skip to main content

Title: Elimination of transforming activity and gene degradation during UV and UV/H 2 O 2 treatment of plasmid-encoded antibiotic resistance genes
To better understand the elimination of transforming activity of antibiotic resistance genes (ARGs), this study investigated the deactivation of transforming activity of an ARG (in Escherichia coli as a host) and ARG degradation (according to quantitative PCR [qPCR] with different amplicon sizes) during UV (254 nm) and UV/H 2 O 2 treatments of plasmid pUC19 containing an ampicillin resistance gene ( amp R ). The required UV fluence for each log 10 reduction of the transforming activity during UV treatment was ∼37 mJ cm −2 for both extra- and intra-cellular pUC19 (the latter within E. coli ). The resulting fluence-based rate constant ( k ) of ∼6.2 × 10 −2 cm 2 mJ −1 was comparable to the k determined previously for transforming activity loss of plasmids using host cells capable of DNA repair, but much lower (∼10-fold) than that for DNA repair-deficient cells. The k value for pUC19 transforming activity loss was similarly much lower than the k calculated for cyclobutane-pyrimidine dimer (CPD) formation in the entire plasmid. These results indicate the significant role of CPD repair in the host cells. The degradation rate constants ( k ) of amp R measured by qPCR increased with increasing target amplicon size (192–851 bp) and were close to the k calculated for the CPD formation in the given amplicons. Further analysis of the degradation kinetics of plasmid-encoded genes from this study and from the literature revealed that qPCR detected most UV-induced DNA damage. In the extracellular plasmid, DNA damage mechanisms other than CPD formation ( e.g. , base oxidation) were detectable by qPCR and gel electrophoresis, especially during UV/H 2 O 2 treatment. Nevertheless, the enhanced DNA damage for the extracellular plasmids did not result in faster elimination of the transforming activity. Our results indicate that calculated CPD formation rates and qPCR analyses are useful for predicting and/or measuring the rate of DNA damage and predicting the efficiency of transforming activity elimination for plasmid-encoded ARGs during UV-based water disinfection and oxidation processes.  more » « less
Award ID(s):
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Environmental Science: Water Research & Technology
Page Range / eLocation ID:
1239 to 1251
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells.

    more » « less
  2. Abstract Background

    There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents.


    The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75–90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40–73 to 31–68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35–13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4–2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination.


    Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention.

    more » « less
  3. The main objective of this study was to investigate the effectiveness of ultraviolet light (UV-C) emitting diodes for the decontamination of stainless steel food contact surfaces. Listeria monocytogenes (ATCC 19115), Escherichia coli (ATCC 25922), and Salmonella enterica serovar Typhimurium (ATCC 700720) were chosen as challenge microorganisms. Target microorganisms were subjected to UV-C dosages of 0, 2, 4, 6, and 8 mJ cm −2 at an average fluence of 0.163 mW/cm 2 using a near-collimated beam operating at 279 nm wavelength. Escherichia coli showed lower sensitivity to UV-C light compared to Salmonella Typhimurium and followed first-order kinetics. Escherichia coli and Salmonella Typhimurium were reduced by more than 3-log 10 cycles at the maximum UV dosage of 12 mJ cm −2 . In contrast, Listeria monocytogenes followed the Weibull model with an apparent shoulder in the initial doses. A maximum reduction of 4.4-log 10 was achieved at the highest exposure level. This study showed that UV-C LED devices represent an excellent alternative for the inactivation of foodborne microorganisms in droplets. Results clearly demonstrate that UV-C LED devices can serve as an additional sanitation method to routine cleaning practices, which are commonly utilized in the food industry. 
    more » « less
  4. After decades of UV disinfection practice and numerous studies on the potential for pathogens to undergo dark or photo-repair after UV exposure, recent advances in UV light emitting diode (LED) technologies prompt renewed attention to bacterial reactivation and regrowth processes after UV exposure. The aspect of photorepair conditions warrants particular attention, because even studies on conventional mercury vapor lamps have not sufficiently characterized these parameters. Wastewater encounters a wide range of environmental conditions upon discharge ( e.g. , solar irradiation and dissolved organics) which may affect repair processes and ultimately lead to overestimations of pathogen removal. Escherichia coli was used here to investigate the impacts of changing reactivation conditions after UV 254 and UV 278 irradiation. UV 254 and UV 278 doses of 13.75 ± 0.4 mJ cm −2 and 28.3 ± 0.8 mJ cm −2 were required to induce a 3.0 log inactivation of E. coli , respectively. Specifically, photoreactivation conditions were varied across dissolved organic matter (DOM) content and photoreactivation wavelengths and intensities. Photoreactivation achieved higher log recoveries than dark repair, ranging from 0.8 to 1.8 log differences, but a secondary disinfection effect occurred under UVA irradiation. During photoreactivation, humic acid inhibited the initial repair of UV 278 -dosed E. coli , but culture media enhanced recovery for both dosage wavelengths. Photoreactivation profiles under UV 395 , UV 365 , and visible light depended on both fluence and time, with more regrowth observed upon exposure to visible light and the least under 365 nm. The susceptibility of E. coli to UVA was increased by prior exposure to UVC. 
    more » « less
  5. Abstract Sequencing of melanomas has identified hundreds of recurrent mutations in both coding and non-coding DNA. These include a number of well-characterized oncogenic driver mutations, such as coding mutations in the BRAF and NRAS oncogenes, and non-coding mutations in the promoter of telomerase reverse transcriptase ( TERT ). However, the molecular etiology and significance of most of these mutations is unknown. Here, we use a new method known as CPD-capture-seq to map UV-induced cyclobutane pyrimidine dimers (CPDs) with high sequencing depth and single nucleotide resolution at sites of recurrent mutations in melanoma. Our data reveal that many previously identified drivers and other recurrent mutations in melanoma occur at CPD hotspots in UV-irradiated melanocytes, often associated with an overlapping binding site of an E26 transformation-specific (ETS) transcription factor. In contrast, recurrent mutations in the promoters of a number of known or suspected cancer genes are not associated with elevated CPD levels. Our data indicate that a subset of recurrent protein-coding mutations are also likely caused by ETS-induced CPD hotspots. This analysis indicates that ETS proteins profoundly shape the mutation landscape of melanoma and reveals a method for distinguishing potential driver mutations from passenger mutations whose recurrence is due to elevated UV damage. 
    more » « less