Pentamidine Sensitized FDA-approved Antibiotics Towards Inhibition Of Gram-negative Pathogens

Compared with Gram-positive bacteria,Gram-negative bacteria is less susceptible to antibiotics due to their impermeable outer membranes.Together with the overuse and misuse of antibiotics,the infections caused by multi-drug resistant negative bacteria is rising which pose a serious threat to public health.In the post-antibiotic era that developing new antibiotics is extremely difficult,a new strategy for the treatment of multidrug-resistant bacteria is to use combination therapy to effectively combine existing antibiotics with a compound called sensitizer to achieve "drug repurposing".Pentamidine is a promising sensitizer.It has been found in previous studies that it can sensitize rifampicin,novobiocin and erythromycin,making these antibiotics have the ability to inhibit a variety of Gram-negative pathogens.However,whether pentamidine has a broad-spectrum sensitizing effect on FDA-approved antibiotics remains to be unknown.This project aim to study the spectrum of sensitization of pentamidine and to investigate its mechanisms of action.The main contents and findings are:(1)We carried out a screening with pentamidine in combination of 174FDA-approved antibiotics for inhibition of E.coli K12,and the 6 best combinations were further tested against 14 laboratory or clinical Gram-negative bacteria,including E.coli,E.cloacae,A.baumannii,P.aeruginosa,and K.pneumoniae.(2)Time kill study showed that the combination of pentamidine and minocycline or nadifloxacin had fast killing kinetics and the in vivo efficacy was further demonstrated with a Caenorhabditis elegans infection model.(3)Preliminary structure-activity relationship(SAR)showed that sensitization by pentamidine was related to the hydrophobicity of antibiotics.We also performed a bacterial uptake assay with dye of various hydrophobicity to validate the above SAR.Further mechanism studies showed that hydrophilic aminoglycosides competed with pentamidine for lipopolysaccharide(LPS)binding sites,which led to their antagonistic effect.Hydrophobic rifampins could synergy with pentamidine to break the membrane.Lastly,we propose a model to explain the selectivity and mechanism of sensitization for hydrophobic substrate of pentamidine sensitization.