Dual population-level processes contribute to polyclonal ceftiofur heteroresistance in swine-derived <i>Escherichia coli</i>

Antimicrobial resistance represents a major global health challenge. In veterinary medicine, ceftiofur is widely used to treat bacterial infections, yet its efficacy has been increasingly compromised by the dissemination of extended-spectrum β-lactamase (ESBL) genes such as blaCTX-M. Although heteroresistance has been widely reported, its role in ceftiofur resistance, particularly in swine-derived Escherichia coli, remains poorly understood. Here, we identified four polyclonal ceftiofur heteroresistance (PCHR) E. coli isolates from swine, each comprising genetically distinct resistant and susceptible subpopulations. Whole-genome sequencing showed that all resistant subpopulations carried blaCTX-M genes, and conjugation assays demonstrated that blaCTX-M-carrying plasmids were transferable. Notably, in the resistant subpopulation EP91A, a chromosomal blaCTX-M-containing fragment was identified in the transconjugant plasmid pTEP91A-1, with sequence features consistent with a possible IS1380-associated recombination event. Under ceftiofur pressure, resistant subpopulations expanded in all four PCHR isolates, although the associated population-level processes differed among isolates, including plasmid-mediated transfer and differential expansion of preexisting resistant subpopulations. Under ceftiofur-free conditions, resistant subpopulations were maintained at different levels, with EP70A and EP91A reaching higher proportions and stabilizing at approximately 43%, suggesting clone-associated population dynamics under antibiotic-free conditions. Transcriptomic analysis further identified clone-associated transcriptional differences between EP91A and EP91B across pathways related to environmental sensing, metabolism, transport, and cellular processes, providing hypothesis-generating observations. Collectively, these findings suggest that PCHR in these selected swine-derived E. coli isolates is associated with genetic heterogeneity, transferable blaCTX-M-carrying plasmids, and distinct population dynamics, providing insights into ceftiofur resistance expansion and maintenance in heterogeneous bacterial populations.