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&#160;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.

1. Wastewater sampling
<ol>
	<li>Dip a 500 mL polypropylene beaker in the open drain or sewage treatment plant (STP) reservoir and collect ~300 mL of wastewater sample.</li>
	<li>Transfer ~250 mL of the sample to a 250 mL autoclaved polypropylene bottle.</li>
	<li>Screw on the cap of the bottle and seal it with a plastic film. Keep the bottle upright in a closed bag.</li>
	<li>Transport the sample upright in a closed container at ambient temperature.</li>
	<li>Heat-inactivate the sample at 70 &#176;C i...

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

The authors declare no conflicts of interest.

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>24-seat microcentrifuge</td>
			<td>Eppendorf Centrifuge 5425 R</td>
			<td>EP5406000046</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute Ethanol (Emsure ACS, ISO, Reag. Ph Eur Ethanol absolute for analysis)</td>
			<td>Supelco</td>
			<td>100983-0511</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Invitrogen</td>
			<td>16500500</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench top refrigerated centrifuge</td>
			<td>Eppendorf Centrifuge 5920 R</td>
			<td>EP5948000131</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemiDoc Imaging System</td>
			<td>BioRad</td>
			<td>12003153</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy PowerSoil Pro Kit</td>
			<td>Qiagen</td>
			<td>47014</td>
			<td></td>
		</tr>
		<tr>
			<td>DNeasy PowerWater Pro Kit</td>
			<td>Qiagen</td>
			<td>14900-100-NF</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (dNTP Mix 10mM Each,0.2 mL, R0191)</td>
			<td>Thermo Fisher</td>
			<td>R0191</td>
			<td></td>
		</tr>
		<tr>
			<td>DreamTaq DNA Polymerase, 5 U/&#181;L + 10x DreamTaq Buffer*</td>
			<td>Thermofscientific</td>
			<td>EP0702</td>
			<td></td>
		</tr>
		<tr>
			<td>E-Gel 1 Kb Plus Express DNA Ladder</td>
			<td>Invitrogen</td>
			<td>10488091</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiamp PCR tubes 0.2 mL</td>
			<td>Tarsons</td>
			<td>510051</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Biology Grade Water for PCR</td>
			<td>HiMedia</td>
			<td>ML065-1.5ML</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop OneC Microvolume UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>13400519</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>S37440</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG-8000</td>
			<td>SRL</td>
			<td>54866</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR &#38; Gel Cleanup Kit</td>
			<td>Qiagen</td>
			<td>28506</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 4 Fluorometer (with WiFi)</td>
			<td>Thermofisher</td>
			<td>Q33238</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Thermofisher</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubitt reagent kit for ds DNA, broad range</td>
			<td>Thermo Scientific</td>
			<td>Q32853 (500 assays)</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>HiMedia</td>
			<td>TC046M-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>BioRad</td>
			<td>1861096</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Cycler (ProFlex 3 x 32-well PCR System)</td>
			<td>Applied Biosystems</td>
			<td>4484073</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard Genomic DNA Purification Kit</td>
			<td>Promega</td>
			<td>A1125</td>
			<td></td>
		</tr>
	</tbody>
</table>

enhanced extraction of low-molecular weight dna from wastewater for comprehensive assessment of antimicrobial resistance 

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,&#160;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.

Author Spotlight: Understanding and Detecting Environmental Antimicrobial Resistance by Combining Culture-Based Techniques and Genomics

Enhanced Extraction of Low-Molecular Weight DNA from Wastewater for Comprehensive Assessment of Antimicrobial Resistance 

We are interested in the evolution, transmission, and molecular mechanisms underlying antibiotic resistance. Specifically, the work that feeds into this paper comes from our interest in environmental resistance. Currently, we're trying to build a local database to track spatial temporal variation in antimicrobial resistance using one year's worth of data.A combination of culture-based techniques and genomics is used to detect and monitor antimicrobial resistance. DNA from samples undergo PCR or shotgun sequencing to profile microbial diversity and detect resistance genes. Additionally, metabarcoding and gene-based AMR panels are used for advanced AMR detection.Fragmented low molecular weight DNA is known to be a reservoir of AMR genes, but there has been little focus on developing methods specific to high-yield extraction of linear and low molecular weight DNA. Our work focuses on addressing this gap. Our protocol introduces a simple pre-processing step to enrich the proportion of low molecular weight DNA extracted from wastewater.As a result, this captures environmental AMR in its entirety without excluding free DNA fractions. This protocol can be developed into a kit-free method with a little more work. In turn, this paves the way for development of cost-effective techniques for capturing environmental AMR.We want to go beyond mutational resistance and explore the contribution of non-genetic mechanisms to antibiotic resistance. We are interested specifically in comparing the relative contributions of horizontal gene transfer and genomic mutations to adaptation to antibiotics in different environments.

Establishment of a protocol for high-yield extraction of DNA from wastewater samples 
A modified version of previously established protocols was used for the extraction of high-quality DNA and RNA from water samples17. The samples were sourced from open drains as well as sewage treatment plants in the Delhi-NCR region of North India. After pre-processing using PEG and NaCl (Figure 1), the samples were processed th...

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.

Environmental surveillance is recognized as an important tool for assessing public health in the post-pandemic era. Water, in particular wastewater, has ...

enhanced-extraction-low-molecular-weight-dna-from-wastewater-for

Research

JoVE Journal

Biology

Optimized Extraction of Low-Molecular Weight DNA from Wastewater Samples

To begin, decant the 27.5 milliliters of the wastewater in 50 milliliter centrifuge tubes. Add 13.5 grams of polyethylene glycol 8000 and three grams of sodium chloride to the tubes and mix well until completely dissolved. Incubate the sample overnight at four degrees Celsius.The next day, centrifuge the sample at 15, 500g for 30 minutes at four degrees Celsius. Discard the supernatant and dissolve the pellet in 800 microliters of the lysis solution from the soil DNA extraction kit. Then add the solution to the mixed zirconium bead tube.Secure the bead tube horizontally on a vortex and vortex at maximum speed for 20 to 30 minutes. Spin down the tubes at 8, 000g for 15 seconds. After removing the froth, transfer the supernatant to a 1.5 milliliter micro centrifuge tube.Centrifuge the supernatant at 15, 000g for one minute. Transfer the supernatant to a clean two milliliters micro centrifuge tube and add 200 microliters of precipitant solution. Vortex at maximum speed for five seconds before incubating at four degrees Celsius for five minutes.Again, centrifuge the mixture and transfer the clear supernatant to a clean two milliliter micro centrifuge tube. Add 600 microliters of binding buffer per 700 microliters of supernatant. After vortexing, load 700 microliters of the lysate onto a silica spin column and incubate for two minutes.Centrifuge the tube at 15, 000g for one minute and discard the flow-through. Now carefully place the spin column into a clean two milliliter collection tube. Add 500 microliters of wash buffer to the spin column and centrifuge at 15, 000g for one minute.Discard the flow-through and return the spin column to the same two milliliter collection tube. Add 500 microliters of ethanol wash buffer to the spin column and centrifuge at 15, 000g for one minute. Discard the flow-through and place the spin column into a new two milliliter collection tube.Centrifuge at 16, 000g for two minutes to remove residual ethanol. Place the spin column into a 1.5 milliliter tube. Add 100 microliters of preheated nuclease-free water to the center of the white filter membrane and incubate for five minutes.Then centrifuge the tube at 15, 000g for one minute. To the eluted DNA, add 10 microliters of three molar sodium chloride and 250 microliters of chilled absolute ethanol and invert to mix well. Spin down the contents at 8, 000g for 15 seconds.Incubate the DNA ethanol mixture at minus 20 degrees Celsius for one hour. Then centrifuge the mixture at 19, 000g for 30 minutes at four degrees Celsius and decant the tube to discard the supernatant. Add 500 microliters of chilled 70%ethanol to the pellet and centrifuge at 19, 000g for 15 minutes at four degrees Celsius.Decant the supernatant and gently tap the tube on tissue paper to remove residual ethanol. Then place the tubes open on a heating block for five to 10 minutes at 37 degrees Celsius to dry the DNA pellet. Resuspend the pellet in 30 to 50 microliters of nuclease-free water and incubate at 37 degrees Celsius for five to 10 minutes.Spin down the DNA at 8, 000g for 15 seconds. On a NanoDrop spectrophotometer under the nucleic acid settings, select the double stranded DNA application. Clean the pedestal with lint-free tissue paper and water.Use nuclease-free water to blank the instrument, then wipe the pedestal and load two microliters of the DNA sample on it. Measure the concentration and absorbance ratios to determine the purity. After measurement, store the sample at minus 20 degrees Celsius.

525 Views.  Ashoka University.  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.

Efficient Technique for Assessing Environmental AMR by Enriching Low-MW DNA

Summary