Ethanol Induces a Persister Cell Phenotype in E. Coli and S. Epidermidis

Persister cells are a subpopulation of bacteria capable of surviving high doses of antibiotics and various stressors, contributing to chronic infections and antibiotic resistance. Ethanol exposure is known to induce significant morphological and physiological changes, such as alterations in fatty acid composition, ion leakage, and increased membrane fluidity, which may promote biofilm formation. In the current study, Escherichia coli and Staphylococcus epidermidis were cultured in LB broth supplemented with varying concentrations of ethanol (0 – 10%). Concurrently, growth curves were recorded, and membrane potential was assessed using flow cytometry to determine the impact of ethanol stress on cellular membrane alterations. Following growth, cells were centrifuged to remove ethanol and resuspended in fresh LB broth for an additional 18-hour static incubation to promote biofilm formation. Biofilms were subsequently fixed, stained, washed, and solubilized in acetic acid for quantification via OD530 measurements. The results demonstrated that bacteria maintain growth under high ethanol stress while exhibiting a significant reduction in membrane potential, consistent with the induction of a dormant persister phenotype. Moreover, ethanol exposure markedly enhanced biofilm formation, particularly in S. epidermidis, with robust biofilm production observed even at ethanol concentrations as high as 10%. In conclusion, these findings provide a comprehensive and quantitative analysis of bacterial responses to ethanol-induced stress, linking decreased membrane potential and enhanced biofilm formation to persister cell development. This work deepens our understanding of the mechanisms underlying bacterial persistence and may inform the design of novel therapeutic strategies to combat chronic infections and curb the emergence of antibiotic resistance.