The plastisphere and river systems as reservoirs for antibiotic resistant bacteria

Antimicrobial resistance (AMR) is a critical global health threat. This phenomenon involves the diffusion of bacteria and genes among humans, animals and the environment. In particular, the presence of third generation cephalosporin (3GC)-resistant Enterobacteriaceae in natural environments is of high concern as they are classified as critical-priority pathogens of public health importance. In this work we studied the relation among plastic pollution in freshwater ecosystems, the spread of multidrug-resistant (MDR) bacteria and diffusion of antibiotic resistance genes (ARGs). Caged plastic fragments were deliberately introduced in a river of central Italy. Plastic samples were collected and analyzed in parallel with river water samples. Out of 267 cefotaxime (CTX) resistant isolates obtained, 65 CTX-resistant Enterobacteriaceae were selected for further analysis. Most of the isolates (75% of plastic-derived and 84% of water-derived isolates) were MDR with seven being carbapenem-resistant enterobacteria (CRE). Five of them synthesize KPC ( Klebsiella pneumoniae carbapenemases) enzymes, and two strains were positive for metallo-β-lactamases (NDM). Among the KPC producers, three isolates were identified as K. pneumoniae sequence type ST1519. Their isolation in a natural ecosystem is alarming because they can potentially re-enter human populations through environmental pathways. Shotgun metagenomic analysis provided a comprehensive snapshot of the microbial communities associated to the plastisphere, revealing dominance of families such as Comamonadaceae , Sphaerotilaceae , and Flavobacteriaceae , which play key roles in environmental biofilm formation and stability. The resistome analysis highlighted the presence of ARGs conferring resistance to clinically important antibiotics, such as beta-lactams, vancomycin, and tetracyclines, alongside mobile genetic elements (MGEs) such as integrons, which facilitate the horizontal transfer of resistance genes. This study provides crucial experimental evidence that riverine plastic debris acts as a genetic reservoir and could act as an efficient vehicle for the accumulation and transfer of clinically relevant resistance determinants.