Periodic Reporting for period 1 - REDTEAR (The Role of free Extracellular DNA in determining The Environmental Antibiotic Resistome)
Période du rapport: 2021-10-01 au 2023-09-30
The escalating emergence of antimicrobial resistance claims worldwide attention, as its anticipated consequences on human health and the global economy are projected to be as severe as those caused by cancer. Over the past few decades, morbidity and mortality linked to multiple antimicrobial resistance (AR) have significantly risen. The use of the few remaining last-resort antibiotics to treat multi-drug resistant (MDR) bacterial infections has become a common medical practice in various regions across the globe. In Europe alone, reports indicate an annual death toll of approximately 25,000 attributed to antimicrobial resistance. Thanks to the One Health concept, it is evident that an urgent response to the spread of AR requires a deeper understanding of the cycle of antimicrobial resistant bacteria (ARB). In addition to intracellular DNA (iDNA, DNA content within bacterial cells), there is another fraction known as extracellular DNA (eDNA, DNA content outside bacterial cells), which remains a potential vector for antimicrobial resistance genes (ARGs), potentially contributing to the dissemination of AR across different sectors (human, animal, environment). This underscores the need for a more comprehensive understanding of the role of eDNA in the dissemination of AR. With the REDTEAR project, we addressed this significant health and economic concern by investigating the diversity and abundance of ARGs in eDNA isolated from aquatic ecosystems. Additionally, through experimental approaches, we assessed the impact of anthropogenic pollution on the dissemination of ARGs facilitated by eDNA transformation.
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.
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.
The developed eDNA extraction protocol proved to be effective for applying shotgun sequencing to DNA samples extracted from various types of water. Its versatility in processing a wide range of samples is particularly noteworthy. In addition to the methodological innovation, the results of the REDTEAR project have significantly enhanced our understanding of the role of eDNA in the dissemination of AR and the impact of anthropogenic pollution on the spread of ARGs. Moreover, anthropogenic pollution was identified as a significant driver of the transformation of ARG-positive plasmids. Overall, the project's findings have substantially advanced our comprehension of the antibiotic resistance cycle, encompassing eDNA. This knowledge will undoubtedly play a crucial role in shaping measures to combat the issue of antibiotic-resistant bacteria, which remains a pressing concern for human health.