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

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 i.......

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