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Darlene Miller, Jorge Maestre-Mesa, Martha Diaz, Edith Perez, Valery Shestopalov, Russell Van Gelder, Eduardo C. Alfonso; Acanthamoeba Associated Microbial Communities. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6145.
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The epidemiology and pathology of Acanthamoeba keratitis is still being deciphered. Acanthamoeba may harbor unique microbiomes which may contribute to the pathology of the disease. We used a combination of metagenomics and next generation sequencing techniques to document the presence, complexity and diversity of Acanthamoeba associated microbial communities in isolates recovered from patients with amoebic keratitis.
DNA from Acanthamoeba hosts recovered from patients with amebic keratitis (N=9) and 1 QC strain (A. polyphaga ATCC) strain was extracted (Chelex), purified (Qaigen purification kit) and then aliquoted and analyzed by metagenomics and next generation deep sequencing: 454 pyrosequencing (N=7) and Biome representational in silico karyotyping -BRISK (N= 4). Results were compared with culture dependent recovery from cornea/contact lens (N=9). Impact of microbial community complexity was correlated with time to clinical/laboratory diagnosis and time to cure.
Complex and diverse microbial communities were documented by both culture independent techniques. More than 10 phyla, 72 genera and 199 different species were recovered from amebic hosts in the 7 samples evaluated by 454 pyrosequencing vs 13 phyla, 105 genera and 290 species identified in samples analyzed by BRISK. The QC strain revealed 6 phyla 10 genera and 19 species. Culture revealed 7 phyla, 5 genera and 12 species. Less than 20% of the species documented by the two culture independent methods were recognized as corneal pathogens. A "core" microbiome however, was common to the three communities and included: Delftia acidovorans, Ochrochrum anthropi, Pseudomonas aeruginosa and Stenotrophomonas maltophilia.There was a moderate correlation (R2=0.413) with increasing community diversity and time to clinical and laboratory diagnosis/confirmation, but a reverse correlation (R2 = -0.464) with community complexity and time to cure.
Culture independent molecular methods reveal complex and diverse Acanthamoeba associated microbial communities in clinical isolates. Community complexity and diversity may impact clinical severity, course and time to cure.
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