»BioFLOW«

Biofilm-associated infections pose a major threat not only in the healthcare but also in agriculture. In clinical settings, biofilms are responsible for up to 80% of human bacterial infections, including chronic lung infections in cystic fibrosis (CF) patients, chronic wounds, and infections on medical implants and devices. Within biofilms, cells are embedded in a dense extracellular matrix that restrict antibiotic penetration and protect them from host immunity. Additionally, most antibiotics fail to eliminate persister cells that form a subpopulation in the inner biofilm layer because many of their molecular targets are inactive during dormancy. These cause the biofilm-embedded cells to be 10- to 1,000-fold more resistant toward antimicrobials compared to their planktonic state.

In the agricultural sector, biofilms also play a crucial role in plant diseases. For example, Xylella fastidiosa, a phytopathogenic bacterium that colonizes plant xylems, develops biofilms inside the plant vessels. This leads to blockage of the water and nutrient transportation and ultimately to the death of the host plants. It has a wide host range, infecting over 700 plant species and causing severe diseases, including olive quick decline syndrome, Pierce's disease in grapes, and almond leaf scorch. Despite the huge economic losses caused by this phytopathogen, there is still no known cure, highlighting the urgent need to find new treatment options for this biofilm-related infection.

In the xylem, the bacteria are exposed to continuous sap flow, shear forces, and nutrient limitations, which can affect biofilm development and susceptibility to antimicrobials. To evaluate the anti-biofilm potential of candidate compounds, reliable testing assays are required. Static microplate-based assays are widely used due to their simplicity and scalability. In the natural environment biofilms often develop under continuous fluid flow, hydrodynamic forces, and oxygen limitation. As a result, some of the promising candidate showed excellent performances in vitro but lose their activities in later stages of development. To close this gap, new physiologically relevant screening approach is required to better mimic in-vivo biofilm conditions.

This project aims to implement a physiologically relevant microfluidic-based biofilm screening assay that imitate the in-vivo flow condition, with a specific focus on Xylella fastidiosa. The established assay allows real-time monitoring of biofilm development, treatment (including structural disruption and viability), as well as potential regrowth under controlled hydrodynamic conditions. 

Since Xylella fastidiosa is a slow-growing bacteria with a typical in-vitro biofilm formation period of 8-14 days, we used fast-growing clinically relevant pathogen Pseudomonas aeruginosa as model organism in the initial developmental stage to enable faster optimization and finalization of the assay workflow.

After the robustness of the assay was validated, we then adjust the assay parameters for Xylella fastidiosa. Finally, the established platforms were used to evaluate commercially available antibiotics as well as newly discovered natural products. In this context, the extensive microbial strain collection from Fraunhofer IME Gießen provide a valuable resource of bioactive compounds that may serve as new anti-biofilm agents. The screening workflow starts with primary screening using static microplate-based biofilm eradication assays to enable testing of multiple compounds and concentrations simultaneously. Promising candidates from the initial screening is then further evaluated using the microfluidic model to assess their antibiofilm activity under hydrodynamic conditions. 

The research is implemented into three stages:

• Development and validation of the microfluidic biofilm screening assay using fast-growing strain
  • In the initial developmental stage, we used fast-growing strain Pseudomonas aeruginosa as model organism for faster workflow optimization. This work resulted in a publication demonstrating the robustness of the platform, including the implementation of sequential multi-dose treatments to improve drug exposure over time.

• Microfluidic assay transfer to Xylella fastidiosa
  • In this phase, the microfluidic assay is adapted to the phytopathogen Xylella fastidiosa. This transfer required optimization of flow rate, long-term biofilm cultivation, and treatment period.

• Screening of antimicrobial compounds using the microfluidic assay against Xylella fastidiosa biofilms
  • The established microfluidic assay is subsequently applied for screening of antimicrobial compounds to see their potential as antibiofilm candidate. After the primary screening using static microplate-based assays, promising hits are then further evaluated using the microfluidic model to assess their antibiofilm activity under hydrodynamic conditions. 

The established workflow for P. aeruginosa was used to evaluate the anti-biofilm activity of colistin and darobactin B, a promising pre-clinical antibiotic candidate with unique mode of action, showing the robustness of the platform to detect both immediate and long-term treatment effects.

By transferring this technology to Xylella fastidiosa, the developed in vivo mimicking assay will help to enhance the transfer rate of positive primary assay results and serve as a basis for in vivo or in planta experiments. Integrating this assay into the running high throughput natural product discovery pipelines can increase the probability of finding a treatment option for xylem-associated biofilm infections, which is critically needed.

One of the key advantages of the microfluidic approach is that this assay requires a lower amount of test compound compared to regular in vivo or in planta assays, making it suitable for testing early-stage natural products with low compound consumption. The microfluidic platform therefore offers a physiologically relevant and resource-saving strategy for biofilm research and antimicrobial discovery. Additionally, the multiple dosage workflow provides a foundation for designing dosing regimens of bioactive compounds in both medical and agricultural application.