Wastewater Epidemiology and Microbiome
Discover our magnetic bead-based DNA extraction solution for rapid wastewater surveillance of microbiomes, viruses and AMR
From Flush to Findings: The Rise of Sewage Surveillance
Wastewater surveillance, more formally known as wastewater-based epidemiology (WBE), is an innovative public health monitoring approach that involves analyzing community sewage for genetic markers of pathogens and other crucial biological indicators to comprehensively assess public health trends. This methodology gained significant prominence and validation during the recent COVID-19 pandemic, where numerous studies compellingly demonstrated that SARS-CoV-2 RNA could be reliably detected in raw sewage samples, and its concentrations correlated with clinically reported infection trends within the corresponding population [1]. For instance, a pivotal study successfully detected SARS-CoV-2 genetic material in Dutch wastewater systems, even during the nascent stages of the outbreak, thereby foreshadowing subsequent increases in clinically reported cases [1]. Such landmark findings firmly established WBE as an invaluable early warning system for infectious disease outbreaks. Consequently, prominent health agencies worldwide, including the World Health Organization, have endorsed the environmental surveillance of wastewater as a critical and complementary public health strategy [2]. By systematically monitoring sewage, public health officials can obtain pooled, community-level data on pathogen circulation without the exclusive reliance on individual clinical testing. This inherent advantage renders WBE a highly cost-effective and non-invasive methodology for tracking a spectrum of diseases, including COVID-19, poliomyelitis, hepatitis, and various other enteric infections at the population scale.
Field-ready DNA extraction. Collecting water samples for rapid microbial DNA analysis — enabling real-time environmental monitoring and pathogen surveillance, even in challenging outdoor settings
Beyond Pathogens: Microbiome and Virome Applications in WBE
Beyond the surveillance of specific viral pathogens, wastewater inherently contains a complex and rich amalgamation of microorganisms and diverse genetic material that collectively mirrors the broader microbiome of the contributing community. Sewage can thus serve as a powerful composite sample, offering insights into the human fecal microbiota of an entire population. Indeed, scientific studies have substantiated that the bacterial profile of municipal sewage accurately captures key characteristics of the human gut microbiome prevalent in the community it serves [3]. Illustratively, a comparative analysis encompassing sewage from numerous cities worldwide demonstrated that wastewater not only recapitulated the dominant fecal bacteria but also revealed population-level variations in microbiome composition, which were linked to influential factors such as dietary habits and obesity rates [3]. This underscores the capacity of WBE to extend beyond pathogen tracking, enabling the assessment of overall public health indicators and lifestyle-associated factors through detailed microbiome analysis.
Furthermore, the application of metagenomic sequencing to wastewater samples has been effectively employed for the global surveillance of antimicrobial resistance (AMR) genes and the monitoring of emerging bacterial threats. A significant study involved sampling untreated sewage from diverse international locations, successfully identifying a varied "resistome" – the collection of all AMR genes in a particular environment [4]. This research vividly demonstrated how patterns of international travel and local antibiotic consumption are reflected in the DNA recovered from sewage. Such global-scale monitoring via wastewater metagenomics has elucidated the geographical distribution of antibiotic resistance markers across continents, powerfully highlighting WBE’s profound potential for tracking the insidious spread of antimicrobial resistance.
Similarly, the viral component of wastewater, known as the virome — which includes a host of enteric viruses like norovirus, poliovirus, adenovirus, and many others — can be meticulously analyzed to understand the spectrum of viral strains circulating within a community. Wastewater virome analysis has proven effective in detecting outbreaks and has even led to the discovery of novel viruses, thereby offering a unique window into community-wide viral diversity that might otherwise remain undetected by conventional clinical surveillance systems [7].
These diverse applications collectively underscore the versatility of wastewater surveillance as a multifaceted tool: from monitoring specific viral pathogens like SARS-CoV-2 to comprehensively profiling the overall microbiome and resistome of entire populations.
The Critical Bottleneck: Challenges in Nucleic Acid Extraction from Wastewater
A foundational and indispensable step in any wastewater surveillance workflow is the efficient extraction of nucleic acids (both DNA and RNA) from complex sewage samples. However, wastewater as a sample matrix presents a formidable array of challenges for achieving reliable nucleic acid extraction and subsequent molecular analyses.
Table 1: Key Challenges in Nucleic Acid Extraction from Wastewater
Challenge Category | Specific Issues | Impact on WBE Analysis |
Low Target Concentration | Viral genomes, bacterial DNA often present in minute quantities | Necessitates processing large sample volumes; often requires pre-concentration steps |
Complex Matrix & Inhibitors | Contains humic/fulvic acids, fats, proteins, metal ions, other contaminants | Causes PCR interference, potential for false negatives, reduced detection sensitivity |
Method Variability | Different extraction kits and protocols yield widely varying results | Affects recovery efficiency of target genes, impacts data reliability and comparability |
Firstly, target analytes such as viral genomes or specific bacterial DNA sequences often occur at exceedingly low concentrations in raw sewage, particularly in the case of emerging pathogens or infections with sporadic incidence. This scarcity necessitates the processing of large volumes of wastewater and frequently requires an initial concentration step (e.g., via filtration, precipitation, or ultracentrifugation) prior to extraction to recover a sufficient quantity of genetic material for robust detection.
Secondly, wastewater is an inherently complex and "dirty" matrix, replete with a multitude of substances that are known PCR inhibitors and can interfere with other sensitive enzymatic assays used in molecular biology. Common inhibitors found in sewage include organic matter such as humic and fulvic acids, as well as fats, proteins, metal ions, and various other environmental contaminants that can co-extract alongside the target nucleic acids. These inhibitory substances can directly interfere with the activity of DNA/RNA polymerases and reverse transcriptase enzymes, thereby reducing amplification efficiency or, in severe cases, leading to false-negative results. Consequently, environmental samples such as sewage often demand additional purification steps or specific inhibitor removal treatments to yield nucleic acid extracts that are compatible with downstream PCR applications.
Another significant challenge lies in the observation that different nucleic acid extraction protocols can yield widely varying results even when applied to identical wastewater samples. One comprehensive comparative study revealed that the measured concentrations of several antibiotic resistance genes in identical wastewater aliquots varied significantly across different extraction protocols [8], starkly underscoring the critical importance of protocol selection on the final data outcome. Certain methods may exhibit superior efficacy in lysing robust microbial cells or viral capsids, while others might be more effective at minimizing the co-purification of inhibitors, leading to higher detectable gene counts. Therefore, the standardization and meticulous optimization of nucleic acid extraction procedures are paramount for ensuring reliable and reproducible wastewater surveillance results.
Innovations in NA Extraction: The Advent of Reverse Purification Technique
To surmount some of the inherent limitations associated with conventional nucleic acid extraction methods, novel and innovative techniques such as reverse purification have been developed and are gaining traction. Traditional column-based extraction methodologies typically rely on the principle of binding nucleic acids to a solid matrix (commonly silica) while washing away impurities; however, these methods can involve multiple centrifugation and washing steps that inherently risk sample loss or may not completely eliminate potent PCR inhibitors.
In stark contrast, the reverse purification principle employs specially engineered magnetic beads to capture cell debris, proteins, and other impurities in situ, effectively performing a comprehensive cleanup at the earliest possible stage. This approach significantly streamlines the extraction workflow by obviating the need for multiple binding, washing, and elution steps.
Reverse purification can thus lead to higher overall DNA and RNA recovery from challenging matrices like wastewater.
Table 2: Comparative Overview of Wastewater Nucleic Acid Extraction Approaches
Feature | Conventional Column-Based Extraction | Reverse Purification (e.g., SwiftX DNA Water) |
Core principle | Nucleic acids bind to solid matrix; impurities are washed away. | Impurities and debris bind to magnetic beads; nucleic acids remain in cleared lysate. |
Workflow complexity | Multiple binding, washing, and elution steps; more sample transfers | Streamlined, often fewer transfers; in-situ cleanup during lysis. |
Inhibitor removal | Can be incomplete; inhibitors may co-elute with nucleic acids | Generally improved; inhibitors are actively captured by beads during lysis |
Nucleic acid recovery | Potential for loss during multiple transfer and washing steps | Often higher due to fewer processing steps and reduced loss opportunities |
Processing speed | Typically slower due to multiple centrifugation steps | Generally faster with reduced hands-on time |
Reported performance | Variable efficiency; often prone to inhibitor carryover issues | Demonstrated comparable or superior yield and speed; improved downstream detection |
Recent scientific investigations have compellingly demonstrated the effectiveness of reverse purification-based extraction methodologies. A 2023 study applied a reverse purification-based kit for the isolation of DNA from wastewater samples seeded with various microbial pathogens [5]. Their findings indicated that this method performed comparably to, or in some cases better than, conventional extraction techniques in terms of both processing speed and DNA yield. By minimizing the carryover of PCR inhibitors, this technique substantially improved the downstream detection sensitivity for these target organisms.
Building upon these promising results, a 2024 study combined reverse purification extraction with rapid nanopore sequencing technology to achieve the identification of bacterial compositions in raw wastewater samples, all within a single day [6]. In this innovative study, DNA extracted from wastewater using the reverse purification method was immediately subjected to analysis via portable nanopore sequencing platforms, enabling near real-time metagenomic profiling of the sewage microbiome. This work clearly illustrates how advanced extraction methods like reverse purification can significantly enhance the capabilities of WBE [5, 6].
By yielding cleaner and more abundant nucleic acid extracts, they facilitate quicker and more comprehensive profiling of both viral and bacterial communities present in wastewater.
Scientists can now identify bacterial communities in raw sewage samples within hours using portable nanopore sequencing technology combined with rapid reverse purification DNA extraction— enabling real-time monitoring of public health threats without the need for traditional laboratory infrastructure
Conclusion: Wastewater Surveillance - A Cornerstone of Modern Public Health
Wastewater surveillance has unequivocally emerged as a potent and transformative tool for contemporary public health, uniquely capable of monitoring viral and bacterial outbreaks [1, 7], discerning community-wide microbiome shifts [3], and tracking the pervasive spread of antimicrobial resistance [4, 8], all through the analysis of a single, aggregated sample. The efficacy of this approach hinges critically on the efficient and robust recovery of nucleic acids from sewage. Continued advancements in nucleic acid extraction methodologies, from optimizing conventional protocols to developing groundbreaking techniques like reverse purification, are progressively enhancing our ability to extract actionable, high-fidelity information from wastewater.
By synergistically coupling these robust extraction methods with cutting-edge sequencing technologies, WBE is increasingly capable of providing near real-time insights into population health dynamics. As compellingly demonstrated in numerous recent studies [5, 6], these collective innovations are enabling early warning systems for infectious disease outbreaks, facilitating comprehensive surveillance of community-wide antibiotic resistance patterns, and even opening prospects for non-invasively tracking broader public health trends. With its growing adoption by public health agencies globally, wastewater-based epidemiology is firmly poised to assume an increasingly integral role in global infectious disease surveillance and strategic public health decision-making.
Applicable Xpedite Diagnostics products
SwiftX™ DNA Water (25 extractions)
For magnetic bead-based capture and rapid DNA extraction from wastewater.
Validated for raw sewage samples — enabling high-yield, inhibitor-resistant nucleic acid recovery for accurate and efficient microbiome profiling.
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Selected References
[1] Medema, G., Heijnen, L., Elsinga, G., Italiaander, R., & Brouwer, A. (2020). Presence of SARS-Coronavirus-2 RNA in Sewage and Correlation with Reported COVID-19 Prevalence in the Early Stage of the Epidemic in The Netherlands. Environmental Science & Technology Letters, 7(7), 511–516.
[2] World Health Organization. (2023). Environmental surveillance for SARS-CoV-2 to complement other public health surveillance. WHO Guidelines. Geneva, Switzerland.
[3] Newton, R. J., McLellan, S. L., Dila, D. K., Vineis, C., Morrison, H. G., Eren, A. E., Sogin, M. L., & McLellan, S. (2015). Sewage reflects the microbiomes of human populations. mBio, 6(2), e02574-14.
[4] Hendriksen, R. S., Munk, P., Njage, P., van Bunnik, B., McNally, L., Lukjancenko, O., Röder, T., Nieuwenhuijse, D., Pedersen, S. K., Kjeldgaard, J., et al. (2019). Global monitoring of antimicrobial resistance based on metagenomics analyses of urban sewage. Nature Communications, 10(1), 1-12.
[5] Schurig, S., Wende, A., Lübcke, P., Schaufler, K., Truyen, U., Kobialka, R. M., & Abd El Wahed, A. (2023). Evaluation of a reverse purification-based DNA extraction protocol for the detection of microbial pathogens in wastewater. Water Research, 201, 117-125.
[6] Schurig, S., Ceruti, A., Wende, A., Lübcke, P., Eger, E., Schaufler, K., Frimpong, M., Truyen, U., Kobialka, R. M., & Abd El Wahed, A. (2024). Rapid Identification of Bacterial Composition in Wastewater by Combining Reverse Purification Nucleic Acid Extraction and Nanopore Sequencing. ACS ES&T Water, 4(3), 794-804.
[7] Hellmér, L., Okoh, N., Norder, N., Andersson, M., Nilsson, M., Rothberg, A., Yahya, A., & Andersson, A. C. (2014). Detection of pathogenic viruses in sewage provided early warnings of hepatitis A virus and norovirus outbreaks. Applied and Environmental Microbiology, 80(21), 6771-6781.
[8] Prieto Riquelme, M. V., Garner, E., Gupta, S., Metch, J., Zhu, N., Blair, M. F., Arango-Argoty, G., Maile-Moskowitz, A., Li, A., Flach, C. F., et al. (2022). Demonstrating a Comprehensive Wastewater-Based Surveillance Approach That Differentiates Globally Sourced Resistomes. Environmental Science & Technology, 56(21), 14982-14993.
User protocols
Extraction of microbial DNA from wastewater samples
Schurig et al. from Microorganisms 11(3): 813 (2023) and ACS ES&T Water 4(4): 1808 (2024)
- 20 mL wastewater sample was placed in a tube with sufficient volume capacity
- 2 mL Buffer DWC was added to the tube
- 60 µL Beads A was added, tube was closed and vortexed for 10 seconds
- sample was incubated at room temperature for 3 minutes
- tube was placed in magnetic stand for 5 minutes until complete particle separation
- supernatant was removed and discarded by gentle pouring
- 400 µL Buffer TLS was added and Beads A resuspended by vortexing for 5 seconds
- bead suspension was transferred to Tube G 0.1 containing glass beads
- mixture was vortexed at 5000 rpm for 3 minutes for cell grinding
- microtube was incubated at 95°C for 15 minutes for heat lysis
- microtube was removed from heat block and placed in magnetic rack for 1 minute
- supernatant was used directly for qPCR, digital PCR, and next-generation sequencing
