Increase in virulence of Sporothrix brasiliensis over five years in a patient with chronic disseminated sporotrichosis

Abstract

The metropolitan region of Rio de Janeiro is hyperendemic for cat-associated sporotrichosis. This study aimed to assess the virulence of serial Sporothrix isolates from a 61-year-old male patient with chronic, destructive disseminated sporotrichosis. Five Sporothrix isolates were cultured from skin exudates and bone samples over a 5-year period, and all were molecularly identified as Sporothrix brasiliensis. The final isolate was significantly more virulent in Galleria mellonella larvae compared to earlier isolates. We conclude that S. brasiliensis has the capacity to increase in virulence in vivo. This finding is significant to clinicians caring for individuals with S. brasiliensis disease and it suggests that further studies are needed to identify the mechanisms underlying pathogenicity enhancement during chronic disease.

Keywords: Brazil; Galleria mellonella; Rio de Janeiro; Sporothrix brasiliensis; T3B PCR; Virulence; chitin synthase; disseminated; reactive nitrogen intermediates; sporotrichosis.

Figure 1.

A sixty-one-year-old patient with disseminated sporotrichosis. (A) Multiple scars are visible on the limbs (arrows), where cutaneous lesions were previously present. (B) Inflammatory cyst on the left wrist. The first 3 isolates were collected from the wrist lesion, by puncture with fine needle at different time points over a 6-month period. C-D) Radiographs showed severe lytic lesions and sclerosis at the left wrist and knees (arrows).

Figure 2.

Representative T3B PCR fingerprinting profiles of the 5 Sporothrix isolates. (1) Negative control. (2 and 12) Molecular marker DNA ladder, 100 bp (Invitrogen). (3) S. brasiliensis (CBS120339). (4) S. mexicana (MUM11.02). (5) S. schenckii (ATCC32286). (6) S. globosa (IPEC27135). (7) IPEC32742. (8) IPEC33070. (9) IPEC33718. (10) IPEC33946. (11) IPEC43174.

Figure 3.

Neighbor-joining phylogram of the partial β-tubulin (A) and Chitin synthase (B) genes obtained for the 5 isolates (IPEC32742, IPEC33070, IPEC33718, IPEC33946, IPEC43174) and S. brasiliensis (CBS120339), S. globosa (FMR8600), S. mexicana (CBS120341), S. pallida (CBS111110) and S. schenckii (FMR8604), reference strains in NCBI public GenBank sequences constructed with MEGA version 4.0.2. Bootstrap values after 1,000 replicates are presented in the branch node.

Figure 4.

Galleria mellonella infection model. (A) Photographs of 2 groups of larvae (PBS [left] and isolate 5 [right]) 2 days after injection; the five dark brown / black larvae were dead. (B) Survival curves of the larvae. Lines represent the percentage of live individuals at each day. Control: uninfected larvae; PBS: larvae inoculated with 10 μL sterile PBS; numeric codes: larvae inoculated with 10⁷ yeasts of each isolate, or with the reference CBS strain of S. brasiliensis in 10 μL sterile PBS. The larvae were maintained at 37°C. N = 60 larvae per group.

Figure 5.

Growth curves of the 5 isolates in BHI medium (Bioscreen C Analyzer).

Figure 6.

Percentage of viable yeasts after 4 h of exposure to different oxidative stress conditions. (A) Exposure to oxygen-derived species. (B) Exposure to reactive nitrogen intermediates. The 5 isolates (10⁷ yeasts / mL) were in the specific media at 37°C and after each time point samples of 100 μL were plated onto PCA for CFU determinations. Points represent medians of 2 experiments performed in triplicate.