Temperature affects the susceptibility of Cryptococcus neoformans biofilms to antifungal agents

The fungal opportunist Cryptococcus neoformans forms biofilms in vitro and in vivo. C. neoformans has an unusual ability to grow over a wide range of temperatures, and is one of only two species in the genus able to grow at 37°C. The optimum growth temperature in the laboratory is 30°C, but Clinical and Laboratory Standards Institute (CLSI) planktonic susceptibility testing is performed at 35°C. We investigated whether these growth temperatures affected C. neoformans biofilm formation and drug resistance. Biofilms of 30 strains of C. neoformans were grown at 30°C or 35°C, and antifungal susceptibilities evaluated at 30°C or 35°C using minimum biofilm eradication endpoints. At 35°C, biofilms from 40% of the strains were more susceptible to flucytosine, 30% were more susceptible to nystatin, 27% were more susceptible to amphotericin, and 20% were more susceptible to fluconazole, as compared to 30°C. The reverse, that is an increased susceptibility at 30°C, only occurred with a single strain using nystatin or fluconazole. For the remaining strains, biofilm susceptibility was equivalent at the two temperatures. Biofilm colony forming units (CFU)s, as measured indirectly by 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5- carboxanilide (XTT) reduction, were greater at 35°C than at 30°C for the majority of the strains. Thus, growth temperature does affect C. neoformans biofilm properties, but factors other than relative biofilm CFUs/ml must be involved in the increased drug susceptibility at 35°C.