Activity in an air-liquid interface lung infection model, feasibility of inhaled delivery, and stability of cell-free supernatants from Lacticaseibacillus rhamnosus against Pseudomonas aeruginosa pulmonary infections

Abstract

Objective: Given the increasing prevalence of multidrug-resistant pathogens and the diminishing efficacy of conventional antibiotics, this study explores the potential of probiotics or their metabolic products as alternative antimicrobial agents. Specifically, we investigated the antibacterial properties of cell-free supernatants (CFS) derived from the probiotic strain Lacticaseibacillus rhamnosus GG for the local treatment of Pseudomonas aeruginosa lung infections.

Methods: To simulate the human respiratory environment, we employed various in vitro models. The cytotoxicity and antibacterial activity of CFS were assessed using an Air-Liquid Interface (ALI) lung infection model based on differentiated NCI-H441 human distal lung epithelial cells cultured on Transwell® inserts. To evaluate the feasibility of aerosol-based delivery, we developed and characterized a liquid formulation of CFS. The aerodynamic performance of nebulized CFS was analyzed using a twin-stage impinger (TSI) and a Next Generation Impactor (NGI), the latter 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.

Results: CFS demonstrated significant antibacterial activity in the ALI model, reducing P. aeruginosa colony-forming units by up to 3 log units after 7 h of incubation, without inducing cytotoxic effects. Scanning electron microscopy confirmed these findings. Aerodynamic testing with the TSI and an Aerogen® mesh nebulizer showed that 76% of the nebulized product was deposited in the second stage, indicating effective deep lung delivery. NGI analysis revealed a favorable 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.

Conclusion: These findings support the potential of L. rhamnosus GG-derived CFS as a promising candidate for inhaled therapy against P. aeruginosa lung infections. Further validation in animal models is warranted to confirm its therapeutic efficacy and safety in vivo, potentially contributing to the development of novel localized treatment strategies for respiratory infections.

Keywords: Lacticaseibacillus rhamnosus; Pseudomonas aeruginosa; cystic fibrosis; lung infections; lung model; solution for inhalation.

Figure 1

P. aeruginosa growth in the ALI model. PA1 was used to infect differentiated lung epithelial NCI-H441 cells at a multiplicity of infection of 1:25 bacteria:cells. (A) At each time point, the content of the wells was collected, serially diluted, and plated on TSA for CFU count. (B) Light microscopy images of NCI-H441 at 7, 17, and 25 h post-infection. CTRL indicates un-infected NCI-H441 cells at 25 h of culture.

Figure 2

Antipseudomonal activity of CFS in the ALI lung infection model. PA1 and PA4 were used to infect differentiated lung epithelial NCI-H441 cells at a multiplicity of infection of 1:25 bacteria:cells. After 7 h of incubation, CFU numbers in CFS-treated and untreated samples were quantified. (A) PA1 non mucoid strain; (B) PA4 mucoid strain. * p < 0.05, *** p < 0.001.

Figure 3

Scanning electron microscopy images of NCI-H441 cells at the ALI. (A) Uninfected and untreated monolayers; (B) monolayers infected with P. aeruginosa strain PA4 (mucoid), untreated; (C) monolayers infected with P. aeruginosa strain PA4 (mucoid) and treated with CFS 1:8.

Figure 4

Flow cytometric determination of the cytotoxic effect of CFS on human lung epithelial cells in the ALI model. The figure shows live cell percentages following 7 h exposure to CFS diluted 8 or 4 times in complete RPMI. Control cells were grown in RPMI, 25% MRSB. Mean values ± SEM are shown, n = 3. *** p < 0.001.

Figure 5

APSD analysis of CFS nebulized with Aerogen® Ultra mesh nebulizer under healthy adult breathing profile (A) and adult CF patient profile (B). Lactic acid was used as a tracer for CFS distribution. Amount recovered in Stages (S1–S7) and micro-orifice collector (MOC) are reported (n = 5).

Figure 6

Physicochemical and antibacterial CFS stability under stressed temperature conditions. (A) Visual appearance of CFS exposed to high temperatures for different days during the stress stability study, highlighting the browning effect. (B) Effect of heat-treatment on antibacterial activity against PA1. Heat-treated CFS were diluted 1:4 in TSB and incubated with PA1 for 45 min at 37°C. Surviving bacteria were quantified by CFU counting on TSA. CTRL represents bacteria treated with MRSB. Data are shown as mean ± SEM from one experiment performed in triplicate. *p < 0.001 vs. CTRL (one-way ANOVA followed by Tukey–Kramer multiple comparisons test).

Figure 7

Plot of lnK vs 1/T based on the Arrhenius equation. The slope (−Ea//R) and intercept (lnA) were used to calculate reaction parameters. Experimental validation at 70°C and 95°C confirmed consistency with the predicted data.

Figure 8

Growth kinetics of PA1 strain in the presence of CFS after 1 year of storage at room temperature (RT), 4°C, and −20°C. Bacteria were incubated with CFS diluted 1:16 and 1:32, and growth was monitored by OD₆₂₀ measurement every 30 min for 24 at 37°C. MRSB indicates bacteria exposed to TSB/MRSB under the same conditions. Data are shown as mean ± SEM from one experiment performed in quadruplicate. (Two-way ANOVA followed by Tukey–Kramer multiple comparisons test).