Molecular and phenotypic reidentification of Sporothrix schenckii clinical isolates preserved under mineral oil for 34 to 64 years in a culture collection in Brazil

Abstract

Sporotrichosis is a subcutaneous mycosis worldwide distributed reaching hyperendemic proportions in Brazil. Many isolates from patients with sporotrichosis are preserved in culture collections by different methods around the world. The preservation methods are used to maintain the viability and the morphophysiological and genetic characteristics of isolates for long periods. In this study, we evaluated 34 isolates, previously, identified as S. schenckii by a classical identification method, initially preserved by periodical subcultures and then under mineral oil at culture collection of Oswaldo Cruz Institute/Fiocruz, to re-identify them by polyphasic identification. Our results showed that seven isolates remained viable for 34 to 64 years under oil, one isolate lost the ability to sporulate which was reverted by using a medium culture supplemented with rosebush branches and all of them were identified as Sporothrix schenckii sensu stricto by morphological, physiological, partial β-tubulin gene sequencing and phylogenetic analysis.

Keywords: Culture collection; Molecular identification and β-tubulin; Morphophysiological stability; Polyphasic identification; Sporothrix schenckii; Sporotrichosis; Viability.

Graphical abstract

Fig. 1

Macromorphological analysis of Sporothrix spp. after 21 days of incubation on PDA at 30 °C. A – IOC 1275; B – IOC 2993; C – IOC 1799; D – IOC 1835; E – IOC 1912; F – IOC 2547; G – IOC 2835.

Fig. 2

Micromorphological analysis of Sporothrix spp. after microculture for 12 days on PDA at 30 °C. The isolates IOC 1275, IOC 1799, IOC 1835, IOC 1912 and IOC 2835 showed sessile conidia (arrowhead) and septate hyaline hyphae (black arrow) containing conidiophores with conidia arranged in the shape of a daisy (red arrow). Isolate IOC 2993 presented only septate hyaline hyphae (black arrows). (1000X magnification).

Fig. 3

Microculture of Sporothrix spp. IOC 2993 for 12 days at 30 °C. A and B show the isolate cultured on PDA producing only thin, hyaline hypha. C and D show the isolates cultured on MEA with rose bush branches presenting sessile conidia and thin, septate hyaline hypha containing conidia arranged in the shape of a daisy at the end of conidiophores (Arrows) (1000X magnification).

Fig. 4

Phylogenetic relationships between the isolates IOC 2547, IOC 1835, IOC 1912, IOC 1799 IOC 1275, IOC 2993 and IOC 2835 with reference strains of the Sporothrix schenckii complex inferred from β-tubulin sequences by Neighbor-Joining method . The optimal tree is shown. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches . The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. This analysis involved 31 nucleotide sequences. There were a total of 428 positions in the final dataset. Evolutionary analyses were conducted in MEGA X . Bootstrap support values above 80% are indicated at the nodes.