Respiratory diseases are a leading cause of death globally, prompting the need for novel therapeutics to combat antibiotic-resistant bacteria and the limitations of existing antibiotics. A significant challenge is the prevalence of biofilms in respiratory infections, particularly in chronic diseases like cystic fibrosis and acute infections like VAP. Biofilms, complex bacterial communities embedded in a protective matrix, exhibit high tolerance to antibiotics, leading to persistent and recurrent infections. The use of endotracheal tubes (ETTs) in mechanically ventilated patients significantly increases VAP risk, with higher incidence and mortality rates observed in patients with conditions like COVID-19 and *P. aeruginosa* biofilm involvement. Current biofilm-reducing interventions often prove ineffective, underscoring the need for alternative approaches. Engineered live bacteria, classified as live biotherapeutic products (LBPs), offer a potential solution with fewer adverse effects compared to traditional antibiotics. While LBPs have been developed for gut diseases, their application to lung infections remains largely unexplored. The choice of bacterial chassis for LBP development is crucial; ideally, the bacterium should be naturally present in the target organ. *M. pneumoniae*, a human lung bacterium with a small genome, mild pathogenicity, and well-characterized genetics, emerges as a suitable chassis. Its lack of a cell wall reduces inflammatory responses and allows combination with cell-wall-targeting antibiotics. This study leverages the advantages of *M. pneumoniae* to engineer a strain capable of treating *P. aeruginosa* infections and biofilms.

The literature extensively highlights the challenges posed by antibiotic resistance and biofilm-associated infections in the respiratory tract. Studies demonstrate the high prevalence of biofilms in various pulmonary diseases, including cystic fibrosis, chronic obstructive pulmonary disease, and ventilator-associated pneumonia (VAP). The biofilm matrix protects bacteria from antibiotics and the host immune system, leading to persistent infections and treatment failure. Existing strategies to combat biofilms, such as aerosolized antibiotics, have shown limited success. The development of live biotherapeutic products (LBPs), genetically engineered bacteria designed to treat diseases, has gained traction, primarily focusing on gut infections. However, the application of this technology to lung infections is still in its early stages. The selection of a suitable bacterial chassis is a key aspect of LBP design, and the properties of *M. pneumoniae*, such as its small genome size and reduced pathogenicity, make it a promising candidate for treating lung infections.

This research employed a multifaceted approach involving *in vivo* and *in vitro* experiments. Initially, the researchers characterized the safety and clearance kinetics of different attenuated *M. pneumoniae* strains in mouse lungs using intratracheal inoculation. They assessed bacterial load in lung tissue and bronchoalveolar lavage fluid (BALF) at various time points (2, 4, and 14 days post-infection). Histopathological analysis and cytokine profiling were used to evaluate lung lesions and inflammatory responses. Genetic engineering techniques were used to generate attenuated *M. pneumoniae* strains by deleting specific genes associated with pathogenicity (*mpn372*, *mpn133*, *mpn453*, and *mpn051*). The optimal chassis was selected based on attenuation and lung colonization capability. Next, the selected attenuated strain (CV2) was further engineered to express genes encoding biofilm-dispersing enzymes (PelAh, PslGh, and A1-II alginate lyase) and bacteriocins (pyocin L1 or pyocin S5) to target *P. aeruginosa*. The efficacy of the engineered strain (CV2_HA_P1) was evaluated *in vitro* using biofilm dispersal and antimicrobial activity assays. Biofilm dispersal was assessed using Crystal Violet and Alcian Blue staining, while antimicrobial activity was determined through growth curves of various *P. aeruginosa* strains in the presence of the engineered strain's supernatant. *Ex vivo* experiments evaluated the effect of CV2_HA_P1 on biofilms formed on endotracheal tubes (ETTs) from VAP patients. Finally, *in vivo* studies used a murine model of acute *P. aeruginosa* lung infection to assess the efficacy of CV2_HA_P1 as a therapeutic and prophylactic agent. Mouse survival rates, bacterial loads in lung tissue, histopathological changes, and inflammatory cytokine levels were monitored. Mass spectrometry (MS) was used for protein quantification in the various *M. pneumoniae* strains. Real-time quantitative PCR (RT-qPCR) analyzed the expression levels of inflammatory markers in lung tissue.

The study demonstrated that the attenuated *M. pneumoniae* strain CV2 exhibited reduced pathogenicity in mice compared to the wild-type strain, showing mild and self-resolving lung lesions. The engineered strain CV2_HA_P1, expressing both biofilm-dispersing enzymes and pyocin L1, effectively reduced *P. aeruginosa* biofilms *in vitro*. Importantly, CV2_HA_P1 significantly reduced *P. aeruginosa* PAO1 load and improved survival in a mouse model of acute pneumonia. In *ex vivo* studies using ETTs from VAP patients, CV2_HA_P1 significantly decreased *P. aeruginosa* burden, demonstrating its efficacy against multidrug-resistant clinical isolates. Furthermore, the combination of CV2_HA_P1 with cell wall-targeting antibiotics showed synergistic effects in reducing bacterial growth. The CV2_HA_P1 strain also demonstrated prophylactic efficacy in preventing *P. aeruginosa* infection in a mouse model.

This study successfully engineered an attenuated *M. pneumoniae* strain (CV2_HA_P1) capable of effectively treating *P. aeruginosa* infections and biofilms in a mouse model and *ex vivo* using ETTs from VAP patients. The results highlight the potential of engineered live bacteria as a novel therapeutic modality for respiratory infections. The ability of CV2_HA_P1 to dissolve biofilms and synergize with antibiotics addresses the limitations of current treatment strategies. The observed attenuation of the *M. pneumoniae* strain minimizes potential adverse effects, making it a promising candidate for clinical translation. The findings demonstrate a new paradigm for tackling biofilm-associated infections, a major challenge in respiratory medicine.

This research demonstrates the successful engineering of an attenuated *M. pneumoniae* strain (CV2_HA_P1) that effectively combats *P. aeruginosa* infections and biofilms in both *in vivo* and *ex vivo* models. This work provides strong evidence supporting the potential of engineered bacteria as a novel therapeutic strategy for respiratory tract infections, particularly those associated with biofilms and antibiotic resistance. Future research could focus on optimizing the engineered strain further, exploring additional antimicrobial agents, and conducting clinical trials to evaluate the safety and efficacy of this LBP in human patients. Expanding the application of the CV2 chassis to other lung diseases is also a promising avenue for future investigation.

The study primarily focused on *P. aeruginosa* PAO1, and further research is needed to evaluate the efficacy of CV2_HA_P1 against other respiratory pathogens and diverse *P. aeruginosa* strains. The mouse model, while useful, may not fully recapitulate the complexities of human lung infections. Long-term effects and potential risks associated with prolonged administration of CV2_HA_P1 also warrant further investigation. The *ex vivo* study utilized ETTs from a limited number of patients, which might not be fully representative of the broader clinical population.