Guitard J, Atanasova R, Brossas J Y, Meyer I, Gits M, Marinach C, Vellaissamy S, Angoulvant A, Mazier D, Hennequin C
Assistance Publique-Hôpitaux de Paris, Hôpital St Antoine, Service de Parasitologie-Mycologie, Paris, France Inserm, U1135, CIMI-Paris, Paris, France CNRS, ERL 8255, CIMI-Paris, Paris, France Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France
Sorbonne Universités, UPMC Univ Paris 06, CR7, Centre d'Immunologie et des Maladies Infectieuses (CIMI-Paris), Paris, France.
J Clin Microbiol. 2015 May;53(5):1655-61. doi: 10.1128/JCM.02913-14. Epub 2015 Mar 11.
Candida inconspicua and Candida (Pichia) norvegensis are two emerging pathogenic species that exhibit reduced susceptibility to azole derivatives. Conventional (biochemical) approaches do not readily differentiate between the two species. The first aim of this work was to analyze the performance of biochemical, proteomic (matrix-assisted laser desorption ionization-time of flight [MALDI-TOF]), and molecular approaches in the precise identification of these species. These results then led us to sequence 3 genomic loci, i.e., the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA), the D1/D2 domain of the 28S rDNA, and the elongation factor 1α (EF-1α) gene, either directly or following cloning, of 13 clinical isolates and 9 reference strains belonging to the 5 species included in the Pichia cactophila clade, namely, Pichia cactophila, Pichia insulana, C. inconspicua, C. norvegensis, and P. pseudocactophila. Finally, isolates of C. inconspicua were challenged for sexual reproduction on the appropriate medium. Our results show that EF-1α sequencing and proteic profiling by MALDI-TOF are the two most efficient approaches to distinguish between C. norvegensis and C. inconspicua. As a characteristic of the P. cactophila clade, we found multiple alleles of the rDNA regions in certain strains belonging to the tested species, making ITS or D1/D2 sequencing not appropriate for identification. Whatever the method of identification, including MALDI-TOF and EF-1α sequencing, none could differentiate C. inconspicua from P. cactophila. The results of phylogenetic analysis and the generation of asci from pure cultures of all C. inconspicua strains both support the identification of P. cactophila as the teleomorph of C. inconspicua.
隐匿念珠菌和挪威念珠菌（毕赤酵母属）是两种新出现的致病菌种，对唑类衍生物的敏感性降低。传统（生化）方法不易区分这两种菌种。这项工作的首要目标是分析生化、蛋白质组学（基质辅助激光解吸电离飞行时间质谱[MALDI-TOF]）和分子方法在精确鉴定这些菌种方面的性能。这些结果随后促使我们对属于嗜仙人掌毕赤酵母分支的5个菌种（即嗜仙人掌毕赤酵母、岛屿毕赤酵母、隐匿念珠菌、挪威念珠菌和拟嗜仙人掌毕赤酵母）的13株临床分离株和9株参考菌株，直接或在克隆后对核糖体DNA（rDNA）的内部转录间隔区（ITS）、28S rDNA的D1/D2结构域以及延伸因子1α（EF-1α）基因的3个基因组位点进行测序。最后，在合适的培养基上对隐匿念珠菌分离株进行有性繁殖挑战。我们的结果表明，EF-1α测序和MALDI-TOF蛋白质谱分析是区分挪威念珠菌和隐匿念珠菌的两种最有效的方法。作为嗜仙人掌毕赤酵母分支的一个特征，我们在某些受试菌种的菌株中发现了rDNA区域的多个等位基因，这使得ITS或D1/D2测序不适用于鉴定。无论采用何种鉴定方法，包括MALDI-TOF和EF-1α测序，都无法区分隐匿念珠菌和嗜仙人掌毕赤酵母。系统发育分析结果以及所有隐匿念珠菌菌株纯培养物中产生子囊的情况均支持将嗜仙人掌毕赤酵母鉴定为隐匿念珠菌的有性型。
