Different components in human serum inhibit multiplication of Cryptococcus neoformans and enhance fluconazole activity

Abstract

The inhibitory effect of human serum on the multiplication of Cryptococcus neoformans and the interaction with fluconazole were studied. Compared with cryptococcal multiplication in RPMI 1640 medium alone, 5% human serum in medium inhibited multiplication by 76% +/- 6% (n = 8). The inhibitory effect of human serum was donor independent, [corrected] heat stable (56 degrees C, 30 min), and not due to albumin or globulin. Bovine and murine sera were not inhibitory at that concentration. A fungistatic concentration of fluconazole (5.0 micrograms/ml) in medium plus 5% human serum resulted in 40% +/- 5% (n = 8) killing (reduction of inoculum CFU) in a 24-h assay. Bovine or murine sera did not have the enhancing effect, and this human serum activity was heat stable and donor independent. At 2.5 micrograms of fluconazole per ml, fungistasis by fluconazole plus human serum was significantly greater than with either alone. Higher serum concentrations [corrected] potentiated fluconazole more. At higher fluconazole concentrations (e.g., 20 micrograms/ml) fluconazole alone could kill, but serum potentiated this. A fluconazole-resistant isolate (MIC, 100 micrograms/ml) was not killed by fluconazole (5.0 micrograms/ml) in 5% human serum, but human serum potentiated the partial fluconazole inhibition. When human serum was dialyzed (molecular weight cutoff, 6,000 to 8,000) against phosphate-buffered saline, it lost the ability to synergize with fluconazole for killing Cryptococcus organisms but not the capacity to inhibit multiplication. Filtration of serum suggested the filtrate with a molecular weight of < 10,000 could interact synergistically with fluconazole for killing but could not inhibit cryptococcal multiplication. These findings indicate that human serum has two components, one (macromolecular) with a unique ability to inhibit C. neoformans and a low-molecular-weight component that enhances fluconazole anticryptococcal activity.