The increasing frequency of antibiotic resistance poses myriad challenges to modern medicine. Environmental survival of multidrug-resistant bacteria in health care facilities, including hospitals, creates reservoirs for transmission of these difficult to treat pathogens. To prevent bacterial colonization, these facilities deploy an array of infection control measures, including bactericidal metals on surfaces, as well as implanted devices. Although antibiotics are routinely used in these health care environments, it is unknown whether and how antibiotic exposure affects metal resistance. We identified a multidrug-resistant Enterobacter clinical isolate that displayed heteroresistance to the antibiotic colistin, where only a minor fraction of cells within the population resist the drug. When this isolate was grown in the presence of colistin, a 9-kb DNA region was duplicated in the surviving resistant subpopulation, but surprisingly, was not required for colistin heteroresistance. Instead, the amplified region included a three-gene locus (ncrABC) that conferred resistance to the bactericidal metal, nickel. ncrABC expression alone was sufficient to confer nickel resistance to E. coli K-12. Due to its selection for the colistin-resistant subpopulation harboring the duplicated 9-kb region that includes ncrABC, colistin treatment led to enhanced nickel resistance. Taken together, these data suggest that the use of antibiotics may inadvertently promote enhanced resistance to antimicrobial metals, with potentially profound implications for bacterial colonization and transmission in the health care environment.IMPORTANCE To inhibit bacterial transmission and infection, health care facilities use bactericidal metal coatings to prevent colonization of surfaces and implanted devices. In these environments, antibiotics are commonly used, but their effect on metal resistance is unclear. The data described here reveal that exposure of a human isolate of Enterobacter cloacae to a last-line antibiotic, colistin, resulted in a DNA amplification that does not confer antibiotic resistance but instead facilitates resistance to the toxic metal nickel. This highlights a novel aspect of antibiotic and metal interplay. Concerningly, these data suggest the use of antibiotics could in some cases promote bacterial survival and colonization in the health care environment and ultimately increase transmission and infection of patients.