Impact of cyst maturity on drug resistance assays in Acanthamoeba Genotype T4

Therapeutic failure in Acanthamoeba Keratitis (AK) is largely driven by the high resistance of cysts to antimicrobial agents. However, the development of effective cysticidal drugs is hindered by the lack of standardized in vitro encystation protocols. Inconsistencies in induction methods can produce immature cells that lack the structural defenses of mature clinical walls, potentially leading to false-positive results in drug screening. This study aims to evaluate the impact of cyst maturity on drug resistance assays by systematically comparing two common liquid media-glucose-supplemented PBS (PBSEB) and alkaline encystation buffer (EB2)-within Acanthamoeba Genotype T4 backgrounds. We assessed morphological transitions, transcriptional profiles (EMSP, ATG8, and CS-I), ultrastructure (TEM), and functional resistance to sodium dodecyl sulfate (SDS) and chlorhexidine gluconate (CHG) using environmental reference and clinical isolates, including the stress-tolerant NCKU_D strain. Although both media induced morphological rounding and upregulated the autophagy marker ATG8, functional and molecular assays revealed distinct developmental outcomes. PBSEB-treated cells failed to significantly upregulate the critical early serine proteinase EMSP and exhibited insufficient CS-I expression, remaining highly susceptible to SDS lysis and lacking intact walls under TEM. Conversely, EB2 triggered a robust 11.8-fold increase in EMSP, yielding structurally mature, SDS-resistant cysts. Importantly, functional assays demonstrated that EB2-induced cysts exhibited significantly higher chemical resistance to CHG than the PBSEB group at 48 h, with this resistance advantage further widening by 72 h of induction. Based on these distinct profiles, we suggest that while PBSEB serves as a useful model for investigating cell detachment kinetics and early autophagic pathways, the EB2 protocol is more suitable for evaluating downstream therapeutic efficacy and product safety where functional, double-walled resistance is required to avoid false-positive outcomes in Genotype T4 drug screening.