Resumo

Background: Candida rugosa is generally isolated from environmental sources and as an agent of bovine mastitis. In humans, it is an emergent pathogen in critically ill patients. Recently, C. rugosa was recognized as a species complex comprising 4 taxa: C. rugosa sensu stricto (Cr), C. pseudorugosa (Cp), C. neorugosa (Cn) and C. mesorugosa (Cm). Although isolates within the complex exhibit low virulence, patients with candidemia due to those species can present mortality rates of 70%, mainly associated with the limited susceptibility to amphotericin B and fluconazole. The aims of this work were to genotype the species within the Cr complex based on phylogenetic analysis of the internal transcribed spacer region (ITS) and generate their proteomic profiles by MALDI-ToF MS. Methods: To perform phylogenetic analysis, ITS sequences were downloaded from GenBank after BLASTn searches using sequence-queries from reference strains Cr CBS613, Cm CBS12656, Cp CBS10433, Cn CBS12627, and Cr (C. pararugosa) CBS1948. Sequences were aligned in SEAVIEW and phylogeny was inferred by MrBayes program. Proteomic analysis was performed with isolates from the reference strains mentioned above, 4 clinical isolates of Cm and C. albicans SC5314. Protein spectra were generated by using the smear protocol, in a Microflex LT mass spectrometer and visualized in MALDI Biotyper 3.0 software that produced the MSP dendrogram. Results: Phylogeny of 72 ITS sequences showed 4 distinct clades inferred with high posterior probabilities, corresponding to the 4 species within the Cr complex. Clade Cr was divided into 2 distinct groups that separated sequences from human or animal sources from environmental isolates. Clades Cp and Cm appeared diverse, with uncharacterized and environmental isolates grouping with humans' isolates. Cn presented only 3 sequences, all from human sources. Protein spectrum profiles differentiated all species with most intense peaks at 3 to 4 and 6 to 8 kDa. Dendrogram successfully discriminated all reference strains from different species and grouped the 5 isolates belonging to the novel Cm species. Conclusions: Phylogenetic and proteomic analyses demonstrate to be useful tools for species identification and genotyping, consolidating the 4 taxa within the Cr complex as distinct species. ITS diversity observed within each clade could reflect an ongoing speciation process.