Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance
Summary
Here, we present a simple technique to assess environmental antimicrobial resistance (AMR) by enhancing the proportion of low-molecular-weight extracellular DNA. Prior treatment with 20%-30% PEG and 1.2 M NaCl allows detection of both genomic and horizontally transferred AMR genes. The protocol lends itself to a kit-free process with additional optimization.
Abstract
Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has emerged as the source of choice to sample pathogen burdens in the environment. Wastewater from open drains and community water treatment plants is a reservoir of both pathogens and antimicrobial resistance (AMR) genes, and frequently comes in contact with humans. While there are many methods of tracking AMR from water, isolating good-quality DNA at high yields from heterogeneous samples remains a challenge. To compensate, sample volumes often need to be high, creating practical constraints. Additionally, environmental DNA is frequently fragmented, and the sources of AMR (plasmids, phages, linear DNA) consist of low-molecular-weight DNA. Yet, few extraction processes have focused on methods for high-yield extraction of linear and low-molecular-weight DNA. Here, a simple method for high-yield linear DNA extraction from small volumes of wastewater using the precipitation properties of polyethylene glycol (PEG) is reported. This study makes a case for increasing overall DNA yields from water samples collected for metagenomic analyses by enriching the proportion of linear DNA. In addition, enhancing low-molecular-weight DNA overcomes the current problem of under-sampling environmental AMR due to a focus on high-molecular-weight and intracellular DNA. This method is expected to be particularly useful when extracellular DNA exists but at low concentrations, such as with effluents from treatment plants. It should also enhance the environmental sampling of AMR gene fragments that spread through horizontal gene transfer.
Introduction
SARS-CoV-2 and its aftermath underlined the importance of environmental surveillance in monitoring and predicting infectious disease outbreaks1,2. While viral pandemics are apparent, the rise of antimicrobial resistance (AMR) is often described as an insidious pandemic and one that constitutes a leading public health concern across the world3,4. Consequently, there is an urgent need for coordinated strategies to understand the evolution and spread of AMR. Water bodies, as well as wastewater, can serve as reservoirs for both pathogens and AMR5,6,7,8. Shared water sources are, therefore, a potent source of disease transmission among humans, particularly in low and middle-income countries (LMIC) where poor hygiene and over-population go hand in hand9,10,11. Testing of water sources has long been employed to assess community health12,13,14. Recently, wastewater from urban sewage treatment plants proved a good advance indicator of COVID cases in the clinic1,2,15,16,17,18.
Compared with monitoring specific diseases, detecting and tracking AMR in the environment poses a more complex problem. The large number of antibiotics in use, diverse resistance genes, different local selection pressures, and horizontal gene transfer among bacteria make it difficult to assess true AMR burden and, once assessed, to correlate it with clinical observations19,20,21,22. As a result, while concerted surveillance of clinical AMR is being carried out by several organizations across the world3,23,24, environmental AMR monitoring is still in its infancy, reviewed in19,25,26.
In recent years, different methods for tracking environmental AMR have been reported5,27, reviewed in28,29. The starting point of most of these is the extraction of good quality DNA from heterogenous environmental samples, in itself a challenge. Additionally, environmental DNA is typically fragmented because of exposure to hostile surroundings. Fragmented extracellular DNA has long been recognized as an important reservoir of AMR genes (reviewed in30,31,32), with the added potential to enter and leave bacteria via horizontal gene transfer. Hence, it is important that any protocol that aims to measure AMR burden in the environment should sample linear and low-molecular-weight DNA as best as possible. Surprisingly, there has been little focus on developing methods specific to high-yield extraction of linear and low-molecular-weight DNA: this work focuses on addressing the gap.
A common and simple method to precipitate DNA is to combine polyethylene glycol (PEG) and salts such as sodium chloride (NaCl)33. PEG is a macromolecular crowding agent used to achieve size-specific precipitation of DNA fragments34,35. The lower the PEG concentration, the higher the molecular weight of DNA that can be efficiently precipitated. Many studies have used PEG during environmental extraction of DNA and RNA1,2 (summarized in Table 117,33,36,37,38,39) either in the final step 33,36,37or to concentrate large water samples for extraction of viral particles as with SARS-CoV-215,40. In the current work, it is found that the PEG concentrations used previously for environmental DNA extractions (largely determined by viral surveillance protocols) do not capture low-molecular weight linear DNA. Therefore, they lose out on sampling short DNA fragments and are unsuitable for assessing AMR content accurately. This study has exploited the properties of polyethylene glycol and sodium chloride to effectively precipitate low-molecular weight linear DNA fragments at a high yield that can, in the future, lead to a cost-effective DNA extraction method. This method can be used to enrich the proportion of fragmented and low-molecular-weight DNA from complex natural samples, thus capturing a more accurate picture of environmental AMR. With a little further refinement, the technique lends itself to easy and low-cost application by local municipal corporations and other government bodies to use as a surveillance tool with minimal technical training.
Protocol
Representative Results
Discussion
AMR is one of the top 10 health threats today, as listed by the WHO, and environmental surveillance for AMR is recognized as an important tool across the world. As mentioned in the introduction, a comprehensive record of environmental AMR includes low-molecular-weight, fragmented, and extracellular DNA. The pre-processing protocol reported here using a high concentration of PEG combined with salt (30% PEG and 1.2 M NaCl) achieves this result by enriching the proportion of low-molecular-weight DNA without impacting extrac…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We acknowledge funding support from the Rockefeller Foundation (Rockefeller Foundation Grant Number 2021 HTH 018) as part of the APSI India team (Alliance for Pathogen Surveillance Innovations https://data.ccmb.res.in/apsi/team/). We also acknowledge the financial aid provided by Axis Bank in supporting this research and the Trivedi School of Biosciences at Ashoka University for equipment and other support.
Materials
| 24-seat microcentrifuge | Eppendorf Centrifuge 5425 R | EP5406000046 | |
| Absolute Ethanol (Emsure ACS, ISO, Reag. Ph Eur Ethanol absolute for analysis) | Supelco | 100983-0511 | |
| Agarose | Invitrogen | 16500500 | |
| Bench top refrigerated centrifuge | Eppendorf Centrifuge 5920 R | EP5948000131 | |
| ChemiDoc Imaging System | BioRad | 12003153 | |
| DNeasy PowerSoil Pro Kit | Qiagen | 47014 | |
| DNeasy PowerWater Pro Kit | Qiagen | 14900-100-NF | |
| dNTPs (dNTP Mix 10mM Each,0.2 mL, R0191) | Thermo Fisher | R0191 | |
| DreamTaq DNA Polymerase, 5 U/µL + 10x DreamTaq Buffer* | Thermofscientific | EP0702 | |
| E-Gel 1 Kb Plus Express DNA Ladder | Invitrogen | 10488091 | |
| Maxiamp PCR tubes 0.2 mL | Tarsons | 510051 | |
| Molecular Biology Grade Water for PCR | HiMedia | ML065-1.5ML | |
| NanoDrop OneC Microvolume UV-Vis Spectrophotometer | Thermo Scientific | 13400519 | |
| Parafilm | Bemis | S37440 | |
| PEG-8000 | SRL | 54866 | |
| QIAquick PCR & Gel Cleanup Kit | Qiagen | 28506 | |
| Qubit 4 Fluorometer (with WiFi) | Thermofisher | Q33238 | |
| Qubit Assay Tubes | Thermofisher | Q32856 | |
| Qubitt reagent kit for ds DNA, broad range | Thermo Scientific | Q32853 (500 assays) | |
| Sodium Chloride | HiMedia | TC046M-500G | |
| SYBR Safe DNA Gel Stain | Invitrogen | S33102 | |
| T100 Thermal Cycler | BioRad | 1861096 | |
| Thermo Cycler (ProFlex 3 x 32-well PCR System) | Applied Biosystems | 4484073 | |
| Wizard Genomic DNA Purification Kit | Promega | A1125 |
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