Evaluation of Plastic Pollution and associated Multidrug Resistant Bacteria in a River of Central Italy

Antimicrobial resistance (AMR) and plastic pollution represent two global environmental and public health challenges. River ecosystems play a central role in the spread of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with plastic debris providing an additional vehicle for their persistence and transmission. This thesis investigates the interplay between ARB colonizing plastic materials and those present in river water, focusing on third-generation cephalosporin (3GC)-resistant Enterobacteriaceae in the Chienti River (Marche, Italy). Using a multidisciplinary approach, which combines microbiological, molecular, and metagenomic analyses, we identified a high prevalence of multidrug-resistant (MDR) profiles in 3GC-resistant Enterobacteriaceae. Additionally, we detected carbapenem-resistant enterobacteria (CRE), including Klebsiella pneumoniae ST1519, recorded for the first time in a natural environment in Italy. The resistome analysis of plastic-associated bacteria revealed a high abundance of ARGs conferring resistance to clinically relevant antibiotics, such as beta-lactams, glycopeptides, and tetracyclines, alongside mobile genetic elements (MGEs) such as integrons, which facilitate their horizontal gene transfer. These findings emphasize the potential of rivers and plastics to act as reservoirs for priority resistant pathogens challenging the public health care system. To address the growing need for a rapid AMR surveillance, this work also contributed to the development of a CRISPR/Cas12a-based molecular detection toolbox capable of identifying ARGs with high sensitivity. This portable and adaptable tool, initially validated in clinical isolates, bridges the gap between laboratory and in-field environmental monitoring, representing a promising solution for real-time AMR detection.