The field of probiotic applications is rapidly expanding, including their use for the control of respiratory tract infections. Nevertheless, probiotics ability to colonize the lung environment and to compete with pulmonary pathogens is still a poorly investigated research area. In this study, we aimed to evaluate the adhesion ability of a number of commercial probiotic strains to the human lung epithelial cell line A549. Furthermore, we assessed probiotic ability to prevent host cell adhesion of one of the major lung pathogens in cystic fibrosis, Pseudomonas aeruginosa, and to reduce the pathogen-induced inflammatory response of human peripheral blood mononuclear cells (PBMCs) in terms of cytokine release. Lactobacillus acidophilus displayed the highest adhesion ability to A549 cells evaluated as percent of adhered bacteria compared to the inoculum. In agreement with such an observation, L. acidophilus was the most efficient in preventing adhesion to A549 cells of a P. aeruginosa isolate from CF sputum. Three-color fluorescence labeling of A549 cells, P. aeruginosa, and L. acidophilus, and confocal microcopy image analyses revealed a likely exclusion effect played by both live and UV-killed L. acidophilus towards P. aeruginosa. Such results were confirmed by CFU count. When co-cultured with PBMCs, both live and UV-killed L. acidophilus reduced the amount of IL-1β and IL-6 in culture supernatants in a statistically significant manner. Overall, the results obtained point to L. acidophilus as an interesting candidate for further studies for a potential aerogenous administration to control P. aeruginosa infections.

Therapy of lung infections sustained by Pseudomonas aeruginosa in cystic fibrosis (CF) patients is challenging due to the presence of a sticky mucus in the airways and the ability of the bacterium to form biofilm, which exhibits increased antibiotic tolerance. A lung-directed bacteriotherapy through the airway administration of probiotics could represent an alternative approach to probiotic diet supplementation to improve the benefits and clinical outcomes of this kind of intervention in CF patients. This study aims to evaluate the ability of probiotic strains to grow in artificial sputum medium (ASM), mimicking the CF lung microenvironment, and to affect the planktonic and biofilm growth of CF clinical strains of P. aeruginosa in the same conditions. The results demonstrate that Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum (LP) can grow in ASM. LP inhibited the planktonic growth of P. aeruginosa, while both lactobacilli reduced the pre-formed biofilm of P. aeruginosa. Interestingly, LP was demonstrated to reduce the amount of polysaccharides in the extracellular matrix of P. aeruginosa biofilms and to potentiate the antibiofilm effects of tobramycin. Overall, the results indicated that LP is a promising candidate as an adjuvant in the antimicrobial therapy of P. aeruginosa infections in CF patients.

It is widely agreed that microbial biofilms play a major role in promoting infection and delaying healing of chronic wounds. In the era of microbial resistance, probiotic strains or their metabolic products are emerging as an innovative approach for the treatment of hard-to-heal (chronic) wounds due to their antimicrobial, healing, and host immune-modulatory effects. In this study, we aimed to investigate the potential of cell-free supernatants (CFS) from Lacticaseibacillus rhamnosus GG against mono- and dual-species biofilms of wound pathogens in a 3D in vitro infection model. Mature biofilms of Pseudomonas aeruginosa and Staphylococcus aureus were obtained on collagen scaffolds in the presence of a simulant wound fluid (SWF) and treated with CFS at different doses and time intervals. At 1:4 dilution in SWF, CFS caused a marked reduction in the colony forming-unit (CFU) numbers of bacteria embedded in mono-species biofilms as well as bacteria released by the biofilms in the supernatant. CFU count and electron microscopy imaging also demonstrated a marked antibiofilm effect against dual-species biofilms starting from 8 h of incubation. Furthermore, CFS exhibited acceptable levels of cytotoxicity at 24 h of incubation against HaCaT cells and, differently from ciprofloxacin, failed to induce resistance after 15 passages at sub-inhibitory concentrations. Overall, the results obtained point to L. rhamnosus GG postbiotics as a promising strategy for the treatment of wound biofilms.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Background/Objectives: Postbiotics derived from lactic acid bacteria are emerging as promising antimicrobial agents due to their antibacterial, antibiofilm, and immunomodulatory properties. Among their metabolites, lactic acid (LA) is thought to play a major role in antimicrobial activity. This study investigated the proteomic response of Pseudomonas aeruginosa and Staphylococcus aureus to Lacticaseibacillus rhamnosus cell-free supernatant (CFS) and compared it with that elicited by LA alone. Methods: Overnight bacterial cultures were exposed to sub-MIC LA or CFS (1:10 for P. aeruginosa and 1:8 for S. aureus; ~12.5–15.6 mM LA) for 6 h at 37 °C. Intracellular proteins were harvested and subsequently quantified and purified to be analysed by HPLC–MS/MS, for quantitative label-free proteomics. Results: Proteomic analysis revealed clear separation of treated samples from controls, with largely overlapping responses to CFS and LA. Hallmark acid-stress adaptations were observed, including urease-mediated pH buffering, confirming that part of the response was driven by mild organic acid. In P. aeruginosa, treatments suppressed virulence pathways (phenazines, T3SS), while shifting metabolism toward lactate utilisation and reinforcing the outer membrane (lipid A, polyamine). In S. aureus, decreased abundance of the SaeRS-regulated immune-evasion factor Sbi, together with changes in envelope, ROS and translation-related proteins, suggested a bacteriostatic-like state. S. aureus differences between CFS and LA were more pronounced; CFS uniquely increased cell-wall defences, oxidative stress (SodA, SodM) and chaperone expression (GroS, GrpE), suggesting stress beyond acidification alone. Conclusions: These findings shed light on the molecular mechanisms underlying bacterial adaptation to CFS and highlight their potential as a novel antimicrobial approach.