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D. R. Ledee, D. Miller, M. E. Fini, E. Alfonso; Detection and Genotyping of Acanthamoeba Isolates and Bacterial Endosymbionts Recovered From Keratitis. Invest. Ophthalmol. Vis. Sci. 2008;49(13):505. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Amebic keratitis (AK) is a recalcitrant and sight threatening infection. Acanthamoeba species, the causative agents are known reservoirs for intracellular bacteria endosymbionts. Several of these intracellular bacteria (Legionella, Pseudomonas, Burkholderia, Mycobacteria) are pathogens of humans, and this symbiosis may enhance infectivity and virulence for both amoeba and bacteria. The role or contributions of these endosymbionts to the protracted clinical course of AK is unknown. Identification and characterization of associated endosymbionts may help decipher the pathology and yield clues to the management of ocular disease.
PCR and sequencing techniques were use to detect the presence of endosymbionts in clinical Acanthamoeba isolates recoved from cornea, contact lens and contact lens cases. Random isolates from consecutive clinical cases (N=29), environmental (N=8) and QC (N=2) were collected and grown axenically in PYG broth or on agar plates supplemented with E. coli for molecular characterization and detection of bacterial symbionts. The genotypes of the Acanthamoeba isolates used were determined by sequencing a highly informative region in the 18S gene, whereas the genotype for the endosymbionts were determined by sequencing the variable 23S-5S internal transcribed spacer region.
DNA sequencing showed all of the clinical isolates for this study belonged to genotype T4, except for 1 with genotype T5. The environmental isolates belonged to genotype T4, T5 and T3. The quality controls were both genotype T4. Bacterial endosymbionts were recovered in 69% (20/29) of the clinical and 62% (5/8) of the environmental Acanthamoeba isolates examined. 18 of the 29 were sequenced and 61% (11/18) are Pseudomonas species and 39% (7/18) are Legionella species. None of the detected intracellular bacteria were recovered in culture.
PCR and sequence analysis confirmed the presence of bacterial endosymbionts in the majority of our clinical Acanthamoeba isolates. The clinical relevance of this symbiosis is uncertain. Additional molecular and microbiological studies are needed to determine the impact of this symbiotic relationship on ocular disease outcomes.
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