Analysis and description of the stages of Aspergillus fumigatus biofilm formation using scanning electron microscopy

Abstract

Background: Biofilms are a highly structured consortia of microorganisms that adhere to a substrate and are encased within an extracellular matrix (ECM) that is produced by the organisms themselves. Aspergillus fumigatus is a biotechnological fungus that has a medical and phytopathogenic significance, and its biofilm occurs in both natural and artificial environments; therefore, studies on the stages observed in biofilm formation are of great significance due to the limited knowledge that exists on this specific topic and because there are multiple applications that are being carried out.

Results: Growth curves were obtained from the soil and clinical isolates of the A. fumigatus biofilm formation. The optimal conditions for both of the isolates were inocula of 1 × 10⁶ conidia/mL, incubated at 28 °C during 24 h; these showed stages similar to those described in classic microbial growth: the lag, exponential, and stationary phases. However, the biofilms formed at 37 °C were uneven. The A. fumigatus biofilm was similar regardless of the isolation source, but differences were presented according to the incubation temperature. The biofilm stages included the following: 1) adhesion to the plate surface (4 h), cell co-aggregation and exopolymeric substance (EPS) production; 2) conidial germination into hyphae (8-12 h), development, hyphal elongation, and expansion with channel formation (16-20 h); and 3) biofilm maturation as follows: mycelia development, hyphal layering networks, and channels formation, and high structural arrangement of the mycelia that included hyphal anastomosis and an extensive production of ECM (24 h); the ECM covered, surrounded and strengthened the mycelial arrangements, particular at 37 °C. In the clinical isolate, irregular fungal structures, such as microhyphae that are short and slender hyphae, occurred; 4) In cell dispersion, the soil isolate exhibited higher conidia than the clinical isolate, which had the capacity to germinate and generate new mycelia growth (24 h). In addition, we present images on the biofilm's structural arrangement and chemical composition using fluorochromes to detect metabolic activity (FUNI) and mark molecules, such as chitin, DNA, mannose, glucose and proteins.

Conclusions: To our knowledge, this is the first time that, in vitro, scanning electronic microscopy (SEM) images of the stages of A. fumigatus biofilm formation have been presented with a particular emphasis on the high hyphal organization and in diverse ECM to observe biofilm maturation.

Keywords: Aspergillus fumigatus biofilm; Microhyphae; Scanning electronic microscopy (SEM); Stages of biofilm.

Fig. 1

Aspergillus fumigatus isolates biofilm growth curves. Biofilm growth was assessed at different inoculum concentrations ranging from 1 × 10⁴ (square), 1 × 10⁵ (triangle) to 1 × 10⁶ (circle) and various incubation times (0, 4, 8, 12, 16, 20 and 24 h) and at two dissimilar temperatures (28 and 37 °C). In addition, two strains isolated from different niches were used (clinical and soil isolates). Significant differences were determined by a Student-Newman-Keuls test, performing a multicomparison of procedures (p < 0.050). The significant difference is described as follows: *comparison of the inocula of the biofilm at 24 h; **Comparison among the inocula at 20 h

Fig. 2

Stages of the Aspergillus fumigatus biofilm isolates. These were described by scanning electronic microscopy (SEM) in both of the A. fumigatus isolates: clinical (AFU) and soil isolates (AFS). Inoculum concentration 1 × 10⁶ conidia/mL, and in various stages of biofilm formation was perceived: i) Adhesion with co-aggregation and exopolymeric substance (EPS) production, 3,000X and 6,000X (4 h); ii) Conidial germination into hyphae, 1,000X (8-12 h) and development, 1000X (16-20 h); and iii) Biofilm maturation, 1,000X (24 h). Blank box, extreme left side: details increasing (3,000X-6,000X)

Fig. 3

Extracellular matrix (ECM) structure of the Aspergillus fumigatus biofilm isolates. Inoculum concentration was 1x10⁶ and at 24 h/37 °C of incubation. The in vitro biofilm was observed by scanning electron microscopy (SEM) and certain types of ECM were differentiated. In the clinical biofilm isolate, a porous ECM (a-b: 1,000X-2,000X; e-f: 1,000X-2,000X) and condensed ECM (c-d: 1,000X-2,000X) was observed. Similarly, the same types of ECM for the soil biofilm isolate were observed. Porous ECM (i-j: 1,000X-2,000X) and condensed ECM (g-h: 1,000X-2,000X; k-l: 1,000X-2,000X). White dotted circles: ECM areas were observed at a higher magnification in the image on the right

Fig. 4

Microhyphae of the Aspergillus fumigatus clinical isolate biofilm. This structure was observed by scanning electron microscopy (SEM) at an inoculum concentration of 1 × 10⁶ conidia/mL only at 20 h/37 °C of incubation on the in vitro biofilm. Comparing the microhyphae with the size and diameter of the normal hyphae are crucial. a microhyphae projecting between the normal hyphae and the extracellular matrix (ECM) (2,700X); b microhyphae exiting and surrounding the normal hyphae (5,000X); c microhyphae on fungal anastomosis (2,500X). d developing microhyphae from the interior of the normal hyphae (5000X). White pointed arrow: normal hyphae; white arrow: microhyphae; white asterisk: porous ECM

Fig. 5

Structural composition of extracellular matrix (ECM) by epifluorescence microscopy (EPM). The EPM images on 12-wells polystyrene plates incubated with RPMI and an inoculum concentration of 1 × 10⁶ microconidia/mL at 24 h at 37 °C in vitro biofilm of Aspergillus fumigatus clinical isolate. a Co-localization of chitin/DNA. Hyphal anastomosis marked with Calcofluor white (chitin), FUN1 (fungal metabolic activity), and DAPI (DNA) (10X); b Detection of metabolic activity and chitin biofilm. Hyphal anastomosis indicated conidia marked with FUN1 and Calcofluor white. The latter exhibited a strong signal of chitin (100X); c Top view of the ECM detecting the co-localization of different molecules. Arrangement of z-stack images showing the dimensions of a section of the fungal biofilm, marked with FUN1, Calcofluor White and Flamingo stain; d Three-dimensional reconstruction of the in vitro biofilm showing molecular components of the ECM. These depicted different images that dissect the ECM and show some of its components marked as chitin (White Calcofluor, D1), hyphae with high metabolic activity (FUN1, D2) and protein (Flamingo, D3). In d, the merged image shows the reconstruction of the entire ECM model of the biofilm of A. fumigatus. All of the fluorochromes were co-localized in the ECM with the hyphae embedded within it. The ECM showed a thickness of <10 μm d. White pointed arrow: hyphae; white arrow: DNA (Signal with DAPI); white dotted circles: colocalization of exopolymers in ECM; c: conidia

Fig. 6

Cell dispersion stage of the Aspergillus fumigatus biofilm isolate. The latest phase of the biofilm formation of A. fumigatus was observed only at 37 °C for both of the isolates. Clinical isolate: a-b developing asynchronous conidia from the hyphae (1,000X-2,000X); c-d dispersion of the planktonic cells (1,000X-2,000X); Soil isolate: e-f presence of conidia released from mature biofilm (1,000X-2,000X). g-h conidial structure assembly during the cell-dispersion phase (1,000X-2,000X). White dotted circles: detailed presence of the dispersed fungal structures that are observed at a higher magnification in the image on the right; c: conidia