Mixed biofilm of Aspergillus fumigatus and Stenotrophomonas maltophilia: a strategy of protection against antimicrobial agents?

Lolita Roisin1, Elise Melloul1, Paul-Louis Woerther1,2, Jean-Winoc Decousser1,2, Jacques Guillot1,3, Eric Dannaoui1,4, Françoise Botterel1,2*

1 EA Dynamyc 7380 UPEC, EnvA, Faculté de Médecine de Créteil, Créteil, France 
2 Département de Microbiologie, DHU VIC, Hôpital Henri Mondor, AP-HP, Créteil, France
3 Ecole nationale vétérinaire d’Alfort, Maisons-Alfort, France
4 Département de Microbiologie, Hôpital Européen Georges Pompidou AP-HP, Paris, France

Purpose: Aspergillus fumigatus (Af), a fungal opportunistic pathogen, and Stenotrophomonas maltophilia (Sm), a gram-negative bacillus, can form biofilms, especially in the respiratory tract of immunocompromised patients or in chronic respiratory diseases. In biofilms, microorganisms are embedded in an extracellular matrix, which may confer protection against antimicrobial treatment. Few data are available on biofilm susceptibility to antimicrobial treatment. We recently developed an in vitro mixed biofilm associating Af and Sm (Melloul et al., 2016) to analyze interactions between different antimicrobial agents against this mixed biofilm. The aim of the present study was to test two antifungal agents and two antibiotics on an Af-Sm mixed biofilms.

Methods: Development of in vitro 24h-old mixed biofilm was performed by simultaneous static co-cultures of Af (ATCC 13073-GFP) and Sm (ATCC 13637) in 96-well plates at 37°C. Two antifungal (amphotericin B and itraconazole) and two antibacterial (levofloxacin and rifampicin) agents were chosen to explore their action on single and mixed 24h-old biofilms. Microscopic (CLSM, SEM), spectrophotometric (metabolic activity with XTT assay) analyses, cell viability (CFU/mL) and qPCR analyses were used to test the activity of antifungal (from 0.25 to 256 µg/mL) and antibacterial (from 0.03 to 32 µg/mL) agents on 24h-old biofilms.

Results: For amphotericin B, the Af biofilm metabolism was altered at 256 µg/mL and we showed an inhibition of fungal growth of 95% at 32 µg/mL in single and mixed biofilms. Furthermore, the hyphae were lysed in single and mixed biofilms after treatment with 64µg/mL of amphotericin B. For itraconazole, there was no effect on Af in single and mixed biofilms until 256 µg/mL. In Sm single biofilm, levofloxacin and rifampicin had no effect on Sm if the concentration was <1 µg/mL. In this case, qPCR analyses showed an inhibition of bacterial growth of 95% at 8 µg/mL. In contrast, in mixed biofilm, levofloxacin and rifampicin hadalow activity on Sm growth, even with high concentration (32 µg/mL).

Conclusion: qPCR analyses allowed us to evaluate biofilm susceptibility to antimicrobial treatment more precisely than metabolic analyses. These results may indicate that A. fumigatus biofilm protects S. maltophilia from antibacterial agents in mixed biofilms, but further studies are needed to precise the role of each microorganisms in mixed biofilm with antimicrobial agents.

Melloul E et al. PLoS One. 2016.21;11(11):e0166325.

Mixed biofilm of Aspergillus fumigatus and Stenotrophomonas maltophilia: a strategy of protection against antimicrobial treatment?