Emergence of PHMB resistance in Acanthamoeba castellanii and observations on cross-resistance to other frontline therapeutics

Purpose: Acanthamoeba keratitis (AK) is a sight threatening infection of the cornea caused by opportunistic pathogens belonging to the genus Acanthamoeba. AK is commonly associated with contact lens use, and treatments are currently limited and ineffective. As such, the emergence of antimicrobial resistance in Acanthamoeba poses a significant challenge to the management of AK. This study investigates the development of polyhexamethylene biguanide (PHMB) resistance, a frontline therapeutic, in Acanthamoeba trophozoites and explores potential cross-resistance to hexamidine and voriconazole. Methods: Acanthamoeba castellanii trophozoites were exposed and maintained in PHMB starting at 2 µg/mL and increasing upon reaching confluence. Cells were subsequently exposed to incrementally higher doses of PHMB in a stepwise manner (2, 4, 5.5, and 7 µg/mL). When sustained growth under PHMB exposure was observed, morphology was assessed by imaging flow cytometry and susceptibility assays were performed by incubating resistant strains with PHMB, hexamidine, and voriconazole for 24 hours, and viability determined using alamarBlue. Results: Trophozoites surviving exposure at 2 µg/mL reached confluence within 11 days. Stepwise increases to 4 µg/mL, 5.5 µg/mL, and 7 µg/mL were achieved within 6 – 7 days at each stage. PHMB-resistant Acanthamoeba strains exhibited a 9-fold increase in resistance to PHMB relative to naïve cells, alongside significant cross-resistance to voriconazole (159-fold) and hexamidine (8.4-fold). No significant change in trophozoite or cyst morphology was observed relative to the naïve cell line. Conclusions: These findings represent the first known laboratory-induced PHMB-resistant Acanthamoeba strains, raising concerns regarding the longevity of current therapeutic options and the potential for cross-resistance to alternative treatments. This highlights the need for clinical vigilance and further investigation into the molecular mechanisms of resistance to better inform treatment strategies.