An effective method to track disease outbreaks and spread of antibiotic resistance in wastewater
Routine wastewater surveillance for microbial DNA/RNA can be an efficient, non-invasive tool to provide a near real-time, cost-effective monitoring for viral or bacterial outbreaks and to survey distribution of antibiotic resistance genes.
Microbial particles are extremely diluted in wastewater; consequently, extracting DNA/RNA from wastewater is challenging.
Concentrating microbial particles is essential for nucleic acid extraction for downstream applications.
NucleoMag DNA/RNA Water or NucleoSpin RNA Virus extraction kits can purify inhibitor-free nucleic acids from concentrated microbial particles. The purified nucleic acids can be directly used in downstream applications, such as qPCR, RT-qPCR, or Next Generation Sequencing (NGS).
High-throughput surveillance for wastewater monitoring can be efficiently managed by performing highly parallel qPCR using the SmartChip Real-Time PCR System.
Large-scale application of diagnostic testing at the individual level has been the main approach in epidemics. However, testing individuals is a slow process requiring extensive resources. Fast surveys of large populations can be better accomplished by wastewater-based surveillance to provide a near real-time, cost-effective monitoring of community-level transmission. Routine wastewater surveillance can be the efficient, noninvasive tool to not only monitor current and future viral or bacterial outbreaks, but also survey the spread or emergence of antibiotic resistance (AR) genes.
In this technical note, we discuss efficient and inhibitor-free nucleic acid purification from pathogens in wastewater using microbial concentration methods in combination with NucleoMag DNA/RNA Water and NucleoSpin RNA Virus extraction kits, with the DNA/RNA used directly for downstream applications (Figure 1). We show that the purified nucleic acids enabled identification of pathogens like MS2 bacteriophage and SARS-COV-2 using qPCR and RT-qPCR. Finally, we discuss how a multi-city surveillance of wastewater for antibiotic resistance prevalence was tackled using the SmartChip Real-Time PCR System, an automation system that enables highly parallel qPCR.
Figure 1. Steps for microbial detection from wastewater.
Microbial concentration
Viruses and bacteria are typically found at low concentrations in wastewater. Consequently, extracting microbial DNA/RNA from wastewater is challenging, as the microbial particles are extremely diluted, with large volumes of wastewater needed for detecting their presence. Commonly used wastewater concentration methods, besides being labor intensive and costly, tend to concentrate PCR inhibitors present in the wastewater. Concentrating microbial particles but not PCR inhibitors is essential for downstream applications. Methods to concentrate microbial particles from wastewater samples for these downstream applications include:
Filtration, ultrafiltration, or PEG precipitation (Figure 2).
Figure 2. Microbial concentration by filtration, ultrafiltration, or PEG.
Nanotrap Magnetic Virus Particles from Ceres Nanosciences (Figure 3 and Table 1).
Figure 3. Nanotrap Magnetic particles for capturing virus.
Nanotrap concentration procedure
Capture and concentrate virus
Add 150 μl of Nanotrap Magnetic Virus Particles to 10 ml sample. Invert sample 5 times to mix and incubate for 10 minutes at room temperature.
Collect Nanotrap particles
Place samples on magnetic rack and allow the Nanotrap particles to come out of solution.
Wash
Remove the supernatant and wash particles with 1 ml of 0.05% Tween‑20 in PCR‑grade water. Transfer sample to fresh 1.5 ml tube.
Lysis
Pellet Nanotrap particles. Remove the supernatant. Resuspend pellet in 500 μl MWA 1. Incubate at room temperature for 10 minutes.
Extraction
Pull Nanotrap particles out of solution with a magnetic rack. Proceed with Step 3 of the NucleoMag DNA/RNA Water protocol using 450 μl of lysate.
Table 1. Procedure for capture and concentration of virus particles.
INNOVAPREP CP‑Select sample concentration device (Figure 4 and Table 2).
Table 2: Specifications of INNOVAPREP CP‑Select sample concentration device.
DNA/RNA isolation
Efficiently isolating inhibitor-free nucleic acid from wastewater is the key to conducting reliable monitoring. NucleoMag DNA/RNA Water and NucleoSpin RNA Virus kits isolate microbial RNA/DNA, with the volume of starting material and the technology preferred influencing the choice (Table 3).
NucleoMag DNA/RNA Water kit isolates microbial DNA/RNA by reversible adsorption of nucleic acids to paramagnetic beads under appropriate buffer conditions. DNA and RNA from bacteria, fungi, virus, and algae are isolated simultaneously for subsequent downstream applications. NucleoMag DNA/RNA Water allows easy automation on common liquid handling instruments or automated magnetic separators.
NucleoSpin RNA Virus kit features a special type of silica membrane with a high binding capacity for viral nucleic acids, enabling the simultaneous purification of viral RNA and DNA. The silica membrane improves the binding and recovery of low-concentration viral nucleic acids. Potential PCR inhibitors like salts, soluble macromolecular cellular components, and other contaminants are easily removed by washing with supplied buffers. The isolated nucleic acid is ready-for-use in subsequent downstream applications.
Nucleic acid isolation and purification
NucleoMag DNA/RNA Water
NucleoSpin RNA Virus
Technology
Magnetic bead technology
Silica membrane technology
Sample material
10–1,000 ml
<150 μl cell-free fluid
Preparation time
40–120 min/96 preps (excluding lysis and sample concentration)
30 min/4–6 preps (excluding lysis and sample concentration)
Fragment size
300 bp–approx. 50 kb
100 bp–approx. 50 kb
Elution volume
50–250 μl
50 μl
Table 3. Technology specifications for NucleoMag DNA/RNA Water and NucleoSpin RNA Virus.
Application data
The success of a reliable wastewater surveillance effort is dependent on identifying pathogens and AR genes, even when present in low abundance. We present data to show that the processes outlined above can be used for routine wastewater surveillance.
RT-qPCR analysis for MS2 bacteriophage and SARS-CoV-2
Microbial particles were concentrated from wastewater samples by PEG precipitation or ultrafiltration for subsequent nucleic acid extraction with NucleoMag DNA/RNA Water kit. MS2 bacteriophage was detected consistently and reliably over a range of dilution series with excellent linearity in wastewater precipitate using PEG/NaCl (Figure 5, Panel A) and ultrafiltration (Figure 5, Panel B).
Figure 5. Detection of MS2 bacteriophage by RT-qPCR in wastewater precipitates using PEG/NaCl (Panel A) and wastewater concentrates by ultrafiltration (Panel B)
Pairing NucleoMag DNA/RNA Water kit with the Nanotrap Magnetic Virus Particles from Ceres Nanosciences for wastewater concentration was an efficient, cost-effective method to extract SARS-CoV-2 viral RNA from wastewater samples in four geographic regions of the United States (Figure 6, Panels A and B).
Figure 6: Detection of SARS-CoV-2 in wastewater samples from four geographic regions of the US. Viral detection was performed using the SARS-CoV-2 Wastewater RT-qPCR Kit (Promega) for both N1 and N2 primers. Samples were from Seattle, WA (1–9), San Bernardino, CA (10–15), Storrs, CT (16–19), and Los Angeles, CA (20–26). Detection as Ct is seen in Panel A. In Panel B, the amount of virus was quantified and normalized to BCoV spike-in control.
qPCR for MS2 bacteriophage
Combining INNOVAPREP’s CP-Select sample concentration device with Nucleospin RNA Virus and NucleoMag DNA/RNA Water nucleic acid purification procedures gave reliable nucleic acid concentration and purification from wastewater samples (Figure 7, Panels A and B).
Figure 7. Consistent detection of MS2 bacteriophage RNA from wastewater concentrates by qPCR. RNA was purified from synthetic wastewater concentrates spiked with different amounts of MS2 phage RNA. INNOVAPREP’s CP-Select device was used to concentrate the samples, followed by nucleic acid isolation using Nucleospin RNA Virus (Panel A) or Nucleomag DNA/RNA Water (Panel B).
Highly parallel qPCR for detecting antibiotic resistance
High-throughput wastewater surveillance entails processing hundreds of samples for the assay of many potential targets with precision, reproducibility, and speed. Researchers used highly parallel qPCR to target 229 antibiotic resistance genes and 25 mobile genetic elements in wastewater from 12 urban wastewater treatment plants located in seven countries (Figure 8). They used the high-throughput, quantitative SmartChip Real-Time PCR system to successfully survey AR distribution and concluded that AR profiles in urban wastewater treatment plants mirrored the AR gradient observed in clinics. The authors suggest that high-throughput data should be routinely obtained from highly parallel qPCR to improve antibiotic resistance control.
Figure 8. Abundance of resistance genes observed in wastewater samples from countries with HAC (high antibiotic consumption) and LAC (low antibiotic consumption). HAC countries were Portugal (PT), Spain (ES), and Cyprus (CYP). LAC countries were Germany (DE), Finland (FI), and Norway (NO). Samples taken in Spring 2016 (C2) and Autumn 2016 (C3). Resistance gene categories: AMG (aminoglycosides), MDR (multidrug resistance), SUL (sulfonamides), BL (β-lactams), MLSB (macrolide, lincosamide and streptogramin B), TET (tetracycline), QUI (quinolones), AMP (amphenicols), and VAN (vancomycin). Image and caption adapted from Pärnänen et al. 2019 and used under Creative Commons Attribution 4.0 International license.
Conclusion
Effective surveillance systems are the key to rapid intervention. Real-time analysis of wastewater samples to track circulating and emerging variants can be the basis for monitoring disease outbreaks and tracking spread of antibiotic resistance.
There are two main challenges to wastewater surveillance: purifying high-quality nucleic acids from large volumes of water containing complex matrices and scaling up sample processing while testing for many clinically relevant genes.
Here, we showed that our nucleic acid purification kits, NucleoMag DNA/RNA Water and NucleoSpin RNA, in combination with microbial concentration, provide an efficient method for extracting nucleic acids for downstream applications like qPCR and RT-qPCR. We discussed how the daunting task of processing hundreds of samples to identify numerous potential targets is efficiently tackled by the SmartChip Real-Time PCR System.
The method presented can be used on a routine basis as an affordable, noninvasive tool for wastewater surveillance.
