EefR mutations drive sanguinarine resistance by activating cryptic multidrug efflux pumps in AcrB-Null <i>Escherichia coli</i>

Antimicrobial resistance is one of the greatest threats to global health. A thorough comprehension of the underlying processes through which bacteria evolve the ability to withstand diverse stressors is vital for the development of efficacious strategies to address this challenge. This study aimed to elucidate the mechanisms underlying the adaptation of an <i>acrB</i>-deficient strain of <i>Escherichia coli</i> ATCC 35218 (35218m) to sanguinarine (SAN), the main component of phytobiotic derived from <i>Macleaya cordata</i>. Analysis of the <i>in vitro</i>-selected SAN-resistant clones of strain 35218m led to the identification of a functionally uncharacterized TetR regulator, EefR. The regulator represses its own expression and that of four neighboring genes forming the <i>eefRABCD</i> operon, encoding components of a tripartite RND-efflux pump (EefABC) and a putative MFS-type exporter (EefD). Overexpression of these pumps reduces the susceptibility of <i>acrB</i>-deficient <i>E. coli</i> to SAN and structurally diverse antibiotics. The <i>eefR-eefABC-eefD</i> cluster, widely distributed across Enterobacteriaceae genomes, is also found on plasmids in several <i>E. coli</i> isolates, indicating a significant risk for the rapid spread of this multidrug resistance mechanism.