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Aminoglycosides are essential components in the available armamentarium to treat bacterial infections. The surge and rapid dissemination of resistance genes strongly reduce their efficiency, compromising public health. Among the multitude of modifying enzymes that confer resistance to aminoglycosides, the aminoglycoside 6'- N -acetyltransferase type Ib [AAC(6')-Ib] is the most prevalent and relevant in the clinical setting as it can inactivate numerous aminoglycosides, such as amikacin. Although the mechanism of action, structure, and biochemical properties of the AAC(6')-Ib protein have been extensively studied, the contribution of the intracellular milieu to its activity remains unclear. In this work, we used a fluorescent-based system to quantify the number of AAC(6')-Ib per cell in Escherichia coli , and we modulated this copy number with the CRISPR interference method. These tools were then used to correlate enzyme concentrations with amikacin resistance levels. Our results show that resistance to amikacin increases linearly with a higher concentration of AAC(6')-Ib until it reaches a plateau at a specific protein concentration. In vivo imaging of this protein shows that it diffuses freely within the cytoplasm of the cell, but it tends to form inclusion bodies at higher concentrations in rich culture media. Addition of a chelating agent completely dissolves these aggregates and partially prevents the plateau in the resistance level, suggesting that AAC(6')-Ib aggregation lowers resistance to amikacin. These results provide the first step in understanding the cellular impact of each AAC(6')-Ib molecule on aminoglycoside resistance. They also highlight the importance of studying its dynamic behavior within the cell.IMPORTANCEAntibiotic resistance is a growing threat to human health. Understanding antibiotic resistance mechanisms can serve as foundation for developing innovative treatment strategies to counter this threat. While numerous studies clarified the genetics and dissemination of resistance genes and explored biochemical and structural features of resistance enzymes, their molecular dynamics and individual contribution to resistance within the cellular context remain unknown. Here, we examined this relationship modulating expression levels of aminoglycoside 6'- N -acetyltransferase type Ib, an enzyme of clinical relevance. We show a linear correlation between copy number of the enzyme per cell and amikacin resistance levels up to a threshold where resistance plateaus. We propose that at concentrations below the threshold, the enzyme diffuses freely in the cytoplasm but aggregates at the cell poles at concentrations over the threshold. This research opens promising avenues for studying enzyme solubility's impact on resistance, creating opportunities for future approaches to counter resistance., Competing Interests: The authors declare no conflict of interest.