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 in a hot air oven for 4 h before processing it for DNA extraction.</li>
</ol>
2. DNA extraction from wastewater samples
<ol>
	<li>Once the wastewater sample is cooled, vortex the samples at maximum speed and let the debris/sludge settle.</li>
	<li>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.</li>
	<li>Incubate the sample overnight (~16-18 h) at 4 &#176;C.</li>
	<li>Centrifuge the sample at 15,500 x g at 4 &#176;C for 30 min.</li>
	<li>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.</li>
	<li>Dissolve the pellet in 800 &#181;L 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 &#176;C for 30 min, discard supernatant from all four tubes. Add 200 &#181;L of CD1 solution to each, re-suspend the pellets, and pool them together in one bead tube.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Centrifuge the supernatant at 15,000 x g for 1 min.</li>
	<li>Transfer the supernatant to a clean 2 mL microcentrifuge tube. The supernatant may still contain some suspended particles.</li>
	<li>Add 200 &#181;L of precipitant solution and vortex at maximum speed for 5 s. Incubate at 4 &#176;C for 5 min. 
	NOTE:&#160;Incubation at 4 &#176;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.</li>
	<li>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.</li>
	<li>Add 600 &#181;L of binding buffer per 700 &#181;L of supernatant and vortex at maximum speed for 5 s.</li>
	<li>Load 700 &#181;L 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.</li>
	<li>Discard the flow-through and repeat step 2.14 until all the lysate has passed through the spin column.</li>
	<li>Carefully place the spin column into a clean 2 mL collection tube. Ensure that no flow-through is splashed onto the spin column.</li>
	<li>Add 500 &#181;L of wash buffer to the spin column and centrifuge the spin column at 15,000 x g for 1 min.</li>
	<li>Discard the flow-through and return the spin column to the same 2 mL collection tube.</li>
	<li>Add 500 &#181;L of ethanol-wash buffer to the spin column. Centrifuge at 15,000 x g for 1 min.</li>
	<li>Discard the flow-through and place the spin column into a new 2 mL collection tube.</li>
	<li>Centrifuge at 16,000 x g for 2 min to remove residual ethanol. Place the spin column into a new 1.5 mL tube.</li>
	<li>Add 100 &#181;L of elution buffer (pre-heated to 55 &#176;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.</li>
	<li>Centrifuge at 15,000 x g for 1 min. Discard the spin column.</li>
	<li>To the eluted DNA, add 10 &#181;L of 3 M NaCl (1/10 volume of DNA eluate) and 250 &#181;L 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.</li>
	<li>Incubate the DNA-ethanol mixture at -20 &#176;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.</li>
	<li>Centrifuge at 19,000 x g at 4 &#176;C for 30 min. Discard the supernatant by decantation.</li>
	<li>Add 500 &#181;L of chilled 70% ethanol to the pellet.</li>
	<li>Centrifuge at 19,000 x g at 4 &#176;C for 15 min. Discard the supernatant by decantation.</li>
	<li>Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.</li>
	<li>Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 &#176;C till all ethanol has evaporated.</li>
	<li>Re-suspend the DNA pellet in 30-50 &#181;L of autoclaved double distilled water (ddH2O). Incubate at 37 &#176;C for 5-10 min.</li>
	<li>Spin down the DNA at 8,000 x g for 15 s.</li>
	<li>Select the dsDNA application under the Nucleic Acids settings in a Nanodrop spectrophotometer.
	<ol>
		<li>Clean the pedestal with lint-free tissue paper and water. Use ddH2O to blank the instrument and then load 2 &#181;L of the DNA sample on the pedestal.</li>
		<li>Measure the concentration and absorbance ratios (A260/280 and A260/230) to determine the purity.</li>
	</ol>
	</li>
	<li>Store the DNA sample at -20 &#176;C.</li>
</ol>
3.&#160;Precipitation of polymerase chain reaction (PCR)-amplified linear DNA to check DNA recovery across a range of molecular weights
<ol>
	<li>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).</li>
	<li>Purify the PCR products using the PCR purification kit and pool the PCR products together.</li>
	<li>Divide the pooled amplicons into 1.5 mL microcentrifuge tubes with equal volumes- label one tube as &#39;Input DNA&#39;.</li>
	<li>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 &#39;Input DNA&#39; and make up the final volume to 1 mL.</li>
	<li>Incubate the tubes overnight (~16-18 h) at 4 &#176;C. 
	NOTE: For the rest of the steps till 3.16, &#39;Input DNA&#39; is kept unchanged in the fridge.</li>
	<li>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 &#176;C.</li>
	<li>Carefully remove 900 &#181;L of the supernatant from the side opposite the pellet using a micropipette.</li>
	<li>First ethanol wash: Add 900 &#181;L of chilled 70% ethanol to the pellet and mix by gently inverting the tubes.</li>
	<li>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 &#176;C.</li>
	<li>Remove the supernatant by decantation.</li>
	<li>Second ethanol wash: Add 500 &#181;L of chilled 70% ethanol to the pellet.</li>
	<li>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&#176;C. Discard the supernatant by decantation.</li>
	<li>Invert and gently tap the tube on tissue paper to remove residual ethanol by capillary action.</li>
	<li>Dry the DNA pellet completely by keeping the tubes open on a heating block for 5-10 min at 37 &#176;C until all ethanol evaporates.</li>
	<li>Re-suspend the pellet in 20 &#181;L of autoclaved double-distilled water.</li>
	<li>Measure the concentration of DNA precipitated by the different conditions and the &#39;Input DNA&#39; using a Nanodrop spectrophotometer or fluorometer.</li>
	<li>Load the precipitated DNA on a 1% agarose gel for DNA visualization.</li>
</ol>
&#160;&#160;

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><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/&amp;micro;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 &amp;amp; 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.

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...

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

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.

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 ...

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

Research

JoVE Journal

Biology

951 Views.  Ashoka University. We zijn geïnteresseerd in de evolutie, overdracht en moleculaire mechanismen die ten grondslag liggen aan antibioticaresistentie. In het bijzonder komt het werk dat in dit artikel wordt gevoed voort uit onze interesse in milieubestendigheid. Momenteel proberen we een lokale database op te bouwen om ruimtelijke temporele variatie in antimicrobiële resistentie te volgen met behulp van gegevens van een jaar.Een combinatie van op cultuur gebaseerde techn...

Video: Auteur in de schijnwerpers: Antimicrobiële resistentie in het milieu begrijpen en detecteren door op cultuur gebaseerde technieken en genomica te combineren

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.

Two complementary analyses of atmospheric biological particles from air sampled filters are described herein: the extraction and detection of endotoxin, and of DNA.

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles ...

Environment

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Evaluating the Impact of Hydraulic Fracturing on Streams using Microbial Molecular Signatures

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil and gas. This process revolutionized the U.S. energy industry, as it gives access to resources that were previously unobtainable and now produces two-thirds of the total natural gas in the United States. Although fracking has had a positive impact on the U.S. economy, several studies have highlighted its detrimental environmental effects. Of particular concern is the effect of fracking on headwater streams, which are especially important due to their disproportionately large impact on the health of the entire watershed. The bacteria within those streams can be used as indicators of stream health, as the bacteria present and their abundance in a disturbed stream would be expected to differ from those in an otherwise comparable but undisturbed stream. Therefore, this protocol aims to use the bacterial community to determine if streams have been impacted by fracking. To this end, sediment, and water samples, from streams near fracking (potentially impacted) and upstream or in a different watershed of fracking activity (unimpacted) must be collected. Those samples are then subjected to nucleic acid extraction, library preparation, and sequencing to investigate microbial community composition. Correlational analysis and machine learning models can subsequently be employed to identify which features are explanative of variation in the community, as well as identification of predictive biomarkers for fracking&#39;s impact. These methods can reveal a variety of differences in the microbial communities among headwater streams, based on the proximity to fracking, and serve as a foundation for future investigations on the environmental impact of fracking activities.

Here, we present a protocol to investigate the impacts of hydraulic fracturing on nearby streams by analyzing their water and sediment microbial communities.

Hydraulic fracturing (HF), commonly called &#34;fracking&#34;, uses a mixture of high-pressure water, sand, and chemicals to fracture rocks, releasing oil ...

DNA Extraction from 0.22 &mu;M Sterivex Filters and Cesium Chloride Density Gradient Centrifugation

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been treated with storage/lysis buffer and archived at -80&deg;C, and to purify this DNA using a cesium chloride density gradient.  The protocol begins with two one-hour incubation steps to liberate DNA from cells and remove RNA.  Next, a series of Phenol:Chloroform and Chloroform extractions are performed followed by centrifugation to remove proteins and cell membrane components, collection of the aqueous DNA extract, and several buffer exchange steps to wash and concentrate the extract.  Part Five describes the optional purification via cesium chloride density gradient.  It is recommended to work with less than 15 samples at one time to avoid confusion and cut down protocol time. The total time required for this protocol depends on the number of samples to be extracted. For 10-15 samples and assuming the proper centrifugation equipment is available, this entire protocol should take 3 days. Make sure you have the hybridization ovens set to temperature at the outset of the process.

We describe a method for extraction of high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;m Sterivex filters, followed by cesium chloride density gradient centrifugation for purification.

This method is used to extract high molecular weight genomic DNA from planktonic biomass concentrated on 0.22 &mu;M Sterivex filters that have been ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Rapid Antimicrobial Susceptibility Testing by Stimulated Raman Scattering Imaging of Deuterium Incorporation in a Single Bacterium

To slow and prevent the spread of antimicrobial resistant infections, rapid antimicrobial susceptibility testing (AST) is in urgent need to &#65279;quantitatively determine the &#65279;antimicrobial effects on pathogens. &#65279;It typically takes days to complete the AST by conventional methods based on the long-time culture, and they do not work directly for clinical samples. Here, we report a rapid AST &#65279;method enabled by stimulated Raman scattering (SRS) imaging of deuterium oxide (D2O) metabolic incorporation. Metabolic incorporation of D2O into biomass and the metabolic activity inhibition upon exposure to antibiotics at the single bacterium level are monitored by SRS imaging. The&#65279; single-cell metabolism inactivation concentration (SC-MIC) of bacteria upon exposure to antibiotics can be obtained after a total of 2.5 h of sample preparation and detection. Furthermore, this rapid AST method is directly applicable to bacterial samples in complex biological environments, such as urine or whole blood. SRS metabolic imaging of deuterium incorporation is transformative for rapid single-cell phenotypic AST in the clinic.

This protocol presents rapid antimicrobial susceptibility testing (AST) assay within 2.5 h by single-cell-stimulated Raman scattering imaging of D2O metabolism. This method applies to bacteria in the urine or whole blood environment, which is transformative for rapid single-cell phenotypic AST in the clinic.

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

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