Assessment of in vitro dynamics of pathogenic environmental Acanthamoeba T4 and T9 genotypes isolated from three recreational lakes in Klang Valley, Malaysia over the HaCaT cell monolayer

Free-living amoebae of the genus Acanthamoeba are causative agents of keratitis and amoebic encephalitis. They are widely found in various ecological environments. Therefore, the present study brings results that can help to better understand the genotypes of the environmental isolates and their pathogenicity. This study procured 26 Acanthamoeba isolates from three recreational lakes in 2022. Polymerase chain reaction amplification was performed on positive Acanthamoeba samples. The thermotolerance, osmotolerance, and cytopathogenicity in human keratinocyte (HaCaT) cells of the samples were also evaluated. The phylogenetic analysis demonstrated that 12 isolates were of genotype T4, two (T9), six (T17), four (T8), and one each from T5 and T11. The thermo- and osmotolerance assays indicated that eight Acanthamoeba samples were potentially pathogenic. Two T4 and one T9 genotype also recorded 33-, 42-, and 133-kDa serine-type proteases, respectively. The HaCaT cell monolayer revealed that three T4 and one T9 samples achieved cytopathic effects within the 50-100% range, hence significantly cytotoxic. The lactate dehydrogenase secretion results demonstrated that three (T4) and one (T9) sample exhibited exceptional toxicity (over 40%) compared to the other samples. The responses of Acanthamoeba members with similar genotypes to pathogenicity indicator assays varied considerably, rendering correlation of pathogenicity with specific genotypes challenging.