Colistin: understanding the mechanism of action and the causes of treatment failure
Creators: name: Sabnis, Akshay
Publisher: name: Imperial College London
Year: 2022

description: The rapid rise in the prevalence of pathogenic bacteria that are resistant to front-line antibiotic drugs has necessitated a simultaneous upsurge in the use of “last-resort” antimicrobial agents. Colistin is one such antibiotic of last-resort that is increasingly used in the clinic as a salvage therapy to treat infections caused by multi-drug resistant Gram-negative microorganisms, including Pseudomonas aeruginosa and Escherichia coli. Unfortunately, despite its growing importance, colistin treatment is toxic, frequently fails, and resistance to the antibiotic is an intensifying threat. There is, therefore, a crucial requirement to augment the efficacy of colistin therapy, but efforts to do so are hampered by a lack of understanding about the drug’s mechanism of action. The work in this thesis initially uncovered a novel process by which colistin-susceptible P. aeruginosa cells survive exposure to the antibiotic, through the extracellular release of lipopolysaccharide (LPS) molecules that inactivate colistin in the external environment. In attempting to overcome this mode of drug tolerance by inhibiting LPS biosynthesis, critical insight into colistin’s bactericidal mechanism was obtained – namely, that the antibiotic kills Gram-negative bacteria by targeting LPS in the cytoplasmic membrane, not by interacting with membrane phospholipids, as previously thought. This finding in turn led to investigations about the site where colistin resistance, mediated by cationic chemical modifications to LPS, was conferred. It was shown that resistance to colistin in P. aeruginosa and E. coli, acquired through the harbouring of diverse plasmid-borne mobile colistin resistance (mcr) genes or chromosomal mutations, was in fact conferred at the cytoplasmic membrane, as opposed to the outer membrane of bacterial cells. Subsequent experiments revealed that intrinsic colistin resistance in Burkholderia cenocepacia was mediated at the outer membrane, and that strains of Enterobacter cloacae possess a unique inducible form of hetero-resistance to colistin. After characterising the mode of action of colistin and a number of potential causes of colistin treatment failure, new combination treatment strategies were designed to improve the effectiveness of colistin therapy. Using murepavadin, an inhibitor of the LPS transport system in pre-clinical development, to accumulate LPS in the cytoplasmic membrane proved to be particularly potent at amplifying the bactericidal activity of colistin in vitro against clinical isolates, in an in vivo murine lung infection model, and for overcoming colistin resistance. Furthermore, the capacity for colistin to permeabilise the outer membrane of colistin-resistant bacteria was exploited to re-sensitise Gram-negative pathogenic organisms to rifampicin, an antibiotic which normally cannot traverse the cell envelope. In summary, this work has identified how colistin works and why it often fails clinically, as well as providing urgently-needed novel solutions to enhance colistin efficacy and patient outcomes.