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Several advances in the medical field are often dependent on the ability to fight infections with the use of antibiotics, including joint replacements, organ transplants, and cancer therapy. The capacity of the bacteria to adapt to and escape from the mechanisms of action of antibiotics makes the antimicrobial resistance a serious public health problem worldwide. Polymyxin E colistin has rarely been used because of its nephrotoxicity and neurotoxicity. More recently, the emergence of multi-drug resistant bacteria as carbapenem-resistant Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa and the re-evaluation of its pharmacokinetic properties have led to a resurgence of colistin as a treatment option, contributing to select resistant strains. Investigating the phenomenon of colistin-resistance in gram-negative bacteria, especially P. aeruginosa, is now mandatory, particularly after identification of a plasmid-mediated mechanism for the resistance to colistin (mcr) in Enterobacteriaceae strains, a mechanism transferable to other species. In this study, we investigated colistin-resistance in gram-negative bacteria isolated from respiratory secretions of cystic fibrosis patients in follow-up at Children’s Hospital Bambino Gesù of Rome.