Antibiotic-Resistance Genes: Playing Hide and Seek in the Soil of a European Capital

This study focuses on metagenomic sequencing conducted in the vicinity of Prague, Czech Republic, specifically exploring diverse environmental sites such as a faculty garden under organic management, an experimental agricultural field, an area with construction material deposition, and soil along the shores of the Vltava River. Given the well-established link between antibiotic resistance and the genes governing resistance mechanisms, this research aims to provide a comprehensive catalog and characterization of Antibiotic Resistance Genes (ARGs) and associated mobile elements within these environmental niches. Prague, situated along the Vltava River—a tributary to the Elbe River with a vast watershed encompassing the Czech Republic, Germany, Poland, and Austria—serves as a crucial study area. The estimated population of 24 million people in this region underscores the potential impact of anthropogenic activities on environmental microbial communities. The dynamics of rain-induced soil deposition, microbial genes, and extracellular DNA into river courses, facilitated by stormwater runoff, contribute to downstream transport. This process, exacerbated by anthropogenic pollution around urban centers, can create hotspots for the selection and dissemination of resistance genes. While the primary objective is to document and understand ARGs and mobile elements, it is imperative to acknowledge the multifaceted functionalities of microbial communities beyond antibiotic resistance. Employing a metagenomics approach allows for a broader assessment of the functional genetic profiles of these microbial communities. The significance of this study lies in its direct relevance to environmental monitoring and its pivotal role in preventing the spread of resistance to clinical settings. Once resistance genes infiltrate hospitals, their permanence becomes inevitable. Resistance genes are intrinsic to the global microbiome, with the environmental compartment serving as a vast reservoir for new genes. Recognizing and comprehending these dynamics are critical steps in developing strategies to mitigate the spread of antibiotic resistance. Libraries were prepared according to in-house standard procedures, using NEBNext Ultra II DNA Library Prep Kit (Illumina, CA, USA) and NEBNext Multiplex Oligos for Illumina (Unique Dual Index Primer Pairs). The quality of the library was assessed using Agilent Bioanalyzer 2100 High sensitivity DNA Kit (Agilent, CA, USA), and quantification was evaluated through Invitrogen Collibri Library Quantification Kit (Invitrogen, MA, USA). Sequencing was carried out at an Illumina NovaSeq 6000 System following a Paired End strategy, 2 x 150 bp, 100 million reads sequencing depth.