Phd in CELL AND DEVELOPMENTAL BIOLOGY

Thesis title: Pharmacological inhibition of colistin resistance in Gram-negative pathogens

Antibiotic resistance in Gram-negative pathogens, especially Pseudomonas aeruginosa and Klebsiella pneumoniae, represents a serious global health threat. Colistin, a last-resort antibiotic against multidrug-resistant (MDR) pathogens, targets the outer membrane (OM) of Gram-negative bacteria, leading to cell death. However, OM remodeling by the ArnT-mediated lipid A aminoarabinosylation, makes the bacteria resistant to colistin. This study explores the use of ArnT inhibitors, as adjuvants to colistin, to restore sensitivity to this antibiotic in otherwise resistant bacteria, in planktonic bacteria and in biofilms. Additionally, we have addressed liposomal formulations as vehicle for the delivery of colistin and its adjuvant. Antimicrobic susceptibility tests and gene expression analysis in P. aeruginosa and K. pneumoniae strains, showed higher resistance to colistin and high level of ArnT expression in biofilms respect to planktonic cultures. According to this observation, the ArnT inhibitors, FDO or FDO-H, used in combination with colistin, significantly enhanced colistin efficacy against biofilms. The ArnT inhibitors we have identified in previous works, are insoluble in water, which restricts their use as colistin adjuvants. To overcome this drawback, liposomal formulations were designed for the co-delivery of colistin and its adjuvants. In particular, colistin was entrapped in the aqueous core of the liposomes while the hydrophobic ArnT inhibitor, isostevic acid, within the lipid bilayer. The resulting dual-loaded liposomes reduced the minimum inhibitory concentration of colistin against a colistin-resistant P. aeruginosa strain and potentiate its bactericidal activity. The development of ArnT inhibitors was further refined through structure-activity relationship (SAR) studies on diterpenoid-based compounds, with a particular focus on the podocarpic acid and its derivatives. By this study, we identified relevant modifications, such as the oxalate-like group at C-16, with improved binding affinity to the ArnT catalytic site, and enhanced activity as colistin adjuvant. Collectively, this research underscores the promise of combining ArnT inhibitors with colistin, particularly through liposomal delivery, as a strategy to potentiate colistin’s efficacy against MDR infections. These results provide a foundation for further in vivo studies aimed at optimizing these formulations and may pave the way for more effective and safer treatments for challenging Gram-negative bacterial infections.