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 outbreaks^1,2. 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 world^3,4. 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 AMR^5,6,7,8. 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 hand^9,10,11. Testing of water sources has long been employed to assess community health^12,13,14. Recently, wastewater from urban sewage treatment plants proved a good advance indicator of COVID cases in the clinic^{1,2,15,16,17,18}.

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 observations^19,20,21,22. As a result, while concerted surveillance of clinical AMR is being carried out by several organizations across the world^3,23,24, environmental AMR monitoring is still in its infancy, reviewed in^19,25,26.

In recent years, different methods for tracking environmental AMR have been reported^5,27, reviewed in^28,29. 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 in^30,31,32), 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 fragments^34,35. 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 RNA^1,2 (summarized in Table 1^{17,33,36,37,38,39}) either in the final step ^33,36,37or to concentrate large water samples for extraction of viral particles as with SARS-CoV-2^15,40. 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

1. Wastewater sampling

1. Dip a 500 mL polypropylene beaker in the open drain or sewage treatment plant (STP) reservoir and collect ~300 mL of wastewater sample.
2. Transfer ~250 mL of the sample to a 250 mL autoclaved polypropylene bottle.
3. Screw on the cap of the bottle and seal it with a plastic film. Keep the bottle upright in a closed bag.
4. Transport the sample upright in a closed container at ambient temperature.
5. Heat-inactivate the sample at 70 °C in a hot air oven for 4 h before processing it for DNA extraction.

2. DNA extraction from wastewater samples

1. Once the wastewater sample is cooled, vortex the samples at maximum speed and let the debris/sludge settle.
2. Decant 27.5 mL of the wastewater in 50 mL centrifuge tubes and add 13.5 g of PEG-8000 (final concentration 30%) and 3 g of NaCl (final concentration 1.2 M) to it and mix well till completely dissolved.
3. Incubate the sample overnight (~16-18 h) at 4 °C.
4. Centrifuge the sample at 15,500 x g at 4 °C for 30 min.
5. Discard the supernatant.
   NOTE: PEG-8000 at 30% is highly viscous, and the pellet can get dislodged while discarding the supernatant. The supernatant should be decanted slowly and carefully to ensure the pellet is not lost. Steps 2.6 to 2.23 are performed using the Soil DNA extraction kit according to the kit instructions with a few modifications.
6. Dissolve the pellet in 800 µL of the lysis solution of the kit and add the solution to the mixed zirconium bead tube.
   NOTE: If processing larger volumes, pool the pellets from the desired number of tubes. For example, if processing 160 mL of sample; there will be four 50 mL tubes containing wastewater with PEG and NaCl. After centrifugation of all four tubes at 15,500 x g at 4 °C for 30 min, discard supernatant from all four tubes. Add 200 µL of CD1 solution to each, re-suspend the pellets, and pool them together in one bead tube.
7. Secure the bead tube horizontally on a vortex. Vortex the tube at maximum speed for 20-30 min.
   NOTE: Prolonged vortexing ensures complete lysis and homogenization of the samples, which improves DNA yield.
8. Spin down the tubes at 8,000 x g for 15 s to settle the beads at the bottom, remove frothing, and transfer the supernatant completely from the tube to a 1.5 mL microcentrifuge tube. Some beads may also get transferred during this step.
9. Centrifuge the supernatant at 15,000 x g for 1 min.
10. Transfer the supernatant to a clean 2 mL microcentrifuge tube. The supernatant may still contain some suspended particles.
11. Add 200 µL of precipitant solution and vortex at maximum speed for 5 s. Incubate at 4 °C for 5 min.
    NOTE: Incubation at 4 °C increases the efficiency of precipitation of proteins and cellular debris while DNA remains in solution. It is possible to pause the protocol at this step for up to 2 h without a significant decrease in the yield and quality of the extracted DNA.
12. Centrifuge at 15,000 x g for 1 min at room temperature (RT). Avoid the pellet and transfer the clear supernatant to a clean 2 mL microcentrifuge tube.
13. Add 600 µL of binding buffer per 700 µL of supernatant and vortex at maximum speed for 5 s.
14. Load 700 µL of the lysate onto a silica spin column, incubate for 2 min, and centrifuge at 15,000 x g for 1 min.
    NOTE: Incubation of lysate on silica column enhances the binding of DNA to the column, resulting in better DNA yield.
15. Discard the flow-through and repeat step 2.14 until all the lysate has passed through the spin column.
16. Carefully place the spin column into a clean 2 mL collection tube. Ensure that no flow-through is splashed onto the spin column.
17. Add 500 µL of wash buffer to the spin column and centrifuge the spin column at 15,000 x g for 1 min.
18. Discard the flow-through and return the spin column to the same 2 mL collection tube.
19. Add 500 µL of ethanol-wash buffer to the spin column. Centrifuge at 15,000 x g for 1 min.
20. Discard the flow-through and place the spin column into a new 2 mL collection tube.
21. Centrifuge at 16,000 x g for 2 min to remove residual ethanol. Place the spin column into a new 1.5 mL tube.
22. Add 100 µL of elution buffer (pre-heated to 55 °C) to the center of the white filter membrane and incubate for 5 min.
    NOTE: Heated elution buffer, along with incubation on the membrane, increases the efficiency of DNA elution, especially high-molecular-weight DNA.
23. Centrifuge at 15,000 x g for 1 min. Discard the spin column.
24. To the eluted DNA, add 10 µL of 3 M NaCl (1/10 volume of DNA eluate) and 250 µL of chilled absolute ethanol (2.5 volume of DNA eluate). Invert to mix well and spin down the contents at 8,000 x g for 15 s.
25. Incubate the DNA-ethanol mixture at -20 °C for at least 1 h.
    NOTE: The incubation of the DNA-ethanol mixture can be increased to 16 h without a significant decrease in the yield or quality of the extracted DNA.
26. Centrifuge at 19,000 x g at 4 °C for 30 min. Discard the supernatant by decantation.
27. Add 500 µL of chilled 70% ethanol to the pellet.
28. Centrifuge at 19,000 x g at 4 °C for 15 min. Discard the supernatant by decantation.
29. Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.
30. Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 °C till all ethanol has evaporated.
31. Re-suspend the DNA pellet in 30-50 µL of autoclaved double distilled water (ddH₂O). Incubate at 37 °C for 5-10 min.
32. Spin down the DNA at 8,000 x g for 15 s.
33. Select the dsDNA application under the Nucleic Acids settings in a Nanodrop spectrophotometer.
    1. Clean the pedestal with lint-free tissue paper and water. Use ddH₂O to blank the instrument and then load 2 µL of the DNA sample on the pedestal.
    2. Measure the concentration and absorbance ratios (A260/280 and A260/230) to determine the purity.
34. Store the DNA sample at -20 °C.

3. Precipitation of polymerase chain reaction (PCR)-amplified linear DNA to check DNA recovery across a range of molecular weights

1. Using genomic DNA from E. coli MG155, amplify gene fragments by PCR to generate a pool of linear fragments from the following genes: fusA, lacZ, gapA, rpsD, 16S rRNA, marR (see Table 2 for primer sequences and PCR conditions).
2. Purify the PCR products using the PCR purification kit and pool the PCR products together.
3. Divide the pooled amplicons into 1.5 mL microcentrifuge tubes with equal volumes- label one tube as 'Input DNA'.
4. Add PEG and NaCl according to the desired conditions (9%, 20%, 30% PEG-8000; 0.3 M, 1.2 M NaCl) to each of the tubes except the one labeled 'Input DNA' and make up the final volume to 1 mL.
5. Incubate the tubes overnight (~16-18 h) at 4 °C.
   NOTE: For the rest of the steps till 3.16, 'Input DNA' is kept unchanged in the fridge.
6. Spin the tubes in a table-top microcentrifuge according to the desired speed (15,000 x g or 20,000 x g) for 1 h at 4 °C.
7. Carefully remove 900 µL of the supernatant from the side opposite the pellet using a micropipette.
8. First ethanol wash: Add 900 µL of chilled 70% ethanol to the pellet and mix by gently inverting the tubes.
9. Spin the tubes at the same speed as used in step 3.6 (15,000 x g or 20,000 x g) for 30 min at 4 °C.
10. Remove the supernatant by decantation.
11. Second ethanol wash: Add 500 µL of chilled 70% ethanol to the pellet.
12. Spin the tubes at the same speed as used in step 3.6 (15,000 x g or 20,000 x g) for 30 min at 4°C. Discard the supernatant by decantation.
13. Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.
14. Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 °C until all ethanol evaporates.
15. Re-suspend the pellet in 20 µL of autoclaved double-distilled water.
16. Measure the concentration of DNA precipitated by the different conditions and the 'Input DNA' using a Nanodrop spectrophotometer or fluorometer.
17. Load the precipitated DNA on a 1% agarose gel for DNA visualization.

  

Results

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...

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...

Materials

Name	Company	Catalog Number	Comments
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