Modulated Response of Aspergillus fumigatus and Stenotrophomonas maltophilia to Antimicrobial Agents in Polymicrobial Biofilm

Abstract

Introduction: The complexity of biofilms constitutes a therapeutic challenge and the antimicrobial susceptibility of fungal-bacterial biofilms remains poorly studied. The filamentous fungus Aspergillus fumigatus (Af) and the Gram-negative bacillus Stenotrophomonas maltophilia (Sm) can form biofilms and can be co-isolated from the airways of cystic fibrosis (CF) patients. We previously developed an in vitro biofilm model which highlighted the antibiosis effect of Sm on Af, which was dependent on the bacterial fitness. The aim of the present study was to investigate the in vitro susceptibility of Af and Sm in mono- or polymicrobial biofilms to five antimicrobial agents alone and in two-drug combinations. Methods: Af and Sm clinical reference strains and two strains from CF sputa were tested through a planktonic and biofilm approaches. Af, Sm, or Af-Sm susceptibilities to amphotericin B (AMB), itraconazole (ITC), voriconazole (VRC), levofloxacin (LVX), and rifampicin (RFN) were evaluated by conventional planktonic techniques, crystal violet, XTT, qPCR, and viable plate count. Results: Af planktonic cells and biofilms in formation were more susceptible to AMB, ITC, and VRC than Af mature biofilms. Af mature biofilms were susceptible to AMB, but not to ITC and VRC. Based on viable plate count, a lower concentration of LVX and RFN was required to reduce Sm cell numbers on biofilms in formation compared with mature biofilms. The antibiosis effect of Sm on Af growth was more pronounced for the association of CF strains that exhibited a higher fitness than the reference strains. In Af-Sm biofilms, the fungal susceptibility to AMB was increased compared with Af biofilms. In contrast, the bacterial susceptibility to LVX decreased in Af-Sm biofilms and was fungal biomass-dependent. The combination of AMB (64 μg/mL) with LVX or RFN (4 μg/mL) was efficient to impair Af and Sm growth in the polymicrobial biofilm. Conclusion: Sm increased the Af susceptibility to AMB, whereas Af protected Sm from LVX. Interactions between Af and Sm within biofilms modulate susceptibility to antimicrobial agents, opening the way to new antimicrobial strategies in CF patients.

Keywords: Aspergillus fumigatus; Stenotrophomonas maltophilia; antibacterial agent; antifungal agent; antimicrobial susceptibility; polymicrobial biofilm.

Figure 1

Antimicrobial susceptibility testing of planktonic cells and biofilm forms. (A,B) Mono-cultures with conidia or bacteria were simultaneously inoculated with antimicrobial agents for 24 h. Then, the optical density (OD) was measured to obtain the MIC (in planktonic cells), and the wells were washed before crystal violet (CV) and XTT analyses to determine the drug concentration that inhibited the biofilm formation (MBIC). (C,D) Mono- and co-cultures were incubated for 24 h to obtain mature mono- and polymicrobial biofilms, and then treated with antimicrobial agents for another 24 h. (C) CV and XTT analyses on monomicrobial biofilm enabled determining the drug concentration that eradicated the mature biofilm (MBEC). The viability of Sm following antibacterial treatment was assessed on planktonic cells (MBC), adherent cells of biofilm in formation (MBIC_CFU), and mature biofilm (MBEC_CFU). (D) Antimicrobial susceptibility of mono- and polymicrobial biofilms was compared using qPCR and viable plate count.

Figure 2

Susceptibilities of biofilms in formation and mature biofilms to drugs. The biomasses and the metabolic activities of biofilms were measured by CV and XTT methods, respectively. Results were expressed in percentages of AF_REF or SM_REF inhibition after antimicrobial treatment compared with untreated controls. AMB, amphotericin B; ITC, itraconazole; VRC, voriconazole; LVX, levofloxacin; RFN, rifampicin.

Figure 3

Quantification of fungal and bacterial concentrations in biofilms and phenotype modifications of A. fumigatus in the presence of S. maltophilia. (A,B) Assessment of Af or Sm growth in mono- and polymicrobial biofilms after 24 h of culture by qPCR. *p < 0.05. (C) AF_REF phenotype in polymicrobial biofilm with SM_REF. (D) AF_CF phenotype in polymicrobial biofilm with SM_CF. (E) AF_REF phenotype in monomicrobial biofilm. (F) AF_CF phenotype in monomicrobial biofilm. Af, A. fumigatus; Sm, S. maltophilia.

Figure 4

Effects of amphotericin B on A. fumigatus in fungal and polymicrobial biofilms. Assessment of AMB activity on AF_REF or AF_CF in fungal (solid line) and polymicrobial (broken line) biofilms by qPCR. Percentages of biomass inhibition were calculated based on untreated biofilms results. *p < 0.05; Af, A. fumigatus; AMB, amphotericin B.

Figure 5

Effects of levofloxacin on SM_REF in bacterial and polymicrobial biofilms. (A) Assessment of LVX activity on SM_REF in bacterial (solid line) and polymicrobial (broken line) biofilms by qPCR. (B) Percentages of SM_REF survival following LVX (1, 4, and 32 μg/mL) treatment in bacterial (light gray) and polymicrobial (dark gray) biofilms using viable plate count. *p < 0.05; LVX, levofloxacin.

Figure 6

Effects of levofloxacin on SM_CF in bacterial and polymicrobial biofilms. Susceptibility of SM_CF to LVX was assessed in bacterial (solid line) and polymicrobial biofilms (broken lines) with an initial AF_CF inoculum of 10⁵ conidia/mL (circle) or 10⁶ conidia/mL (triangle) by qPCR. *p < 0.05; LVX, levofloxacin.

Figure 7

TEM observations of A. fumigatus-S. maltophilia biofilms exposed to levofloxacin. (A) AF_REF + SM_REF untreated biofilm. (B) AF_REF + SM_REF biofilm treated with 8 μg/mL LVX. (C) AF_CF + SM_CF untreated biofilm. (D) AF_CF + SM_CF biofilm treated with 8 μg/mL LVX. H, hypha; B, bacteria. The white arrows show damages to bacterial cells.

Figure 8

Effects of A. fumigatus matrix on S. maltophilia biofilm susceptibility to levofloxacin. Bacterial and polymicrobial biofilms were treated with or without proteinase K (50 μg/mL) for 2 h before LVX treatment at 1 μg/mL. Percentages of SM_REF survival was assessed using viable plate count. *p < 0.05; LVX, levofloxacin; PK, proteinase K.

Figure 9

Effects of antifungal-antibacterial combination on A. fumigatus-S. maltophilia biofilm. (A) Susceptibility of SM_REF in bacterial (light gray) and polymicrobial (dark gray) biofilms to LVX and RFN alone or in two-drug combination with AMB assessed by qPCR. (B) Percentages of SM_REF survival following LVX alone or in two-drug combination with AMB treatment in bacterial and polymicrobial biofilms using viable plate count. *p < 0.05; AMB_64, amphotericin B (64 μg/mL); LVX_4, levofloxacin (4 μg/mL); RFN_4, rifampicin (4 μg/mL).