Anlaysis of the role of plastics in spreading potential pathogenic microorganisms and their antibiotic-resistance genes in aquatic ecosystems using microbiological and molecular methods

Antimicrobial resistance, a silent pandemic, poses a significant global health threat, as the proliferation of antibiotic-resistant pathogenic bacteria contributes to high mortality and morbidity rates. The spread of resistant microbial species and the association of genetic determinants at the human-animal-environment interface can alter microbial genomes, resulting in resistant superbugs, which can colonize various environmental niches. Many common human pathogens, including Enterococcus faecium, Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and other Enterobacter species, are key actors of antimicrobial resistance (AMR). A great deal of these bacteria and/or their modes of resistance, mediated by antibiotic resistance genes (ARGs), come from the natural environment, including bacteria within soils and water. In addition, our planet is facing the pervasive problem of plastic pollution, with aquatic ecosystems bearing a substantial burden on it. The environmental risks caused by antibiotic resistance genes and plastic pollution have drawn considerable attention from researchers worldwide, particularly in the transdisciplinary approach of the One-Health framework. The global challenge of antimicrobial resistance in pathogenic bacteria is anticipated to lead to approximately 10 million annual deaths by 2050. The intricate mechanisms behind antibiotic resistance involve the exchange of genetic materials, Horizontal Gene Transfer (HGT), and selective pressures, leading to the dissemination of multidrug-resistant bacteria in various ecological habitats. Bacteria use three mechanisms for HGT: conjugation, transduction, and transformation. Bacterial conjugation involves specific apparatus like conjugative pili for DNA transfer from donor to recipient cells; transduction is the process in which bacterial DNA is moved from one bacterium to another by a bacteriophage, and transformation is the uptake by the recipient cell of extracellular DNA (eDNA). In the environment, eDNA can be naturally supplied from dead cells to neighboring cells within the same habitat. Even in antibiotic-free environments, the environmental microbiota possesses an enormous number and diversity of antibiotic-resistance genes, some very similar to the genes circulating in pathogenic microbiota. This topic has drawn significant attention from the World Health Organization (WHO), the European Centre for Disease Prevention and Control (ECDC), and the Food Agricultural Organization (FAO) as they emphasize that the aquatic ecosystem is one potential route for transferring ARGs from the environment to humans and animals. Hence, studying the role of the aquatic ecosystem as a reservoir of antibiotic-resistant bacteria and ARGs is very important from the One-Health perspective, as it could contribute to a better understanding of the events that lead to the dissemination of ARGs. The omnipresence of plastic debris, originating from diverse anthropogenic activities, has adverse impacts on microbial communities and ecosystem dynamics of terrestrial and aquatic ecosystems. Plastic fragments serve as resilient substrates and reservoirs for microbial colonization in aquatic habitats, fostering the adhesion and proliferation of diverse microbial communities. These anthropogenic pollutants are emerging as a new substrate for biofilm formation, where high bacterial cell density and metabolic activity provide a stable physical environment for cell-to-cell gene exchange and are a potential hot-spot for the horizontal gene transfer (HGT) of antibiotic resistance genes (ARGs). This phenomenon will likely show a worsening picture if we consider that bacteria in benthic states can exhibit up to 1,000 times more antibiotic resistance than in their planktonic state. Nevertheless, the acquisition of ARGs in these biofilms via natural transformation mediated by extracellular DNA (eDNA) has been rarely explored. To adequately tackle the environmental and health consequences associated with plastic pollution and AMR, it is essential to have a comprehensive understanding of the complex interactions between plastics and microbial communities. This doctoral research work is focused on analyzing the role of plastics in spreading potential pathogenic microorganisms and their antibiotic-resistance genes in aquatic ecosystems using microbiological and molecular methods. These findings are crucial for evaluating the risks of disseminating multidrug-resistant bacteria and their associated antibiotic-resistance genes via plastic fragments and provide a vital research basis for proposing strategies to mitigate the risks of AMR. Research activities were conducted at the School of Biosciences and Veterinary Medicine, University of Camerino, Italy, and the Department of Environment and Primary Prevention, National Institute of Health, Rome, Italy. Over the course of the three-year Ph.D. (from October 2020 to January 2024), Ifra Ferheen spent six months as part of an international mobility program at BC3 Basque Centre for Climate Change, Klima Aldaketa Ikergai, Bilbao, Spain. During her Ph.D., Ifra Ferheen was involved in the European Union's BlueAdapt Project, and her Ph.D. was funded by a scholarship from the National Institute of Health, Rome, Italy.