Acanthamoeba is an opportunistic free-living heterotrophic protist that causes devastating corneal infection. This study investigated the types of intracellular bacteria in Acanthamoeba species recovered from the home environment of Acanthamoeba keratitis (AK) patients and examined the effect of an Acanthamoeba harbouring P. aeruginosa, on corneal infection.
Acanthamoeba strains and genotypes were confirmed by PCR followed by sequencing. Acanthamoeba isolates were assessed for the presence of bacterial endosymbionts using PCR, 16S rRNA sequencing and FISH. The type of Pseudomonas aeruginosa was assessed by examining possession of Type III secretion-associated genes. The viability of the bacteria in Acanthamoeba was assessed by labelling with alkyne-functionalized D-alanine (alkDala). Electron microscopy was used for ultrastructural characterization of bacterial endocytobionts. Human monocyte derived macrophages (hMDMs) were used to compare the intracellular proliferation of the endosymbiotic P. aeruginosa and a wild type strain. Wistar rats eyes were challenged with Acanthamoeba containing or devoid of endosymbiotic P. aeruginosa and evaluated for the clinical response.
The domestic water supply of three AK patients (37.5%, 3/8) were positive for Acanthamoeba spp. and all Acanthamoeba belonged to genotype T4 clade. One of the Acanthamoeba isolates harboured intracellular P. aeruginosa and bacterial cells were seen throughout the Acanthamoeba’s cytoplasm by fluorescence in situ hybridization (FISH). The bacterium was classified as cytotoxic as it contained exoU gene. It was metabolically active as cells stained with alkDala and could be seen undergoing binary fission inside presumed phagolysosomal membranes in the Acanthamoeba. This strain was able to replicate in hMDMs to a greater degree than PAO1 (p<0.05). Inoculation of Acanthamoeba containing P. aeruginosa endosymbionts in rats' eyes resulted in severe keratitis, in contrast, Acanthamoeba alone did not induce overt keratitis (mean clinical score, 20.2 vs 3.4; p<0.05).
One Acanthamoeba strain contained viable P. aeruginosa that showed enhanced intracellular replication in human macrophages. Upon infection with the Acanthamoeba strain containing intracellular P. aeruginosa, acute keratitis occurred, but infection with the strain devoid of endosymbionts was very mild. As P. aeruginosa is a known ocular pathogen, this may indicate the potential for these intracellular bacteria in Acanthamoeba to cause mixed and severe infections during keratitis.