Elevated temperature enhances rpoE/degP-dependent bacterial membrane vesicle biogenesis and blaNDM-5 dissemination in pig-derived carbapenem-resistant Escherichia coli.

The emergence and dissemination of carbapenem-resistant Enterobacteriaceae in livestock production systems pose a serious threat to animal health and food safety. Bacterial membrane vesicles (BMVs) have recently been recognized as effective vehicles for the horizontal dissemination of antimicrobial resistance genes. However, beyond antibiotic exposure, the contribution of host-derived physiological cues associated with infection, particularly elevated temperature, modeled here as an in vitro high-temperature condition, to BMV biogenesis and vesicle-mediated resistance dissemination remains poorly understood. Here, using a pig-derived carbapenem-resistant Escherichia coli (E. coli) strain, we investigated the effects of elevated temperature conditions (37 ℃ and 42 ℃) on BMV production, vesicular DNA cargo loading, and resistance gene transfer efficiency. Exposure to elevated temperature (42 ℃) significantly increased BMV release by approximately 1.47-fold (P < 0.001) without affecting bacterial growth or vesicle size distribution. BMVs produced under elevated-temperature conditions exhibited a pronounced enrichment of the carbapenem resistance gene blaNDM-5, with vesicular gene copy numbers increasing over two-fold and vesicle-mediated transfer frequency enhanced by approximately 4-5 fold. Transcriptomic profiling revealed that elevated temperature induced a coordinated transcriptional response characterized by remodeling of cell envelope-associated functions and enhanced energy metabolism. Functional genetic analyses further identified the rpoE/degP envelope regulatory axis as a critical determinant linking elevated temperature to increased vesiculation and amplified resistance dissemination. Collectively, these findings demonstrate that elevated temperature can act as a potent non-antibiotic driver of BMV-mediated antimicrobial resistance spread in livestock-associated bacteria under in vitro conditions. These results provide new mechanistic insight into resistance dissemination under disease-relevant physiological conditions.