Sanguinarine Reduces Conjugative Transfer of mcr-1-Positive Plasmids in Escherichia coli

Abstract

Background/Objectives: IncI2 and IncX4 plasmids are important vehicles for dissemination of the mobile colistin resistance gene mcr-1. We evaluated whether sanguinarine (SA) reduces conjugative transfer of these two mcr-1-carrying plasmid types and examined biological changes accompanying the transfer phenotype. Methods: Conjugation was measured in Escherichia coli carrying pIncI2-mcr-1 or pIncX4-mcr-1 after exposure to 2–8 µg/mL SA. Growth-related measurements, intracellular ATP signal, glycogen content, enzyme activities, targeted energy metabolomics, RNA sequencing, reactive oxygen species (ROS), and membrane permeability were assessed, with biological profiling focused on the IncI2 system. pIncI2-mcr-1 transfer was also examined in a mouse-gut model. Results: SA caused no detectable reduction in the reported OD600-based growth curves or viable donor/recipient counts under the tested conditions. At 8 µg/mL, SA reduced IncI2-mcr-1 and IncX4-mcr-1 conjugation frequency by approximately 176-fold and 117-fold, respectively. In the IncI2 system, lower luciferase-based ATP signals, glycogen accumulation, altered GAPDH and G6PDH activities, and decreased abundance of transfer-related transcripts accompanied the reduction in transfer. Targeted LC–MS/MS independently confirmed a dose-associated decrease in ATP and identified lower pools of several glycolysis-associated metabolites. In mice, the mean IncI2 transfer-frequency estimate was 8.41 × 10−3 in controls and 2.49 × 10−3 with SA (n = 10 per group; p < 0.0001). Conclusions: SA reduced transfer of the two tested mcr-1-carrying plasmids under the experimental conditions. The metabolic and transcriptional observations support an energy-associated explanatory model for the IncI2 system and highlight SA’s potential as a conjugation inhibitor to curb the spread of colistin resistance.