Probiotics and Postbiotics: Translating Beneficial Microbes into Therapeutic Innovations for Difficult-to-Treat Infections

Antimicrobial resistance to the current arsenal of antibiotics has emerged as one of the leading public health threats of the 21st century, stimulating the search for new antimicrobial strategies as alternatives or adjuvants to antibiotic use. Among these strategies, the local application of harmless microbes (probiotics) or their inanimate derivatives (postbiotics) is emerging as a viable approach. The rationale behind this strategy is to exploit the natural competition among microbes, using beneficial, non-pathogenic bacteria to outcompete pathogenic ones and contrast their growth and/or expression of their virulence traits. So far, the main fields of anti-infective application of probiotics remain the gastrointestinal tract, but evidence is accumulating pointing to the possible use of probiotics or their derivatives in extra-intestinal districts such as the respiratory system or the skin. In diseases like cystic fibrosis and non-healing wounds, bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus are hard to eliminate because they form protective biofilms and dormant persister cells, adapt their metabolism, and resist many antibiotics, leading to prolonged inflammation, tissue damage, and recurrent exacerbations. This thesis aims to explore the antimicrobial potential of live probiotics and postbiotics against difficult-to-treat pathogens involved in cystic fibrosis and chronic infections, with particular emphasis on the cell-free supernatants (CFS) produced by Lactiplantibacillus plantarum and Lacticaseibacillus rhamnosus. Live preparations of lactobacilli strains demonstrated strong antibacterial and antibiofilm effects against clinical isolates of P. aeruginosa, reducing biofilm matrix formation and interfering with the pathogen’s adhesion to lung epithelial cells. Moreover, their CFS exhibited a broad spectrum of activity, including antimicrobial, antibiofilm, and anti-persister effects. In addition, they displayed immunomodulatory and antivirulence properties without promoting the development of resistance, in contrast to conventional antibiotics. Proteomic analysis showed distinct adaptive responses in P. aeruginosa and S. aureus exposed to CFS, involving metabolic reprogramming, stress adaptation, and modulation of virulence-related pathways. The biological relevance of these findings was further validated in physiologically representative infection models, including an air–liquid interface (ALI) lung epithelial model, and a dual-species biofilm wound infection model. Finally, testing CFS in the Galleria mellonella infection model confirmed the protective and anti-infective potential of CFS. To evaluate the feasibility of aerosol delivery of CFS to the lung, a liquid formulation of CFS was developed and characterised. The aerodynamic performance of nebulised CFS was analysed using a Next Generation Impactor (NGI) equipped with a breathing simulator to mimic respiratory profiles of both healthy individuals and cystic fibrosis patients. Additionally, the physicochemical and biological stability of CFS was assessed under various storage conditions. NGI analysis revealed a favourable aerodynamic particle size distribution (APSD), with a fine particle fraction (FPF) exceeding 60% and a mass median aerodynamic diameter (MMAD) suitable for deep airway deposition. Physicochemical stability studies under stressed temperature conditions predicted prolonged physical stability for CFS at 25°C and demonstrated that they retained anti-pseudomonal activity after 1 year of storage at room temperature, 4°C, and -20°C. Overall, the results obtained support the use of beneficial microbes and/or their inanimate derivatives as new opportunities to tackle chronic infections from resistant bacteria in districts like the lung or the skin. The experimental workflow established through this research could provide a versatile and reproducible platform for the preclinical evaluation of newly identified or engineered probiotic strains, thereby contributing to translating beneficial microbes into therapeutic innovations for difficult-to-treat infections.