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 meta bar coding, 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 notational 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. To begin, decant the 27.5 milliliters of the wastewater in 50 milliliters centrifuge tubes.

Add 13.5 grams of polyethylene glycol 8, 000, 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, 500 G 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, 000 G for 15 seconds. After removing the froth, transfer the supernatant to a 1.5 milliliter micro centrifuge tube. Centrifuge the supernatant at 15, 000 G 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, 000 G 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, 000 G 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, 000 G for one minute. Discard the flow through, and place the spin column into a new, two milliliter collection tube. Centrifuge at 16, 000 G for two minutes to remove residual the 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, 000 G for one minute.

To the eluded 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, 000 G four 15 seconds. Incubate the DNA ethanol mixture at minus 20 degrees celsius for one hour.

Then, centrifuge the mixture at 19, 000 G 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, 000 G 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 the 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, 000 G 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.