Investigating adaptation of Staphylococcus aureus to hand sanitisers and subsequent antibiotic co-selection via experimental evolution

Abstract

One of the most notable results of the COVID-19 pandemic is the escalation in the use of hand sanitisers as personal hygiene products as well as in healthcare settings. Besides devising effective infection control strategies, understanding how bacterial strains adapt to these agents is crucial, especially due to the possibility of an unwanted evolution of antibiotic-resistant strains. This study explored how Staphylococcus aureus responds to some common hand sanitiser formulations. We investigated microbial tolerance acquisition and potential antibiotic co-selection through experimental evolution. S. aureus was exposed to escalating subinhibitory hand sanitiser concentrations of Hexisol® (0.5% chlorhexidine gluconate & 70% isopropyl alcohol), Sepnil® (70% ethanol, carbomer, glycerin, polyethylene glycol, TEA, aqua, and perfume), and 70% Ethanol. We compared the Minimum Inhibitory Concentrations (MIC) between evolved and parent strains, performed antibiotic susceptibility testing, assessed fitness using a growth curve assay, and subcultured the evolved strains in sanitiser-free broth for 10 days to assess the stability of the acquired tolerance. Hexisol® showed a 4-fold increase in MIC against the adapted strain, co-selection for macrolide and β-lactam antibiotics, and increased susceptibility to other antibiotics. The growth capacity of the adapted strain was significantly decreased. Tolerance to Hexisol® remained stable, but antibiotic co-selection reversed after 10 days. 70% ethanol and Sepnil® showed similar results. The MIC of these hand sanitisers against the adapted strain did not significantly increase; however, it exceeded the recommended dose. No antibiotic co-selection occurred, but susceptibility to certain antibiotics increased. The growth capacity of the adapted strain was significantly decreased. Bacterial tolerance to 70% ethanol and Sepnil® remained stable after 10 subcultures. The findings shed light on the emergence of potential public health hazards resulting from microbial adaptation to hand sanitisers, emphasizing the need for reevaluation of their long-term impact on antibiotic resistance and infection control strategies.