The incidence of infection by nontuberculous mycobacteria, mainly Mycobacterium abscessus, is increasing in patients with cystic fibrosis and other chronic pulmonary diseases, leading to an accelerated lung function decline. In most cases, M. abscessus coinfects Pseudomonas aeruginosa, the most common pathogen in these conditions. However, how these two bacterial species interact during infection remains poorly understood. This study explored their behaviour in three relevant pathogenic settings: dual-species biofilm development using a recently developed method to monitor individual species in dual-species biofilms, coinfection in bronchial epithelial cells, and in vivo coinfection in the Galleria mellonella model. The results demonstrated that both species form stable mixed biofilms and reciprocally inhibit single-biofilm progression. Coinfections in bronchial epithelial cells significantly decreased cell viability, whereas in G. mellonella, coinfections induced lower survival rates than individual infections. Analysis of the immune response triggered by each bacterium in bronchial epithelial cell assays and G. mellonella larvae revealed that P. aeruginosa induces the overexpression of proinflammatory and melanization cascade responses, respectively. In contrast, M. abscessus and P. aeruginosa coinfection significantly inhibited the immune response in both models, resulting in worse consequences for the host than those generated by a single P. aeruginosa infection. Overall, this study highlights the novel role of M. abscessus in suppressing immune responses during coinfection with P. aeruginosa, emphasizing the clinical implications for the management of cystic fibrosis and other pulmonary diseases. Understanding these interactions could inform the development of new therapeutic strategies to mitigate the severity of coinfections in vulnerable patients.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the CF transmembrane conductance (CFTR) gene, resulting in the secretion of hyperviscous mucus. Infective exacerbations are a major determinant of morbidity and mortality in CF patients. These infections are clinically challenging, and antimicrobial treatment should effectively target the organisms and be delivered early to improve survival. Ceftobiprole is a fifth-generation cephalosporin antibiotic that is not indicated for the treatment of CF. However, due to its activity against common causes of infective exacerbations in CF such as Staphylococcus aureus, including MRSA, and Pseudomonas aeruginosa where resistance has not developed, it has utility for managing infective exacerbations.

To describe the use of ceftobiprole in the treatment of infective exacerbations in CF.

Ten patients with CF (age 24-63; six male and four female) were treated with ceftobiprole for infective exacerbations following discussion within the multi-disciplinary team. In most patients, ceftobiprole was given concomitantly with other antibiotics.

All patients had positive sputum cultures for S. aureus (including nine MRSA), and seven patients had concomitant P. aeruginosa infection. Ceftobiprole treatment was associated with improved lung function, and markers of systemic inflammation decreased for most patients, with some variation. There was good tolerability in all but four patients.

Ceftobiprole presents a therapeutic option for susceptible infections in CF patients with limited treatment options. Its broad-spectrum coverage may help to reduce polypharmacy. However, further clinical studies are needed.

Pseudomonas aeruginosa and Staphylococcus aureus are bacterial pathogens of major clinical concern that cause polymicrobial infections in diverse patient populations. Human calprotectin (CP; S100A8/S100A9 heterooligomer, MRP8/MRP14 heterooligomer) is a host-defense protein that contributes to nutritional immunity by sequestering multiple nutrient metal ions including Mn(II), Fe(II), and Zn(II). Here, we examine the consequences of metal availability and CP treatment on cocultures of P. aeruginosa and S. aureus. We report that CP elicits Fe-starvation responses in both P. aeruginosa and S. aureus in coculture, including the upregulation of genes involved in Fe uptake by both organisms. Moreover, analyses of pseudomonal metabolites in coculture supernatants further demonstrate Fe-starvation responses, showing that CP treatment leads to increased siderophore levels and reduced phenazine levels. Consistent with prior studies, growth under conditions of Fe depletion accelerated P. aeruginosa killing of S. aureus in coculture, but treatment with CP promoted S. aureus survival. Treatment with CP site variants lacking functional transition-metal-binding sites and metalated CP also enhanced S. aureus survival in coculture with P. aeruginosa, revealing that this consequence of CP treatment is independent of its canonical metal-sequestering function. Thus, the protective effects of CP treatment during coculture appear to override the observed Fe-starvation effects that make P. aeruginosa more virulent toward S. aureus. This work highlights an unappreciated facet of how CP contributes to host-pathogen and pathogen-pathogen interactions that are relevant to human infectious disease.

The current working model that describes how the innate immune protein calprotectin (CP) protects the host against bacterial pathogens focuses on its capacity to sequester multiple essential metal nutrients in a process called nutritional immunity. Our study further explores this function by focusing on the effects of metal availability and CP treatment on the dynamics of Pseudomonas aeruginosa and Staphylococcus aureus grown in coculture. These two bacterial pathogens are of significant clinical concern and colocalize with CP at infection sites. This work reveals that CP modulates P. aeruginosa/S. aureus coculture dynamics in a manner that is independent of its ability to sequester nutrient metal ions. This surprising result is important because it demonstrates that CP has metal-independent function and thus contributes to the host-pathogen and pathogen-pathogen interactions in ways that are not accounted for in the current working model focused on metal sequestration.

The incidence of infections attributed to antimicrobial-resistant (AMR) pathogens has increased exponentially over the recent decades reaching 1.27 million deaths worldwide in 2019. Without intervention, these infections are predicted to cause up to 10 million deaths a year and incur costs of up to 100 trillion US dollars globally by 2050. The emergence of AMR bacteria such as the ESKAPEE pathogens, and in particular Pseudomonas aeruginosa and species from the genus Burkholderia, underscores an urgent need for new therapeutic strategies. Monoclonal antibody (mAb) therapy offers a promising alternative to treat and prevent bacterial infections. In this study, we used peptides from highly conserved areas of the bacterial flagellin to generate monoclonal antibodies capable of broad binding to flagellated Gram-negative bacteria. We generated a broadly reactive IgG2bĸ mAb (WVDC-2109) that recognizes P. aeruginosa, Burkholderia sp., and other Gram-negative pathogens of interest. Characterization of the therapeutic potential of this antibody was determined using P. aeruginosa as model. In vitro characterization of WVDC-2109 demonstrated complement-mediated bactericidal activity and enhanced opsonophagocytosis of P. aeruginosa. Prophylactic administration of WVDC-2109 markedly improved survival and outcome in a lethal sepsis model and a sub-lethal murine pneumonia model of P. aeruginosa infection, reducing bacterial burden and inflammation. These findings suggest that WVDC-2109 and similar FliC-targeting antibodies could be valuable in preventing or treating diseases caused by P. aeruginosa as well as other life-threatening diseases of concern.IMPORTANCEAntimicrobial resistance (AMR) costs hundreds of thousands of lives and billions of dollars annually. To protect the population against these infections, it is imperative to develop new medical countermeasures targeting AMR pathogens like P. aeruginosa and Burkholderia sp. The administration of broadly reactive monoclonal antibodies can represent an alternative to treat and prevent infections caused by multi-drug-resistant bacteria. Unlike vaccines, antibodies can provide protection regardless of the immune status of the infected host. In this study, we generated an antibody capable of recognizing flagellin from P. aeruginosa and B. pseudomallei along with other Gram-negative pathogens of concern. Our findings demonstrate that the administration of the monoclonal antibody WVDC-2109 enhances survival rates and outcomes in different murine models of P. aeruginosa infection. These results carry significant implications in the field given that there are no available vaccines for P. aeruginosa.

The pharmacodynamic response of bacterial pathogens to antibiotics can be influenced by interactions with other bacterial species in polymicrobial infections (PMIs). Understanding the complex eco-evolutionary dynamics of PMIs and their impact on antimicrobial treatment response represents a step towards developing improved treatment strategies for PMIs. Here, we investigated how interspecies interactions in a multi-species bacterial community affect the pharmacodynamic response to antimicrobial treatment. To this end, we developed an in silico model which combined agent-based modeling with ordinary differential equations. Our analyses suggest that both interspecies interactions, modifying either drug sensitivity or bacterial growth rate, and drug-specific pharmacological properties drive the bacterial pharmacodynamic response. Furthermore, lifestyle of the bacterial population and the range of interactions can influence the impact of species interactions. In conclusion, this study provides a foundation for the design of antimicrobial treatment strategies for PMIs which leverage the effects of interspecies interactions.

Burkholderia cenocepacia H111 is an obligate aerobic bacterium which has been isolated from a cystic fibrosis (CF) patient. In CF lungs the environment is considered micro-oxic or even oxygen-depleted due to bacterial activities and limited oxygen diffusion in the mucus layer. To adapt to low oxygen concentrations, bacteria possess multiple terminal oxidases. In this study, we identified six terminal oxidases of B. cenocepacia H111 and constructed reporter strains to monitor their expression in different environments. While the heme-copper oxidase aa3 (cta) was constitutively expressed, the bd-1 oxidase (cyd) was induced under oxygen-limited growth conditions. The cyanide-insensitive bd-type terminal oxidase (cio-1) was mainly expressed in cells grown on the surface of solid medium or in liquid cultures in presence of cyanide, which is known to be produced in the CF lung by the often co-residing CF pathogen Pseudomonas aeruginosa. Indeed, a cio-1 insertional mutant was not able to grow in the presence of cyanide confirming the important role of Cio-1 in cyanide resistance. The caa3 oxidase (caa), was only expressed under nutrient limitation when cells were grown on the surface of solid medium. We also investigated the involvement of two regulatory systems, Anr and RoxS/RoxR, in the expression of cio-1 and cyd. Our data suggest, that, given that Cio-1 is only present in prokaryotes and plays an important role in the defense against cyanide-producing P. aeruginosa, it may be a valuable drug target for treatment of polymicrobial infections in CF patients.

Chronic infections represent a significant global health and economic challenge. Biofilms, which are bacterial communities encased in an extracellular polysaccharide matrix, contribute to approximately 80% of these infections. In particular, pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from the sputum of patients with cystic fibrosis and are commonly found in chronic wound infections. Within biofilms, bacteria demonstrate a remarkable increase in resistance and tolerance to antimicrobial treatment. We investigated the efficacy of combining the last-line antibiotic colistin with a membrane- and stringent stress response-targeting anti-biofilm peptide DJK-5 against co-biofilms comprised of multidrug-resistant P. aeruginosa and methicillin-resistant S. aureus (MRSA). Colistin lacks canonical activity against S. aureus. However, our study revealed that under co-biofilm conditions, the antibiofilm peptide DJK-5 synergized with colistin against S. aureus. Similar enhancement was observed when daptomycin, a cyclic lipopeptide against Gram-positive bacteria, was combined with DJK-5, resulting in increased activity against P. aeruginosa. The combinatorial treatment induced morphological changes in both P. aeruginosa and S. aureus cell shape and size within co-biofilms. Importantly, our findings also demonstrate synergistic activity against both P. aeruginosa and S. aureus in a murine subcutaneous biofilm-like abscess model. In conclusion, combinatorial treatments with colistin or daptomycin and the anti-biofilm peptide DJK-5 show significant potential for targeting co-biofilm infections. These findings offer promising avenues for developing new therapeutic approaches to combat complex chronic infections.

The lungs of patients with cystic fibrosis (CF) are vulnerable to persistent polymicrobial colonization by bacterial pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, and the non-tuberculous mycobacterium (NTM) Mycobacterium abscessus. The polymicrobial milieu within the CF lung impacts individual species fitness, influences biofilm-forming capabilities, pathogenicity, production of virulence factors and even antimicrobial responses, all potentially compromising therapeutic success. Interaction studies among these CF pathogens are very limited, especially studies on the influences of P. aeruginosa and S. aureus on M. abscessus co-existence and virulence. Based on the little known thus far about coinfection of these pathogens, we hypothesize that the co-existence of P. aeruginosa and S. aureus alters M. abscessus virulence and phenotypic characteristics. We evaluated the direct (co-culture) and indirect (using supernatant) effects of P. aeruginosa and S. aureus on M. abscessus growth rate, biofilm formation, macrophage internalization and glycopeptidolipids (GPL) expression. Our observations indicate that P. aeruginosa and S. aureus exert a competitive behavior toward M. abscessus during direct contact or indirect interaction in-vitro, probably as is the case of polymicrobial infections in the lungs of patients with CF. This is the first report that demonstrates S. aureus inhibitory effects on M. abscessus growth and biofilm forming capabilities. Collectively, co-culture studies enhance our understanding of polymicrobial interactions during coinfection and can guide to establish better management of coinfections and treatment strategies for M. abscessus.

Polymicrobial infections are infections that are caused by multiple pathogens and are common in patients with cystic fibrosis (CF). Although polymicrobial infections are associated with poor treatment responses in CF, the effects of the ecological interactions between co-infecting pathogens on antibiotic sensitivity and treatment outcome are poorly characterized. To this end, we systematically quantified the impact of these effects on the antibiotic sensitivity of Pseudomonas aeruginosa for nine antibiotics in medium conditioned by 13 secondary cystic fibrosis-associated bacterial and fungal pathogens through time-kill assays. We fitted pharmacodynamic models to these kill curves for each antibiotic-species combination and found that interspecies interactions changing the antibiotic sensitivity of P. aeruginosa are abundant. Interactions that lower antibiotic sensitivity are more common than those that increase it, with generally more substantial reductions than increases in sensitivity. For a selection of co-infecting species, we performed pharmacokinetic-pharmacodynamic modeling of P. aeruginosa treatment. We predicted that interspecies interactions can either improve or reduce treatment response to the extent that treatment is rendered ineffective from a previously effective antibiotic dosing schedule and vice versa. In summary, we show that quantifying the ecological interaction effects as pharmacodynamic parameters is necessary to determine the abundance and the extent to which these interactions affect antibiotic sensitivity in polymicrobial infections.IMPORTANCEIn cystic fibrosis (CF) patients, chronic respiratory tract infections are often polymicrobial, involving multiple pathogens simultaneously. Polymicrobial infections are difficult to treat as they often respond unexpectedly to antibiotic treatment, which might possibly be explained because co-infecting pathogens can influence each other's antibiotic sensitivity, but it is unknown to what extent such effects occur. To investigate this, we systematically quantified the impact of co-infecting species on antibiotic sensitivity, focusing on P. aeruginosa, a common CF pathogen. We studied for a large set co-infecting species and antibiotics whether changes in antibiotic response occur. Based on these experiments, we used mathematical modeling to simulate P. aeruginosa's response to colistin and tobramycin treatment in the presence of multiple pathogens. This study offers comprehensive data on altered antibiotic sensitivity of P. aeruginosa in polymicrobial infections, serves as a foundation for optimizing treatment of such infections, and consolidates the importance of considering co-infecting pathogens.

Tackling the challenge created by antibiotic resistance requires understanding the mechanisms behind its evolution. Like any evolutionary process, the evolution of antimicrobial resistance (AMR) is driven by the underlying variation in a bacterial population and the selective pressures acting upon it. Importantly, both selection and variation will depend on the scale at which resistance evolution is considered (from evolution within a single patient to the host population level). While laboratory experiments have generated fundamental insights into the mechanisms underlying antibiotic resistance evolution, the technological advances in whole genome sequencing now allow us to probe antibiotic resistance evolution beyond the lab and directly record it in individual patients and host populations. Here we review the evolutionary forces driving antibiotic resistance at each of these scales, highlight gaps in our current understanding of AMR evolution, and discuss future steps toward evolution-guided interventions.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens ubiquitous in the environment. Mycobacteroides abscessus is a relatively new pathogen associated with underlying lung chronic pathologies, accounting for most of the pulmonary infections linked to the rapidly growing mycobacteria group. This includes chronic obstructive pulmonary disease, bronchiectasis, or cystic fibrosis. Patient outcomes from M. abscessus infections are poor due to complicated treatments and other factors. Intrinsic drug resistance plays an important role. The M. abscessus toolbox of resistance is varied leading to complex strategies for treatment. Mechanisms include waxy cell walls, drug export mechanisms, and acquired resistance. Many studies have also shown the impact of extracellular DNA found in the biofilm matrix during early infection and its possible advantage in pathogenicity. In this review, we discuss the current knowledge of early infection focusing on biofilm formation, an environmental strategy, and which treatments prevent its formation improving current antibiotic treatment outcomes in preliminary studies.

Bacterial infections often involve more than one pathogen. While it is well established that polymicrobial infections can impact disease outcomes, we know little about how pathogens interact and affect each other's behaviour and fitness. Here, we used a microscopy approach to explore interactions between Pseudomonas aeruginosa and six human opportunistic pathogens that often co-occur in polymicrobial infections: Acinetobacter baumannii, Burkholderia cenocepacia, Escherichia coli, Enterococcus faecium, Klebsiella pneumoniae, and Staphylococcus aureus. When following growing microcolonies on agarose pads over time, we observed a broad spectrum of species-specific ecological interactions, ranging from mutualism to antagonism. For example, P. aeruginosa engaged in a mutually beneficial interaction with E. faecium but suffered from antagonism by E. coli. While we found little evidence for active directional growth towards or away from cohabitants, we observed that some pathogens increased growth in double layers in response to competition and that physical forces due to fast colony expansion had a major impact on fitness. Overall, our work provides an atlas of pathogen interactions, highlighting the diversity of potential species dynamics that may occur in polymicrobial infections. We discuss possible mechanisms driving pathogen interactions and offer predictions of how the different ecological interactions could affect virulence.

The global crisis of antimicrobial resistance (AMR) necessitates the development of broad-spectrum antibacterial drugs effective against multi-drug resistant (MDR) pathogens. BWC0977, a Novel Bacterial Topoisomerase Inhibitor (NBTI) selectively inhibits bacterial DNA replication via inhibition of DNA gyrase and topoisomerase IV. BWC0977 exhibited a minimum inhibitory concentration (MIC90) of 0.03-2 µg/mL against a global panel of MDR Gram-negative bacteria including Enterobacterales and non-fermenters, Gram-positive bacteria, anaerobes and biothreat pathogens. BWC0977 retains activity against isolates resistant to fluoroquinolones (FQs), carbapenems and colistin and demonstrates efficacy against multiple pathogens in two rodent species with significantly higher drug levels in the epithelial lining fluid of infected lungs. In healthy volunteers, single-ascending doses of BWC0977 administered intravenously ( https://clinicaltrials.gov/study/NCT05088421 ) was found to be safe, well tolerated (primary endpoint) and achieved dose-proportional exposures (secondary endpoint) consistent with modelled data from preclinical studies. Here, we show that BWC0977 has the potential to treat a range of critical-care infections including MDR bacterial pneumonias.

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili (TFP)-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of TFP motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. Reduced invasion leads to the formation of denser and thicker S. aureus colonies with increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate treatment strategies. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies expand our understanding of how P. aeruginosa TFP-mediated interspecies chemotaxis facilitates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.

The polymicrobial nature of many chronic infections makes their eradication challenging. Particularly, coisolation of Pseudomonas aeruginosa and Staphylococcus aureus from airways of people with cystic fibrosis and chronic wound infections is common and associated with severe clinical outcomes. The complex interplay between these pathogens is not fully understood, highlighting the need for continued research to improve management of chronic infections. Our study unveils that P. aeruginosa is attracted to S. aureus, invades into neighboring colonies, and secretes anti-staphylococcal factors into the interior of the colony. Upon inhibition of P. aeruginosa motility and thus invasion, S. aureus colony architecture changes dramatically, whereby S. aureus is protected from P. aeruginosa antagonism and responds through physiological alterations that may further hamper treatment. These studies reinforce accumulating evidence that spatial structuring can dictate community resilience and reveal that motility and chemotaxis are critical drivers of interspecies competition.

Polymicrobial biofilms are among the leading causes of antimicrobial treatment failure. In these biofilms, bacterial and fungal pathogens interact synergistically at the interspecies, intraspecies, and interkingdom levels. Consequently, combating polymicrobial biofilms is substantially more difficult compared to single-species biofilms due to their distinct properties and the resulting potential variation in antimicrobial drug efficiency. In recent years, there has been an increased focus on developing alternative strategies for controlling polymicrobial biofilms formed by bacterial and fungal pathogens. Current approaches for controlling polymicrobial biofilms include monotherapy (using either natural or synthetic compounds), combination treatments, and nanomaterials. Here, a comprehensive review of different types of polymicrobial interactions between pathogenic bacterial species or bacteria and fungi is provided along with a discussion of their relevance. The mechanisms of action of individual compounds, combination treatments, and nanomaterials against polymicrobial biofilms are thoroughly explored. This review provides various future perspectives that can advance the strategies used to control polymicrobial biofilms and their likely modes of action. Since the majority of research on combating polymicrobial biofilms has been conducted in vitro, it would be an essential step in performing in vivo tests to determine the clinical effectiveness of different treatments against polymicrobial biofilms.

Chronic infections harbor multiple pathogens where dynamic interactions between members of the polymicrobial community play a major role in determining the infection outcome. For example, in a nutrient-rich polymicrobial infection, bacteria have the potential to undergo evolutionary changes that impair their ability to synthesize essential metabolites. This adaptation may facilitate metabolic interdependencies between neighboring pathogens and lead to difficult-to-treat chronic infections. Our research group previously demonstrated that Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), typically considered classical competitors, can adopt a cooperative lifestyle through bi-directional purine exchange medicated by exogenous DNA (eDNA) release. To further validate our initial findings, in this study, we investigated the potential exchange of pyrimidine between PA and other pathogens, which is another constituent of DNA. In our findings, we observed that a pyrimidine-deficient transposon mutant strain of PA showed improved growth when co-cultured with wild-type PA, SA, Acinetobacter baumannii (AB), and Enterococcus faecalis (EF). Additionally, improved fitness of pyrimidine-deficient PA was further observed in chemical complementation with eDNA and uridine-5'-monophosphate. Interestingly, the rescue of PA growth through eDNA complementation is not as effective as in intact cells, such as SA, AB, EF, and wild-type PA, implying that eDNA is a lesser contributor to this metabolic complementation. Also, the exchange mechanism between pathogens involves more active mechanisms beyond simple eDNA or metabolite release. Our data further highlights the importance of cell-to-cell contact for effective and increased metabolic complementation.

This research holds crucial implications for combating chronic infections, where multiple pathogens coexist and interact within the same environment. By uncovering the dynamic exchange of pyrimidines between Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA), our study reveals a previously unrecognized aspect of interspecies cooperation. The observed enhanced growth of a pyrimidine-deficient PA strain when co-cultured with SA suggests potential avenues for understanding and disrupting bacterial metabolic interdependencies in chronic infection settings. Furthermore, our findings highlight the mechanisms involved in metabolic exchange, emphasizing the importance of cell-to-cell contact. This research explored essential metabolic interactions to address the challenges posed by difficult-to-treat chronic infections.

Cystic fibrosis (CF), an inherited genetic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, results in sticky and thick mucosal fluids. This environment facilitates the colonization of various microorganisms, some of which can cause acute and chronic lung infections, while others may positively impact the disease. Rothia mucilaginosa, an oral commensal, is relatively abundant in the lungs of CF patients. Recent studies have unveiled its anti-inflammatory properties using in vitro three-dimensional lung epithelial cell cultures and in vivo mouse models relevant to chronic lung diseases. Apart from this, R. mucilaginosa has been associated with severe infections. However, its metabolic capabilities and genotype-phenotype relationships remain largely unknown. To gain insights into its cellular metabolism and genetic content, we developed the first manually curated genome-scale metabolic model, iRM23NL. Through growth kinetics and high-throughput phenotypic microarray testings, we defined its complete catabolic phenome. Subsequently, we assessed the model's effectiveness in accurately predicting growth behaviors and utilizing multiple substrates. We used constraint-based modeling techniques to formulate novel hypotheses that could expedite the development of antimicrobial strategies. More specifically, we detected putative essential genes and assessed their effect on metabolism under varying nutritional conditions. These predictions could offer novel potential antimicrobial targets without laborious large-scale screening of knockouts and mutant transposon libraries. Overall, iRM23NL demonstrates a solid capability to predict cellular phenotypes and holds immense potential as a valuable resource for accurate predictions in advancing antimicrobial therapies. Moreover, it can guide metabolic engineering to tailor R. mucilaginosa's metabolism for desired performance.IMPORTANCECystic fibrosis (CF) is a genetic disorder characterized by thick mucosal secretions, leading to chronic lung infections. Rothia mucilaginosa is a common bacterium found in various parts of the human body, acting as a normal part of the flora. In people with weakened immune systems, it can become an opportunistic pathogen, while it is prevalent and active in CF airways. Recent studies have highlighted its anti-inflammatory properties in the lower pulmonary system, indicating the intricate relationship between microbes and human health. Herein, we have developed the first manually curated metabolic model of R. mucilaginosa. Our study examined the previously unknown relationships between the bacterium's genotype and phenotype and identified essential genes that impact the metabolism under various conditions. With this, we opt for paving the way for developing new strategies in antimicrobial therapy and metabolic engineering, leading to enhanced therapeutic outcomes in cystic fibrosis and related conditions.

Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human pathogens, Pseudomonas aeruginosa and Staphylococcus aureus commonly coexist in the bacterial infections for hospitalized and/or immunocompromised patients. Here, we observed that autolysis of the P. aeruginosa quorum-sensing (QS) mutant (lasRmvfR) was suppressed by the presence of the S. aureus cells in vitro. The QS mutant still displayed killing against S. aureus cells, suggesting the link between the S. aureus-killing activity and the autolysis suppression. Independent screens of the P. aeruginosa transposon mutants defective in the S. aureus-killing and the S. aureus transposon mutants devoid of the autolysis suppression revealed the genetic link between both phenotypes, suggesting that the iron-dependent metabolism involving S. aureus exoproteins might be central to both phenotypes. The autolysis was suppressed by iron treatment as well. These results suggest that the interaction between P. aeruginosa and S. aureus might be governed by mechanisms that necessitate the QS circuitry as well as the metabolism involving the extracellular iron resources during the polymicrobial infections in the human airway.

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of type IV pilus motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. P. aeruginosa reduced invasion leads to the formation of denser and thicker S. aureus colonies with significantly increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate the effective treatment of infections. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies build on our understanding of how P. aeruginosa type IV pili-mediated interspecies chemotaxis mediates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.

Co-infections with Staphylococcus aureus and Pseudomonas aeruginosa are common in patients with chronic wounds, but little is known about their synergistic effect mediated by extracellular vesicles (EVs). In this study, we investigated the effect of EVs derived from S. aureus (SaEVs) on the pathogenicity of P. aeruginosa. By using lipophilic dye, we could confirm the fusion between SaEV and P. aeruginosa membranes. However, SaEVs did not alter the growth and antibiotic susceptible pattern of P. aeruginosa. Differential proteomic analysis between SaEV-treated and non-treated P. aeruginosa was performed, and the results revealed that lipopolysaccharide (LPS) biosynthesis protein in P. aeruginosa significantly increased after SaEV-treatment. Regarding this result, we also found that SaEVs promoted LPS production, biofilm formation, and expression of polysaccharide polymerization-related genes in P. aeruginosa. Furthermore, invasion of epithelial cells by SaEV-pretreated P. aeruginosa was enhanced. On the other hand, uptake of P. aeruginosa by RAW 264.7 macrophages was impaired after pretreatment P. aeruginosa with SaEVs. Proteomic analysis SaEVs revealed that SaEVs contain the proteins involving in host cell colonization, inhibition of host immune response, anti-phagocytosis of the macrophages, and protein translocation and iron uptake of S. aureus. In conclusion, SaEVs serve as a mediator that promote P. aeruginosa pathogenicity by enhancing LPS biosynthesis, biofilm formation, epithelial cell invasion, and macrophage uptake impairment.

Despite advances in therapies, bacterial chronic respiratory infections persist as life-threatening to patients suffering from cystic fibrosis (CF). Pseudomonas aeruginosa and bacteria of the Burkholderia cepacia complex are among the most difficult of these infections to treat, due to factors like their resistance to multiple antibiotics and ability to form biofilms. The lack of effective antimicrobial strategies prompted our search for alternative immunotherapies that can effectively control and reduce those infections among CF patients. Previous work from our group showed that the anti-BCAL2645 goat polyclonal antibody strongly inhibited Burkholderia cenocepacia to adhere and invade cultured epithelial cells. In this work, we showed that the polyclonal antibody anti-BCAL2645 also strongly inhibited the ability of P. aeruginosa to form biofilms, and to adhere and invade the human bronchial epithelial cell line CFBE41o-. The polyclonal antibody also inhibited, to a lesser extent, the ability of B. multivorans to adhere and invade the human bronchial epithelial cell line CFBE41o. We also show that the ability of B. cenocepacia, P. aeruginosa and B. multivorans to kill larvae of the Galleria mellonella model of infection was impaired when bacteria were incubated with the anti-BCAL2645 antibody prior to the infection. Our findings show that an antibody against BCAL2645 possesses a significant potential for the development of new immunotherapies against these three important bacterial species capable of causing devastating and often lethal infections among CF patients.

Persons with cystic fibrosis (CF), starting in early life, show intestinal microbiome dysbiosis characterized in part by a decreased relative abundance of the genus Bacteroides. Bacteroides is a major producer of the intestinal short chain fatty acid propionate. We demonstrate here that cystic fibrosis transmembrane conductance regulator-defective (CFTR-/-) Caco-2 intestinal epithelial cells are responsive to the anti-inflammatory effects of propionate. Furthermore, Bacteroides isolates inhibit the IL-1β-induced inflammatory response of CFTR-/- Caco-2 intestinal epithelial cells and do so in a propionate-dependent manner. The introduction of Bacteroides-supplemented stool from infants with cystic fibrosis into the gut of CftrF508del mice results in higher propionate in the stool as well as the reduction in several systemic pro-inflammatory cytokines. Bacteroides supplementation also reduced the fecal relative abundance of Escherichia coli, indicating a potential interaction between these two microbes, consistent with previous clinical studies. For a Bacteroides propionate mutant in the mouse model, pro-inflammatory cytokine KC is higher in the airway and serum compared with the wild-type (WT) strain, with no significant difference in the absolute abundance of these two strains. Taken together, our data indicate the potential multiple roles of Bacteroides-derived propionate in the modulation of systemic and airway inflammation and mediating the intestinal ecology of infants and children with CF. The roles of Bacteroides and the propionate it produces may help explain the observed gut-lung axis in CF and could guide the development of probiotics to mitigate systemic and airway inflammation for persons with CF.IMPORTANCEThe composition of the gut microbiome in persons with CF is correlated with lung health outcomes, a phenomenon referred to as the gut-lung axis. Here, we demonstrate that the intestinal microbe Bacteroides decreases inflammation through the production of the short-chain fatty acid propionate. Supplementing the levels of Bacteroides in an animal model of CF is associated with reduced systemic inflammation and reduction in the relative abundance of the opportunistically pathogenic group Escherichia/Shigella in the gut. Taken together, these data demonstrate a key role for Bacteroides and microbially produced propionate in modulating inflammation, gut microbial ecology, and the gut-lung axis in cystic fibrosis. These data support the role of Bacteroides as a potential probiotic in CF.

Microbial biofilms, as a hallmark of cystic fibrosis (CF) lung disease and other chronic infections, remain a desirable target for antimicrobial therapy. These biopolymer-based viscoelastic structures protect pathogenic organisms from immune responses and antibiotics. Consequently, treatments directed at disrupting biofilms represent a promising strategy for combating biofilm-associated infections. In CF patients, the viscoelasticity of biofilms is determined mainly by their polymicrobial nature and species-specific traits, such as Pseudomonas aeruginosa filamentous (Pf) bacteriophages. Therefore, we examined the impact of microbicidal ceragenins (CSAs) supported by mucolytic agents-DNase I and poly-aspartic acid (pASP), on the viability and viscoelasticity of mono- and bispecies biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains co-cultured with Staphylococcus aureus or Candida albicans.

The in vitro antimicrobial activity of ceragenins against P. aeruginosa in mono- and dual-species cultures was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Inhibition of P. aeruginosa mono- and dual-species biofilms formation by ceragenins alone and in combination with DNase I or poly-aspartic acid (pASP) was estimated by the crystal violet assay. Additionally, the viability of the biofilms was measured by colony-forming unit (CFU) counting. Finally, the biofilms' viscoelastic properties characterized by shear storage (G') and loss moduli (G"), were analyzed with a rotational rheometer.

Our results demonstrated that ceragenin CSA-13 inhibits biofilm formation and increases its fluidity regardless of the Pf-profile and species composition; however, the Pf-positive biofilms are characterized by elevated viscosity and elasticity parameters.

Due to its microbicidal and viscoelasticity-modifying properties, CSA-13 displays therapeutic potential in biofilm-associated infections, especially when combined with mucolytic agents.

Chronic rhinosinusitis (CRS) is a debilitating condition characterized by long-lasting inflammation of the paranasal sinuses. It affects a significant portion of the population, causing a considerable burden on individuals and healthcare systems. The pathogenesis of CRS is multifactorial, with bacterial infections playing a crucial role in CRS development and persistence. In recent years, the presence of biofilms has emerged as a key contributor to the chronicity of sinusitis, further complicating treatment and exacerbating symptoms. This review aims to explore the role of biofilms in CRS, focusing on the involvement of the bacterial species Staphylococcus aureus and Pseudomonas aeruginosa, their interactions in chronic infections, and model systems for studying biofilms in CRS. These species serve as an example of how microbial interplay can influence disease progression and exemplify the need for continued investigation and innovation in CRS research.

Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long infections of the respiratory mucosa caused by a diverse array of opportunists, which are leading causes of morbidity and mortality. In recent years, there has been increased appreciation for the range and diversity of microbes causing CF-related respiratory infections. The introduction of new therapeutics and improved detection methodology has revealed CF-related opportunists such as Achromobacter xylosoxidans (Ax). Ax is a Gram-negative bacterial species which is widely distributed in environmental sources and has been increasingly observed in sputa and other samples from pwCF, typically in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors including flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized cultures of CFBE41o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization of cell layers. Ax colonized and persisted in mouse lungs for up to 72 h post infection, with inflammatory consequences that include increased neutrophil influx in the lung, lung damage, cytokine production, and mortality. We also identified genes that are differentially expressed in synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.

Cystic fibrosis (CF) is widely known as a disease of the lung, even though it is in truth a systemic disease, whose symptoms typically manifest in gastrointestinal dysfunction first. CF ultimately impairs not only the pancreas and intestine but also the lungs, gonads, liver, kidneys, bones, and the cardiovascular system. It is caused by one of several mutations in the gene of the epithelial ion channel protein CFTR. Intense research and improved antimicrobial treatments during the past eight decades have steadily increased the predicted life expectancy of a person with CF (pwCF) from a few weeks to over 50 years. Moreover, several drugs ameliorating the sequelae of the disease have become available in recent years, and notable treatments of the root cause of the disease have recently generated substantial improvements in health for some but not all pwCF. Yet, numerous fundamental questions remain unanswered. Complicating CF, for instance in the lung, is the fact that the associated insufficient chloride secretion typically perturbs the electrochemical balance across epithelia and, in the airways, leads to the accumulation of thick, viscous mucus and mucus plaques that cannot be cleared effectively and provide a rich breeding ground for a spectrum of bacterial and fungal communities. The subsequent infections often become chronic and respond poorly to antibiotic treatments, with outcomes sometimes only weakly correlated with the drug susceptibility of the target pathogen. Furthermore, in contrast to rapidly resolved acute infections with a single target pathogen, chronic infections commonly involve multi-species bacterial communities, called "infection microbiomes," that develop their own ecological and evolutionary dynamics. It is presently impossible to devise mathematical models of CF in its entirety, but it is feasible to design models for many of the distinct drivers of the disease. Building upon these growing yet isolated modeling efforts, we discuss in the following the feasibility of a multi-scale modeling framework, known as template-and-anchor modeling, that allows the gradual integration of refined sub-models with different granularity. The article first reviews the most important biomedical aspects of CF and subsequently describes mathematical modeling approaches that already exist or have the potential to deepen our understanding of the multitude aspects of the disease and their interrelationships. The conceptual ideas behind the approaches proposed here do not only pertain to CF but are translatable to other systemic diseases. This article is categorized under: Congenital Diseases > Computational Models.

PwCF commonly test positive for pathogenic fungi, and more than 90% of the cystic fibrosis patient population is approved for the modulator treatment, Trikafta. Therefore, it is critical to understand how fungal communities, specifically A. fumigatus, respond to Trikafta exposure. Therefore, we sought to determine whether Trikafta impacted the biology of A. fumigatus biofilms. Our data demonstrate that Trikafta reduces biomass in several laboratory strains as well as clinical strains isolated from the expectorated sputum of pwCF. Furthermore, Trikafta reduces fungal viability and the capacity of biofilms to recover following treatment. Of particular importance, Trikafta affects how A. fumigatus biofilms respond to cell wall stressors, suggesting that Trikafta modulates components of the cell wall. Since the cell wall directly affects how a host immune system will respond to and effectively neutralize pathogens, our work, demonstrating that Trikafta impacts the A. fumigatus cell wall, is potentially highly relevant to fungal-induced disease pathogenesis.

Mucins are important glycoproteins that form a protective layer throughout the gastrointestinal and respiratory tracts. There is scientific evidence of increase in phage-resistance in the presence of mucin for some bacterial pathogens. Manipulation in mucin composition may ultimately influence the effectiveness of phage therapy. In this work, two clinical strains of K. pneumoniae (K3574 and K3325), were exposed to the lytic bacteriophage vB_KpnS-VAC35 in the presence and absence of mucin on a long-term co-evolution assay, in an attempt to mimic in vitro the exposure to mucins that bacteria and their phages face in vivo. Enumerations of the bacterial and phage counts at regular time intervals were conducted, and extraction of the genomic DNA of co-evolved bacteria to the phage, the mucin and both was performed. We determined the frequency of phage-resistant mutants in the presence and absence of mucin and including a mucolytic agent (N-acetyl L-cysteine, NAC), and sequenced them using Nanopore. We phenotypically demonstrated that the presence of mucin induces the emergence of bacterial resistance against lytic phages, effectively decreased in the presence of NAC. In addition, the genomic analysis revealed some of the genes relevant to the development of phage resistance in long-term co-evolution, with a special focus on the mucoid environment. Genes involved in the metabolism of carbohydrates were mutated in the presence of mucin. In conclusion, the use of mucolytic agents prior to the administration of lytic phages could be an interesting therapeutic option when addressing K. pneumoniae infections in environments where mucin is overproduced.

Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and an increasingly frequent cause of opportunistic infections. Mycobacterium abscessus complex (MABC) is one of the major NTM lung pathogens that disproportionately colonize and infect the lungs of individuals with cystic fibrosis (CF). MABC infection can persist for years, and antimicrobial treatment is frequently ineffective.

We sequenced the genomes of 175 isolates longitudinally collected from 30 patients with MABC lung infection. We contextualized our cohort amidst the broader MABC phylogeny and investigated genes undergoing parallel adaptation across patients. Finally, we tested the phenotypic consequences of parallel mutations by conducting antimicrobial resistance and mercury-resistance assays.

We identified highly related isolate pairs across hospital centers with low likelihood of transmission. We further annotated nonrandom parallel mutations in 22 genes and demonstrated altered macrolide susceptibility co-occurring with a nonsynonymous whiB1 mutation. Finally, we highlighted a 23-kb mercury-resistance plasmid whose loss during chronic infection conferred phenotypic susceptibility to organic and nonorganic mercury compounds.

We characterized parallel genomic processes through which MABC is adapting to promote survival within the host. The within-lineage polymorphisms we observed have phenotypic effects, potentially benefiting fitness in the host at the putative detriment of environmental survival.

This review addresses the topic of biofilms, including their development and the interaction between different counterparts. There is evidence that various diseases, such as cystic fibrosis, otitis media, diabetic foot wound infections, and certain cancers, are promoted and aggravated by the presence of polymicrobial biofilms. Biofilms are composed by heterogeneous communities of microorganisms protected by a matrix of polysaccharides. The different types of interactions between microorganisms gives rise to an increased resistance to antimicrobials and to the host's defense mechanisms, with the consequent worsening of disease symptoms. Therefore, infections caused by polymicrobial biofilms affecting different human organs and systems will be discussed, as well as the role of the interactions between the gram-negative bacteria Pseudomonas aeruginosa, which is at the base of major polymicrobial infections, and other bacteria, fungi, and viruses in the establishment of human infections and diseases. Considering that polymicrobial biofilms are key to bacterial pathogenicity, it is fundamental to evaluate which microbes are involved in a certain disease to convey an appropriate and efficacious antimicrobial therapy.

The polymicrobial biofilm (PMBF) is formed when microbes from multiple species co-aggregate into an envelope made of extra polymeric substances (EPS) that keep the microbes safe from external stresses. The formation of PMBF has been linked to a variety of human infections, including cystic fibrosis, dental caries, urinary tract infections, etc. Multiple microbial species co-aggregation during an infection results in a recalcitrant biofilm formation, which is a seriously threatening phenomenon. It is challenging to treat polymicrobial biofilms since they contain multiple microbes which show drug resistance to various antibiotics/antifungals. The present study discusses various approaches by which an antibiofilm compound works. Depending on their mode of action, antibiofilm compounds can block the adhesion of cells to one another, modify membranes/walls, or disrupt quorum-sensing systems.

SARS-CoV-2, the etiological cause of the COVID-19 pandemic, can cause severe illness in certain at-risk populations, including people with cystic fibrosis (pwCF). Nevertheless, several studies indicated that pwCF do not have higher risks of SARS-CoV-2 infection nor do they demonstrate worse clinical outcomes than those of the general population. Recent in vitro studies indicate cellular and molecular processes to be significant drivers in pwCF lower infection rates and milder symptoms than expected in cases of SARS-CoV-2 infection. These range from cytokine releases to biochemical alterations leading to morphological rearrangements inside the cells associated with CFTR impairment. Based on available data, the reported low incidence of SARS-CoV-2 infection among pwCF is likely a result of several variables linked to CFTR dysfunction, such as thick mucus, IL-6 reduction, altered ACE2 and TMPRSS2 processing and/or functioning, defective anions exchange, and autophagosome formation. An extensive analysis of the relation between SARS-CoV-2 infection and pwCF is essential to elucidate the mechanisms involved in this lower-than-expected infection impact and to possibly suggest potential new antiviral strategies.

Cystic fibrosis (CF) is a genetic disease affecting epithelial ion transport, resulting in thickened mucus and impaired mucociliary clearance. Persons with CF (pwCF) experience life-long respiratory mucosal infections caused by a diverse array of opportunists, and these infections are a leading cause of morbidity and mortality for pwCF. In recent years, there has been increased appreciation for the range and diversity of microbes in CF-related respiratory infections. Introduction of new therapeutics and improved detection methodology has revealed CF related opportunists such as Achromobacter xylosoxidans (Ax). Ax is a Gram-negative bacterial species that is widely distributed in the environment and has been increasingly observed in sputa and other samples from pwCF; typically Ax infections occur in patients in later stages of CF disease. In this study, we characterized CF clinical isolates of Ax and tested colonization and persistence of Ax in respiratory infection using immortalized human CF respiratory epithelial cells and BALB/c mice. Genomic analyses of clinical Ax isolates showed homologs for factors involved in flagellar synthesis, antibiotic resistance, and toxin secretion systems. Ax isolates adhered to polarized CFBE14o- human immortalized CF bronchial epithelial cells and caused significant cytotoxicity and depolarization. Ax colonized and persisted in mouse lung for up to 72 hours post infection, with inflammatory consequences that include increased neutrophilia, lung damage, cytokine production, and mortality. Transcript profiling reveled differential expression of Ax genes during growth in SCFM2 synthetic CF sputum media. Based on these results, we conclude that Ax is an opportunistic pathogen of significance in CF.

Antibiotic resistance is a major medical and public health challenge, characterized by global increases in the prevalence of resistant strains. The conventional view is that all antibiotic resistance is problematic, even when not in pathogens. Resistance in commensal bacteria poses risks, as resistant organisms can provide a reservoir of resistance genes that can be horizontally transferred to pathogens or may themselves cause opportunistic infections in the future. While these risks are real, we propose that commensal resistance can also generate benefits during antibiotic treatment of human infection, by promoting continued ecological suppression of pathogens. To define and illustrate this alternative conceptual perspective, we use a two-species mathematical model to identify the necessary and sufficient ecological conditions for beneficial resistance. We show that the benefits are limited to species (or strain) interactions where commensals suppress pathogen growth and are maximized when commensals compete with, rather than prey on or otherwise exploit pathogens. By identifying benefits of commensal resistance, we propose that rather than strictly minimizing all resistance, resistance management may be better viewed as an optimization problem. We discuss implications in two applied contexts: bystander (nontarget) selection within commensal microbiomes and pathogen treatment given polymicrobial infections. IMPORTANCE Antibiotic resistance is commonly viewed as universally costly, regardless of which bacterial cells express resistance. Here, we derive an opposing logic, where resistance in commensal bacteria can lead to reductions in pathogen density and improved outcomes on both the patient and public health scales. We use a mathematical model of commensal-pathogen interactions to define the necessary and sufficient conditions for beneficial resistance, highlighting the importance of reciprocal ecological inhibition to maximize the benefits of resistance. More broadly, we argue that determining the benefits as well as the costs of resistances in human microbiomes can transform resistance management from a minimization to an optimization problem. We discuss applied contexts and close with a review of key resistance optimization dimensions, including the magnitude, spectrum, and mechanism of resistance.

Several reports have indicated that SARS-CoV-2 infection displays unexpected mild clinical manifestations in people with cystic fibrosis (pwCF), suggesting that CFTR expression and function may be involved in the SARS-CoV-2 life cycle. To evaluate the possible association of CFTR activity with SARS-CoV-2 replication, we tested the antiviral activity of two well-known CFTR inhibitors (IOWH-032 and PPQ-102) in wild type (WT)-CFTR bronchial cells. SARS-CoV-2 replication was inhibited by IOWH-032 treatment, with an IC₅₀ of 4.52 μM, and by PPQ-102, with an IC₅₀ of 15.92 μM. We confirmed this antiviral effect on primary cells (MucilAir^TM wt-CFTR) using 10 μM IOWH-032. According to our results, CFTR inhibition can effectively tackle SARS-CoV-2 infection, suggesting that CFTR expression and function might play an important role in SARS-CoV-2 replication, revealing new perspectives on the mechanisms governing SARS-CoV-2 infection in both normal and CF individuals, as well as leading to potential novel treatments.