Degradation kinetics and efficiency of antibiotic resistance gene mecA of methicillin resistant Staphylococcus aureus (MRSA) during water treatments with FAC, O3 and UV

Abstract

Increasing antibiotic resistance is a growing public health issue worldwide, as it has lowered the therapeutic efficacy of antibiotics.2, 3 Rising antibiotic resistance is typically associated with overuse and misuse of antibiotics in healthcare or agricultural practice, which can select antibiotic resistant bacteria (ARB) carrying genes (ARGs) responsible for antibiotic resistance traits.4 The methicillin resistant Staphylococcus aureus (MRSA) is one of representative ARBs and this bacteria chromosomally encoded ARG, mecA gene. Some previous studies consider mecA gene as indicator to assess the antibiotic resistance status in environmental settings.5 The MRSA and mecA gene was used as representative ARB and ARG to investigate this thesis. Bacteria can also share mobile ARGs in forms of plasmids, chromosomal DNA, and bacteriophages through horizontal gene transfer (HGT) processes, leading to dissemination of antibiotic resistance among bacterial populations.6 Since HGT easily transfer antibiotic resistance to non-resistant bacteria, it greatly contribute to antibiotic resistance dissemination. Treatment of ARGs could play major role in mitigating antibiotic resistance dissemination. In urban water cycle, wastewater treatment plant (WWTP) considered as hot spot of antibiotic resistance dissemination. Water treatment could be used to treat ARGs in WWTP. So, water treatment could act as a barrier of antibiotic resistance dissemination. Some previous studies investigated about treatment of ARGs in water treatment.7-9 Chlorine, ozone (O3), ultraviolet (UV) were known as effective oxidants for ARG treatment. Interestingly, Chemical oxidants such as chlorine and O3 need diffusion to react with intracellular-DNA. This means, position of DNA could control the degradation kinetic of DNA during chemical oxidant treatments. So, degradation kinetics for intracellular-DNA and extracellular-DNA (intra- and extra-DNA) could be different. Further investigation was needed to check this. Researches are ongoing to present a prediction model for ARG degradation kinetic with oxidants.7 In case of chlorine, FAC two-hit model was presented to explain prediction model for ARG degradation kinetic.7 In case of O3, It is known that DNA degradation kinetic by O3 is following second-order linear kinetic.7 In case of UV, however, model for partial prediction was presented. Kinetic-based model for initial-linear kinetic was only presented by previous studies.7 This could overestimate the DNA degradation by UV. So, kinetic-based model to predict overall kinetic during UV treatment is needed. Some previous studies showed that CPD formation and its photo-reversal could be affected by adjacent base pairs of bipyrimidine doublets. Reflecting this information on the prediction model could be used to improve the accuracy of the model for a various genes. This doctoral thesis aims to assess the degradation efficiency of mecA gene by oxidants and to present prediction models for ARG degradation during water treatment. The thesis focuses on the following three chapters: 1) investigation of degradation kinetics of MRSA and mecA gene during water treatment and presentation of kinetic-based model of mecA gene during water treatments, 2) presenting kinetic information for effect of water quality on mecA gene water treatment, 3) presenting kinetic information for effect of adjacent base pairs to improve the kinetic-based UV model. In chapter 1, degradation kinetics of mecA gene of MRSA by chlorine, O3, and UV treatment were investigated in phosphate buffered solutions. For chlorine, degradation rates of extra-mecA accelerated with increasing chlorine exposure, which could be explained by a FAC two-hit model. Degradation of extra-mecA by O3 treatment followed second-order reaction kinetics. Degradation of extra-mecA by UV treatment exhibited tailing kinetics, which could be explained by newly presented kinetic-based UV model considering cyclobutane pyrimidine dimer (CPD) formation, its photoreversal, and irreversible (6-4) photo-products formation. Calculated rate constants for extra-mecA increased linearly with amplicon length for chlorine and O3, or with number of intrastrand pyrimidine doublets for UV, which enabled prediction of degradation rate constants of extra-mecA amplicons based on sequence length and/or composition. Compared to extra-mecA, observed degradation rates of intra-mecA were faster for FAC and O3 at low oxidant exposures, but significantly slower at high exposures for chlorine and UV. Differences in observed extra- and intra-mecA kinetics could be due to decreased DNA recovery efficiency and/or the presence of MRSA aggregates protected from disinfectants. In chapter 2, degradation of mecA gene of MRSA by UV and O3 in wastewater matrix was investigated. Degradation efficiency of extra-mecA by UV treatment in wastewater matrix was decreased as the of total solid (TS) concentration increased. The decrease of UV treatment efficiency affected by TS was quantified in terms of light screening factor calculated based on UV254 absorbance of matrix and it was applied to kinetic-based UV model in chapter 1. The kinetics presented by revised model made a good agreement with experimental data. Decreased light screening factor could be improved by removal of total solid through wastewater filtration. Degradation efficiency of extra-mecA was also improved after wastewater filtration. There were no significant degradation kinetic differences between extra- and intra-mecA during UV treatment. Degradation of extra-mecA by O3 treatment in wastewater matrix was expressed as a function of specific ozone dose. The experimental data as a function of specific ozone dose followed two-phase kinetic consisting of initial-lag and subsequent-linear kinetic. The initial-lag kinetic could be caused by initial rapid O3 consumption by materials in wastewater (such as dissolved organic carbon (DOC)) and subsequent-linear kinetic could be caused by residual O3. The empirical O3 model was presented based on this two-phase kinetic. The kinetics presented by empirical O3 model made a good agreement with experimental data. The slope of subsequent-linear kinetic was decreased as the of TS concentration increased. Decreased slope of subsequent-linear kinetic could be improved by removal of total solid through wastewater filtration. This suggested that difference in slope of subsequent-linear kinetic could be related with TS concentration. Degradation kinetic of intra-mecA during O3 treatment was different with that of extra-mecA. Further investigation is needed to explain this. In chapter 3, effect of adjacent base pairs on CPD formation and its photo-reversal was investigated. Proper experimental system was needed to investigate this. 16 plasmids were prepared for this. The prepared 16 plasmids had a synthesized DNA sequence (140 bp) with only one TT dimer and one of 16 adjacent base pair combinations in its sequence. DNA damage by UV treatment was measured by qPCR and qPCR efficiency increase during UV treatment was corrected with experimental data with plasmid which did not had TT dimer in its sequence. With proper scheme and formula, modeling was done and rate constants of CPD formation and its photo-reversal were calculated for 16 plasmids. It was found that the rate constants of TT-CPD formation for 16 plasmids varied from 0.4-4 times and rate constants of TT-CPD photo-reversal for 16 plasmids varied from 2-4 times. Analysis was done to investigate the effect of A,T,G,C base pair on CPD formation and its photo-reversal. The rate constant of CPD formation was increased in order of A, C, G, T an adjacent base pair and rate constant of CPD photo-reversal was increased in order of C, T, A, G as an adjacent base pair. These results made a good agreement with previous studies. Additional analysis was done to investigate the effect of adjacent bp combinations on CPD formation and its photo-reversal. Different tendencies was shown for combinations of A, T, G, C base pair compared with previous analysis. There were no regularity for effect of bp combination on CPD formation and its photo-reversal. Further investigation is needed to explain the non-regularity. This thesis provided degradation kinetic information of mecA gene and MRSA. This thesis also confirmed the applicability of previously presented kinetic-based model for chlorine and O3 treatments and newly presented kinetic-based model for UV treatment. The potential for application to wastewater was investigated for tested kinetic-based models and attempts to improve the presented kinetic-based UV model by studying degradation mechanisms of DNA during UV treatment were done. This information can be useful in terms of efficient control of ARG dissemination and elimination of potential threat caused by ARB and ARG dissemination in environment.