Subinhibitory concentrations of micafungin exert species-dependent differential effects on Candida and modulate the antifungal activity of human neutrophils against mature biofilms

Maria Simitsopoulou, Theodoros Karampatakis, Daniela Kyrpitzi, Anastasia Geladari, Paschalis Kadiltzoglou, Charalampos Antachopoulos, Thomas J. Walsh, Emmanuel Roilides


Background: Biofilms (BF) represent the most resistant structures of fungal growth able to develop on intravenous catheters and indwelling medical devices. Candida albicans (CA) and Candida parapsilosis (CP) constitute the predominant fungi responsible for bloodstream infections and BF formation. We hypothesized that pre-incubation of CA and CP cells with sub-MIC concentrations of micafungin (MFG) affect biofilm formation. Human neutrophils (PMN) are critical innate immune cells of antifungal host defenses. The aim of this study was to investigate whether pre-exposure of CA and CP to sub-MIC MFG concentrations could subsequently modify PMN activity against mature biofilms.

Material/methods: 106 blastoconidia/mL of three CA and four CP BF-producing strains were grown in RPMI at 37°C for 24h with MFG at 0.125xMIC, 0.25xMIC and 0.5xMIC for CA and at 0.001xMIC, 0.015xMIC, 0.06xMIC, 0.25xMIC and 0.5xMIC for CP. Pretreated and untreated isolates were then grown in fresh RPMI at 37°C for 48h (CA) and 72h (CP) to produce mature biofilms. PMN were isolated from healthy donors by dextran sedimentation/ficoll centrifugation. Biofilms were incubated with PMN at effector-to-target ratio 5:1 for 24h. To test the combinational interactive effect on untreated BF, intact BF were exposed to the simultaneous action of PMN (5:1 or 3:1) and MFG (0.007- 64mg/L) for 24h. The PMN-induced BF damage and MIC of MFG for BF measured as %-reduction of metabolic activity were assessed by XTT reduction assay. MFG MIC was determined as the minimum concentration that caused ≥50% BF damage. PMN-induced %BF damage of MFG-pretreated BF was compared to untreated BF by ANOVA with Dunnett’s post-test. P<0.05 was considered significant. Four independent experiments were performed.

Results: MFG MIC for BF of CA and CP isolates were 0.25mg/L and 4mg/L, respectively. MFG used at 0.125xMIC, 0.25xMIC and 0.5xMIC concentrations caused 9.8%±2.6, 7%±2.9 and 18%±3 BF damage of CA, respectively. When PMN were added to drug-pretreated BF there was a significant augmentation of PMN-induced %-damage against CA exposed to 0.5xMIC compared to druguntreated isolates (81%±1.8vs60%±3.5; p<0.01). In contrast, the highest BF damage caused by MFG against BF of CP was 29%±10 at 0.015xMIC; whereas, PMN addition to drug-pretreated BF exhibited similar %-damage compared to the damage induced by PMN alone on drug-untreated isolates (28%±5.3, 36%±6.4 and 21%±3.8, respectively; p=ns). The BF damage of CA and CP induced by the simultaneous combined treatment of PMN with MFG (0.007-4mg/L; 54%-65% for CA and 0.06- 64mg/L; 0%-79.5% for CP) against intact BF was not significantly different from that of PMN alone (48%±5.6 for CA; 46%±10 for CP).

Conclusions: Micafungin sub-MIC concentrations increase susceptibility of CA-biofilms to PMN but not to CP-biofilms. These findings implicate a potentially clinically significant immunomodulatory role of micafungin on PMNs in treatment of CA-biofilms and warrant further study in animal models.


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26th European Congress of Clinical Microbiology and Infectious Diseases
    • ECCMID 26th (2016)