The role of naturally acquired intracellular Pseudomonas aeruginosa in the development of Acanthamoeba keratitis in an animal model

Background Acanthamoeba is an environmental host for various microorganisms. Acanthamoeba is also becoming an increasingly important pathogen as a cause of keratitis. In Acanthamoeba keratitis (AK), coinfections involving pathogenic bacteria have been reported, potentially attributed to the carriage of microbes by Acanthamoeba. This study assessed the presence of intracellular bacteria in Acanthamoeba species recovered from domestic tap water and corneas of two different AK patients and examined the impact of naturally occurring intracellular bacteria within Acanthamoeba on the severity of corneal infections in rats. Methodology/Principal findingsHousehold water and corneal swabs were collected from AK patients. Acanthamoebastrains and genotypes were confirmed by sequencing. Acanthamoeba isolates wereassessed for the presence of intracellular bacteria using sequencing, fluorescence in situhybridization (FISH), and electron microscopy. The viability of the bacteria in Acanthamoebawas assessed by labelling with alkyne–functionalized D–alanine (alkDala). Primaryhuman macrophages were used to compare the intracellular survival and replicationof the endosymbiotic Pseudomonas aeruginosa and a wild type strain. Eyes of rats werechallenged intrastromally with Acanthamoeba containing or devoid of P. aeruginosa andevaluated for the clinical response. Domestic water and corneal swabs were positive forAcanthamoeba. Both strains belonged to genotype T4F. One of the Acanthamoeba isolatesharboured P. aeruginosa which was seen throughout the Acanthamoeba’s cytoplasm.It was metabolically active and could be seen undergoing binary fission. Thismotile strain was able to replicate in macrophage to a greater degree than strain PAO1(p<0.05). Inoculation of Acanthamoeba containing the intracellular P. aeruginosa in ratseyes resulted in a severe keratitis with increased neutrophil response. Acanthamoebaalone induced milder keratitis. Conclusions/SignificanceOur findings indicate the presence of live intracellular bacteria in Acanthamoeba canincrease the severity of acute keratitis in vivo. As P. aeruginosa is a common cause of keratitis,this may indicate the potential for these intracellular bacteria in Acanthamoeba to leadto severe polymicrobial keratitis.