Prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus combined with therapeutic Lactiplantibacillus plantarum cell-free supernatant protects against Pseudomonas aeruginosa lung infection in mice

Background Pseudomonas aeruginosa is a leading cause of severe respiratory infections in immunocompromised individuals, and its increasing antibiotic resistance poses a major global health challenge. Antibiotic-resistant strains exhibit enhanced biofilm-forming capacity, contributing to persistent infection, immune evasion, and poor clinical outcomes. Innovative non-antibiotic strategies that simultaneously enhance host immunity and directly target bacterial virulence are urgently needed. Methods In this study, we evaluated a dual non-viable microbial therapeutic strategy combining prophylactic immune priming with heat-killed Lacticaseibacillus rhamnosus CRL1505 (HK1505) and therapeutic aerosol administration of Lactiplantibacillus plantarum ATCC ® 10241™ cell-free culture supernatant (LpCFS) in murine models of lung infection caused by antibiotic-sensitive (PaS) and multidrug-resistant (PaR) P. aeruginosa . Pulmonary bacterial burden, systemic dissemination, and lung injury markers were assessed. In addition, cytokine/chemokine profiles were evaluated in the respiratory tract in vivo and in primary cultures of broncho-alveolar lavage (BAL) macrophage-enriched adherent cells ex vivo . HK1505 and LpCFS treatments were evaluated separately and in combination. Results LpCFS did not directly alter cytokine or chemokine production by BAL macrophage-enriched adherent cells, indicating the absence of intrinsic immunostimulatory activity. However, therapeutic aerosol administration of LpCFS significantly reduced pulmonary and systemic PaS and PaR loads, attenuated lung damage, and modulated the inflammatory response by decreasing pro-inflammatory cytokines while increasing IL-10 during infections. Prophylactic administration of HK1505 effectively primed BAL macrophage-enriched adherent cells, enhancing their production of IL-1β, IL-6, IFN-γ, and IL-27 while reducing TNF-α and chemokine expression (CCL2, CXCL2, and CXCL10), thereby promoting efficient bacterial clearance with limited immunopathology. In this set of experiments HK1505 was compared with the live L. rhamnosus CRL1505 and notably, HK1505 retained the immunostimulatory efficacy of the viable bacteria. In addition, the combination of the prophylactic HK1505 administration and the therapeutic LpCFS treatment provided superior protection against the respiratory infections than individual treatments. The combined approach completely prevented lung infection and bacteremia in PaS-infected mice and significantly improved the outcomes in PaR infection. Conclusion Our findings demonstrate that a non-viable probiotic-based strategy integrating prophylactic immune priming with HK1505 and therapeutic antibiofilm intervention with LpCFS effectively protects against antibiotic-resistant P. aeruginosa lung infection. This approach highlights the translational potential of postbiotic immunomodulation as a safe and innovative alternative for managing multidrug-resistant respiratory infections.