April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
Methods for Detection of Bacterial Endosymbionts in Acanthamoeba Isolates
Author Affiliations & Notes
  • A. Iovieno
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida
  • D. Miller
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida
  • D. R. Ledee
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida
  • E. C. Alfonso
    Ophthalmology, Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida
  • Footnotes
    Commercial Relationships  A. Iovieno, None; D. Miller, None; D.R. Ledee, None; E.C. Alfonso, None.
  • Footnotes
    Support  University of Miami Wallace H. Coulter Center for Translational Research; Research to Prevent Blindness; National Eye Institute’s grant P30 EY014801 provided core support; Gift from Bausch&Lomb Inc.
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 5122. doi:
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    • Get Citation

      A. Iovieno, D. Miller, D. R. Ledee, E. C. Alfonso; Methods for Detection of Bacterial Endosymbionts in Acanthamoeba Isolates. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5122.

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Abstract

Purpose: : Acanthamoeba can cause a rare and sight-threatening cornea infection. Acanthamoeba species are known to harbor intracellular bacteria endosymbionts. The purpose of our study was to detect the presence of bacterial endosymbionts in isolates obtained from patients with Acanthamoeba keratitis and from the environment.

Methods: : Clinical isolates recovered from cornea, contact lenses and contact lens cases (n=38) and environmental sources (n=12) were grown axenically in PYG broth and passaged in Page’s saline solution. The presence of microbial endosymbionts in Acanthamoeba was detected using PCR and sequencing techniques. Fluorescence in situ hybridization (FISH) and electron microscopy were used to directly visualize the endosymbionts.

Results: : Four different species of bacterial endosymbionts were detected in 28/38 (74%) clinical and 6/12 (50%) environmental Acanthamoeba isolates by PCR. Pseudomonas species were detected in 18/50 (36%), Legionella species other than pneumophila in 7/50 (14%), Mycobacterium other than tuberculosis in 15/50 (30%) and Chlamydia trachomatis in 1/50 (2%). 7/50 (14%) of the isolates had more than one endosymbiont.FISH (Image 1) confirmed molecular identification of endosymbionts. Intracellular localization of endosymbionts was showed by electron microscopy images(Image 2) .

Conclusions: : The majority of our clinical isolates hosted one or more bacterial endosymbionts compared to environmental isolates. FISH and electron microscopy may be used to visualize and confirm the presence of bacterial endosymbionts in Acanthamoeba. This might have clinical relevance for the management and treatment of Acanthamoeba keratitis.

Keywords: amoeba • microscopy: light/fluorescence/immunohistochemistry • microscopy: electron microscopy 
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