β,β-Dimethylacrylalkannin Restores Colistin Efficacy Against mcr- and TCS-Mediated Resistant Gram-Negative Bacteria via Membrane Disturbance

<b>Background:</b> The reintroduction of colistin has led to the rapid emergence of colistin-resistant strains, significantly diminishing its therapeutic efficacy. This presents a need for effective adjuvants to restore colistin efficacy. <b>Approach</b>: We screened the colistin adjuvants through a high-throughput method and then evaluated their synergistic effects and underlying mechanisms. <b>Results</b>: We identified β,β-dimethylacrylalkannin (β,β-Dim), a naphthoquinone compound derived from Lithospermum erythrorhizon, as a potent colistin adjuvant (fractional inhibitory concentration index (FICI) < 0.5). β,β-Dim enhanced colistin activity against 4 of 6 susceptible strains and all 18 colistin-resistant strains carrying either plasmid-borne <i>mcr</i> genes (<i>mcr-1</i>, <i>mcr-3</i>, <i>mcr-8</i>, and <i>mcr-9</i>) or chromosomal two-component system (TCS) mutations (<i>pmrA</i>/<i>B</i>, <i>phoP</i>, and <i>mgrB</i>). These strains included <i>Klebsiella pneumoniae</i>, <i>Escherichia coli</i>, <i>Salmonella</i> Typhimurium, <i>Pseudomonas aeruginosa</i>, and <i>Acinetobacter baumannii</i>. The combination reduced the minimum inhibitory concentrations (MICs) of colistin by 4-1024-fold (from 512 to ≤2 µg/mL). Mechanistically, colistin-mediated outer membrane permeabilization facilitates β,β-Dim entry. Once internalized, β,β-Dim interacts with cytoplasmic membrane phospholipids and disrupts membrane biofunction. Further analysis showed that LPS transport and efflux pump activity were impaired, leading to LPS accumulation in the cytoplasmic membrane and increased intracellular colistin content. These processes elevated reactive oxygen species (ROS) production and markedly reduced ATP levels. In a murine infection model, β,β-Dim (2 mg/kg) combined with colistin (0.2 mg/kg) markedly increased survival from 20% (colistin alone) to 80%. <b>Conclusions:</b> These findings highlight that β,β-Dim combined with colistin is a promising therapeutic strategy for infections caused by colistin-resistant pathogens.