Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
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.
Keywords: Acanthamoeba • cornea: basic science • clinical laboratory testing