Interactions of Aspergillus fumigatus and Stenotrophomonas maltophilia in an in vitro Mixed Biofilm Model: Does the Strain Matter?

Abstract

Introduction: Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are pathogenic microorganisms, which coexist in the respiratory tract of cystic fibrosis (CF) patients. We recently developed an in vitro model of mixed biofilm associating Af ATCC 13073-GFP (Af13073) and Sm ATCC 13637 (Sm13637) and described an antibiosis effect. The present study aim was to assess the antibiosis of Sm on Af using different strains and to analyze the potential synergistic virulence of these strains in an in vivo Galleria mellonella model. Methods: The effect of Sm13637 was evaluated on eight Af strains and the effect of nine Sm strains was evaluated on Af13073. The strains originated from clinical cases (human and animal) and from environment. Fungal and bacterial inocula were simultaneously inoculated to initiate mixed biofilm formation. Fungal growth inhibition was analyzed by qPCR and CLSM and the fungal cell wall modifications by TEM analysis. The virulence of different Sm strains was assessed in association with Af in G. mellonella larvae. Results: All strains of Af and Sm were able to produce single and mixed biofilms. The antibiosis effect of Sm13637 was similar whatever the Af strain tested. On the other hand, the antibiosis effect of Sm strains was bacterial-fitness and strain dependent. One strain (1/9) originated from animal clinical case was never able to induce an antibiosis, even with high bacterial concentration. In the G. mellonella model, co-inoculation with Sm13637 and Af13073 showed synergism since the mortality was 50%, i.e., more than the summed virulence of both. Conclusion: Human clinical strains of Sm yielded in higher antibiosis effect on Af and in a thinner mixed biofilm, probably due to an adaptive effect of these strains. Further research covering Af increased wall thickness in the presence of Sm strains, and its correlation with modified antifungal susceptibility is encouraged in patients with chronic respiratory infections by these 2 microorganisms.

Keywords: Aspergillus fumigatus; Galleria mellonella; Stenotrophomonas maltophilia; antibiosis; bacterial-fungal interactions; mixed biofilm.

FIGURE 1

A. fumigatus phenotype and thickness of single and mixed biofilms by fungal origin. (A) Percent of mixed biofilms thickness (compared with single fungal biofilms) by A. fumigatus origin: human (black), animal (gray) and environment (white). (B) Example of single fungal biofilm of Af_H1. (C) Example of mixed biofilm of Af_H1 and Sm13637. (C’) Zoom of shorter and atrophied ramifications and highly branched hyphae (white circles). The letters indicate the phenotype observed: b, photo B; c, photo C. ^∗p < 0.05 compared with single fungal biofilms; Af, A. fumigatus.

FIGURE 2

Modification and measurement of cell wall thickness of A. fumigatus in single and mixed biofilms, by fungal strains. (A,A’) Single fungal biofilm. (B,B’) Mixed biofilm with Sm13637 bacteria (white arrows). (A’,B’) Zoom of fungal cell wall. (C) TEM, measurement of fungal cell wall thickness by A. fumigatus origin: human (black), animal (gray) and environment (white). ECM, extracellular matrix; CW, cell wall. ^∗p < 0.05 compared with single fungal biofilms; Af, A. fumigatus.

FIGURE 3

Reduction of mixed biofilms thicknesses and modification of Af13073 phenotype according to S. maltophilia strains. (A) Thicknesses of mixed biofilms compared with thickness of Af13073 vs. S. maltophilia strains: human (black), animal (gray) and environment (white). (B) Wild type phenotype of Af13073 in the presence of S. maltophilia (C) Modified phenotype of Af13073 in the presence of S. maltophilia. The letters indicate the phenotype observed: b, photo B; c, photo C. ^∗p < 0.05 compared with single fungal biofilm; Sm, S. maltophilia.

FIGURE 4

Quantification of fungal and bacterial DNA in mixed biofilms by S. maltophilia strains. (A) Quantification by qPCR of Af13073 (Log CE/mL) in single and mixed biofilms with Sm strains from human (black), animal (gray), and environment (white) (at 10⁶ cells/mL). (B) Quantification by qPCR of Sm (Log BE/mL) in the mixed biofilms (Af13073 + Sm at 10⁶ cells/mL); ^∗p < 0.05; Af, A. fumigatus; Sm, S. maltophilia.

FIGURE 5

Growth, adhesion, and biofilm formation of S. maltophilia strains by temperature degree. Culture of S. maltophilia strains originated from human (black), animal (gray), and environment (white), in RPMI + FBS media at 25 and 37°C. (A) Planktonic bacterial growth measured by absorbance (OD 600 nm) after 24 h of culture. (B) Bacterial adhesion on polystyrene support after 4 h measured by crystal violet (OD 550 nm). (C) Biofilm formation after 24 h of culture measured by crystal violet (OD 550 nm); ^∗p < 0.05.

FIGURE 6

Dose-dependent fungal biomass inhibition in mixed biofilms. (A) Af13073 phenotype in mixed biofilm with S. maltophilia strains from animal (gray) or environment (white). (B) Quantification by qPCR of Af13073 in fungal and mixed biofilms with 10⁸ bacteria/mL; ^∗p < 0.001 compared with Af13073 biofilm; Af, A. fumigatus, Sm, S. maltophilia.

FIGURE 7

Survival curves after single and co-inoculation in G. mellonella. Inoculation of ten larvae per group with S. maltophilia strain (blue line), Af13073 (black line), or both of them (red line) depending on S. maltophilia strains. (A) Co-inoculation of Sm strains of genogroup 6 (human and animal clinical strains). (B) Co-inoculation of Sm strains of genogroup 2 (animal clinical strains). (C) Co-inoculation of Sm strains of genogroup D (environmental strains); Af, A. fumigatus; Sm, S. maltophilia.