Detection and identification of Cryptococcus neoformans var gattii complex in novel environmental samples using physiological and genetic tools

Cryptococcus neoformans/gattii complex (C.neo/gat ) are opportunistic, encapsulated yeasts that adversely affect primarily immune-suppressed patients. The yeasts live freely in the environment, mainly associated to pigeon droppings and to eucalyptus, almond or other tree detritus. At present, the varieties belonging to C.neo/gat have been divided into two species according to their environmental niches and to physiological and molecular differences. Few studies on the ecology of C.neo/gat have been conducted in Puerto Rico. Therefore, we decided to determine the presence of the yeast on novel and diverse environmental sources. Detritus and surface samples from trees and succulent plants from western Puerto Rico were collected as well as pigeon droppings. The C.neo/gat isolates were preliminarily identified by melanin production on Niger Seed Agar. The identity of the isolates was confirmed using microscopical and biochemical tests and by the in silico analysis of the small ribosomal subunit 18S rDNA, intergenic spacer (IGS) and internal transcribed spacer (ITS) of the isolates. Genetic differences between the isolates were identified by a phylogenetic analysis using the 18S rDNA sequences, showing five homology groups. Also, strains and environmental isolates from Puerto Rico, belonging to the C.neo/gat complex, were analyzed using the BIOLOG Microstation System, to identify five specific common carbon sources for members of C. neo/gat. This research also included a preliminary phage display analysis. Through the interaction of C. gattii C77 strain with human brain and lung genomic libraries fused on the T7 phage, it was determined that two main peptides, which coded to nectin and telecephalin, interacted. The peptides can potentially serve as vaccine precursors to develop as a new anticryptococcal therapy and also be used to identify the interaction of the capsule with the target organs.;To the best of our knowledge, the findings present in this research correspond to the first report ever on the isolation of C. gattii from cacti, and from tree detritus in Puerto Rico. Also, this research corresponded to the first study of the reliability of the BIOLOG Microstation System for the rapid identification of the C. neo/gat complex and on the interaction of whole cells of C. gattii to T7 premade human genomic libraries of lung and brains.