Investigating the Development of Alcohol Tolerance in the ESKAPE Pathogens

Antiseptics and disinfectants are used globally to kill or inhibit the growth of bacteria to prevent infection. Considering the well-documented development of antibiotic resistance, the potential for evolved tolerance to alcohol-based antimicrobial agents raises concern. The ESKAPE bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are a major cause of healthcare-associated mortality and present a threat to public health due to the progression of multi-drug resistance. We conducted an experiment to determine if the ESKAPE pathogens develop an increased tolerance to ethanol upon repeated exposure by selecting for survivors after a two- to four-log reduction in cells. After a 20-day cycle of ethanol exposure, we used whole genome sequencing to identify arisen mutations and analyze the genes and mechanisms that potentially contribute to ethanol tolerance. We discovered that the Gram-positive species, E. faecium and S. aureus, survived at higher ethanol concentrations and developed increased ethanol tolerance after repeated daily exposure. Ethanol tolerance was not observed in the Gram-negative species K. pneumoniae and P. aeruginosa. Genetic sequencing indicated that mutations in genes involved in peptidoglycan synthesis, two-component regulatory systems, and response to environmental stress are likely involved in adaptation to ethanol exposure.