Efflux Pump Activation Confers Mupirocin Resistance and Enhances Rhizosphere Fitness in Pseudomonas

Mupirocin (Mup) is a polyketide antibiotic for clinical MRSA treatment and agricultural biocontrol, acting by binding to IleRS to inhibit protein synthesis. Here, we demonstrate that Pseudomonas viciae 11K1 acquires resistance to mupirocin not through canonical mutations in ileS, the gene encoding the drug target, but via single nucleotide polymorphisms (SNPs) of EmhR, a transcriptional repressor. These SNPs attenuate EmhRs DNA-binding ability, resulting in derepression of the resistance-nodulation-division (RND) efflux pump EmhABC. This leads to a 7-fold (to 800 g/mL) increase in the mupirocin minimum inhibitory concentration (MIC), and confers cross-resistance to multiple other antibiotics. Critically, 11K1 strains harboring EmhR mutations exhibit enhanced competitive fitness in colonizing wheat rhizospheres against the mupirocin-producing Pseudomonas bijieensis 2P24. The conservation of this regulatory mechanism in Pseudomonas aeruginosa, mediated by the EmhR ortholog NalD, underscores its broader biological significance. Our findings establish a direct link between efflux pump regulation and ecological adaptation, highlighting a key mechanism contributing to environmental antimicrobial resistance with important implications for the clinical and agricultural use of mupirocin. IMPORTANCEMupirocin and its producing Pseudomonas strains are widely used in both clinical and agricultural contexts, making mupirocin resistance a significant concern for public health and food security. While canonical mupirocin resistance is primarily attributed to mutations in ileS, the gene encoding the drug target isoleucyl-tRNA synthetase (IleRS), our study identifies a novel resistance mechanism in Pseudomonas viciae strain 11K1 mediated by SNPs in the transcriptional repressor EmhR. These SNPs derepress the RND efflux system EmhABC, and reduce intracellular mupirocin levels. This mechanism not only enhances tolerance to mupirocin but also confers cross-resistance to multiple antimicrobial agents, raising the risk of multidrug-resistant strain spread. EmhR-mutant 11K1 strains exhibit enhanced rhizosphere competitiveness in the presence of mupirocin-producing Pseudomonas bijieensis strain 2P24, indicating an ecological fitness advantage that could promote resistant population expansion in agriculture environments. The same regulatory pathway is conserved in human pathogen Pseudomonas aeruginosa PAO1 via the functional ortholog NalD, suggesting that this mechanism may be widespread in Pseudomonas. These findings fill a critical gap in understanding non-target-based mupirocin resistance, clarify the ecological drivers of antimicrobial resistance (AMR), and offer practical insight for improving mupirocin application to limit the emergence and spread of resistant pathogens.