Phylogenetic Analysis of Phenotypically Characterized Cryptococcus laurentii Isolates Reveals High Frequency of Cryptic Species

Abstract

BackgroundAlthough Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.MethodsIn this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.ResultsBLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99–100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.ConclusionsSequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.

MLST-Based Population Genetic Analysis in a Global Context Reveals Clonality amongst Cryptococcus neoformans var. grubii VNI Isolates from HIV Patients in Southeastern Brazil

“…In this analysis, differences between STs are presented as single locus variant (SLV), double locus variant (DLV), and triple locus variant (TLV) respectively. The concept of a clonal complex (CC) was adopted when a SLV linkage with the founder ST was observed [49, 50]. …”

Molecular identification, genotypic heterogeneity and comparative pathogenicity of environmental isolates of Papiliotrema laurentii

“…Infections caused by these two species, particularly P. laurentii , have increased considerably in recent years, particularly in patients with impaired cell-mediated immunity and prior exposure to antifungal drugs [7, 8]. A wide spectrum of diseases has been attributed to P. laurentii in recent years, including fungaemia, meningitis and meningoencephalitis [10–17]. P. laurentii is considered aheterogeneous group since phylogenetic studies based on sequence analysis of the internal transcribed spacer (ITS) region and D1/D2 domains of rDNA revealed that P. laurentii represents a species complex comprising P. aureus , P. aspenensis , P. terresteris , P. flavescens and P. laurentii [9, 15–17].…”

“…The major virulence factors of Cryptococcus neoformans include its ability to grow at 37 °C, production of a polysaccharide capsule and the expression of laccase enzyme, which forms melanin in the presence of phenolic compounds or their precursors, such as catecholamines in the human brain [18, 19]. It has been shown that several non- Cryptococcus neoformans / Cryptococcus gattii species including P. laurentii can also express laccase or synthesize melanin in the presence of suitable substrates, even though naturally occurring melanin-forming strains of P. laurentii are rare [17, 20]. The first two melanin-forming strains of P. laurentii isolated from plant debris in the USA were reported by Kwon-Chung et al .…”

Prevalence of Decreased Susceptibility of Cryptococcus spp. Isolates to Fluconazole in Urban Sources of Presidente Prudente

“…However, in the last years it was verified that these non-pathogenic species can cause injuries to individuals with some types of immune deficiency, compromising the central nervous system leading to fungemia [16][17][18].…”