1) Fingolimod, a drug approved for the treatment of multiple sclerosis, was found to exhibit notable anti-biofilm activity against three clinically relevant pathogens, Staphylococcus aureus, Staphylococcus epidermidis and Acinetobacter baumannii. Interference of the conserved communication system known to trigger biofilm formation in bacteria, was demonstrated as one of the underlying mechanisms-of-action. Fingolimod could be used to target both the Gram-positive and -negative bacteria, and therefore, was integrated also into the medical devices, such as endotracheal catheters, for further studies.
2) A wound dressing, containing a fosfidomycin prodrug C366 or niclosamide, were developed by melt electrowriting. Drug release from the dressing showed 70-80% release after 24 hours. The dressings were tested in an artificial dermis model, where the dressing was able to resist A. baumannii infection and cause a 1-2 log or 2-3 log reduction in bacterial load inside the artificial dermis itself for CC366 or niclosamide, respectively.
3) A new bioink formulation for drop-on-demand (DoD) 3D printing technology was developed and optimized. The formulation is based on poly(lactic-co-glycolide) acid or polylactic acid, and it incorporates various antimicrobial agents. Some formulations incorporating rifampicin, niclosamide or C11 diterpenoid showed high efficacy in preventing the biofilm formation and reducing planktonic bacteria.
4) New in vitro systems, more closely resembling those of bacterial biofilms in vivo, were developed. These included e.g. the co-culture of SaOS-2, osteogenic cells and S. aureus on a titanium surface, indicating the applicability of newly identified biofilm inhibitors as part of anti-infective implantable devices, giving information on their anti-biofilm effects in clinically relevant surfaces and effects in tissue integration.
5) A ventilator associated-pneumonia (VAP) mouse model was established to demonstrate the in vivo antimicrobial activity of 3D-printed catheters and formulations developed and tested in vitro against S. aureus. The manufactured ciprofloxacin-loaded endotracheal tubes were produced by hot melt extrusion of polyurethane. Tubes loaded with 5% ciprofloxacin showed a 1.9 log reduction of S. aureus ATCC25923 strain in comparison with the non-loaded tubes. More importantly, this reduction prevented infection in vivo.
6) An anti-biofilm peptide SAAP-148 was incorporated in poly(lactic-co-glycolic acid) and poly(lactic-co-glycolide) by the DoD additive manufacturing technique. The release kinetics of the antimicrobial peptide can be tailored based on the number of layers of coating and the type and ratio of hydrophilic polymer. The antimicrobial activity of these novel constructs against S. aureus was confirmed.
7) Thermoplastic polyurethane catheters loaded with 2 and 5% of niclosamide were highly effective at inhibiting bacterial colonization on implants for S. aureus ATCC25923 and 33591 using a murine foreign body infection model. A significant prolonged reduction in bacterial burden of implants was observed from day 1 post infection, and soft tissue infection was reduced in mice carrying niclosamide catheters. Niclosamide-loaded catheters represent an alternative to control catheter-associated infections.
8) The Print-Aid project resulted in the minimum information guidelines for spectrophotometric and fluorometric methods to assess the biofilm formation in microscale format (96-well microplates). The combined efforts enabled to establish standardized methods for three commonly utilized biofilm protocols, which resulted in a consortium-wide ‘ring-trial’. Expansion of the experimental trial to laboratories outside the PRINT-AID consortium is envisioned in the future.
