Degradation of Extracellular Antibiotic Resistance Genes with UV254

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Degradation of extracellular antibiotic resistance genes with UV treatment Pin Hsuan Chang, Brianna Juhrend, Terese M Olson, Carl F. Marrs, and Krista R. Wigginton Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 05 May 2017 Downloaded from http://pubs.acs.org on May 7, 2017

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Environmental Science & Technology

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Degradation of extracellular antibiotic resistance

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genes with UV254 treatment

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Pin Hsuan Chang,† Brianna Juhrend,† Terese M. Olson, † Carl F. Marrs, ‡ and Krista R.

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Wigginton†* †

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University of Michigan, Ann Arbor, Michigan 48109, United States

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Department of Civil and Environmental Engineering,

‡

Epidemiology Department, University of Michigan, 1415 Washington Heights, Ann Arbor, Michigan 48109, United States

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_____________________________________ *corresponding author: [email protected] Tel. (734) 763-9661 Fax. (734) 764-4292

Word Counts 243 words in Abstract + 4,326 words in Text + 1,500 words in Figures + 600 words in Tables = 6,669

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Abstract: Disinfected wastewater effluent contains a complex mixture of biomolecules including

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DNA. If intact genes conveying antibiotic resistance survive the disinfection process,

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environmental bacteria may take them up. We treated plasmid pWH1266, which contains

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ampicillin resistance gene blaTEM-1 and tetracycline resistance gene tetA, with UV254 doses up to

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430 mJ/cm2 and studied the ability of those genes to be acquired by Acinetobacter baylyi. The

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plasmids required approximately 20-25 mJ/cm2 per log10 loss of transformation efficiency. We

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monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short

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amplicons (~200 bps, representative of ARG amplicon lengths commonly used for

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environmental monitoring) and long amplicons (800-1200 bps, designed to cover the entire

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resistance genes). The rate of transformability loss due to UV254 treatment was approximately

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20× and 2× larger than the rate of gene degradation measured with the short and long amplicons

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qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid,

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the qPCR rate constants were 2–7× larger than the rate constants measured with transformation

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assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating

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mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for

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a gene to be transformed by environmental bacteria following UV254 treatment.

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Introduction The proliferation of bacterial resistance to antibiotics results in more than two million

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illnesses and 23,000 deaths each year in the U.S. alone.1 To combat the continued emergence and

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spread of resistant bacteria, the World Health Organization (WHO) announced a global action

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plan in 2015 that urges international participants to take action in controlling and monitoring the

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spread of all forms of antimicrobial resistance.2

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Although links between the release of antibacterial resistance genes into the environment and

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the emergence of new resistant pathogens in the clinic are not fully developed, resistant bacteria

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can transfer resistance genes to human pathogenic species, compromising antibiotic treatment

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effectiveness.3,4 Experts therefore recommend minimizing the release of antibacterial resistance

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to the environment. Municipal wastewater and livestock wastes have been identified as

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significant sources of antibiotic resistant bacteria and their associated antibiotic resistance genes

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(ARGs) in the environment,5,6 with wastewater treatment plant (WWTP) effluent leading to

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increased levels of resistance genes and resistant bacteria downstream of effluent discharges.7–9

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Antibiotic resistance is passed by either vertical gene transmission, where genetic information

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is inherited from parent cells, or horizontal gene transfer (HGT), where a bacterium without

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resistance acquires the necessary genes from mobile genetic elements. HGT can occur by



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conjugation (in which DNA is passed from a donor cell to an acceptor cell through direct cell-

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cell contact), transduction (where bacteriophage introduce ARGs into microbial cells), or

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transformation (where competent microbes take up free DNA from their surroundings).10,11

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Wastewater carries different forms of ARGs, including DNA carried within bacteria and viruses,

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and extracellular DNA.12 Each form has the potential to transfer resistance by the different

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mechanisms described above. For either vertical gene transmission or conjugation to occur, the

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bacterium carrying the gene must be viable so that it can pass the gene onto its daughter cells or

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to another organism. Likewise, for transduction to occur, the virus containing the genes must be

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infective.13 Transformation, however, does not require a viable or infective donor

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microorganism; in this case, competent bacteria in the environment may pick up a gene present

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in extracellular DNA. Consequently, wastewater treatment processes that kill bacteria containing

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ARGs do not necessarily eliminate the potential for ARG transfer downstream. Indeed, most

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wastewater treatment and animal waste processes are not designed to destroy nucleic acids in the

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waste.

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To track antibacterial resistance through wastewater treatment, both culture-based and

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molecular-based methods are employed.14 Culture methods, like broth dilution, disk diffusion,

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and selective plating, detect the presence of culturable antibiotic resistance bacteria.15,16 These

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methods do not detect ARGs in viruses, extracellular DNA, or nonculturable bacteria.

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Molecular-based quantitative PCR (qPCR), on the other hand, detects pieces of the ARGs,

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regardless of whether they are in a viable cell.17 However, qPCR amplicons rarely encompass the

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entire ARG, not to mention regions outside of the gene that are necessary for gene transfer

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pathways.18 Furthermore, qPCR may not detect certain damaged ARGs that can be repaired by

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the bacteria that pick up the DNA. Finally, enzymes involved with the ARG transfer pathways in


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the cells may have different fidelities to damaged DNA than polymerase enzymes used in

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qPCR.19

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The relationship between concentrations of ARGs detected by qPCR and the concentration of

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ARGs present in extracellular DNA capable of transformation by bacteria in the environment is

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unknown. To address this, we compared the kinetics of ARG inactivation by ultraviolet (UV254)

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disinfection using qPCR and transformation assays. UV254 is increasingly used to treat

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wastewater effluent and is known to target nucleic acids. We studied plasmid transformation in

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Acinetobacter species as a proxy for transformation of extracellular DNA in the environment.

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Acinetobacter species are ubiquitous in soil and aquatic environments, undergo spontaneous

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gene transformation at high frequency,20,21 and can be opportunistic human pathogens.22

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Experimental Methods

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General Experimental Approach

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Solutions of purified plasmids that contain antibiotic resistance genes (ARGs) were exposed to

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different doses of UV254 (Figure 1). The treated plasmids were then subjected to transformation

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assays to establish the reaction kinetics for the loss of ARG transformation ability. The same

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samples were assayed by qPCR to track the reaction kinetics of the plasmid ARG regions. Short

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amplicon qPCR was conducted to represent qPCR assays that are commonly conducted to track

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ARGs in water treatment and the environment. Long amplicon qPCR was conducted to track the

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modification of the entire resistance genes. The same samples were analyzed by gel

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electrophoresis to detect plasmid nicks and breaks following UV treatment. Ultimately, the

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kinetics of the ARG transformation loss and qPCR amplicon reactions were compared to assess

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the effectiveness of qPCR at tracking the destruction of ARG transformation ability.



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Model transformation system

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We adopted a model transformation system to mimic environmental transformation events

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downstream of WWTP effluents. The system (kindly provided by Dr. Chuanwu Xi, University of

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Michigan), was comprised of the A. baylyi strain AC811, a derivative of Acinetobacter sp. strain

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BD413,20,23 and plasmid pWH1266 which is a construct of the E. coli plasmid pBR322 (4.4

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kbps) and the cryptic plasmid pWH1277 (4.5 kbps) isolated from A. lwoffi (Figure 2).24 The

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pBR322 portion of the plasmid carries the tetA tetracycline resistance gene and the ampicillin

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resistance gene blaTEM-1 that encodes TEM-1 beta-lactamase (Figure 2).24–26 The pWH1266 was

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propagated in E. coli strain TOP10.

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Plasmid Extraction

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Host E. coli TOP10 cells carrying the plasmid were incubated in 5 mL LB broth (Sigma-

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Aldrich, St. Louis, MO) containing 20 µg/L tetracycline with shaking (180 rpm) at 37 °C until

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they reached stationary growth phase (~ 16 hr). The cells were then treated with QIAprep Spin

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Miniprep Kits (Qiagen, Valencia, CA)15 per the manufacturer’s instruction. Several extractions

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were conducted concurrently to prepare the plasmid stocks. The resulting purified plasmid

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solutions contained 20-60 ng/µL plasmid DNA, as measured by a NanoDrop ND-1000 (SI

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Figure 1; ThermoFisher Scientific, Waltham, MA).

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Transformation assays Transformation assays were conducted to quantify the ability A. baylyi to acquire antibiotic

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resistance from the plasmid.15 Transformation efficiency of the plasmid/A. baylyi system was

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determined as the ratio of the antibiotic resistant bacteria (ARB) colonies detected on selective 6 ACS Paragon Plus Environment

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plates to the total colonies detected on nonselective plates, as expressed by the following

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equation:

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=

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where CFU stands for colony forming units.

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Experiments were conducted to establish the optimal transformation efficiencies, including

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plasmid concentration, the point in the A. baylyi growth curve the cells were inoculated with

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plasmids, and the time to incubate the plasmids with the bacteria. Details of these experiments

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are reported in the SI. Once optimized, the transformation assays were always conducted in the

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same manner. In brief, aliquots of A. baylyi strain AC811 stored at -80 °C were thawed and

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streaked on LB agar plates, and incubated overnight at 30 °C. Three colonies of A. baylyi were

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inoculated in 5 mL LB broth and incubated for 18 hours at 30 °C and 140 rpm in order to reach

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stationary growth phase (SI Figure S2). Subsequently, 0.5 mL of the cell suspension was diluted

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10× in LB broth and incubated at 30 °C and 140 rpm for another 8 hours to reach late

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exponential phase. In a sterile glass tube, 480 µL of the bacteria was combined with 20 µL

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untreated or UV-treated plasmid solution (10 ng/µL), resulting in a final ARG concentration of

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0.4 ng/µL. The mixture was incubated at 30 °C and 140 rpm for 24 hours. Serial dilutions of the

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suspension were plated onto selective media plates (10 µg/mL tetracycline or 100 µg/mL

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ampicillin) for transformant counts27,28 and onto LB plates for total cell counts.

(1)

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Gel Electrophoresis

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Potential plasmid breakage after UV254 treatment and enzyme digestion was assessed with DNA

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gel electrophoresis. The pWH1266 plasmids were incubated with restriction enzymes BamHI

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and PvuI (Thermo Fisher Scientific, Waltham, MA) at 37 °C for 1 hour to cut the plasmids inside



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and outside of the tetA gene, respectively. Gel electrophoresis of the plasmid DNA before and

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after UV254 treatment and restriction enzyme treatment was conducted on 0.5% agarose gels at 3

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V/cm for 60 minutes. The bands were visualized by SYBR Safe DNA gel staining (Life

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Technologies, Carlsbad, CA) alongside the GeneRuler 1kb DNA ladders (ThermoFisher

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Scientific, Waltham, MA).

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qPCR measurements

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The tetA gene (1191 bps) and blaTEM-1 gene (861 bps) on the pWH1266 plasmid were quantified

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with both short amplicon and long amplicon qPCR (Table 1). The short amplicons covered a

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fraction of the resistance genes (~200 bps), whereas the long amplicons were designed to cover

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the entire tetA and blaTEM-1 gene sequences. Amplicon and primer sequences were identified

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using the plasmid pBR322 sequence, which is the portion of plasmid pWH1266 that carries both

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resistance genes. Sequences were acquired from the NCBI GenBank database (J01749.1). All

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primers were designed with NCBI Primer-BLAST tool.

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The qPCR measurements were performed on a Mastercycler RealPlex 2 (Eppendorf, Hamburg,

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Germany) using a Fast EvaGreen qPCR Master Mix (Biotium). Standard curves were conducted

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in triplicate, with 10-fold dilutions covering 5 orders of magnitude. Each 10 µL qPCR reaction

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contained 5 µL of 2X Master Mix, 0.5 µL of forward and reverse primers at 100 µM, 0.1 µL of

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DNA template, and 4.4 µL sterile DNase free water. The temperature profile included one cycle

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at 95 °C for 2 minutes, 40 cycles at 95 °C for 5 seconds, the annealing temperature (TA, Table 1)

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for 15 seconds, 72 °C for either 15 seconds (short amplicons) or 60 seconds (long amplicons),

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and then a melt curve to verify specificity. qPCR assays amplifying short and long amplicons


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were successfully developed for both tetA and blaTEM-1 genes. The amplification efficiency was

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0.90 ± 0.05 for tetA short amplicon (216 bps), 0.82 ± 0.08 for blaTEM-1 short amplicon (209 bps),

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0.71 ± 0.05 for tetA long amplicon (1200 bps), and 0.74 ± 0.04 for blaTEM-1 long amplicon (861

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bps). The average R2 value across all qPCR assays was 0.995 ± 0.003 (mean ± SD).

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UV disinfection experiments The UV disinfection experiments were conducted in a collimated beam reactor (SI Figure S3)

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housing four lamps that emit 254 nm germicidal UV254 (Philips TUV G15T8, Amsterdam,

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Netherlands). The UV irradiance was 0.18 ± 0.01 mW/cm2 (mean ± SD) as determined with

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chemical actinometry using potassium iodide. 29 The chemical actinometry and UV experiments

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were conducted in the same 96-well plates and the UV intensity was similar across all plate

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wells. For UV experiments, the plasmid stocks were diluted to 10 ng/µL in 50 µL DNase free

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water in 96-well plates. At this concentration and sample depth, the transmittance of UV254 was

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approximately 93%, thus shielding corrections were not deemed necessary. The samples were

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exposed to UV254 for 1, 3, 5, 10, 20 and 40 minutes, corresponding to UV254 doses of 11, 32, 54,

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108, 215, and 430 mJ/cm2. At each sample point, the remaining ARG amplicon concentration

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was quantified with qPCR and the remaining transformation efficiency was measured with

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transformation assays. Inactivation kinetics were established for the transformation and qPCR

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data. Kinetics were expressed as first-order (ln C/C0 versus UV254 dose).

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Statistical analyses

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Statistical analyses were conducted in Microsoft Excel 2016 and StatPlus (AnalystSoft Inc.,

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Walnut, CA). Data from replicate experiments were pooled for linear regression analyses. The



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first order reaction rate constants from different types of experiments were compared with

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multiple linear regression analyses using StatPlus (AnalystSoft Inc., Walnut, CA). The null

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hypothesis in all of the experimental comparisons (e.g., transformation assay vs. long amplicon

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qPCR) was that the first order rate constants were not significantly different. The P values were

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computed at a confidence level of 95%.

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Results and Discussion After optimization, the transformation efficiencies of both tetracycline resistance from tetA

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and ampicillin resistance from blaTEM-1 were consistently between 10-4 and 10-5. In other words,

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one out of 10,000 or 100,000 A. baylyi cells attained resistance when exposed to the plasmids.

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Loss of transformation efficiency during UV treatment. Transformation efficiencies of both tetracycline and ampicillin resistance decreased with

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increasing UV254 doses (Figure 3). At 54 mJ/cm2, the transformation efficiencies dropped to

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approximately 10-7 or lower, and beyond this dose, they were below the assay detection limit

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(90% reductions in concentration

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(Figure S6). For this reason, rate constants for the qPCR experiments were determined using

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only data points where C/C0 > 0.1. Deviations from first order kinetics measured with qPCR

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have previously been reported with UV treatment.17,39 Reaction kinetics measured with qPCR

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did not change with replicate experiments on different days. For this reason, qPCR data was

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pooled to find the first order rate constants.

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For both genes, the LA first-order rate constants were larger than the SA first-order rate

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constants (Figure 5). This was expected due to the larger number of targets in the LA sequence



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compared to the SA sequence. Both SAs and LAs had reaction kinetics that were slower than the

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inactivation kinetics of antibiotic resistance transformation. For example, at a dose of 108

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mJ/cm2, the tetA SA had decreased about 0.25 log10 units, whereas the LA had decreased about

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1.5 log10 units; extrapolating the inactivation kinetics of tetracycline resistance transformation to

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this dose results in a 5-log10 loss in the gene transformation efficiency. These results highlight

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that qPCR of the entire resistance gene will underestimate the loss in a gene’s ability to be

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transformed. Ultimately, the results demonstrate that using qPCR results to track ARGs through

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a UV treatment process will provide conservative conclusions on the ability of DNA to transfer

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resistance. For the case of our two ARGs, this is true even when the entire ARG is covered by

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the selected amplicon.

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The slower LA-qPCR reaction kinetics compared to that of the antibiotic resistance

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transformation could be explained by the fact that modifications outside the tetA and blaTEM-1

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genes are critical for ARG transformation and expression. In an earlier study of the shuttle

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plasmid pWH1266, deletion and insertion analyses were conducted to identify specific plasmid

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regions that were necessary for the transformation of ampicillin and tetracycline resistance in A.

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calcoaceticus BD413.23 They identified a 1350-bp region in the cryptic pWH1277 portion of the

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pW1266 plasmid construct that was critical for transformation of blaTEM-1 and tetA in A.

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calcoaceticus BD413. The study did not attempt to identify elements on the pBR322 region of

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the pW1266 plasmid construct that were critical for transformation in A. calcoaceticus BD413.

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The rate of transformation efficiency loss with UV treatment was the same for ampicillin and

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tetracycline resistance genes on the same plasmid, despite the fact that the blaTEM-1 and tetA

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genes degraded at different rates, based on LA-qPCR results (Figures 5, Table S2). This suggests

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that the transformation inactivation mechanism may be the same for both resistance types. 14 ACS Paragon Plus Environment

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To test if modifications anywhere in the plasmid (and detectable by qPCR) would obstruct

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antibiotic resistance transformation, we extrapolated the damage measured by qPCR in a specific

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amplicon to the theoretical damage across the entire plasmid based on the following relationship:

 C ′ C plasmid size  log  = log × C0 amplicon size  C0 

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"

(2)

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where ! is the extrapolated value of normalized ARG copies, and is the

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normalized values of detected amplicon copies. This approach has been applied to track the

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infectivity of RNA viruses with RT-qPCR following UV254 treatment.18 The extrapolation

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approach assumes that modifications in the plasmid are randomly distributed, or that damage in

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the measured amplicon is representative of damage anywhere in the plasmid. By covering

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approximately 25% of the plasmid sequence with our two long amplicons, we aimed to negate

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issues raised when different regions have different susceptibilities to UV254.18 Indeed, our results

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suggested that the blaTEM-1 gene reacted faster with UV than the tetA according to LA-qPCR

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(Figure 3), despite the fact that the tetA gene was 339 bps larger than the blaTEM-1 gene.

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We applied the extrapolation approach on qPCR data from all four of the measured amplicons

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(tetA SA, tetA LA, blaTEM-1 SA, blaTEM-1, LA) to assess if any of the extrapolated qPCR rate

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constants were in the general range of the transformation efficiency rate constants. For the

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extrapolations, each qPCR data point was treated with equation 2 and linear regressions were

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conducted on the extrapolated data, resulting in four new predicted rate constants (tetA SA-

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extrap, tetA LA-extrap, blaTEM-1 SA-extrap, blaTEM-1 LA-extrap) and associated standard errors

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(Figures 5, Table S1). For both genes, extrapolated LA-qPCR rate constants were larger than the

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transformation assay rate constants (Figure 5,Table S2). Although the rate constants of the



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extrapolated SA-qPCR data were not statistically different than the transformation rate constants

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for both genes (Table S2, p > 0.05), t-tests on replicate data collected at the largest tested dose

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(54 mJ/cm2) did suggest that the extrapolated SA-qPCR overestimated the loss of gene

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transformation (p-values = 3.4×10-3 for tetA comparison and 5.0×10-3 for blaTEM-1 comparison).

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In summary, extrapolating any of the four amplicon first order rate constants measured by qPCR

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to the entire plasmid overestimated the loss in a gene’s ability to be transformed. The

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overestimation of the extrapolated qPCR results in predicting ARG transformation loss could be

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due to certain regions of the plasmid not being critical for the effective transformation of the

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antibiotic resistance genes.23 Another possible explanation is that the 25% of the plasmid

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measured by qPCR is much more sensitive to UV254 than the rest of the plasmid, and thus our

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extrapolated reaction rate constants overestimate the reaction rate constant of the entire plasmid.

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It is also possible that the A. baylyi cells repair the UV-damaged DNA upon uptake; if this is the

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case, then the damage we detect with qPCR does not necessarily inactivate the plasmid’s ability

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to be transformed in A. baylyi. Bacteriophage DNA damaged by UV254 can be repaired when it

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enters its host cell40 and the same may occur with plasmid DNA that enters the bacterial cell. A

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number of potential DNA repair mechanisms have been identified in Acinetobacter species.41,42

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When qPCR is applied to predict the inactivation of ARGs, we assume that the polymerase

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enzymes in the qPCR technique (Taq polymerase in this study) detect the same DNA

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modifications that inhibit the cellular mechanisms responsible for plasmid transformation and

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ARG expression. Research on the chemistry of DNA photolysis with UV254 suggests that

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cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproduct (6-4PP) are the major DNA

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products.43 Both modifications occur between two adjacent pyrimidines, with thymine-thymine

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(T-T) CPDs being the most prevalent product.43,44 The ability of polymerase enzymes in qPCR to 16 ACS Paragon Plus Environment

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read over DNA adducts depends on the type of modification, with T-T dimers having the biggest

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impact on amplification efficiency.45 It is also dependent on the polymerase used for

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amplification; Taq polymerases, which were used here, do not read over pyrimidine dimers.46

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The impacts of specific photoproducts on DNA transformation mechanisms have not been

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identified.

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To summarize the qPCR and transformation results, the ARG transformation inactivation rate

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constants did not correspond to the ARG rate constants measured by LA-qPCR. In this case, the

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LA-qPCR results underestimated ARG transformation loss. The transformation inactivation rate

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constants also did not correspond to the qPCR reaction rate constants when the results were

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extrapolated to the size of entire plasmid. In this case, the extrapolated qPCR results

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overestimated ARG transformation loss.

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As mentioned above, the blaTEM-1 gene first order reaction rate measured with qPCR were faster

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than the first order reaction rate of the tetA gene, despite the fact that the tetA gene is larger than

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the blaTEM-1 gene. Previously, McKinney and Pruden studied reactions in ARGs due to UV254

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radiation using qPCR and demonstrated that the disappearance of ARG amplicons correlated

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strongly with the number of adjacent T-T bases in the amplicon targets (r = -0.93). 17 In their

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analysis, neighboring thymidine bases on one of the two DNA strands were enumerated. We

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applied the same approach, but summed up adjacent pyrimidine bases on both DNA strands due

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to the fact that qPCR measures both strands (Table 2). Interestingly, the blaTEM-1 gene contains

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~1.5× the number of adjacent T-T bases than tetA, but is only 72% the length of tetA. The higher

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T-T base content of blaTEM-1 explains the LA-qPCR results; the blaTEM-1 gene reacted ~1.2 faster

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than tetA (Table 2; p < 0.05). Our analysis demonstrates that the T-T base content of DNA is

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arguably more important than DNA size when predicting a genome’s reactivity with UV254. 17 ACS Paragon Plus Environment


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Environmental Implications

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In this study, qPCR underestimated the loss of the DNA’s ability to be transformed into the

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Acinetobacter species, even when the entire resistance gene was amplified. In other words, qPCR

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detection of an ARG represents a conservative assessment of the gene’s ability to be transformed

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when it is released into the environment. When the qPCR results were extrapolated to the length

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of the entire plasmid, the results overestimated the loss of transformation efficiency. Future

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research should expand to other transformation models to see if the inactivation kinetics differs

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amongst bacterial species or types of extracellular DNA carrying the genes. When qPCR is

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employed to track the loss of ARGs capable of being transformed into competent bacterial cells,

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amplicons should therefore cover the entire gene sequence when possible. Although this will

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result in conservative estimates of the ability of the gene to be transformed, it will be far less

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conservative than when only a fragment of the gene is monitored.

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Supporting Information Available. Details on how plasmid concentrations were measured

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including figure of QubitTM and NanodropTM measurement comparison; details of the

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transformation assay optimization experiments including figure of A. baylyi growth curve, figure

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of plasmid concentration vs. transformation efficiency, and figure of transformation efficiency

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vs. incubation time; schematic of the UV disinfection apparatus; figure of ARG reaction kinetics

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measured with qPCR; tables of measured first-order rate constants and corresponding P-values

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from multiple linear regressions. This information is available free of charge via the Internet at

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http://pubs.acs.org.

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Author Information

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Corresponding author


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*Phone: +1- (734)-763-9661; e-mail: [email protected]

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Acknowledgments

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Funding for this research was provided through the University of Michigan MCubed program.

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We would also like to acknowledge the laboratory of Dr. Chuanwu Xi’s for assistance with

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transformation assays. The findings in this study do not represent the views of the sponsors.

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Table 1. Primers used in this study. Primer

Sequence (5’- 3’)

Primer Length

Amplicon Size

Annealing Temperature

Melting Temperature (℃ ℃)

(℃ ℃) tetA Long FW

CGTGTATGAAATCTAACAATGCGCT

25

tetA Long RV

CCATTCAGGTCGAGGTGGC

19

tetA Short FW

GACTATCGTCGCCGCACTTA

20

tetA Short RV

ATAATGGCCTGCTTCTCGCC

20

blaTEM-1 Long FW

TTACCAATGCTTAATCAGTGAGGC

24

blaTEM-1 Long RV

ATGAGTATTCAACATTTCCGTGTCG

25

blaTEM-1 Short FW

AATAAACCAGCCAGCCGGAA

20

blaTEM-1 Short RV

TTGATCGTTGGGAACCGGAG

20

1200

51.9

56.9 60.6

216

53.9

58.9 59.8

861

51.4

56.6 56.4

209

53.8

58.8 59.2

533 Table 2. Number of neighboring pyrimidine bases in amplicons. Dimer count*

Dimer count/amplicon length (%)

Amplicon Name (size bps)

TT

TC

CT

CC

total

TT

TC

CT

CC

Total

blaTEM-1 short (209)

30

21

35

41

127

14.4

10.0

16.7

19.6

60.8

tetA short (216)

18

32

26

26

102

8.3

14.8

12.0

12.0

47.2

blaTEM-1 long (861)

123

106

105

97

431

14.3

12.3

12.2

11.3

50.1

tetA long (1200)

80

149

132

199

560

6.7

12.4

11.0

16.6

46.7

* The TTTT sequence was counted as 3 neighboring thymidines (T1T2T3T)


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Figure 1. Schematic depicting experimental approach. Purified plasmids containing ARGs are treated with UV254. The treated plasmids are subjected to transformation assays in Acinetobacter bayli to measure the extent that UV254 treatment has reduced the transformation efficiency of the ARGs on the plasmids. The treated plasmid samples are also analyzed with short amplicon- and long amplicon-qPCR to measure the reaction kinetics of ARGs on the plasmids. The qPCR and transformation reaction kinetics are compared. Gel electrophoresis is applied to assess the extent of plasmid DNA breaks following UV254 treatment.



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Figure 2. Plasmid 1266, which is a construct of E. coli plasmid pBR322 and A. calcoaceticus cryptic plasmid pWH1277 (grey portion). 534 535 536 537 538 539 540 541


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Figure 3. tetA and blaTEM-1 degradation versus UV dose measured with qPCR short amplicon (SA), long amplicon (LA), and transformation assays. The error bars indicate one standard deviation from the mean (n≥3). For visibility, data has been pooled. 542



Figure 4. DNA electrophoresis gel of pHW1266 plasmids treated with 0 (control), 11, 32, 54, 108 mJ/cm2 UV or with restriction enzymes BamHI and PvuI. The transformation efficiencies of the samples are labeled at the bottom of the gel. 543


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544 1.0

1.0

tetA

0.8

0.4

k (cm2/mJ)

k (cm2/mJ)

0.8 0.6

blaTEM-1

0.6 0.4 0.2

0.2

0.0

0.0

Figure 5. Rate constants corresponding to SA-qPCR, LA-qPCR, extrapolated qPCR, and transformation assays. The values of k are included in the SI Table S1. The error bars represent 95% confidence intervals. qPCR rate constants were analyzed over the first 90% decrease in copy number concentrations. The rate constant values and statistical differences between rate constants are presented in Tables S1 and S2. 545 546


Degradation of Extracellular Antibiotic Resistance Genes with UV254

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