Sub-inhibitory gentamicin promotes extracellular vesicles biogenesis and blaNDM dissemination in carbapenem-resistant Escherichia coli via mrdA/mrdB pathway

The increasing prevalence of carbapenem-resistant Escherichia coli (CRE) in swine production poses a significant public health threat, largely driven by the misuse of Antibiotics. Recent studies highlight extracellular vesicles (EVs) as emerging mediators of horizontal gene transfer and Antibiotic resistance dissemination. In this study, we investigated the regulatory effects of sub-inhibitory concentrations of gentamicin (GEN), a commonly used Antibiotic in pig farms, on EVs production and blaNDM gene transfer in CRE isolates. EVs purified from porcine CRE strains exhibited typical spherical morphology with average diameters around 100 nm and particle concentrations exceeding 2.0 × 10 ¹¹ particles/mL. Treatment with 1/64 minimum inhibitory concentration (MIC) GEN significantly increased EV secretion and enhanced the protective effect of EVs against meropenem in both intra-species (E. coli ATCC 25922) and inter-species recipient strains (S. Typhimurium ATCC 14028, P. aeruginosa ATCC 15692, K. pneumoniae CMCC 46117, L. monocytogenes ATCC 19115, and O. burkholderi ATCC 25416), in a dose- and time-dependent manner. Moreover, GEN-induced EVs facilitated blaNDM-5 (New Delhi metallo-β-lactamase-5) transfer preferentially to E. coli strains. Transcriptomic analysis revealed that GEN treatment led to differential expression of multiple genes, among which mrdA and mrdB were identified as key regulators of EVs biogenesis. Targeted deletion of mrdA or mrdB markedly reduced EVs production and blaNDM transfer frequency. These findings suggest that the mrdA/mrdB pathway plays a crucial role in GEN-mediated EVs formation and resistance gene dissemination, providing novel insights into the molecular mechanisms by which sub-inhibitory Antibiotic exposure promotes antimicrobial resistance propagation. Our work may inform future strategies for controlling resistance dissemination in livestock production.

Escherichia coli; Extracellular vesicles; Sub-inhibitory antibiotics; blaNDM gene; mrdA/mrdB pathway.