Ferreira-Paim Kennio, Ferreira Thatiana Bragine, Andrade-Silva Leonardo, Mora Delio Jose, Springer Deborah J, Heitman Joseph, Fonseca Fernanda Machado, Matos Dulcilena, Melhem Márcia Souza Carvalho, Silva-Vergara Mario León
Department of Infectious and Parasitic Diseases, Triangulo Mineiro Federal University, Uberaba, Minas Gerais, Brazil.
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, United States of America.
PLoS One. 2014 Sep 24;9(9):e108633. doi: 10.1371/journal.pone.0108633. eCollection 2014.
Although 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.
In 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.
BLAST 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.
Sequencing 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.
尽管罗伦隐球酵母一直被认为是腐生菌，其分类仍在描述中，但已有多例人类感染病例的报道。本研究旨在评估来自巴西、博茨瓦纳、加拿大和美国的罗伦隐球酵母分离株的分子特征。
在本研究中，通过对18S核糖体小亚基rRNA基因（18S - SSU）、28S核糖体大亚基rRNA基因的D1/D2区域（28S - LSU）以及核糖体区域的内部转录间隔区（ITS）进行测序，对100株经表型鉴定的罗伦隐球酵母分离株进行了评估。
使用从18S - SSU、28S - LSU和ITS区域获得的550 bp、650 bp和550 bp测序扩增子进行BLAST搜索，鉴定出75株罗伦隐球酵母菌株，它们与罗伦隐球酵母CBS 139具有99% - 100%的同一性。共有9株分离株与布勒酵母属VY - 68和罗伦隐球酵母RY1均具有99%的同一性。根据28S - LSU和ITS区域，1株分离株与拉贾斯坦隐球酵母CBS 10406具有99%的同一性，8株分离株与隐球酵母属APSS 862具有100%的同一性，并被指定为新种阿斯彭隐球酵母（CBS 13867）。根据18S - SSU序列，16株分离株与淡黄隐球酵母CBS 942具有99%的同一性，但使用28S - LSU和ITS区域序列仅确认了6株。其余10株与巴西最近描述报道的陆地隐球酵母CBS 10810具有99%的同一性。通过串联序列分析，在罗伦隐球酵母中鉴定出7种序列类型，在淡黄隐球酵母中鉴定出3种，在陆地隐球酵母中鉴定出1种，在新种阿斯彭隐球酵母中鉴定出1种。
测序使得对来自不同地理区域的75%的环境罗伦隐球酵母分离株进行了特征描述，并鉴定出该物种的7种单倍型。在测序区域中，与18S - SSU和28S - LSU区域相比，ITS区域具有更高的变异性，这增强了其作为DNA条形码的适用性。
