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Researchers from 11 European institutions, led by the DTU National Food Institute, have developed an advanced method to analyze wastewater data, providing insights into the origins of disease-causing bacteria, viruses, and antimicrobial resistance. This method allows the simultaneous detection of thousands of potential health threats, including cholera and antimicrobial resistance, making it a powerful tool for public health monitoring. The findings have been published in Nature Communications.
A New Approach to Wastewater Surveillance
Wastewater analysis has become increasingly important for tracking health trends in urban populations. The challenge lies in interpreting the complex data generated from untreated wastewater, which can contain both known and unknown microbes from a variety of sources, such as human activity, industry, and environmental runoff. As Patrick Munk, an assistant professor at DTU National Food Institute, explained, "Untreated wastewater is increasingly becoming a vital source for anonymous health and disease surveillance in large urban populations. However, extracting valuable data from it is not straightforward, as the wastewater contains both known and unknown bacteria from a variety of sources, such as humans, plants, animals, rainwater, dishwashing, etc. "
To tackle this challenge, researchers employed metagenomics—a technique that allows for the simultaneous analysis of DNA from multiple organisms. Metagenomics has been shown to hold vast potential for wastewater monitoring.
"While this method is more expensive than PCR testing, which proved highly effective during the COVID-19 pandemic, PCR only screens for one threat at a time. Metagenomics-based wastewater monitoring can assess thousands of threats simultaneously. Additionally, the value of each individual sample increases the more samples are collected over time, as historical data enhances the value of new analyses," said Frank Aarestrup, professor at DTU and leader of the study.
A Three-Year Study Across Europe
The study involved analyzing wastewater samples collected over three years (2019-2021) from seven wastewater treatment plants in five major European cities: Bologna, Budapest, Copenhagen, Rome, and Rotterdam. The researchers processed 278 samples, analyzing billions of DNA sequences to assemble genomes from thousands of bacterial species, including 1,334 previously unknown species.
This massive amount of data was processed using software developed by the University of Bologna, one of the project’s partners. The software identified species that behave similarly over time and grouped them based on these behaviors. This innovative approach allowed the team to identify bacterial species and trace their origins. Munk noted that the bacteria grouped into distinct clusters, prompting further investigation into the reasons behind these groupings. "We began to wonder why and how the groups were formed. Initially, we thought the clusters might represent microbes collaborating with each other, but that was a dead end," said Munk. Eventually, the researchers determined that some groups were made up of bacteria originating from human feces, while others came from environmental sources or biofilms within the wastewater infrastructure.
Identifying Human and Environmental Sources
The method developed in this study has greatly improved the researchers' ability to identify the origins of bacteria in wastewater. Using this approach, they were able to assign approximately 70% of the DNA from their samples to known bacterial species, a significant increase from the previous 10% success rate. By distinguishing between bacteria of human, animal, and environmental origin, the researchers aim to develop more effective surveillance systems. These systems could combine metagenomics-based monitoring with PCR testing for targeted threats, as Munk suggested.
Cholera Bacteria Found in Copenhagen Wastewater
One unexpected discovery during the research was the presence of cholera bacteria in wastewater from the Avedøre Wastewater Treatment Plant in Copenhagen. Although the amounts were small and there have been no cholera cases in Denmark for over 150 years, the finding still raised questions. The bacteria were likely brought to the facility by someone from a region where cholera is endemic, after which the bacteria settled in the pipes of the treatment facility and began to replicate there.
This discovery illustrates the versatility of the new method in tracking bacteria not just in human waste but also in biofilms formed in the wastewater infrastructure itself. The cholera bacteria found in Copenhagen did not pose an immediate public health risk, as they were not linked to active infections. However, the study demonstrated how this technique could help authorities detect and trace the origins of potentially dangerous pathogens before they spread.
Future Potential for Monitoring Antimicrobial Resistance
The study’s findings are particularly relevant in the context of a new EU directive requiring major cities to monitor antimicrobial resistance in wastewater. As antimicrobial resistance becomes a growing global threat, the ability to detect new bacteria carrying resistance genes is critical. This new metagenomics approach can reveal previously unknown bacterial species that could harbor these resistance genes, enabling more proactive public health responses.
The researchers acknowledge that this observational study has some limitations, as the results are based on naturally occurring bacteria in untreated wastewater, without controlled conditions. To improve accuracy and reliability, they plan to create synthetic datasets where they can manipulate conditions and bacterial species.
"We don’t have a final success rate for this method yet, but it’s clear that we’re onto something significant," said Munk, highlighting the need for further optimization of the technique.
Publication Details
Becsei, Á., Fuschi, A., Otani, S. et al. Time-series sewage metagenomics distinguishes seasonal, human-derived and environmental microbial communities potentially allowing source-attributed surveillance. Nat Commun 15, 7551 (2024). https://doi.org/10.1038/s41467-024-51957-8
A New Approach to Wastewater Surveillance
Wastewater analysis has become increasingly important for tracking health trends in urban populations. The challenge lies in interpreting the complex data generated from untreated wastewater, which can contain both known and unknown microbes from a variety of sources, such as human activity, industry, and environmental runoff. As Patrick Munk, an assistant professor at DTU National Food Institute, explained, "Untreated wastewater is increasingly becoming a vital source for anonymous health and disease surveillance in large urban populations. However, extracting valuable data from it is not straightforward, as the wastewater contains both known and unknown bacteria from a variety of sources, such as humans, plants, animals, rainwater, dishwashing, etc. "
To tackle this challenge, researchers employed metagenomics—a technique that allows for the simultaneous analysis of DNA from multiple organisms. Metagenomics has been shown to hold vast potential for wastewater monitoring.
"While this method is more expensive than PCR testing, which proved highly effective during the COVID-19 pandemic, PCR only screens for one threat at a time. Metagenomics-based wastewater monitoring can assess thousands of threats simultaneously. Additionally, the value of each individual sample increases the more samples are collected over time, as historical data enhances the value of new analyses," said Frank Aarestrup, professor at DTU and leader of the study.
A Three-Year Study Across Europe
The study involved analyzing wastewater samples collected over three years (2019-2021) from seven wastewater treatment plants in five major European cities: Bologna, Budapest, Copenhagen, Rome, and Rotterdam. The researchers processed 278 samples, analyzing billions of DNA sequences to assemble genomes from thousands of bacterial species, including 1,334 previously unknown species.
This massive amount of data was processed using software developed by the University of Bologna, one of the project’s partners. The software identified species that behave similarly over time and grouped them based on these behaviors. This innovative approach allowed the team to identify bacterial species and trace their origins. Munk noted that the bacteria grouped into distinct clusters, prompting further investigation into the reasons behind these groupings. "We began to wonder why and how the groups were formed. Initially, we thought the clusters might represent microbes collaborating with each other, but that was a dead end," said Munk. Eventually, the researchers determined that some groups were made up of bacteria originating from human feces, while others came from environmental sources or biofilms within the wastewater infrastructure.
Identifying Human and Environmental Sources
The method developed in this study has greatly improved the researchers' ability to identify the origins of bacteria in wastewater. Using this approach, they were able to assign approximately 70% of the DNA from their samples to known bacterial species, a significant increase from the previous 10% success rate. By distinguishing between bacteria of human, animal, and environmental origin, the researchers aim to develop more effective surveillance systems. These systems could combine metagenomics-based monitoring with PCR testing for targeted threats, as Munk suggested.
Cholera Bacteria Found in Copenhagen Wastewater
One unexpected discovery during the research was the presence of cholera bacteria in wastewater from the Avedøre Wastewater Treatment Plant in Copenhagen. Although the amounts were small and there have been no cholera cases in Denmark for over 150 years, the finding still raised questions. The bacteria were likely brought to the facility by someone from a region where cholera is endemic, after which the bacteria settled in the pipes of the treatment facility and began to replicate there.
This discovery illustrates the versatility of the new method in tracking bacteria not just in human waste but also in biofilms formed in the wastewater infrastructure itself. The cholera bacteria found in Copenhagen did not pose an immediate public health risk, as they were not linked to active infections. However, the study demonstrated how this technique could help authorities detect and trace the origins of potentially dangerous pathogens before they spread.
Future Potential for Monitoring Antimicrobial Resistance
The study’s findings are particularly relevant in the context of a new EU directive requiring major cities to monitor antimicrobial resistance in wastewater. As antimicrobial resistance becomes a growing global threat, the ability to detect new bacteria carrying resistance genes is critical. This new metagenomics approach can reveal previously unknown bacterial species that could harbor these resistance genes, enabling more proactive public health responses.
The researchers acknowledge that this observational study has some limitations, as the results are based on naturally occurring bacteria in untreated wastewater, without controlled conditions. To improve accuracy and reliability, they plan to create synthetic datasets where they can manipulate conditions and bacterial species.
"We don’t have a final success rate for this method yet, but it’s clear that we’re onto something significant," said Munk, highlighting the need for further optimization of the technique.
Publication Details
Becsei, Á., Fuschi, A., Otani, S. et al. Time-series sewage metagenomics distinguishes seasonal, human-derived and environmental microbial communities potentially allowing source-attributed surveillance. Nat Commun 15, 7551 (2024). https://doi.org/10.1038/s41467-024-51957-8