Effects of subinhibitory concentrations of menthol on adaptation, morphological, and gene expression changes in enterohemorrhagic Escherichia coli

Menthol (C₁₀H₂₀O) possesses antibacterial activity; nevertheless, bacterial adaptation to this compound has never been studied. Here we report that precultivation of enterohemorrhagic Escherichia coli (EHEC) strains in increasing subinhibitory (SI) concentrations of menthol significantly elevates (4- to 16-fold) their resistance to menthol. Concomitant morphological alterations included the appearance of mucoid colonies and reduced biofilm production. Scanning electron microscopy (SEM) examination revealed suppressed curli formation in menthol-adapted cells. Expression of the gene cpsB10 (encoding one of the enzymes responsible for colanic acid production) was elevated in response to SI concentrations of menthol in a laboratory E. coli strain, whereas expression in an rcsC null mutant was reduced, implicating a partial role for the Rcs phosphorelay system in mediating the menthol signal. Adaptation to menthol also reduced expression of the locus of enterocyte effacement-encoded regulator (Ler). This reduction, together with reduced curli and biofilm formation and elevated mucoidity, suggests a general reduction in bacterial virulence following adaptation to menthol. Our results thus suggest menthol as a potential lead in the recently emerging alternative strategy of targeting bacterial virulencefactors to develop new types of anti-infective agents.