In vitro growth characteristics of Cryptococcus gattii VGI/VGII and their virulence diversity to Galleria mellonella
Objective 
To understand the in vitro growth characteristics of Cryptococcus gattii VGⅠ and VGⅡ isolated in China and the diversity in their virulence to Galleria mellonella. 
 
 
Methods 
Based on the results of multilocus sequence typing for eight strains of Cryptococcus gattii isolated in China, the strains were cultured in vitro to draw growth curves, observe the melanin production and measure the capsule thickness. The median lethal time (LT50) and median lethal dose (LC50) at 48 h of Cryptococcus gattii were calculated using Galleria mellonella infection test. Fourteen strains of Cryptococcus neoformans were studied for comparison. 
 
 
Results 
The eight Cryptococcus gattii strains were six VGⅠ and two VGⅡ. The growth curves of Cryptococcus gattii VGⅠ and VGⅡ were similar to that of Cryptococcus neoformans when culture at 30℃. The total number for each of them could reach 108 CFU/ml at 96 h under 30℃. However, the total number at any time point at 37℃ was less than that at 30℃. There was no significant difference in the amount of melanin produced by Cryptococcus neoformans under 30℃ and 37℃, but both VGⅠand VGⅡ types of Cryptococcus gattii could produce more amount of melanin under 37℃ than under 30℃. The ratio of capsule/cell wall diameter of Cryptococcus gattii VGⅠwas greater at 37℃ than that at 30℃ with statistical significance (P<0.001). Cryptococcus neoformans showed the longest LT50, followed by VGⅠand VGⅡ types of Cryptococcus gattii. The LT50 of Cryptococcus gattii VGⅡ at the concentration of 1×106 CFU/ml was 72 h, and its LC50 at 48 h was 1×108 CFU/ml. 
 
 
Conclusions 
Like Cryptococcus neoformans, Cryptococcus gattii VGⅠ and VGⅡ grew faster under 30℃ than under 37℃, but more melanin was produced and thicker capsule was formed under 37℃ than under 30℃. Among Cryptococcus neoformans and VGⅠ and VGⅡ types of Cryptococcus gattii, Cryptococcus gattii VGⅡ showed the shortest LT50 and the strongest virulence to Galleria mellonella. 
 
 
Key words: 
Cryptococcus gattii; Molecular type; Growth characteristics; Galleria mellonella; Virulence