Periodic Reporting for period 1 - VIRULENCE (Assessing the (co)dissemination of virulence and antibiotic resistance genes in resource recovery applications)
Reporting period: 2023-06-01 to 2025-05-31
The current linear economic model of production and consumption leads to significant resource depletion and waste accumulation, highlighting the urgent need to transition toward a circular economy. In this context, recovering resources by using treated organic waste and reclaimed water in agriculture offers great potential for recycling nutrients and water. However, these practices also carry risks by potentially spreading antibiotic-resistant pathogens, and their associated virulence genes and antibiotic resistance genes, which worsen the global health crisis. While efforts exist to control antimicrobial resistance, the role and fate of virulence genes remain largely unexplored. This project aims to address critical knowledge gaps about how new resource recovery technologies affect the dissemination and mitigation of virulence genes and antibiotic resistance genes. Specifically, the project pursued the following objectives:
1) Characterize the presence, co-occurrence, and persistence of virulence and antibiotic resistance genes throughout wastewater treatment plants, examining how different treatment processes influence their removal or dissemination.
2) Evaluate the effectiveness of solid waste treatment technologies (e.g. composting and anaerobic digestion) in reducing virulence and antibiotic resistance genes, and study the risks linked to their presence and transfer after applying treated biosolids to agricultural soils.
3) Investigate the horizontal gene transfer processes that allow virulence and antibiotic resistance genes to move from microbial communities in treatment systems to environmental bacteria.
By understanding the fate of these genes during waste and wastewater treatment and their potential impact on ecosystems, this project has helped to develop safer and more effective strategies to manage health risks while promoting sustainable resource recovery.
1) Characterize the presence, co-occurrence, and persistence of virulence and antibiotic resistance genes throughout wastewater treatment plants, examining how different treatment processes influence their removal or dissemination.
2) Evaluate the effectiveness of solid waste treatment technologies (e.g. composting and anaerobic digestion) in reducing virulence and antibiotic resistance genes, and study the risks linked to their presence and transfer after applying treated biosolids to agricultural soils.
3) Investigate the horizontal gene transfer processes that allow virulence and antibiotic resistance genes to move from microbial communities in treatment systems to environmental bacteria.
By understanding the fate of these genes during waste and wastewater treatment and their potential impact on ecosystems, this project has helped to develop safer and more effective strategies to manage health risks while promoting sustainable resource recovery.
Water and sewage sludge samples were collected from four wastewater treatment plants varying in scale and technology, including conventional and advanced treatment processes. Sampling was carried out at multiple stages along each treatment line to monitor the dynamics of genes throughout the entire process.
In parallel, controlled laboratory-scale experiments simulated anaerobic digestion and composting of solid waste under different operational conditions. Shotgun metagenomic sequencing was applied to all samples, enabling detailed characterization of antibiotic resistance genes, virulence genes, and microbial community shifts. This integrated approach provided valuable insights into how treatment influence gene fate and microbial populations.
Additionally, soil microcosm experiments using plants grown in soils amended with treated residues or irrigated with treated wastewater enabled the evaluation of gene persistence and potential mobility in agricultural settings.
In total, 28 metagenomes from wastewater treatment samples and five metagenomes from solid waste samples were generated to support this analysis and improve our understanding of microbial risks associated with resource recovery. A major achievement of the project was the development of a bioinformatic pipeline designed to detect the presence and co-occurrence of resistance and virulence genes in metagenomic datasets. This tool enhances the capacity to identify microbial hosts carrying multiple risk factors, providing a critical resource for risk evaluation and management strategies.
In parallel, controlled laboratory-scale experiments simulated anaerobic digestion and composting of solid waste under different operational conditions. Shotgun metagenomic sequencing was applied to all samples, enabling detailed characterization of antibiotic resistance genes, virulence genes, and microbial community shifts. This integrated approach provided valuable insights into how treatment influence gene fate and microbial populations.
Additionally, soil microcosm experiments using plants grown in soils amended with treated residues or irrigated with treated wastewater enabled the evaluation of gene persistence and potential mobility in agricultural settings.
In total, 28 metagenomes from wastewater treatment samples and five metagenomes from solid waste samples were generated to support this analysis and improve our understanding of microbial risks associated with resource recovery. A major achievement of the project was the development of a bioinformatic pipeline designed to detect the presence and co-occurrence of resistance and virulence genes in metagenomic datasets. This tool enhances the capacity to identify microbial hosts carrying multiple risk factors, providing a critical resource for risk evaluation and management strategies.
This project represents the first extensive analysis of virulence genes across multiple wastewater treatment plants and solid waste treatment processes, including anaerobic digestion and composting. While antibiotic resistance genes have been widely studied, integrating virulence gene analysis offers a more holistic assessment of microbial risks associated with waste and wastewater treatment, and resource recovery.
A key novel finding is the potential co-occurrence of resistance and virulence traits within the same microbial hosts. Taxonomic profiling revealed that the microbial genera carrying antibiotic resistance genes and those carrying virulence genes were highly similar,, suggesting that both types of genes often coexist within the same microorganisms. This increases the potential health risks posed by these organisms to both human and environmental.
The development of a dedicated bioinformatic framework to detect gene co-occurrence marks a significant advancement, offering a powerful tool.
Collectively, these results deepen the current understanding by integrating virulence and resistance gene monitoring, uncovering complex microbial dynamics and potential resource recovery impacts that had not been previously characterized.
However, the potential for horizontal gene transfer and the long-term persistence of these genes in environmental and agricultural settings remain poorly understood. Further research is needed to:
- Investigate the conditions that favor the retention or attenuation of ARGs and VGs
- Develop treatment-specific indicators or thresholds for safe reuse practices.
- Explore the role of mobile genetic elements and their contribution to gene dissemination.
Expanding this knowledge is essential for designing effective, science-based policies and treatment technologies that protect public health while advancing circular economy goals.
A key novel finding is the potential co-occurrence of resistance and virulence traits within the same microbial hosts. Taxonomic profiling revealed that the microbial genera carrying antibiotic resistance genes and those carrying virulence genes were highly similar,, suggesting that both types of genes often coexist within the same microorganisms. This increases the potential health risks posed by these organisms to both human and environmental.
The development of a dedicated bioinformatic framework to detect gene co-occurrence marks a significant advancement, offering a powerful tool.
Collectively, these results deepen the current understanding by integrating virulence and resistance gene monitoring, uncovering complex microbial dynamics and potential resource recovery impacts that had not been previously characterized.
However, the potential for horizontal gene transfer and the long-term persistence of these genes in environmental and agricultural settings remain poorly understood. Further research is needed to:
- Investigate the conditions that favor the retention or attenuation of ARGs and VGs
- Develop treatment-specific indicators or thresholds for safe reuse practices.
- Explore the role of mobile genetic elements and their contribution to gene dissemination.
Expanding this knowledge is essential for designing effective, science-based policies and treatment technologies that protect public health while advancing circular economy goals.