Periodic Reporting for period 1 - RESOLVE (antibiotic RESistance and mObile genetic eLements dissemination in enVironmEntal conditions) Reporting period: 2016-08-01 to 2018-07-31 Summary of the context and overall objectives of the project Antimicrobial resistance (AMR) is one of the most concerning public health threads of our times. Horizontal gene transfer (HGT) via mobile genetic elements is a key element for the acquisition/dissemination of AMR genes by bacteria. The environment constitutes a large reservoir of AMR genes and there is evidence that at environmentally relevant concentrations, some antibiotics may mediate HGT. Biofilms, which are the principal mode of life of bacteria in the environment, have been proposed to be structures promoting gene transfer. However, the demonstration of the actual transfer, the dynamics and regulation of such MGEs in a complex environmental matrix, is yet experimentally challenging. This left important questions unanswered, such as, how and when transfer and dissemination of ARGs actually occur in nature. The overall aim of this project was to investigate whether wastewaters, as an environmental parameter, are hot-spots for horizontal gene transfer. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Two biofilm models were employed in order to tackle the main research objectives of this project: Biofilm model 1- Monospecies biofilms composed of one strain carrying a known MGE-promoter-lux fusion allowing by means of measuring luminescence whether the expression of these MGE-promoters is affected by different wastewater effluents. Biofilm model 2- Natural biofilms grown from wastewaters (hospital, urban). Those biofilms are likely to be composed of multispecies and closely model environmental in vivo situations. Main objectives were to determine the bacterial compositions of those biofilms, screen for MGEs and ARG content and test whether the expression of MGE-promoters is affected by antibiotics by means of meta-transcriptomics. The aim of implementing biofilm model 1 was to assess the potential inductive role of antibiotics and effluents in HGT associated with AMR, in biofilm cultures. A screening method using a luminescence reporter system has been implemented to screen for the expression of eleven promoters of genes carried by the SGI1 element of Salmonella enterica, which is associated with AMRs. The chosen promoters are known or suspected to be involved in transfer function. The luminescence expression has been measured in Escherichia coli and S. enterica strains carrying the plasmids with the reporter fusions. The effect of exposure to ciprofloxacin and hospital effluents was tested. Our preliminary results indicate that SGI1 promoters involved in HGT are differently expressed in biofilm, while expression of some promoters was further induced in SGI1 positive clones. Remarkably, ciprofloxacin and hospital wastewater mediated promoter gene expression seems to be repressed in SGI1 positive clones, indicating that the presence of a chromosomal copy of the SGI1 element interferes with potential regulatory induction of SGI1 promoters by ciprofloxacin and hospital wastewater. To address the questions as to whether multi-species biofilms (Biofilm model 2; grown in hospital and urban wastewater and natural surface water environments) play an important role in the dissemination of ARGs, a new model has been implemented. Natural biofilms from wastewaters (hospital, urban) and river water were grown on polystyrene slides for 7 days, directly in situ in the water sources of the respective sites (hospital wastewater pipe, urban wastewater treatment plant, natural river) and in the lab by means of a continuous culturing system using water collected from the various sites. Biofilms grown in the lab from each water type were compared to their lab replicate treated with antibiotics and to their in situ counterparts with respect to their: bacterial composition, MGEs and ARGs content, and their transcriptomes focusing on the expression of MGEs and ARGs. Distinct signatures for the studied biofilms and waters were identified, clearly separating the different sources according to their microbial make up. Preliminary analysis indicates that biofilms do share the microbiota with their originating respective water sources (hospital, urban and natural river wastewater), but in differential proportional abundance giving clues on which bacterial taxa are more successful and dominant in the respective biofilms. The resistome (representing 15 resistance gene classes, heavy metals, mobile genetic elements and integrons) analysis indicated that for the hospital biofilms, all but one of the detected ARG classes (tetracycline) were more abundant in biofilms from hospital wastewater compared to hospital wastewater itself. For Urban wastewaters this trend was similar; for 10 ARG classes the in situ urban biofilm was richer in ARG content compared the urban wastewater. The river biofilms and river water contained significantly lower levels of ARGs compared to the urban and hospital wastewater biofilms. Strikingly, MGEs and integrons are highest in all biofilms from all sources compared to the wastewaters, indicating that integrons are abundant in wastewater and natural biofilms. Finally, the 16S rRNA data and resistome data will also serve as a tool to deepen and correlate the ongoing meta-transcriptomic analysis. In addition, an approach by confocal microscopy was developed in order to visualize and study structural matrix components and the architecture of wastewater (hospital, urban) and river biofilms. Extracellular polymeric substances (EPS) such as external DNA (eDNA), which are important structural components of the biofilm matrix, are associated with resistance to antibiotics and the promotion of horizontal gene transfer. Preliminary results clearly showed that the matrix and structural architecture of these mature multi-species biofilms differs according to their environment and is significantly impacted when exposed to antibiotics (ciprofloxacin). Complementing our genomic approaches by advancing high-resolution confocal microscopy is a novel approach and as we identified, an important gap in unravelling the role of wastewater and river biofilms as environmental hotspots for the dissemination of ARGs. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) The progress beyond the state of the art and expected potential impact are highlighted by the main findings and innovative approaches applied during this fellowship. A specific website for this project has not been implemented. However, the specific urban and hospital wastewater resistome and microbiome dynamics and signature identified here may present considerable targets when establishing environmental policies with respect to wastewater treatment and the associated risks of wastewater effluents with the spread of ARGs into the environment. Furthermore, our in-depth and comparative biofilm studies highlight natural biofilms from wastewaters as accumulative hot-spots for ARGs and MGEs. In the face of the global crisis of antimicrobial resistance, our findings lay important groundwork which will add to the fundamental knowledge of the mechanism of ARGs dissemination by means of water bodies and natural biofilms. We also make important connections in our studies by measuring and assessing anthropogenic pollution in the studied wastewaters and water systems, which will allow us to identify putative key players that drive the selection and maintenance for ARGs in aquatic and terrestrial environments. matrix of hospital wastewater biofilms by confocal microscopy microbiome and resistome of complex multispecies biofilms
