Assessing the Bioactive Profile of Antifungal-Loaded Calcium Sulfate against Fungal Biofilms

Abstract

Calcium sulfate (CS) has been used clinically as a bone- or void-filling biomaterial, and its resorptive properties have provided the prospect for its use as a release mechanism for local antibiotics to control biofilms. Here, we aimed to test CS beads loaded with three antifungal drugs against planktonic and sessile fungal species to assess whether these antifungal beads could be harnessed to provide consistent release of antifungals at biofilm-inhibitory doses. A panel of different fungal species (n = 15) were selected for planktonic broth microdilution testing with fluconazole (FLZ), amphotericin B (AMB), and caspofungin (CSP). After establishing planktonic inhibition, antifungal CS beads were introduced to fungal biofilms (n = 5) to assess biofilm formation and cell viability through a combination of standard quantitative and qualitative biofilm assays. Inoculation of a hydrogel substrate, packed with antifungal CS beads, was also used to assess diffusion through a semidry material, to mimic active infection in vivo In general, antifungals released from loaded CS beads were all effective at inhibiting the pathogenic fungi over 7 days within standard MIC ranges for these fungi. We observed a significant reduction of pregrown fungal biofilms across key fungal pathogens following treatment, with visually observable changes in cell morphology and biofilm coverage provided by scanning electron microscopy. Assessment of biofilm inhibition also revealed reductions in total and viable cells across all organisms tested. These data show that antifungal-loaded CS beads produce a sustained antimicrobial effect that inhibits and kills clinically relevant fungal species in vitro as planktonic and biofilm cells.

Keywords: Candida; antimicrobial agents; biofilm; fungal; joint infections; wound management.

FIG 1

Schematic outline of study methods. (A) Antifungal CS beads loaded with FLZ (80 mg), CSP (35 mg), and AMB (25 mg) are incubated in RPMI for 7 days to test the eluent antifungal effect against planktonic fungal species. (B) Pregrown fungal biofilms are incubated alongside CS loaded with antifungal agents and evaluated via XTT, CV, qPCR, and SEM. (C) The fungal inoculum is introduced to our bespoke hydrogels alongside CS beads to determine the capacity for inhibition of growing biofilm, as evaluated via Live/Dead qPCR and SEM.

FIG 2

Inhibition of fungal biomass by antifungal CS beads. Shown are results from crystal violet assays of biofilm biomass across 5 fungal species (A to E) 24 h postexposure to FLZ (80 mg)-, CSP (35 mg)-, and AMB (25 mg)-loaded antifungal beads. Results are displayed as a percentage of reduction of biomass compared with unloaded control beads. Significant reductions in biomass (P < 0.0001) were recorded across all species (A to E) for CSP- and AMB-loaded beads, while C. tropicalis (C), A. fumigatus (D), and A. brasiliensis (E) biofilms displayed resistance to FLZ released from CS beads.

FIG 3

Inhibition of sessile cell viability by antifungal CS beads. Shown are results from a quantitative XTT assay of sessile cell metabolism across 5 fungal species (A to E) 24 h postexposure to FLZ (80 mg)-, CSP (35 mg)-, and AMB (25 mg)-loaded antifungal beads. Results are displayed as percentage of viability compared with biofilms exposed to unloaded control beads. Significant reductions (P < 0.0001) in viability were seen for C. albicans (A) and C. auris (B) across all treatments.

FIG 4

Total qPCR of fungal biofilm treated with antifungal CS after 48 h. Bars represent log CFE of 5 tested fungal isolates (A to E). Results indicate a variable but statistically significant reduction in total CFE for all but one isolate when exposed to CSP- and AMB-loaded beads, while FLZ-loaded beads only proved effective (P < 0.005) versus C. albicans SC5314 (A), C. auris 8973 (B), and C. tropicalis (C). In A. brasiliensis (E), no significant change (P < 0.05) was determined.

FIG 5

Heat map comparison of viability in comparative live versus total CFE for biofilm treatment. Shown are the results of heat map analysis of live versus total PCR samples from biofilm treatment tests. The scale represents a range from 100% viability (red) to 0% viability (green) when comparing CFEs extrapolated from PMA (live)-treated samples to untreated (total) samples. CTRL, control; C. trop, C. tropicalis; A. fum, A. fumigatus; A. brasi, A. brasiliensis.

FIG 6

Scanning electron micrography of fungal biofilms post-antifungal-loaded CS exposure. Images depict fungal biofilms challenged with FLZ (80 mg), AMB (25 mg), and CSP (35 mg) beads after 72 h of growth at ×800 magnification and ×3,500 magnification (insets). (A) C. albicans. (B) C. auris. (C) C. tropicalis. (D) A. fumigatus. (E) A. brasiliensis.

FIG 7

Total qPCR of fungal biofilm inhibited by antifungal CS beads on semidry hydrogels over 48 h. Bars indicate log CFE of 5 tested fungal isolates (A to E), postinoculation on cellulose matrix placed atop hydrogels, including FLZ (80 mg)-, CSP (35 mg)-, and AMB (25 mg)-loaded CS beads. The overall results display a significant reduction of biofilm in samples exposed to AMB-loaded beads. This is also the case for CSP-loaded beads, with the exception of C. auris 8973. FLZ-loaded beads also reduced CFE/ml for all samples, with exception of A brasiliensis.

FIG 8

Heat map comparison of viability in comparative live versus total CFE for hydrogel inhibition. Shown are the results from heat map analysis of live versus total PCR samples from hydrogel inhibition tests. The scale represents a range from 100% viability (red) to 0% viability (green) when comparing CFEs extrapolated from PMA (live)-treated samples to untreated (total) samples. CTRL, control; C. trop, C. tropicalis; A. fum, A. fumigatus; A. brasi, A. brasiliensis.