Environmental Science: Water Research & Technology Elimination of transforming activity and gene degradation during UV and UV/H2O2 treatment of plasmid-encoded antibiotic resistance genes Journal: Environmental Science: Water Research & Technology Manuscript ID EW-ART-03-2018-000200.R2 Article Type: Paper Date Submitted by the Author: 27-May-2018 Complete List of Authors: Yoon, Younggun; Gwangju Institute of Science and Technology, School of Earth Sciences and Environmental Engineering Dodd, Michael; University of Washington, Civil and Environmental Engineering Lee, Yunho; Gwangju Institute of Science and Technology, Environmental Science and Engineering Page 1 of 40 Environmental Science: Water Research & Technology Water Impact Statement The efficiency and mode of actions for deactivating and degrading antibiotic resistance genes (ARGs) during water treatment with UV (254 nm) and UV/H2O2 have been poorly understood. Here, we show that efficiency of elimination of the transforming activity for a plasmid-encoded ARG during the UV-based treatments depends on the rate of formation of cyclobutane-pyrimidine dimers (CPDs) in the plasmid and the repair of such DNA damage during the transformation process in host cells. This work has important contributions to optimizing the monitoring and operation of UV-based water disinfection and oxidation processes for removing ARGs. Environmental Science: Water Research & Technology Page 2 of 40 1 Elimination of transforming activity and gene degradation during 2 UV and UV/H2O2 treatment of plasmid-encoded antibiotic 3 resistance genes 4 5 Younggun Yoon1, Michael C. Dodd2, Yunho Lee1* 6 7 1School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and 8 Technology (GIST), Gwangju 61005, Republic of Korea 9 2Department of Civil and Environmental Engineering, University of Washington, Seattle, WA 10 98195, USA 11 12 13 *Corresponding author: Yunho Lee; Phone: +82 62715-2468; Fax: +82 62-7152434; Email: 14 [email protected] 15 16 17 Submitted to Environmental Science: Water Research & Technology 18 19 1 Page 3 of 40 Environmental Science: Water Research & Technology 20 Abstract 21 To better understand the elimination of transforming activity of antibiotic resistance genes 22 (ARGs), this study determined deactivation of transforming activity of an ARG (in Escherichia 23 coli as a host) and the ARG degradation (according to quantitative PCR [qPCR] with different 24 amplicon sizes) during UV (254 nm) and UV/H2O2 treatment of plasmid pUC19 containing an R 25 ampicillin resistance gene (amp ). The required UV fluence for each log10 reduction of the 26 transforming activity during UV treatment was ∼37 mJ/cm2 for both extra- and intra-cellular 27 pUC19 (the latter within E. coli). The resulting fluence-based rate constant (k) of ∼6.2×10-2 28 cm2/mJ was comparable to the k determined previously for transforming activity loss of 29 plasmids using host cells capable of DNA repair, but much lower (∼10fold) than that for DNA 30 repair–deficient cells. The k value for pUC19 transforming activity loss was similarly much 31 lower than the k calculated for cyclobutane-pyrimidine dimer (CPD) formation in the entire 32 plasmid. These results indicate a significant role of CPD repair in the host cells. The 33 degradation rate constants (k) of ampR measured by qPCR increased with the increasing target 34 amplicon size (192–851 bp) and were close to the k calculated for the CPD formation in the 35 given amplicons. Further analysis of the degradation kinetics of plasmid-encoded genes from 36 this study and from the literature revealed that qPCR detected most UV-induced DNA damage. 37 In the extracellular plasmid, DNA damage mechanisms other than CPD formation (e.g., base 38 oxidation) were detectable by qPCR and gel electrophoresis, especially during UV/H2O2 39 treatment. Nevertheless, the enhanced DNA damage for the extracellular plasmids did not result 40 in faster elimination of the transforming activity. Our results indicate that calculated CPD 41 formation rates and qPCR analyses are useful for predicting and/or measuring the rate of DNA 42 damage and predicting the efficiency of transforming activity elimination for plasmidencoded 43 ARGs during UV-based water disinfection and oxidation processes. 44 2 Environmental Science: Water Research & Technology Page 4 of 40 45 Keywords: antibiotic resistance, ampicillin resistance gene, qPCR, transforming activity, UV, 46 cyclobutane-pyrimidine dimer 47 48 Introduction 49 Increasing antibiotic resistance is a major threat to human and animal health, as it can lower 50 the therapeutic potential of antibiotics against bacterial infections (WHO, 2014). Although 51 antibiotic resistance can occur naturally, overuse or misuse of antibiotics in modern society is 52 associated with increased antibiotic resistance (Allen et al., 2010). Antibiotics can select 53 antibiotic-resistant bacteria (ARB) that carry genes (ARGs) responsible for antibiotic resistance 54 mechanisms. The presence of ARB and ARGs in aquatic environments is a concern because it 55 can promote the spread of antibiotic resistance through natural and anthropogenic water cycles 56 (Berendonk et al., 2015; Pruden, 2014). In addition, antibiotic resistance can be disseminated 57 among bacterial populations by sharing (mobile) ARGs through horizontal gene transfer (HGT) 58 processes (Thomas and Nielsen, 2005). To minimize dissemination of environmental sources of 59 antibiotic resistance, the necessity of coordinated national and international strategies has been 60 advised for monitoring, risk assessment, and mitigation of antibiotic resistance (Vikesland et al., 61 2017). 62 Municipal wastewater has been identified as one of the hotspots that release ARB and ARGs 63 into aquatic environments (Rizzo et al., 2013; Tan and Shuai, 2015). Conventional wastewater 64 treatment does not fully eliminate ARB and ARGs (Chen and Zhang, 2013; Rizzo et al., 2013). 65 Biological treatment processes (e.g., activation of sludge) can significantly reduce the load of 66 ARB but may select highly (or multi-) resistant bacterial species (Czekalski et al., 2012). 67 Disinfection of wastewater effluents with chlorine or ultraviolet irradiation (UV) has been 68 widely practiced for water resource protection (Jacangelo and Trussell, 2002). Ozonation has 69 recently received renewed attention as an option for treating municipal wastewater effluents to 3 Page 5 of 40 Environmental Science: Water Research & Technology 70 eliminate organic micropollutants (Lee and von Gunten, 2016). Advanced oxidation processes 71 such as UV/H2O2 treatment have also been tested to achieve the same goal (Gerrity et al., 2016; 72 Miklos et al., 2018). There has been growing interest in the efficiency of wastewater 73 disinfection and oxidation processes to lower the levels of ARB and ARGs, in addition to 74 micropollutant elimination (Alexander et al., 2016; Czekalski et al., 2016; Dodd, 2012; Ferro et 75 al., 2017; Lüddeke et al., 2015; McKinney and Pruden, 2012; Pak et al., 2016; Sousaa et al., 76 2017; Yoon et al., 2017). 77 ARGs in wastewaters exist in different forms such as intracellular (within bacteria) and 78 extracellular, as free DNA and viruses (Colomer-Lluch et al., 2011; Zhang et al., 2018). ARGs 79 can transfer resistance by HGT mechanisms such as conjugation, transduction, and 80 transformation. Among these HGT mechanisms, transformation requires only intact ARGs for 81 the resistance transfer, as extracellular ARGs can be taken up and incorporated into the genomes 82 of competent bacteria even in the absence of the original donor ARB cell (Thomas and Nielsen, 83 2005). This mechanism is therefore different from conjugation or transduction in which viable 84 donor cells or infective viruses containing ARGs are needed. Considering the potential for ARG 85 transfer via transformation, it is necessary to assess the efficiency of disinfectants at destroying 86 ARGs and eliminating their associated transforming activities (Dodd, 2012). 87 Molecular mechanisms of DNA damage induced by UV or by hydroxyl radicals (•OH) are 88 well established. UV (particularly UVC) mainly generates DNA base lesions such as 89 cyclobutane-pyrimidine dimers (CPDs) and pyrimidine (64) pyrimidone adducts [(6-4) 90 photoproducts] (Görner, 1994; Sinha and Häder, 2002). Good correlations have been found 91 between the UV-induced degradation rate of ARGs and the number of adjacent pyrimidine 92 dimer sites (Chang et al., 2017; Destiani et al., 2017; McKinney and Pruden, 2012). Genomic 93 modeling, an approach to predict the UV sensitivity of microorganisms based on their DNA 94 sequence characteristics, has been tested to predict the degradation efficiency of ARGs 4 Environmental Science: Water Research & Technology Page 6 of 40 95 (Destiani et al., 2017). For •OH, the DNA damage can range from base oxidation to sugar 96 backbone breakages, where the latter can lead to single strand (ss) and double strand (ds) breaks 97 (von Sonntag, 2006). Despite this knowledge, it is unclear how different types of DNA damage 98 resulting from reactions with disinfectants (e.g., UV, •OH) are related to the loss of ARG 99 transforming activity. 100 Quantitative polymerase chain reaction (qPCR) has been widely used to detect and quantify 101 ARGs present in aquatic environments (Luby et al., 2016). The qPCR method has also been 102 employed to assess the efficacy of disinfection processes for ARG elimination by quantifying 103 target qPCR amplicons (Alexander