Colistin is an antibiotic of last resort, but has poor efficacy and resistance is a growing problem. Whilst it is well established that colistin disrupts the bacterial outer membrane by selectively targeting lipopolysaccharide (LPS), it was unclear how this led to bacterial killing. We discovered that MCR-1 mediated colistin resistance in Escherichia coli is due to modified LPS at the cytoplasmic rather than outer membrane. In doing so, we also demonstrated that colistin exerts bactericidal activity by targeting LPS in the cytoplasmic membrane. We then exploited this information to devise a new therapeutic approach. Using the LPS transport inhibitor murepavadin, we were able to cause LPS accumulation in the cytoplasmic membrane of Pseudomonas aeruginosa, which resulted in increased susceptibility to colistin in vitro and improved treatment efficacy in vivo. These findings reveal new insight into the mechanism by which colistin kills bacteria, providing the foundations for novel approaches to enhance therapeutic outcomes.

Tel: 0044 (0)207 594 2072 23 Fax: 0044 (0)207 594 3096 24 a.edwards@imperial.ac.uk 25 26 Keywords: Colistin / polymyxin / Pseudomonas / E. coli / Klebsiella / lipopolysaccharide / resistance / MCR-1 27 28 Running title: Colistin mechanism of action 29 30 31 32 33 2 Summary 34 Colistin is an antibiotic of last resort for infections caused by drug-resistant Gram-negative pathogens such 35as Pseudomonas aeruginosa and Escherichia coli. For this reason, high rates of treatment failure and 36 increasing resistance to this antibiotic are very concerning and attempts to resolve these issues are hampered 37 by a poor understanding of colistin's mode of action. Whilst it is well established that colistin binds to 38 lipopolysaccharide in the bacterial outer membrane, it was unclear how this led to bacterial killing. Here, we 39show that colistin also targets lipopolysaccharide in the cytoplasmic membrane and that this interaction is 40 essential for cytoplasmic membrane permeabilisation, cell lysis and the bactericidal activity of the antibiotic. 41We also found that MCR-1-mediated colistin resistance confers protection against the antibiotic via the 42 presence of modified lipopolysaccharide within the cytoplasmic membrane, rather than the outer 43 membrane. These findings reveal key details about the mechanism by which colistin kills bacteria, providing 44 the foundations for the development of new approaches to enhance therapeutic outcomes.

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

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