Zeta potential measurements of Escherichia coli to evaluate colistin susceptibility and gain insight in resistance mechanisms

Colistin resistance in Escherichia coli arises from outer membrane (OM) remodeling that reduces surface charge and thereby lowers drug binding affinity. In this study, we investigated the zeta potential of E. coli strains with colistin minimum inhibitory concentrations (MICs) ranging from 0.125 to 16 mg/L, following EDTA treatment to chelate divalent cations and unmask intrinsic surface charge. Zeta potential was measured across pH values from 3 to 7, revealing consistent pH-dependent trends and an average 18.5 mV reduction in surface charge with increasing MIC. At pH 7, zeta potential strongly correlated with colistin resistance (R2 = 0.919), and this correlation was further strengthened following exposure to sub-inhibitory colistin (1/8 MIC; R2 = 0.9975). Notably, resistant strains carrying mcr-1 or mcr-4 exhibited significant shifts toward less negative surface charges after sub-MIC exposure, whereas the susceptible parental strain remained unchanged. However, mcr-1 mRNA expression did not consistently increase under these conditions, highlighting a disconnect between transcriptional responses and phenotypic charge alterations. These findings suggest that regulatory pathways beyond mcr-1 transcription, including stress-induced lipid A remodeling, contribute to OM charge modulation. Overall, zeta potential profiling provides a rapid and sensitive readout of resistance-associated membrane alterations and complements molecular assays by capturing functional phenotypes. This approach offers a valuable research tool for dissecting colistin resistance mechanisms and may inform future strategies for monitoring bacterial adaptation to last-resort antibiotics.