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The emergence of bacterial antibiotic resistance is a growing problem, yet the variables that influence the rate of emergence of resistance are not well understood. In a microfluidic device designed to mimic naturally occurring bacterial niches, resistance of Escherichia coli to the antibiotic ciprofloxacin developed within 10 hours. Resistance emerged with as few as 100 bacteria in the initial inoculation. Whole-genome sequencing of the resistant organisms revealed that four functional single-nucleotide polymorphisms attained fixation. Knowledge about the rapid emergence of antibiotic resistance in the heterogeneous conditions within the mammalian body may be helpful in understanding the emergence of drug resistance during cancer chemotherapy.