Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance. This study identifies three primary drivers of gene dosage-dependent heteroresistance in a multi-resistant Klebsiella pneumoniae isolate: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms impose fitness costs and are genetically unstable, leading to rapid reversion to susceptibility in the absence of antibiotics. Using a mouse gut colonization model, the study demonstrates that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. The three mechanisms are prevalent among Escherichia coli bloodstream isolates. The findings highlight the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations. Antibiotic resistance is a significant global health concern, contributing to an estimated 4.95 million antibiotic resistance-associated deaths worldwide in 2019. Effective antimicrobial treatment relies on accurate identification of the causative pathogen and its antibiotic susceptibility, determined by antimicrobial susceptibility tests (ASTs). However, ASTs are often inadequate for detecting rare resistant cells within a predominantly susceptible population. This limitation is particularly evident for bacterial isolates that show single-cell phenotypic heterogeneity due to phenomena like tolerance, persistence, and heteroresistance. Heteroresistance (HR), a phenotype characterized by small subpopulations of resistant bacteria present within a main susceptible population, may ascend to higher frequencies during antibiotic exposure. HR has been observed in various bacterial species and antibiotic classes. It can lead to clinical complications when the resistant subpopulation is selected during antimicrobial treatment. These resistant subpopulations are present at low frequencies (typically 10^-7 to 10^-4), which makes HR detection using standard AST methods challenging. HR prevalence ranges from undetectable levels to >50% depending on the bacterial species and specific antibiotic. HR can result from several types of mechanisms, including point mutations that increase the minimal inhibitory concentration (MIC) in a subpopulation of the cells. Depending on the fitness cost of these mutations, they cause either unstable (does revert to susceptibility) or stable (does not revert to susceptibility) HR phenotypes. Another common mechanism responsible for generating the resistant subpopulations is tandem gene amplification, where an increased dosage of resistance genes results in elevated MICs, potentially reaching clinical breakpoint levels. However, these amplifications are typically quickly lost in the absence of antibiotic selection, leading to reversion to antibiotic susceptibility and an unstable HR phenotype. The formation of tandem amplifications depends on specific genetic contexts, including the presence of direct repeat sequences flanking the resistance gene, which serve as substrates for the initial duplication event. In Enterobacteriaceae and other bacterial species, resistance genes and repeat sequences (often IS elements or transposase genes) are frequently located on large low copy number plasmids.Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance. This study identifies three primary drivers of gene dosage-dependent heteroresistance in a multi-resistant Klebsiella pneumoniae isolate: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms impose fitness costs and are genetically unstable, leading to rapid reversion to susceptibility in the absence of antibiotics. Using a mouse gut colonization model, the study demonstrates that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. The three mechanisms are prevalent among Escherichia coli bloodstream isolates. The findings highlight the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations. Antibiotic resistance is a significant global health concern, contributing to an estimated 4.95 million antibiotic resistance-associated deaths worldwide in 2019. Effective antimicrobial treatment relies on accurate identification of the causative pathogen and its antibiotic susceptibility, determined by antimicrobial susceptibility tests (ASTs). However, ASTs are often inadequate for detecting rare resistant cells within a predominantly susceptible population. This limitation is particularly evident for bacterial isolates that show single-cell phenotypic heterogeneity due to phenomena like tolerance, persistence, and heteroresistance. Heteroresistance (HR), a phenotype characterized by small subpopulations of resistant bacteria present within a main susceptible population, may ascend to higher frequencies during antibiotic exposure. HR has been observed in various bacterial species and antibiotic classes. It can lead to clinical complications when the resistant subpopulation is selected during antimicrobial treatment. These resistant subpopulations are present at low frequencies (typically 10^-7 to 10^-4), which makes HR detection using standard AST methods challenging. HR prevalence ranges from undetectable levels to >50% depending on the bacterial species and specific antibiotic. HR can result from several types of mechanisms, including point mutations that increase the minimal inhibitory concentration (MIC) in a subpopulation of the cells. Depending on the fitness cost of these mutations, they cause either unstable (does revert to susceptibility) or stable (does not revert to susceptibility) HR phenotypes. Another common mechanism responsible for generating the resistant subpopulations is tandem gene amplification, where an increased dosage of resistance genes results in elevated MICs, potentially reaching clinical breakpoint levels. However, these amplifications are typically quickly lost in the absence of antibiotic selection, leading to reversion to antibiotic susceptibility and an unstable HR phenotype. The formation of tandem amplifications depends on specific genetic contexts, including the presence of direct repeat sequences flanking the resistance gene, which serve as substrates for the initial duplication event. In Enterobacteriaceae and other bacterial species, resistance genes and repeat sequences (often IS elements or transposase genes) are frequently located on large low copy number plasmids.