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Spatial organization and Horizontal gene transfer of Antibiotic Resistance by ESBL in WasteWater

Periodic Reporting for period 1 - SHARE_WW (Spatial organization and Horizontal gene transfer of Antibiotic Resistance by ESBL in WasteWater)

Reporting period: 2018-08-01 to 2020-07-31

SHARE-WW project is particularly timely due to the current situation of antibiotic resistance, already responsible for an estimated 25,000 deaths per year in the EU. Wastewater treatment plants (WWTP) are often pointed out as hotspots for antibiotic resistance dissemination from problematic pathogens to environmental bacteria. Furthermore, it is described as inefficient in eliminating antibiotic resistance bacteria and genes from the treated effluent. Beta-lactams are a widely used class of antibiotics, some being the last resource in hospitals to treat multidrug resistant infections. Unfortunately, a fast growing resistance has been observed by different bacteria, widely detected in wastewater, mainly due to the production of β-lactamases, enzymes that degrade the beta-lactam antibiotics, and by growing as aggregates, known as biofilms. Therefore, it is urgent to uncover the relation between the biofilm bacteria’s strategy of growing as biofilms and the resistance to β-lactam. Consequently, one can define the role of wastewater treatment plants (and the environmental contribution) in promoting such fast growing resistance.
This innovative proposal aimed to 1) analyse the role of sharing antibiotic resistance genes between different bacteria growing close together in biofilms, and its contribution to a collective β-lactam resistance; 2) Evaluate the impact of sharing the genes coding for the β-lactamase enzymes, by following its expression and localization in the biofilm; 3) Evaluate the role of WWTP in dissemination of beta-lactam resistance.
As conclusions: the results gave deep insights to not only the mechanisms of spreading antibiotic resistance (the expression of beta-lactamases at the intracellular and extracellular level), but also the conditions underlying such events (species diversity in mixed biofilm is correlated with increased antibiotic resistance). Furthermore, it shows the importance of sensitive bacteria in producing a protective biofilm and shift the focus from focusing only on the resistant bacteria as solo individuals to include the mixed bacterial community, that demonstrate a cooperative behavior. Finally, one can conclude that, in the daily routine of human kind, WWTP have great impact on the selection of which beta-lactamase antibiotic resistance genes and pathogenic bacteria enters back in the antimicrobial resistant (One Health) cycle.
With this project it was possible for the first time, to the best of my knowledge, to construct a stable multi-species consortium of 4 different wastewater bacteria species. This consortium of 4 strains was selected from an initial sample of 63 isolates by the following an approach designed by me tha tincliuded several steps from identification of the species to evaluate the different interactions types occurring between all, by analysing different combinations of 1 to 4 different strains.
The presence and activity of the beta-lactamases enzymes was accessed by a novel approach (construction of a fusion protein including the gene for a beta-lactamase protein and the gene of a reporter protein) being both expressed as a unique protein under the control of the beta-lactamase native promotor. The expression of such fusion protein enables to reveal that beta-lactamases are expressed and active at the periplasm of the producer cells and also in the extracellular, degrading the beta-lactam antibiotic outside of the cells.
Each strain was able to transfer beta-lactamases genes for all other 3 strains of the consortium by horizontal gene transfer. Furthermore, by increasing the number of strains in the biofilm consortium demonstrated that the complexity of the species diversity of the biofilm is correlated with the ability of the bacteria escape from the AB action.
From the thorough analysis of the beta-lactam resistome and microbiome from each treatment points of 2 full-scale WWTP, it was possible to pinpointing the exact treatments (secondary treatment and the disinfection steps) that impact on the dissemination of antibiotic resistance. These results were obtained for the discharged effluent and reused effluent, by analyzing genomic DNA and eDNA.
Exploitation and dissemination:
During the action, the outputs of the project were exploited and disseminated by different means, in all with the reference of the EU funding:
Scientific conferences and symposiums:
• BAGECO 15, 26-30 May 2019, Lisbon, Portugal;
• FEMS 2019 7-11th July 2019, Glasgow, Scotland
• The Danish Microbial Society Annual Congress 2019, 11th November 2019, Copenhagen, Denmark
• Biofilm minisymposium, 13 – 14 August 2018, The Maersk tower, University of Copenhagen, Denmark;
Scientific manuscripts for publication in Peer review scientific journals
• A manuscript entitled “The environmental contribution to carbapenem resistance dissemination by discharged and reused wastewater effluents: the role of cellular and extracellular DNA” is in the process or replying to the reviewer’s comments after submission in the journal Water Research
Outreach activities and other means than scientific publications
• Dansk Naturvidenskabsfestival, visiting the NGG International School, Hørshom,
• Interview to the University Journal (Uniavisen, Copenhagen University);
• Event days to introduce the activities and projects (including my MSCA IF project) developed at the Section of Microbiology, KU, to high school students and bachelor students searching for a Master theme
• Section of Microbiology webpage at KU website
• Twitter account (@AnaSilv82987273) and LinkedIn ( pages
The project results full-filled crucial knowledge gaps in the beta-lactam resistance by biofilms in the environmental framework. Furthermore, such results have impact in the socio-economic (water industry) and societal implications (human health and policy making):
1) The knowledge that the complexity of wastewater biofilms is correlated with the increased ability to resist to beta-lactam antibiotics and that the localization of the beta-lactamase enzyme can be transferred to clinical diagnosis and treatment. Such knowledge reveals the importance of targeting the neighbours of the antibiotic resistant bacteria and the extracellular hydrolyzing enzyme . 2) For the Industry the knowledge of which wastewater treatment steps are involved in shaping the dissemination of AB resistances can be used to tailored treatments, especially when reusing the treated effluent for anthropogenic activities. In fact, close collaboration has been conducted between 2 wastewater treatment plants, that have showed in deep interest in the results obtained, once they are published. Furthermore, the information on the number of AB resistant gene copy numbers for beta-lactam and the diversity of pathogenic bacteria discharged in the treated effluents are crucial knowledge for improvement of EU Water Directives.