The REDTEAR project was organized into four work packages (WPs). Initially, all the effort was focused on optimizing the eDNA extraction protocol by amalgamating and enhancing various steps taken from previously published protocols. To fine-tune the protocol, we analyzed samples collected both before and after disinfection from various wastewater treatment plants (WWTPs). This analysis also provided insights into the impact of chemical disinfection on the diversity and abundance of the antimicrobial resistome (the total content of antibiotic resistance genes), taking into account both intracellular DNA (iDNA) and extracellular DNA (eDNA). The antimicrobial resistome within iDNA exhibited significantly greater diversity and abundance when compared to eDNA. Moreover, the various disinfection methods tested did not have any discernible effect on the content of ARGs in either eDNA or iDNA. Lastly, an innovative bioinformatic approach was employed to characterize the phageome (the total content of bacteriophages), which was found to have limited quantitative relevance in the dissemination of ARGs.
Successively, samples from different water sources, influenced by a diverse anthropogenic pollution, were collected and chemically characterized. Both DNA fractions were extracted (i.e. iDNA and eDNA) and processed by the shotgun sequencing to unveil the microbial community and associated resistome composition. The anthropogenic pollution resulted to be the main driver of the antimicrobial resistome and among the mobile genetic elements (MGEs), the plasmids were the main carriers of ARGs in both DNA fractions.
Finally, we conducted a series of experiments to assess the impact of anthropogenic pollution on the transformation rate and the fitness of transformant bacteria in the environment. To elaborate, in the initial experiment, we created a gradient of aquatic anthropogenic pollution under antibiotic pressure and separately introduced two GFP-plasmids into the experimental vessels containing the water samples. Subsequently, the experiment was concluded, and samples were filtered to recover bacterial cells. The quantity of the GFP gene within these bacterial cells was measured using qPCR, allowing us to determine the rate of transformation and the selection of plasmids within the cells. The results demonstrated that anthropogenic pollution significantly favored both the transformation rate and plasmid selection. Finally, we carried out a series of experiments to assess the adaptability of the plasmids within the transformed environmental bacteria. The plasmid fitness costs were particularly significant, suggesting a low likelihood for these bacteria to thrive in the environment.
