Periodic Reporting for period 1 - ENVIROSTOME (Exploring the contribution of bacteriophages to the emergence and spread of antibiotic resistance in environmental settings)
Reporting period: 2019-04-01 to 2021-03-31
Antimicrobial resistance (AMR) is a global health crisis and is acknowledged by the World Health Organization (WHO) as the silent pandemic. AMR has been widely studied in clinical settings, but its transmission from the environment to clinical settings is not well-understood.
Therefore, the ENVIROSTOME project was set to understand further the role of bacteriophages (phages) in this spread, and to advise on actions to mitigate AMR spread by phage in the water sector.
Water pollution by antimicrobials, industrial and domestic wastewater discharges, agricultural runoff, and animal feeding operations may provide the ideal conditions for the horizontal exchange of antibiotic resistance genes (ARGs) into antibiotic resistant bacteria (ARB). Previously, our research group in ICRA documented that environmental phages contain a large diversity of ARGs and can transmit this resistance between bacteria (Figure 1).
ENVIROSTOME’s aims were to:
- determine the contribution of phages to the horizontal transfer of ARGs in water,
- explore strategies to mitigate this problem, particularly using disinfection.
Also, an intersectoral collaboration with the water sector in Europe, allowed the project to:
- deliver actionable insights on the status of applicable technologies to treat phage/ARG contamination by WWTPs, and
- provide advice and advance policy and regulations of AMR in the water sector.
Therefore, the ENVIROSTOME project was set to understand further the role of bacteriophages (phages) in this spread, and to advise on actions to mitigate AMR spread by phage in the water sector.
Water pollution by antimicrobials, industrial and domestic wastewater discharges, agricultural runoff, and animal feeding operations may provide the ideal conditions for the horizontal exchange of antibiotic resistance genes (ARGs) into antibiotic resistant bacteria (ARB). Previously, our research group in ICRA documented that environmental phages contain a large diversity of ARGs and can transmit this resistance between bacteria (Figure 1).
ENVIROSTOME’s aims were to:
- determine the contribution of phages to the horizontal transfer of ARGs in water,
- explore strategies to mitigate this problem, particularly using disinfection.
Also, an intersectoral collaboration with the water sector in Europe, allowed the project to:
- deliver actionable insights on the status of applicable technologies to treat phage/ARG contamination by WWTPs, and
- provide advice and advance policy and regulations of AMR in the water sector.
This research delivered critical insights for tackling the global AMR crisis and was divided into three parts: biological, and secondment/regulatory results.
Biological results:
- Firstly, water samples from impacted urban locations at the Ter watershed in Catalunya (Spain) were analyzed for the presence of AMR. Culture technique (standard double agar overlay method) was used to isolate phage in several bacteria (such as: Escherichia coli WG5, and E. coli and Aeromonas spp.). A collection of 87 phages, mostly lysogenic, was found and further scanned for the presence of ARGs by PCR. During this work, we discovered the lytic phage vB_EcoM_C2-3 with biocontrol potential (e.g. phage therapy) against multidrug-resistant strains of E. coli (doi.org/10.1016/j.virusres.2021.198623) (open depository at: https://dugi-doc.udg.edu/handle/10256/20502 AND https://zenodo.org/record/7966128).
- A second strategy was to extract phage DNA directly from water samples, without a cultivation step, and perform real-time PCR (qPCR). This method was used to quantify changes in ARGs in both phage and bacteria in upstream and downstream samples of an urban area (Girona, Spain). We selected eight ARGs (which confer resistance to various antibiotic classes, such as sul1 and sul2 (sulfonamides), ermB (macrolides), tetW (tetracycline), among others. In general terms, upstream and downstream values for ARGs ranged at values 1 Log higher in bacteria, with a noticeable increase of sulfonamide genes downstream.
- Thirdly, we performed a metagenomic analysis of bacterial populations in the samples. For doing that, bacterial DNA was sequenced to determine the presence of AMR determinants and more than 12 families of ARG were detected (e.g. bacitracin, fluoroquinolone, and macrolide). Mobile genetic elements (MGEs) related to the spread of AMR in environmental settings were also abundant in these samples. We are analyzing these findings for peer-reviewed publishing.
- Finally, we took the opportunity to investigate the stress-response caused by phage in Enteric bacteria. This is critical for AMR to spread not only in clinical but also environmental settings and to the best of our knowledge it has not been reported before. The work “Stress induced response by phage in E. coli: a complete transcriptome analysis”, is also being produced for peer-reviewed publishing.
Secondment/Regulatory results
- ENVIROSTOME had a secondment organized with a water intelligence company in Ireland. We produced from this industry contact several outputs including: the articles
(i) “How does the emergence of antimicrobial resistance affect policy making?”
(doi: 10.1016/j.watres.2021.117772)
(Open depository: https://dugi-doc.udg.edu/handle/10256/20501 and https://zenodo.org/record/7966090)
(ii) “Antimicrobial Resistance and Bacteriophages: An Overlooked Intersection in Water Disinfection” (doi.org/10.1016/j.tim.2020.12.011)
(Open depository: https://dugi-doc.udg.edu/handle/10256/19014 and https://zenodo.org/record/7965961).
(iii) a full report including interviews with companies, insights, produced between the secondment and ICRA,
(iv) a technical presentation,
(v) an article to the Marie Curie Alumni newsletter “How does collaboration between industry and academia advances research” (September 2020, ISSN 2663-9483).
- Our interaction and collaboration with water industry practitioners, regulators, and the laboratory activities in the Envirostome, resulted in an advice paper for policymakers, entitled: “Standard method for the examination of antimicrobial resistance in environmental settings: where are we now?” (Accepted by Trends in Microbiology).
Exploitation:
- ENVIROSTOME was shared using the hashtag #envirostome online. More than one hundred broad-audience articles, and posts were shared by the fellow on Twitter and LinkedIn, as well as videos on YouTube, about her work with AMR and Envirostome.
- We reached citizens in Europe and beyond in the topics of AMR research and water and established a close collaboration with the water and regulatory sectors.
- We presented at various events, including: “Water innovation and phage research: industry and academia working together to fight antimicrobial resistance” ICRA Spain(2019), the Water research in perspective: beyond 2020 Icra, Spain (2020), the MSCA IF Cluster event on Antimicrobial Resistance, and evaluation of project on European’s Commission Innovation Radar in Barcelona, Spain (2019), the EU Water Innovation Conference, industry collaboration. Zaragoza, Spain (2019), and the RCS Twitter poster conference (2020).
- We demonstrated the topic of AMR and phage research at the Researcher's Night with the University Girona (2019) to over one hundred citizens in Girona, Spain.
- We produced with EURAXESS Brazil & LAC an educational online seminar to over 120 international PhDs, helping them to better connect their research to the job market.
- Finally, in 2023, the fellow reflected on the experience of this MSCA fellowship to the Marie Curie Alumni Association Brazilian Chapter and produced with EURAXESS Latim Americ and Caribbean Area a promotional video for the Marie Curie funding (to be published on YouTube soon).
Biological results:
- Firstly, water samples from impacted urban locations at the Ter watershed in Catalunya (Spain) were analyzed for the presence of AMR. Culture technique (standard double agar overlay method) was used to isolate phage in several bacteria (such as: Escherichia coli WG5, and E. coli and Aeromonas spp.). A collection of 87 phages, mostly lysogenic, was found and further scanned for the presence of ARGs by PCR. During this work, we discovered the lytic phage vB_EcoM_C2-3 with biocontrol potential (e.g. phage therapy) against multidrug-resistant strains of E. coli (doi.org/10.1016/j.virusres.2021.198623) (open depository at: https://dugi-doc.udg.edu/handle/10256/20502 AND https://zenodo.org/record/7966128).
- A second strategy was to extract phage DNA directly from water samples, without a cultivation step, and perform real-time PCR (qPCR). This method was used to quantify changes in ARGs in both phage and bacteria in upstream and downstream samples of an urban area (Girona, Spain). We selected eight ARGs (which confer resistance to various antibiotic classes, such as sul1 and sul2 (sulfonamides), ermB (macrolides), tetW (tetracycline), among others. In general terms, upstream and downstream values for ARGs ranged at values 1 Log higher in bacteria, with a noticeable increase of sulfonamide genes downstream.
- Thirdly, we performed a metagenomic analysis of bacterial populations in the samples. For doing that, bacterial DNA was sequenced to determine the presence of AMR determinants and more than 12 families of ARG were detected (e.g. bacitracin, fluoroquinolone, and macrolide). Mobile genetic elements (MGEs) related to the spread of AMR in environmental settings were also abundant in these samples. We are analyzing these findings for peer-reviewed publishing.
- Finally, we took the opportunity to investigate the stress-response caused by phage in Enteric bacteria. This is critical for AMR to spread not only in clinical but also environmental settings and to the best of our knowledge it has not been reported before. The work “Stress induced response by phage in E. coli: a complete transcriptome analysis”, is also being produced for peer-reviewed publishing.
Secondment/Regulatory results
- ENVIROSTOME had a secondment organized with a water intelligence company in Ireland. We produced from this industry contact several outputs including: the articles
(i) “How does the emergence of antimicrobial resistance affect policy making?”
(doi: 10.1016/j.watres.2021.117772)
(Open depository: https://dugi-doc.udg.edu/handle/10256/20501 and https://zenodo.org/record/7966090)
(ii) “Antimicrobial Resistance and Bacteriophages: An Overlooked Intersection in Water Disinfection” (doi.org/10.1016/j.tim.2020.12.011)
(Open depository: https://dugi-doc.udg.edu/handle/10256/19014 and https://zenodo.org/record/7965961).
(iii) a full report including interviews with companies, insights, produced between the secondment and ICRA,
(iv) a technical presentation,
(v) an article to the Marie Curie Alumni newsletter “How does collaboration between industry and academia advances research” (September 2020, ISSN 2663-9483).
- Our interaction and collaboration with water industry practitioners, regulators, and the laboratory activities in the Envirostome, resulted in an advice paper for policymakers, entitled: “Standard method for the examination of antimicrobial resistance in environmental settings: where are we now?” (Accepted by Trends in Microbiology).
Exploitation:
- ENVIROSTOME was shared using the hashtag #envirostome online. More than one hundred broad-audience articles, and posts were shared by the fellow on Twitter and LinkedIn, as well as videos on YouTube, about her work with AMR and Envirostome.
- We reached citizens in Europe and beyond in the topics of AMR research and water and established a close collaboration with the water and regulatory sectors.
- We presented at various events, including: “Water innovation and phage research: industry and academia working together to fight antimicrobial resistance” ICRA Spain(2019), the Water research in perspective: beyond 2020 Icra, Spain (2020), the MSCA IF Cluster event on Antimicrobial Resistance, and evaluation of project on European’s Commission Innovation Radar in Barcelona, Spain (2019), the EU Water Innovation Conference, industry collaboration. Zaragoza, Spain (2019), and the RCS Twitter poster conference (2020).
- We demonstrated the topic of AMR and phage research at the Researcher's Night with the University Girona (2019) to over one hundred citizens in Girona, Spain.
- We produced with EURAXESS Brazil & LAC an educational online seminar to over 120 international PhDs, helping them to better connect their research to the job market.
- Finally, in 2023, the fellow reflected on the experience of this MSCA fellowship to the Marie Curie Alumni Association Brazilian Chapter and produced with EURAXESS Latim Americ and Caribbean Area a promotional video for the Marie Curie funding (to be published on YouTube soon).
- Our work demonstrates that phage and bacteria in environmental settings carry a large pool of ARGs and MGEs. Thus, they pose a significant risk for the water sector because of the risk of AMR transmission from the environment to clinical settings.
-From a precautionary viewpoint, monitoring of phages and ARGs should be included when designing and developing new disinfection treatments.
Investments in upgrading wastewater treatment plants to decrease AMR risk are on the horizon for the water industry.
- Scientific leadership needs to advocate and collaborate better with policymakers for the immediate regulation of precautionary AMR indicators and accessible methodologies to detect their risk on human health.
-From a precautionary viewpoint, monitoring of phages and ARGs should be included when designing and developing new disinfection treatments.
Investments in upgrading wastewater treatment plants to decrease AMR risk are on the horizon for the water industry.
- Scientific leadership needs to advocate and collaborate better with policymakers for the immediate regulation of precautionary AMR indicators and accessible methodologies to detect their risk on human health.