Table 1.
Clinical isolates.
Table 2.
Aspergillus fumigatus clinical isolates.
Fig 1.
Experimental design (full details are provided in Methods).
To form Af biofilm, polystyrene disks were placed in tissue culture plates with conidia and media. Disks were incubated to allow the conidia to attach. Following the attachment phase, disks were transferred to new plates containing and incubated for an additional 24 h (line I). Direct interaction of live Pa on Af biofilm formation: Suspensions of conidia and bacteria were combined in tissue culture plates for 16 h. Disks were then rinsed gently, transferred to new plates, and incubated for an additional 24 h (line II). Direct interaction of live Pa on preformed Af biofilm: Fungal biofilms were formed as described. After 16 h the disks were rinsed, transferred to new plates containing Pa suspension and incubated an additional 24 h (lines II and IV). Pa planktonic supernatant assay: To obtain planktonic culture filtrates, Pa suspension was incubated in conical tubes for 24 h. The spent medium was centrifuged to remove suspended cells or debris. The supernatant was filter sterilized and added to wells of a tissue culture plate previously inoculated with Af suspension. Fungal biofilms attached and formed. Disks containing biofilms were washed, transferred to a new plate, and incubated for an additional 24 h (line III). For the preformed biofilm assay, filtered supernatant was added to wells. Disks containing Af preformed biofilms were washed, transferred to the plate containing the bacterial filtrates, and incubated an additional 24 h. Af wells without bacterial supernatant were included as controls (line V). To obtain Pa biofilm filtrates, a suspension of Pa adhered to tissue culture flasks for 2 h (attachment phase). The flasks were rinsed, fresh RPMI-1640 added to the flask and adhered cells formed Pa biofilms for 22 h. The spent medium was then removed. Af conidia forming biofilms or preformed Af biofilms were challenged with the Pa biofilm culture filtrate (lines III and V).
Fig 2.
Pa inhibition of Af during biofilm formation.
The data presented show the activity of each live Pa isolate or planktonic- or biofilm-generated Pa culture filtrate on Af during formation of biofilm by Af. Individual isolate results are shown in comparison with their respective Af controls (n = 6 for each). There were five non-CF, five mucoid CF and five non-mucoid CF isolates tested; isolate designations are shown on the x-axis. The bars on the far right of each panel represent the pooled data for each type of isolate (n = 30). Bars represent the mean ± SD of the XTT reduction read at 490 nm. (A) Af conidia were exposed to live Pa cells for 16 h. (B) Af conidia were exposed to Pa planktonic supernatant for 16 h. (C) Af conidia were exposed to Pa biofilm supernatant for 16 h. The resulting readings were determined. “XTT metabolic activity” refers to the spectrophotometric absorbance of the formazan reduction product of XTT at 490 nm. Assays were performed in blocks with two runs of triplicates done on different days, for each Pa-Af combination. Panel (A) All individual Pa isolates, noted on the x-axis, were significantly inhibitory compared to controls (P<0.001). The four bars on the right side show the pooled data. CF mucoid and CF non-mucoid cells were more inhibitory than non-CF Pa cells (P<0.001, both comparisons), with CF non-mucoid cells more inhibitory than CF mucoid cells (P<0.001). Panel (B) Planktonic culture filtrate from all individual Pa isolates, were significantly inhibitory compared to controls (P<0.001). Pooled data analysis showed both CF mucoid or CF non-mucoid isolate culture filtrate was more inhibitory than that from non-CF isolates (P<0.001, both comparisons) and non-mucoid was more inhibitory than mucoid (P < 0.001). Panel (C) Culture filtrates from all Pa isolates grown as biofilm were significantly inhibitory (P<0.001). Pooled data showed culture filtrate from CF non-mucoid isolates under biofilm conditions was more inhibitory than that from CF mucoid or non-CF isolates under the same conditions (P<0.001, both comparisons), and that non-mucoid CF filtrates were more inhibitory than mucoid isolate filtrates (P < 0.001).
Fig 3.
The data presented show the activity of each live Pa isolate or planktonic or biofilm generated culture filtrate on preformed biofilm of Af. Individual isolate results are shown in comparison with their respective Af controls (n = 6 for each). There were five non-CF, five mucoid CF and five non-mucoid CF isolates tested; isolate designations are shown on the x-axis. The bars on the far right of each panel represent the pooled data for each isolate (n = 30). Bars represent the mean ± SD of the XTT reduction at read at 490 nm. Panel (A) Af preformed biofilms exposed to live Pa cells for 24 h. (B) Af preformed biofilms were exposed to Pa planktonic spent supernatant for 24 h. (C) Af preformed biofilms were exposed to Pa biofilm spent supernatant for 24 h. Each data point represents the XTT metabolic activity obtained spectrophotometrically at 490 nm. Assays were performed in triplicate and the results are pooled from two experiments for each Pa-Af combination. The asterisk indicates a significant P value (< 0.001) for the XTT metabolic activity compared to the positive control, using the same analytic methodology as in Fig 2. Panel (A) Individual Pa isolate comparisons showed that only a one non-CF isolate, Pa19, inhibited (P<0.01), whereas all mucoid or non-mucoid CF isolates were inhibitory (P<0.001, all comparisons). Pooled data analysis showed that both mucoid and non-mucoid CF isolates were significantly inhibitory (P<0.001, both comparisons) and that non-mucoid CF isolates were more inhibitory than non-CF or mucoid Pa isolates (P< 0.001, both comparisons). Panel (B) Planktonic spent medium from a single non-CF isolate, Pa19, was inhibitory (P<0.05), whereas all mucoid and non-mucoid CF were significantly inhibitory (P < 0.001). Pooled data analysis showed that planktonic culture filtrate from CF non-mucoid or mucoid isolates was inhibitory (P<0.001, both comparisons). Non-mucoid CF isolates were more inhibitory than mucoid CF isolates (P<0.001). Panel (C) Biofilm culture filtrate from non-CF isolates of Pa was not inhibitory, whereas the culture filtrate from each mucoid or non-mucoid CF Pa isolates was inhibitory (P< 0.001, all comparisons). Pooled data analysis showed that biofilm culture filtrate from CF non-mucoid or mucoid isolates was inhibitory (P<0.001). Non-mucoid CF isolate filtrates were more inhibitory than mucoid CF isolates (P<0.001).
Fig 4.
Pa supernatant affects Af biofilm thickness.
Af biofilms were formed and stained as described. Sections of the xy plane were taken at 1 μm intervals along the z-axis to determine the depth of the biofilms. (A) Af conidia were exposed to planktonic Pa spent supernatant or (B) biofilm Pa spent supernatant, for 16 h. (C) Af preformed biofilms were exposed to planktonic Pa spent supernatant or (D) biofilm Pa spent supernatant, for 24 h. Assays were performed in triplicate and images were taken from three different fields from each sample. The results are representatives of two different experiments for each Pa-Af combination. One asterisk indicates a P value (<0.01), and two asterisks indicates a P value (<0.001) for the biofilm thickness compared to the untreated control.
Fig 5.
CLSM images of Af biofilm after challenging conidia for 16h with biofilm Pa spent supernatant.
Horizontal (xy) view of reconstructed 3-dimensional images of FUN1-stained biofilms, with filter set to capture green fluorescence. Thickness of the biofilm can be observed in the side view of the reconstruction (extreme right and lower panels in each picture). (A) Untreated control. (B) Af conidia exposed to spent supernatant of a non-CF Pa grown as biofilm for 16 h, or (C) exposed to mucoid CF Pa biofilm spent supernatant or (D) exposed to non-mucoid CF Pa biofilm spent supernatant. Arrows show “bulge-like” structures and deposition of amorphous material in treated cultures. Magnification, ×63. Bar, 50 μm.
Fig 6.
CLSM images of Af biofilm after challenging preformed Af biofilm for 24 h with Pa biofilm spent supernatant.
Horizontal (xy) view of reconstructed 3-dimensional images of FUN1-stained biofilms, with filter set to capture green fluorescence. Thickness of the biofilm can be observed in the side view of the reconstruction (extreme right and lower panels in each picture). (A) Untreated control. (B) Af preformed biofilm exposed to spent supernatant of a non-CF Pa biofilm spent supernatant for 16 h or (C) exposed to mucoid CF Pa biofilm spent supernatant or (D) exposed to non-mucoid CF Pa biofilm spent supernatant. Arrows show the amorphous structures distributed throughout the intertwined altered filamentous networks in treated cultures. Magnification, ×63. Bar, 50 μm.
Fig 7.
A. Pa biofilm supernatant inhibits Af biofilm formation. Af conidia were exposed to serial dilutions of Pa spent medium or sterile distilled water, mixed with fresh RPMI supplemented with 10% serum during Af biofilm formation as described in Methods and Fig 1III. The percent of fresh medium in each test situation was decreased as the spent medium or sterile distilled water increased. Then the resulting XTT readings were quantified, and the results expressed as percent of control with medium alone (no supernatant or distilled water). Results are presented as the mean of three replicates from three strains of Pa performed on two separate occasions. Error bars represent the SD of the mean. B. Pa biofilm supernatant inhibits planktonic Af. Af conidia were exposed to serial dilutions of Pa spent medium or sterile distilled water, mixed with fresh RPMI, and grown planktonically in tubes, as described in Methods. The percent of fresh medium in each tube was decreased as the spent medium or sterile distilled water increased. Then the resulting XTT readings were quantified, and the results expressed as percent of control with medium alone (no supernatant or distilled water). The results shown are with the non-mucoid CF Pa isolate supernatant; the mucoid CF Pa isolate and the non-CF Pa isolate were studied concurrently, and the results were not different; only the non-mucoid Pa result is shown to avoid clutter.
Fig 8.
The effect of FeCl3 on the inhibition of Af biofilm formation, by filtrate from a CF non-mucoid Pa grown under biofilm conditions.
Conidia formed biofilms on polystyrene disks (AF) during 16 h exposure to Pa filtrate (PA), filtrate + varying concentrations of FeCl3 (shown as the concentration, uM = μM), or varying concentrations of FeCl3 only. After 16 h of challenge, disks were transferred to fresh RPMI-1640 for 24 h of further growth before biofilm formation was quantified via XTT assay, and the results expressed as percent of control with medium alone (no supernatant or FeCl3). Asterisks denote P <0.001 for comparisons to the experimental control containing Af with RPMI-1640 (AF; no test materials). Daggers denote P <0.001 for comparisons with the experimental control containing Af +filtrate (AF + PA). Results with AF + PA + 11, 33 or 99 μM FeCl3 were not different than AF + PA; AF + 11, 33, or 99 μM FeCl3 were not different than AF + 297 μM FeCl3; and these results are not shown.