Hinokitiol disrupts iron homeostasis and reduces virulence in Acinetobacter baumannii

Multidrug-resistant gram-negative bacterial infections pose a serious threat to human health. Given that iron levels affect bacterial pathogenesis, iron homeostasis is a promising target for antimicrobial therapies. We investigated the effects and mechanisms of action of hinokitiol against Acinetobacter baumannii, a notorious opportunistic pathogen that causes nosocomial infections worldwide.

The minimum inhibitory concentration was determined using the broth microdilution method. The inhibition of A. baumannii isolates was determined by growth kinetics analysis and viable counts with or without iron supplementation following hinokitiol exposure. The expression status of acinetobactin-related genes and a motility-related gene was evaluated using RT-qPCR. The bioenergetics-related activity of hinokitiol-treated bacteria was assessed by determining the intracellular ATP levels and bacterial motility. The fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate was used to detect intracellular reactive oxygen species.

Hinokitiol decreased the number of viable bacteria in time- and concentration-dependent manners against A. baumannii. Hinokitiol-treated bacteria exhibited reduced intracellular iron levels and elevated expression of acinetobactin-related genes compared with untreated cells. Intracellular ATP levels, motility, and virulence-related characteristics were reduced; however, intracellular reactive oxygen species levels increased in A. baumannii following hinokitiol exposure. More survival nematodes were recorded for the Caenorhabditis elegans infection model using hinokitiol.

Hinokitiol affects bacterial iron homeostasis. Impaired bioenergetics-related activity and induced oxidative stress after hinokitiol treatment may contribute to the reduced viability of A. baumannii. Hinokitiol reduced bacterial adhesion, invasion, and cytotoxicity, which may be beneficial for treating A. baumannii infections.