463 (recA-), AB2480 (uvrA- recA-), and DH5α (recA-) were used to assess the deactivation during chlorine and ozone treatment. In summary, it was found that mostly the rate of transforming activity loss of plasmid was directly proportional to the host cell’s DNA repair ability with the exception of E.coli DH5α. E.coli DH5α was observed to possess a higher survival rate for transforming activity with damaged plasmid DNA, compared to another bacterial strain which had proficient repair genes during all disinfection processes. This implied that bacterial replication systems should also be considered an important factor to ARG dissemination. These interesting results suggested a relationship between the genotyping of recipient host cells and antibiotic resistance dissemination.