ABSTRACTAt a human/livestock/wildlife interface,Escherichia colipopulations were used to assess the risk of bacterial and antibiotic resistance dissemination between hosts. We used phenotypic and genotypic characterization techniques to describe the structure and the level of antibiotic resistance ofE. colicommensal populations and the resistantEnterobacteriaceaecarriage of sympatric African buffalo (Syncerus caffer caffer) and cattle populations characterized by their contact patterns in the southern part of Hwange ecosystem in Zimbabwe. Our results (i) confirmed our assumption that buffalo and cattle share similar phylogroup profiles, dominated by B1 (44.5%) and E (29.0%) phylogroups, with some variability in A phylogroup presence (from 1.9 to 12%); (ii) identified a significant gradient of antibiotic resistance from isolated buffalo to buffalo in contact with cattle and cattle populations expressed as the Murray score amongEnterobacteriaceae(0.146, 0.258, and 0.340, respectively) and as the presence of tetracycline-, trimethoprim-, and amoxicillin-resistant subdominantE. colistrains (0, 5.7, and 38%, respectively); (iii) evidenced the dissemination of tetracycline, trimethoprim, and amoxicillin resistance genes (tet,dfrA, andblaTEM-1) in 26 isolated subdominantE. colistrains between nearby buffalo and cattle populations, that led us (iv) to hypothesize the role of the human/animal interface in the dissemination of genetic material from human to cattle and toward wildlife. The study of antibiotic resistance dissemination in multihost systems and at anthropized/natural interface is necessary to better understand and mitigate its multiple threats. These results also contribute to attempts aiming at usingE. colias a tool for the identification of pathogen transmission pathway in multihost systems.