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Frontiers of Environmental Science & Engineering

Front. Environ. Sci. Eng.    2019, Vol. 13 Issue (3) : 38
Degradation of extracellular genomic, plasmid DNA and specific antibiotic resistance genes by chlorination
Menglu Zhang1,2, Sheng Chen1,2, Xin Yu2, Peter Vikesland3(), Amy Pruden3()
1. Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
2. University of Chinese Academy of Science, Beijing 100049, China
3. Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA 24060, USA
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Extracellular DNA structure damaged by chlorination was characterized.

Integrity of extracellular ARG genetic information after chlorination was determined.

Typical chlorine doses will likely effectively diminish extracellular DNA and ARGs.

Plasmid DNA/ARGs were less readily broken down than genomic DNA.

The Bioanalyzer methodology effectively documented damage incurred to DNA.

There is a need to improve understanding of the effect of chlorine disinfection on antibiotic resistance genes (ARGs) in order to advance relevant drinking water, wastewater, and reuse treatments. However, few studies have explicitly assessed the physical effects on the DNA. Here we examined the effects of free chlorine (1–20 mg Cl2/L) on extracellular genomic, plasmid DNA and select ARGs. Chlorination was found to decrease the fluorometric signal of extracellular genomic and plasmid DNA (ranging from 0.005 to 0.05 mg/mL) by 70%, relative to a no-chlorine control. Resulting DNA was further subject to a fragment analysis using a Bioanalyzer, indicating that chlorination resulted in fragmentation. Moreover, chlorine also effectively deactivated both chromosomal- and plasmid-borne ARGs, mecA and tetA, respectively. For concentrations >2 mg Cl2//L × 30 min, chlorine efficiently reduced the qPCR signal when the initial concentration of ARGs was 105 copies/mL or less. Notably, genomic DNA and mecA gene signals were more readily reduced by chlorine than the plasmid-borne tetA gene (by ~2 fold). Based on the results of qPCR with short (~200 bps) and long amplicons (~1200 bps), chlorination could destroy the integrity of ARGs, which likely reduces the possibility of natural transformation. Overall, our findings strongly illustrate that chlorination could be an effective method for inactivating extracellular chromosomal- and plasmid-borne DNA and ARGs.

Keywords Antibiotic resistance      Antibiotic resistance genes (ARGs)      Extracellular DNA/ARGs      Chlorination     
This article is part of themed collection: Environmental Antibiotics and Antibiotic Resistance (Xin Yu, Hui Li & Virender K. Sharma)
Corresponding Authors: Peter Vikesland,Amy Pruden   
Issue Date: 06 June 2019
 Cite this article:   
Menglu Zhang,Sheng Chen,Xin Yu, et al. Degradation of extracellular genomic, plasmid DNA and specific antibiotic resistance genes by chlorination[J]. Front. Environ. Sci. Eng., 2019, 13(3): 38.
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Menglu Zhang
Sheng Chen
Xin Yu
Peter Vikesland
Amy Pruden
Primer TA(℃) Amplicon length (bps) Forward primer Reserve primer Reference
mecA-long 53.5 1018 CGCAACGTTCAATTT AAT TTT GTT AA CCACTTCATATCTTG TAA CG McKinney and Pruden (2012)
mecA-short 53.5 92 CGCAACGTTCAATTTAAT TGGTCTTTCTGGATTCCTGGA Volkmann et al. (2007)
tetA- long 56.5 1054 GTA ATT CTG AGC
McKinney and Pruden (2012)
Tab.1  Primers used in this study. TA means annealing temperature
Fig.1  Removal efficiency based on fluorometric quantification of extracellular genomic and plasmid DNA with increasing chlorine dose. (a) Genomic DNA; (b) First-order reaction kinetics of genomic DNA; (c) Plasmid DNA; (d) First-order reaction kinetics of plasmid DNA. The initial DNA concentration was 5 mg/mL for all experiments.
Chlorine concentration
DNA concentration (mg/mL)
Genomic DNA Plasmid DNA
0.5 0.05 0.5 0.05 0.005
1 66.98±5.03 18.65±2.01 23.87±10.76 NA
2 64.04±5.78 22.17±11.95 38.74±7.65 NA
4 62.19±0.46 25.61±16.13 72.88±7.14 NA
20 98.09±2.70 40.85±13.73 NA NA
Tab.2  Chlorine removal efficiency of lower concentration extracellular DNA after 30 min contact time. All of the samples were conducted in triplicate
Fig.2  Range of genomic DNA fragment size after chlorination for 30 min: (a) 0.5 mg/mL; (b) 0.05 mg/mL.
Fig.3  Range of plasmid DNA fragment sizes after chlorination for 30 min: (a) 0.5 mg/mL; (b) 0.05 mg/mL.
DNA concentration
Number of mecA gene
(log10 copies/mL)
Number of tetA gene
(log10 copies/mL)
0.5 6.39±0.30 6.21±0.11
0.05 5.56±0.34 5.49±0.19
0.005 4.74±0.36 4.62±0.12
Tab.3  Corresponding gene copies for each DNA concentration. Gene copies were determined by qPCR with short amplicons, respectively. Each test was conducted in triplicate
Fig.4  Reduction in mecA short and long amplicon qPCR signal with increased chlorine dose applied for 30 min. Initial gene copy concentrations for experiments (a)–(f) are indicated the different initial DNA concentrations during chlorination: (a), (b) 0.5 mg/mL; (c), (d) 0.05 mg/mL; (e), (f) 0.005 mg/mL. Error bars represent standard deviation of experimental triplicates. Data below the qPCR detection limit is not plotted.
Fig.5  Reduction in tetA short and long amplicon qPCR signal with increased chlorine dose applied for 30 min. Initial gene copy concentrations for experiments (a)–(f) are indicated the different initial DNA concentrations during chlorination: (a), (b) 0.5 mg/mL; (c), (d) 0.05 mg/mL; (e), (f) 0.005 mg/mL. Error bars represent standard deviation of experimental triplicates. Data below the qPCR detection limit is not plotted.
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