In vitro interaction between alginate lyase and amphotericin B against Aspergillus fumigatus biofilm determined by different methods

Abstract

Aspergillus fumigatus biofilms represent a problematic clinical entity, especially because of their recalcitrance to antifungal drugs, which poses a number of therapeutic implications for invasive aspergillosis, the most difficult-to-treat Aspergillus-related disease. While the antibiofilm activities of amphotericin B (AMB) deoxycholate and its lipid formulations (e.g., liposomal AMB [LAMB]) are well documented, the effectiveness of these drugs in combination with nonantifungal agents is poorly understood. In the present study, in vitro interactions between polyene antifungals (AMB and LAMB) and alginate lyase (AlgL), an enzyme degrading the polysaccharides produced as extracellular polymeric substances (EPSs) within the biofilm matrix, against A. fumigatus biofilms were evaluated by using the checkerboard microdilution and the time-kill assays. Furthermore, atomic force microscopy (AFM) was used to image and quantify the effects of AlgL-antifungal combinations on biofilm-growing hyphal cells. On the basis of fractional inhibitory concentration index values, synergy was found between both AMB formulations and AlgL, and this finding was also confirmed by the time-kill test. Finally, AFM analysis showed that when A. fumigatus biofilms were treated with AlgL or polyene alone, as well as with their combination, both a reduction of hyphal thicknesses and an increase of adhesive forces were observed compared to the findings for untreated controls, probably owing to the different action by the enzyme or the antifungal compounds. Interestingly, marked physical changes were noticed in A. fumigatus biofilms exposed to the AlgL-antifungal combinations compared with the physical characteristics detected after exposure to the antifungals alone, indicating that AlgL may enhance the antibiofilm activity of both AMB and LAMB, perhaps by disrupting the hypha-embedding EPSs and thus facilitating the drugs to reach biofilm cells. Taken together, our results suggest that a combination of AlgL and a polyene antifungal may prove to be a new therapeutic strategy for invasive aspergillosis, while reinforcing the EPS as a valuable antibiofilm drug target.

Fig 1

Time-kill curves of AlgL (1 and 10 U/ml) alone and in combination with AMB or LAMB at 8 μg/ml (A), 16 μg/ml (B), or 32 μg/ml (C) against biofilm cells of the A. fumigatus type strain Af293. The results are the averages of three replicates carried out on two separate occasions and are expressed as percent cellular viability determined by the XTT reduction assay. Error bars represent the standard errors of the means.

Fig 2

Time-kill curves of AlgL (1 and 10 U/ml) alone and in combination with AMB or LAMB at 8 μg/ml (A), 16 μg/ml (B), or 32 μg/ml (C) against biofilm cells of a clinical A. fumigatus isolate (CG261). The results are the averages of three replicates carried out on two separate occasions and are expressed as percent cellular viability determined by the XTT reduction assay. Error bars represent the standard errors of the means.

Fig 3

Atomic force micrographs of mature A. fumigatus Af293 biofilms imaged before treatment (A) and after treatment with AlgL (B), AlgL-AMB (C), or AlgL-LAMB (D). Topographic images (top) show hyphae from each experimental condition, as indicated, after they were grown as sessile cells for 24 h at 37°C in static and aerial environments. Deflection images (middle) reveal more details in hyphal cell texture, whereas three-dimensional (3-D) topographic images (bottom) depict clear differences in hyphal thickness between treated (B to D) and untreated control (A) samples.

Fig 4

Biophysical properties of untreated 24-h-old A. fumigatus Af293 biofilms in RPMI 1640 medium and of biofilms treated for a further 24 h with AlgL or a polyene antifungal (AMB or LAMB) alone and with the AlgL-antifungal combinations, as measured by AFM. Averages of hyphal height (A) and adhesion force (B) measurements from three independent experiments are shown. Asterisks indicate statistically significant differences (*, P < 0.05; **, P < 0.01; ***, P < 0.001). NS, no significance (P > 0.05). Error bars indicate the standard errors of the measurements.