Colistin exerts potent activity against mcr+ Enterobacteriaceae via synergistic interactions with the host defense

Abstract

Colistin (COL) is a cationic cyclic peptide that disrupts the membranes of Gram-negative bacteria and is often used as a last resort antibiotic against multidrug-resistant strains. The emergence of plasmid-borne mcr genes, which confer transferable COL resistance, has raised serious concerns, particularly in strains also carrying extended-spectrum β-lactamase and carbapenemase genes. Standard antimicrobial susceptibility testing (AST), performed in enriched bacteriological media, indicates no activity of COL against mcr+ strains, leading to its exclusion from treatment regimens. However, these media poorly reflect in vivo physiology and lack host immune components. Here we show that COL retained bactericidal activity against mcr-1+ Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica when tested in tissue culture medium containing physiological bicarbonate. COL enhanced serum complement deposition on bacterial surfaces and synergized with human serum to kill pathogens. At clinically achievable concentrations, COL killed mcr-1+ strains in freshly isolated human blood and was effective as monotherapy in a murine E. coli bacteremia model. These findings suggest that COL, currently dismissed based on conventional AST, may offer clinical benefit against mcr-1+ infections when evaluated under more physiological conditions - warranting reconsideration in clinical microbiology practices and future trials for high-risk patients.

Keywords: Bacterial infections; Drug therapy; Infectious disease; Microbiology.

Figure 1. COL bactericidal activity against mcr-1⁺ Gram-negative pathogens in physiological medium is dependent on bicarbonate and involves membrane permeabilization.

(A) Kinetic kill curves show COL minimum bactericidal concentration against E. coli, K. pneumoniae, and S. enterica (defined as a reduction in viable bacteria ≥3 log₁₀ CFU/mL at 24 hours vs. starting inoculum) when assessed in supplemented mammalian tissue culture RPMI(10%LB) versus standard bacteriological medium CA-MHB; limit of detection <50 CFU/mL. (B) Bactericidal activity of COL in RPMI(10%LB) or CA-MHB medium amended with 0, 10, or 25 mM HCO₃^–; limit of detection <50 CFU/mL. (C) COL-mediated outer membrane permeabilization of mcr-1⁺ E. coli assessed using the nonpolar compound 1-N-phenylnaphthylamine (NPN) that fluoresces strongly in phospholipid environments in RPMI(10%LB) or CA-MHB. (D) Relative bacterial surface charge estimated by cationic cytochrome c binding to mcr-1⁺ E. coli following growth in CA-MHB or RPMI(10%LB). Data represent the mean ± SEM from the combination of 3 experiments performed in triplicate. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 2. COL accelerates killing of mcr-1⁺ Gram-negative bacteria in human blood and serum.

(A) Bacterial survival in freshly isolated human whole blood with or without COL at concentrations representing ¼×, ½×, and 1× MIC for each mcr-1⁺ bacterial strain. (B) Bacterial survival in 10% human serum with or without COL at concentrations representing ¼×, ½×, and 1× MIC for each mcr-1⁺ bacterial strain. Data are represented as percentage viable CFU versus initial inoculum and mean ± SEM from a combination of 3 experiments performed in triplicate using blood or serum from 3 different donors. ****P ≤ 0.0001 by 1-way ANOVA.

Figure 3. COL promotes C3 deposition on the mcr-1⁺ Gram-negative bacterial surface.

(A) C3 protein deposition on the surface of E. coli, K. pneumoniae, and S. enterica as detected by flow cytometry. Median fluorescence intensity (MFI) of bacteria-bound C3 protein shown as fold change versus untreated control bacteria of each species. Flow cytometry data are representative of 2 independent experiments conducted in triplicate; 10,000 cells were counted per experimental replicate, and analyses were performed using serum from 2 different donors. (B) Confocal microscopy images (average-intensity Z projections) of E. coli in the presence or absence of serum and presence or absence of 1 μg/mL COL; scale bars: 2 μm. DNA staining by Hoechst dye (blue); bound C3 protein detected using an anti-C3 antibody and fluorescent secondary antibody (Alexa Fluor 488, green). (C) Quantification of C3 fluorescence signal (average-intensity Z projections) from confocal microscopy shown as fold change versus untreated control bacteria. Bar graph generated from unbiased analysis of multiple random microscopy fields with more than 100 cells counted per condition. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 by 2-way ANOVA.

Figure 4. Impacts of polymyxin monotherapy on mcr-1⁺ E. coli bacteremia outcomes in normal and neutropenic mice.

(A) C57BL/6J mice were infected i.v. with 2 × 10⁷ CFU of E. coli (mcr-1⁺) and treated subcutaneously with PBS (100 μL, triangles), CTX (50 mg/kg, squares), or COL (20 mg/kg, circles) every 12 hours for 2 doses (n = 6 per group). Bacterial loads were recovered from spleen or kidney at 24 hours and are plotted as CFU/g organ tissue. (B) Survival of C57BL/6J mice infected i.v. with 1 × 10⁹ CFU of E. coli (mcr-1) and treated subcutaneously with a single dose of PBS (100 μL, triangles), CTX (50 mg/kg, squares), or COL (20 mg/kg, circles) 1 hour after infection (n = 13 per group). *P ≤ 0.05, ****P ≤ 0.0001 by 1-way ANOVA for CFU studies and log-rank test for survival. (C) Neutrophil-depleted C57BL/6J mice were infected with 2.5 × 10⁵ CFU of E. coli (mcr-1) and treated subcutaneously with PBS (100 μL, triangles), CTX (50 mg/kg, squares), COL (20 mg/kg, circles), or PMB (20 mg/kg, diamonds) every 12 hours for 2 doses (n = 10 per group). Bacterial loads were recovered from spleen, kidney, lung, or blood at 24 hours and are plotted as CFU/g organ tissue. ***P ≤ 0.001, ****P ≤ 0.0001 by 1-way ANOVA.