Abstract
Purpose :
To perform molecular characterization and phylogenetic studies on putative Pythium insidiosum isolates, recovered from corneal ulcers in India
Methods :
Fourteen isolates from human corneal ulcer scrapings were initially morphologically characterized (colony morphology on Blood Agar and/or Sabouraud dextrose agar) as Pythium sp. Isolates were tested for zoospore production using the Rice Husk Induction method, and also underwent a variety of biochemical tests. ITS and COXII universal primers were used to amplify and then sequence their corresponding genes in the investigated isolates, followed by their annotations. The DNA sequences of the 14 investigated isolates were also submitted to the National Center for Biotechnology Information (NCBI) via Basic Local Alignment Search Tool (BLAST). Phylogenetic analysis using Maximum Parsimony analysis (MEGA 7) was carried out for 77 Pythium insidiosum isolates recovered worldwide from environmental, arthropod, lower animal, and human sources (including the Indian ocular isolates).
Results :
The 14 Pythium isolates from India were culturally and biochemically identified as Pythium sp. The DNA amplified sequences submitted for BLAST analysis showed 100% identity with the DNA sequences of P. insidiosum from Asia. However, evolutionary history, inferred using the Maximum Parsimony method, confirmed via the BLAST analysis, revealed the clustering of these 77 P. insidiosum isolates into four different Clades. The 14 interrogated ocular isolates were placed in Clades II and IV.
Conclusions :
A very robust phylogenetic analysis of the largest number of Pythium spp. isolates, reveals intriguing information on the phylogenetic relationships of the interrogated isolates. Of the Fourteen corneal ulcer isolates from India, 10 belong to Clade II, while the 4 other isolates were placed in a more distant Clade IV. Our phylogenetic analysis reveals interesting genetic divergences among the known P. insidiosum isolates. This finding would be of enormous significance to understand the biology and epidemiology as well as the evolutionary history of Pythium species; more importantly, it could provide clues to explain the pathogenicity as well as the reported differential therapeutic response of medical/ ocular isolates.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.