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 or cystic fibrosis patients. In biofilms, microorganisms are embedded in an extracellular matrix, which may confers protection against antimicrobial drugs. We recently developed an in vitro mixed biofilm (Melloul et al. 2016) to analyse interactions between Af and Sm. The aim of the present study was to test the in vitro antimicrobial susceptibility of single and mixed biofilms associating Af and Sm.
Methods: Development of in vitro mixed biofilm was performed by simultaneous static co-cultures of Af (ATCC 13073-GFP) and Sm (ATCC 13637) in 96-well plates for 24 h at 37°C. The effect of antimicrobial drugs was assessed on planktonic cells (conidia and/or bacteria) and on single and mixed 24h-old biofilms. Antimicrobial susceptibility was evaluated for 5 antifungal (amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin) and 4 antibacterial drugs (trimethoprim-sulfamethoxazole, levofloxacin, rifampicin and colistin). To evaluate the effects of the drugs (range of 0.0156-16 µg/mL) on planktonic cells, we used the EUCAST methodological recommendations for the determination of the minimal inhibitory concentration (MIC), or minimum effective concentration (MEC) for echinocandins. To evaluate the effects of the drugs (range of 0.0156-64 µg/mL) on 24 h-old biofilms, we used microscopic (CLSM, SEM), spectrophotometric (cell viability with XTT assay for the determination of the minimal biofilm inhibitory concentration (MBIC)) and qPCR analyses.
Results: The analysis of Af cell viability showed a higher activity of amphotericin B, azoles and caspofunginin on fungal cells (MIC ≤1 µg/mL) compared to 24 h-old fungal biofilm (MBIC ≥16 µg/mL). In parallel, all antibacterial drugs had similar effect on bacterial cells and on bacterial 24 h-old biofilm (MIC ≤4µg/mL). qPCR analyses of mixed biofilm revealed no activity of antibacterial drugs on fungal (107conidia/mL) and bacterial (108bacteria/mL) growth, even with high concentration (32 µg/mL). Conversely, antifungal drugs ≥16 µg/mL in mixed biofilm induced an average decrease of 5 log conidia/mL and 4 log bacteria/mL compared to control without treatment.
Conclusion: Quantification by qPCR allowed us to evaluate fungal and bacterial growth more precisely in order to estimate the effect of antimicrobial drugs on each species. The 24 h-old biofilms were more tolerant to antimicrobial drugs than planktonic cells. These preliminary results may indicate that A. fumigatus protects S. maltophilia from antibacterial drugs in mixed biofilms, but further studies are needed to precise the role of each microorganisms in mixed biofilm.
Melloul E et al.PLoS One. 2016.21;11(11):e0166325.
Full conference title:
- AAA 8th (2018)