Influence of shaking on antifungal susceptibility testing of Cryptococcus neoformans: a comparison of the NCCLS standard M27A medium, buffered yeast nitrogen base, and RPMI-2% glucose

Abstract

Cryptococcus neoformans is a nonfermentative yeast that requires oxygen for growth. The shaking of culture media achieves good oxygenation, promoting the growth of cryptococci. In this study, three test media (RPMI 1640, RPMI 1640-2% glucose, and buffered yeast nitrogen base ¿BYNB) recommended in the National Committee for Clinical Laboratory Standards M27A standard were examined. Growth abilities and minimum inhibitory concentrations (MICs) in microplates incubated at 35 degrees C for 48 h were determined. The results indicated that shaking and an inoculum size of 10(5) CFU/ml yielded optimal growth of this yeast. Compared to RPMI 1640, supplementation of RPMI 1640 with 2% glucose did not significantly improve growth of C. neoformans and resulted in an 8.7-h delay of exponential growth. Cryptococcal growth in RPMI 1640 at 24 h was notably better than that in RPMI-2% glucose, although by 48 h the growths were comparable. The MIC range of amphotericin B observed for the C. neoformans strains grown in RPMI 1640 with or without glucose was too narrow to allow the separation of susceptible and resistant strains based on clinical outcome. The widest ranges of MICs of flucytosine and fluconazole were obtained with BYNB. This work demonstrates the need for a new antifungal susceptibility test for C. neoformans.

FIG. 1

Mean growth curve of 16 C. neoformans isolates incubated at 35°C in static or shaken culture media at 1,400 rpm.

FIG. 2

Mean curve of seven inhibition curves of C. neoformans ATCC 90112 produced by amphotericin B, flucytosine, fluconazole, and itraconazole in static and 350-rpm-agitated microplates incubated for 48 h at 35°C.