Background:Air-liquid interface (ALI) cell culture systems have improved the study of host-pathogen interactions in respiratory infections. However, most ALI models lack immune components, limiting their ability to capture epithelial-immune crosstalk. To address this, we developed a dual-cell ALI model incorporating human peripheral blood monocyte-derived macrophages beneath differentiated airway epithelial cells. Methodology:Macrophages were seeded on the basolateral side of transwell inserts using fibronectin coating. Model characterization included transepithelial electrical resistance (TEER) to assess epithelial barrier integrity, IL-8 secretion as a marker of epithelial inflammatory signaling, and confocal microscopy to evaluate cellular architecture before and after infection. Mono- and dual-cell cultures were infected with the laboratory strain Pseudomonas aeruginosa PAO1. Results:Macrophages adhered stably to the basolateral surface without compromising epithelial barrier integrity. Following infection, IL-8 secretion was elevated in epithelial monocultures compared to dual-cell cultures, suggesting early immune modulation in the presence of macrophages. While overall bacterial burden was comparable, confocal imaging revealed clustered bacterial growth in monocultures and a more dispersed spatial distribution in dual-cell cultures. Conclusions:This dual-cell ALI model enables investigation of early epithelial-immune interactions, inflammatory modulation, and bacterial colonization dynamics during airway infection. The system provides a versatile and human-relevant platform for studying respiratory host-pathogen interactions.

Background: Air-liquid interface (ALI) cell culture systems have improved the study of host-microbe interactions in respiratory infections. However, most ALI models lack immune components, limiting their ability to capture epithelial-immune crosstalk. To address this, we developed a dual-cell ALI model incorporating human peripheral blood monocyte-derived macrophages beneath differentiated airway epithelial cells. Methodology: Macrophages were seeded on the basolateral side of transwell inserts using fibronectin coating. Model characterization included transepithelial electrical resistance (TEER) to assess epithelial barrier integrity, IL-8 secretion as a marker of epithelial inflammatory signaling, and confocal microscopy to evaluate cellular architecture before and after infection. Mono- and dual-cell cultures were infected with the laboratory strain Pseudomonas aeruginosa PAO1. Results: Macrophages adhered stably to the basolateral surface without compromising epithelial barrier integrity. Following infection, IL-8 secretion was elevated in epithelial monocultures compared to dual-cell cultures, suggesting early immune modulation in the presence of macrophages. While overall bacterial burden was comparable, confocal imaging revealed clustered bacterial growth in monocultures and a more dispersed spatial distribution in dual-cell cultures. Conclusions: This dual-cell ALI model enables investigation of early epithelial-immune interactions, inflammatory modulation, and bacterial colonization dynamics during airway infection. The system provides a versatile and human-relevant platform for studying respiratory host-pathogen interactions. ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, Ref. nr.: NNF10CC1016517, NNF19OC0056411 The John and Birthe Meyer Foundation CAG - Greater Copenhagen Health - Science - Partners 2020 (GCHSP)

The increasing prevalence of antibiotic-resistant bacterial infections highlights the need for physiologically relevant in vitro models that recapitulate host pathogen interactions. Pseudomonas aeruginosa is a clinically important opportunistic pathogen associated with hospital-acquired infections and chronic airway diseases, including cystic fibrosis, where dysregulated inflammatory responses contribute to disease progression. While air liquid interface (ALI) models have advanced the study of airway epithelium, most of these modes lack immune components, limiting their ability to capture immune epithelial interactions. Here, we expanded a previously established dual-cell ALI model incorporating human monocyte-derived macrophages to investigate how immune context, bacterial dose, and time influence early infection dynamics. Standard BCi-NS1.1 epithelial monocultures and macrophage co-cultures were infected with P. aeruginosa (PAO1) at low (100 colony-forming units (CFU) and high (1000 CFU) inoculum and analyzed over 10, 16, and 24 h post-infection (hpi). Macrophage presence did not significantly alter total bacterial burden but markedly influenced cytokine responses and bacterial spatial organization. Pro-inflammatory cytokines (interleukin (IL)-1a, IL-1b, Tumor Necrosis Factor (TNF)- a) were enhanced in dual-cell culture models, while IL-6 exhibited a threshold-dependent response detectable only at higher bacterial loads. Confocal imaging revealed that macrophages altered bacterial distribution, promoting a more dispersed pattern compared to the clustered organization observed in epithelial monocultures. These effects were most pronounced at lower bacterial inocula. Together, our findings demonstrate that macrophages reshape early infection dynamics by modulating inflammatory signaling and bacterial spatial organization without affecting overall bacterial burden. This study highlights the importance of incorporating immune cells into in vitro airway models.

Persistent bacterial infections pose major clinical challenges, particularly in people with cystic fibrosis (pwCF), in whom Pseudomonas aeruginosa can persist for decades despite antibiotic treatment. To investigate the host-pathogen interactions influencing infection outcomes, we modeled P. aeruginosa infection using human airway epithelial cells cultured at the air-liquid interface from both CF and non-CF donors and the BCi-NS1.1 cell line. Infection assays with reference and clinical strains revealed four distinct infection clusters based on virulence, epithelial damage, and localization, independent of model type or strain lineage. Correction of CF transmembrane conductance regulator protein (CFTR) channel function did not alter infection outcomes. Dual RNA sequencing showed conserved host inflammatory responses across models, while bacterial transcriptional profiles varied by host context, particularly in CF models. These findings demonstrate that while infection outcomes and host transcriptional responses are consistent across models, bacterial adaptation in the CF airways drives transcriptional reprogramming linked to persistence in pwCF.

Antibiotic resistance is a major global health threat. While its role in reducing drug susceptibility is well established, the broader consequences of resistance mutations on bacterial physiology and phenotype during infection remain poorly understood. Carbapenem-resistant Pseudomonas aeruginosa is considered among the highest-priority bacterial threats, with resistance commonly driven by loss-of-function mutations in the carbapenem uptake porin OprD. Here we show that such mutations can arise in clinical isolates even without prior carbapenem treatment, suggesting that their biological impact during infection is not limited to antibiotic resistance. Consistent with this, we found that oprD mutants exhibit enhanced early attachment to and translocation across airway epithelial barriers in an in vitro human infection model, an effect observed across strains with distinct clinical genomic backgrounds and infection dynamics. Our findings indicate that loss of OprD alters the bacterial outer membrane charge and reduces mucus entrapment, thereby facilitating epithelial barrier colonization. Overall, these results illustrate how antibiotic resistance mutations can directly shape infection dynamics, extending their impact well beyond antimicrobial susceptibility.

Chronic infections by Pseudomonas aeruginosa in people with cystic fibrosis are characterized by persistent inflammation and oxidative stress, yet the mechanisms enabling bacterial persistence are not fully understood. Here, we identify a persistence mechanism mediated by pyruvate secretion resulting from mutations in the pyruvate dehydrogenase complex in clinical isolates of P. aeruginosa, with putative analogous mutations also identified in Staphylococcus aureus, Haemophilus influenzae and Stenotrophomonas maltophilia. These mutations lead to elevated extracellular pyruvate, which dampens host inflammatory responses and favors bacterial persistence. Pyruvate exerts multiple roles: scavenges reactive oxygen species such as H2O2, suppresses host immune activation both in airway epithelial cells and macrophages, and increases bacterial survival during phagocytosis. This metabolic crosstalk promotes bacterial persistence while reducing epithelial and macrophage inflammatory responses. Our findings reveal pyruvate as a bacterial immunometabolite that mimics host antioxidant defenses, reshaping the infection niche to favor long-term colonization. This work highlights the broader role of secreted metabolites in host-pathogen interactions and suggests new strategies targeting metabolic pathways to manage chronic infections.

Objectives Pseudomonas aeruginosa is the leading cause of death in cystic fibrosis (CF) patients. The production of virulence factors and the complex adaptation to the host play a crucial role in its pathogenesis. However, knowledge of the genetic determinants that drive P. aeruginosa persistence in the host environment is limited. Based on previously collected transcriptional data from human samples, in this study was investigated the role of sirB2, a P. aeruginosa PA14 gene with unknow function and whose expression is enhanced in the CF lung environment. Methods In silico promoter analysis, molecular genetics and biochemical approaches were used to decipher the sirB2 gene regulation. The generation of PA14 mutants and whole-genome sequencing analysis allowed to investigate the role of sirB2 on heterogenous phenotypes known to be essential for P. aeruginosa persistence (i.e. biofilm formation, in vitro and in vivo virulence assays on different infection models). Results In P. aeruginosa, sirB2 gene belongs to the Vfr and AmrZ regulons, and its inactivation increases P. aeruginosa pathogenic potential in Galleria melonella. Similarly, gene deletion stimulates transepithelial migration and biofilm formation in an infection model based on air-liquid interface cultures of the airway epithelium. In this context the lack of sirB2 promotes the production of virulence determinants and the emergence of rugose small colony variants (RSCVs). RSCVs appearance depends on an increased rate of mutations in the wsp regulatory circuit, leading to increased c-di-GMP levels. Conclusions Our data identified the sirB2 gene as a novel genetic determinant underlying the appearance of heterogenous phenotypes typical of infections, through a mechanism involving specific genetic rearrangement in the PA14 genome.

Antibiotic-resistant bacteria could be tackled by identifying trade-offs of evolution, such as high fitness costs, which may be harnessed to force reversion to susceptibility. A decline in antimicrobial resistance can occur through compensatory mutations or by genetic reversion to the wild-type allele, which reduce fitness costs associated with resistance. We analyse here the impact of antibiotic-free environments on declining ciprofloxacin resistance in eight nfxB defective clinical strains of Pseudomonas aeruginosa spanning varied clone types and ciprofloxacin resistance levels. Ciprofloxacin resistance declines in just 100 generations, which is mainly caused by newly acquired mutations in the genes encoding the overproduced efflux pump MexCD-OprJ and not by the reversion of nfxB mutations of the parental strains. The rapid reversion of ciprofloxacin resistance in P. aeruginosa suggests the potential for reusing this essential antibiotic and underlines the need to implement evolution-based approaches against nfxB defective resistant mutant strains.

Despite advances in healthcare, bacterial pathogens remain a severe global health threat, exacerbated by rising antibiotic resistance. Lower respiratory tract infections, with their high death toll, are of particular concern. Accurately replicating host-pathogen interactions in laboratory models is crucial for understanding these diseases and evaluating new therapies. In this communication, we briefly present existing in vivo models for cystic fibrosis and their limitations in replicating human respiratory infections. We then present a novel, 3D-printed, cytocompatible microfluidic lung-on-a-chip device, designed to simulate the human lung environment, and with possible use in recapitulating general infectious diseases.Our device enables the colonisation of fully differentiated lung epithelia at an air-liquid interface with Pseudomonas aeruginosa, a key pathogen in many severe infections. By incorporating dynamic flow, we replicate the clearance of bacterial toxins and planktonic cells, simulating both acute and chronic infections. This platform supports real-time monitoring of therapeutic interventions, mimics repeated drug administrations as in clinical settings, and facilitates the analysis of colony-forming units and cytokine secretion over time. Our findings indicate that this lung-on-a-chip device has significant potential for advancing infectious disease research, in optimizing treatment strategies against infections and in developing novel treatments.

The persistence of Pseudomonas aeruginosa in chronic infections extends beyond the issue of antibiotic resistance. A critical, yet unresolved question is why antibiotics fail to eradicate all infecting bacteria, despite P. aeruginosa often being phenotypically susceptible. This highlights the need for a comprehensive understanding of persistence mechanisms, which we consider to be directly rooted in host-pathogen interactions and which are frequently overlooked. We propose that both gene regulatory adaptation and adaptive genetic evolution play fundamental roles in the long-term persistence of P. aeruginosa. Elucidating these complex interactions has profound clinical implications, but their elucidation depends on access to advanced and innovative model systems that accurately replicate host-pathogen relationships.

Pseudomonas aeruginosa uses multiple type VI secretion systems (T6SSs) to manipulate eukaryotic cells, kill competing microbes and take up nutrients. Bacterial strains are known to differ in their T6SS apparatus and the toxic effector proteins responsible for killing. The ability to eliminate competitors has been repeatedly demonstrated in lab studies, but much less is known about effector genotypes during infection. We used comparative genomics to test for the presence and absence of T6SS effector genes in over 450 clinical P. aeruginosa isolates from people with cystic fibrosis in Copenhagen (Denmark) and complemented these findings with data of 52 isolates from people with cystic fibrosis in London (UK). We found natural variation in the occurrence and combination of effector genes. Patients were typically infected with isolates that differ in their effector gene sets but show no statistically significant association between the number of effector genes and chronic infection. Isolates with a pair of T6SS effector and immunity genes and isolates without these genes, which would be expected to kill each other based on existing work in the laboratory, were found on the same individual. Taking the isolates' phylogeny and sampling times into account, we identified five putative loss events of effector genes during infection. Although the impact of our findings for infected individuals will require further investigation, we demonstrate the extent of strain-level variation in T6SS effector genes in clinical isolates.

Pseudomonas aeruginosa is the leading cause of death in cystic fibrosis (CF) patients, yet the genetic mechanisms driving its fitness in the host remain poorly defined. Previously collected transcriptomic data of clinical samples showed that expression of the gene PA14_RS04555 (sirB2) is stimulated in the CF lung environment. In this work, we show that sirB2 is regulated by the global transcriptional regulators Vfr and AmrZ. Loss of sirB2 markedly enhanced P. aeruginosa pathogenicity, increasing virulence in Galleria mellonella, and promoting bacterial translocation and biofilm formation in a differentiated airway epithelial infection model. Deletion of sirB2 triggered the emergence of biofilm-proficient rugose small colony variants (RSCVs), driven by elevated c-di-GMP and increased Pel polysaccharide production when cultures were grown in static conditions. The RSCV phenotype depends on suppressor mutations in the wsp operon, possibly as a response to redox imbalance caused by the lack of sirB2 under oxygen-limited conditions. Indeed, the sirB2 mutant exhibited impaired fitness during anaerobic respiration when nitrate was the sole electron acceptor, in a manner independent of the ubiquinone pool. Our findings show that sirB2 inactivation promotes RSCV emergence and identify sirB2 as a novel genetic determinant of metabolic fitness under host-relevant conditions, thereby underscoring the role of redox balance in chronic CF infections.

Pseudomonas aeruginosa is an opportunistic pathogen of major clinical importance, which frequently gives rise to persistent antibiotic resilient infections. To investigate the connection between the bacterial physiological activity and the host environment during the early stages of bacterial infections, we have established a dual-fluorescent reporter system to monitor growth activity of P. aeruginosa during infection of a human airway epithelial model. This approach enables quantitative and spatially resolved analysis of bacterial growth within distinct infection micro-niches. Using this infection model, we compared the infection routes for the reference strain PAO1 as well as for two patho-adaptive mutants, PAO1 ΔpscC ( ΔpscC ) and PAO1 ΔmexZ ( ΔmexZ ). All three strains colonized apical-, intracellular-, interepithelial-, and epithelial barrier breach sites, but with strain-specific patterns of localisation. PAO1 was rarely observed intracellularly, ΔpscC was detected in few cases at epithelial-barrier breach- and interepithelial sites, and ΔmexZ colonized apical and intracellular niches in only few cases. Measurements of bacterial growth activities in these niches further revealed distinct hierarchies of bacterial growth activity among the tissue sites: PAO1 bacteria were most active at epithelial-barrier breach sites, ΔpscC at interepithelial sites, and ΔmexZ in intracellular niches. Taken together, these data support a model in which P. aeruginosa follows a progressive infection continuum from apical colonization to barrier breach, involving interepithelial spread, whereas the mutant strains represented truncated or altered versions of this infection program. More broadly, this study demonstrates the utility of unstable fluorescent reporters for capturing dynamic, niche-specific growth activity patterns during host-pathogen interactions, with implications for both basic pathogenesis research and therapeutic development. Author Summary In this study, we set out to better understand how bacterial infections develop and progress on human tissues. To do so, we created fluorescent “reporter” strains of Pseudomonas aeruginosa , a bacterium that commonly infects the lungs of people with cystic fibrosis and other chronic lung diseases. These genetically modified bacteria allow us to directly observe their growth activity in living human cell cultures using advanced microscopy. Here, we can see how the bacteria grow and spread at different locations within the tissue, and if there are any sites they prefer relative to others. We can also see if those patterns change, depending on any mutations the bacteria might have. In the future, the same genetic engineering can be applied to other bacteria, and the fluorescence can inform us of a range of important bacterial functions; for instance, at precisely what point of the infection process – and where in the tissues – the bacteria produce toxins, are stressed, produce antibiotic-resistance proteins or divide, for example. Our experiments revealed that some genetic mutations, that are often found in bacterial isolates from hospital infections, change the preference of colonization to distinct tissue sites and confer specialized and unique patterns – and peaks – of growth activity. Some bacterial strains tend to grow on the surface of the tissue, while others are more likely to move between or inside human cells. These behaviours reflect how bacteria evolve during long-term infections and adapt to different environments within the body tissues. The approach we present here provides a new tool for studying how bacterial infections unfold and may ultimately help identify more effective ways to treat or control them. ### Competing Interest Statement The authors have declared no competing interest.

Pseudomonas aeruginosa has increasing clinical relevance and commonly occupies the cystic fibrosis (CF) airways. Its ability to colonize and persist in diverse niches is attributed to its large accessory genome, where prophages represent a common feature and may contribute to its fitness and persistence. We focused on the CF airways niche and used 197 longitudinal isolates from 12 patients persistently infected by P. aeruginosa. We computationally predicted intact prophages for each longitudinal group and scored their long-term persistence. We then confirmed prophage inducibility and mapped their location in the host chromosome with lysate sequencing. Using comparative genomics, we evaluated prophage genomic diversity, long-term persistence, and level of genomic maintenance. Our findings support previous findings that most P. aeruginosa genomes harbour prophages some of which can self-induce, and that a common CF-treating antibiotic, ciprofloxacin, can induce prophages. Induced prophage genomes displayed high diversity and even genomic novelty. Finally, all induced prophages persisted long-term with their genomes avoiding gene loss and degradation over 4 years of host replication in the stressful CF airways niche. This and our detection of phage genes, which contribute to host competitiveness and adaptation, lends support to our hypothesis that the vast majority of prophages detected as intact and inducible in this study facilitated their host fitness and persistence.

Antibiotic-resistant bacterial infections represent a critical global health challenge, increased by the lack of new antimicrobial agents and the rapid emergence of antimicrobial resistance. Among the most concerning pathogens is Pseudomonas aeruginosa, a bacterium renowned for its vast ability to develop resistance during persistent lung infections, primarily via mutations in antibiotic resistance determinants. While the role of these determinants in reducing antibiotic efficacy is well studied, their broader contributions to bacterial physiology and bacterial host colonization remain highly underexplored.Using an air-liquid interface cell culture model that mimics human airway conditions, we observed that mutations in the carbapenem entrance porin, OprD, enhance bacterial penetration through the epithelial barrier. We hypothesize that an increased secretion of Type-3-Secretion-related toxins detected in oprD mutants may drive this phenotype, potentially triggered by membrane reorganization resulting from the absence of the OprD porin. Notably, we found that clinical isolates harboring these carbapenem resistance mutations are sometimes found in patients who have not undergone carbapenem treatment, further suggesting that these mutations may provide advantages unrelated to antibiotic exposure.Our findings reveal that mutations in antimicrobial resistance determinants can significantly influence bacterial pathogenesis, underscoring the need to study the consequences of antibiotic resistance within the broader framework of host-microbe interactions during infection.

Persistent bacterial infections constitute an increasing health problem, often associated with antibiotic resistance. However, despite extensive antibiotic treatment, Pseudomonas aeruginosa persists for decades in people with cystic fibrosis (pwCF), remaining susceptible. Host-pathogen interactions during infection may, therefore, be a major contributing factor to treatment failure. Using an infection model based on human airway epithelial cells, cultured at the air-liquid interface (ALI), we simulated the infection process of P. aeruginosa to investigate the colonization dynamics and virulence potential during infection in ALI models from non-CF and CF donors, and the BCi-NS1.1 cell line. Infections by reference strains and clinical isolates from pwCF revealed four infection clusters based on virulence, epithelial damage, and localization within the epithelium, in a strain-specific manner regardless of the type of ALI model or clonal lineage. Modulator treatment to restore CFTR channel function did not change infection patterns in CF ALI models. Dual RNA-seq revealed that bacterial colonization of ALI models significantly upregulated host inflammatory pathways, dependent on the strain’s virulence. Simultaneously, while bacterial gene expression was similar in non-CF and BCi-NS1.1 ALI models, CF models promoted differential regulation in a type III secretion mutant. Altogether, our results profile key infection dynamics occurring in pwCF and provide ground knowledge on the interplay between the airway epithelium and P. aeruginosa . ### Competing Interest Statement The authors have declared no competing interest. Novo Nordisk Foundation, https://ror.org/04txyc737, NNF19OC0056411

Pseudomonas aeruginosa is a bacterium with increasing relevance in clinical settings and among the most common bacteria occupying the cystic fibrosis (CF) lung niche. Its ability to colonize and persist in diverse niches is attributed to this bacterium’s large accessory genome. In P. aeruginosa, prophages represent a common feature of a strain’s accessory genome. Hence, we hypothesized that prophages play a role in the bacterium’s fitness and persistence in CF. We focused on the CF niche and used longitudinal isolates of patients persistently infected by P. aeruginosa. Via in silico analysis we predicted intact prophages in the genomes of each longitudinal isolate group and scored their long-term persistence. We then confirmed whether they are inducible and where they reside by induction experiments and lysate sequencing. Lastly, we performed comparative genomics to evaluate prophage diversity and confirm their predicted long-term persistence and level of genomic maintenance. In concurrence with other studies, our findings support that most P. aeruginosa harbour prophages, some of which can self-induce. We also found ciprofloxacin, an antibiotic commonly used for P. aeruginosa treatment in CF, to induce prophages. The induced prophage genomes displayed a high degree of diversity and instances of genomic novelty. Finally, we discovered that all induced prophages persisted long-term with their genomes virtually unchanged, suggesting that they likely assist host persistence. In addition to elucidating the role of prophages in P. aeruginosa, we expect our findings to aid in developing novel diagnostics and phage-based therapies for P. aeruginosa infections.