Abstract title	PHENOTYFIPICATION AND GENOTYPIFICATION OF COLOMBIAN CLINICAL ISOLATES OF Sporothrix spp FROM THE MEDICAL MYCOLOGY LABORATORY, UNIVERSIDAD DE ANTIOQUIA, MEDELLIN, COLOMBIA.

Objectives:
General goal:

To do the phenotypication and genotyping of clinical isolates of Sporothrix spp complex obtained from patients on the Medical Mycology Laboratory, Universidad de Antioquia, from 2004 to 2017.

Specific Goals:

To determine in the Sporothrix spp clinical isolates the phenotypic characteristics as: growth rate, pigment, texture and carbohydrate assimilation.

To identify the different species in the Sporothrix spp clinical isolates, by the calmoduline gene sequencing.

To correlate the phenotypic, genotypic and clinical findings for each one of the Colombian Sporothrix spp clinical isolates.

Methods:

A-Phenotypification of the 41 clinical isolates at days: 7, 14, 21, 28 and 40 of culture.

A1-Macroscopic morphology:

Growth: The diameter was measured at the different time points culturing them on potate dextrose agar (PDA) and cornmeal agar (CMA) at 25 °C.

Pigment and texture were evaluated culturing the isolates on PDA at 25 °C.

The inoculum of each isolate to evaluate growth, pigment and texture was 5x105 conidia/ml.

Transition from the mold to yeast phase was determined in two media culture: 1) brain heart infusion (BHI) supplemented with 2% glucose and 5% CO2 (BHI-1); 2) BHI supplemented with 2% glucose, 5% human blood, 0.1% L-cysteine and 5% CO2 (BHI-2).

Carbohydrate assimilation was determined using the kit api 20 C AUX (Biomerieux®; Marcy-l´Etoile-France). The reading was done after 8 days of incubation at 30°C.

A2-Microscopic morphology:

Microcultures in PDA at 25 °C were made for all isolates; the morphology, diameter and type of sporulation were evaluated using the Micrometrics SE Premium 4 software.

B-Genotypication:

DNA was extracted from the yeast phase for all isolates using the Fungi/Yeast Genomic DNA isolation kit (Norgen Biotek Corporation). The calmoduline gene was amplified by PCR with the primers described by O`Donnell 2000. The amplification products were sequenced by Macrogen (Korea) and the sequences analysis was done with Genious Software.

Results:

A1: The range of growth at the mycelial phase of all isolates was similar in both media. The pigment varied from beige to black; the pigment distribution was: homogeneous, spotted heterogeneous, central heterogeneous, sectorized heterogeneous and dotted. The isolates texture was varied between them: velvety, central vellus, cerebriform and membranous.

The transition from mold to yeast was 100% using the BHI-2 and 90% with the medium BHI-1.

There were differences on the carbohydrate assimilation specifically of sucrose, raffinose and adonitol between all isolates.

A2: All isolates produced oval conidia without pigment; some had elongated oval conidia, other globoses with botrios sympodial sporulation, however not all isolates produced pigmented conidia and some of them were triangular with sesil sporulation.

B-The genetic analysis suggests that the species population of Sporothrix complex in Colombia is polyphyletic.

Conclusion:

The Sporothrix spp isolates studied were obtained from patients with different clinical forms of sporotrichosis. We found phenotypic differences between Colombian Sporothrix spp isolates, however it is neccesary to correlate these findings with their genotypic characterization. The differences found on this study are key to seek for the diversity of Sporothrix species in Colombia.

3 / PHENOTYFIPICATION AND GENOTYPIFICATION OF COLOMBIAN CLINICAL ISOLATES OF Sporothrix spp FROM THE MEDICAL MYCOLOGY LABORATORY, UNIVERSIDAD DE ANTIOQUIA, MEDELLIN, COLOMBIA