Infections In Cystic Fibrosis Patients Research Articles

Structural determinants of protein kinase A essential for CFTR channel activation

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R) domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction essential for these processes. By comparing CFTR current activation in the presence of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate peptides of various lengths, we identify two distinct specific regions of the PKA-C surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation, achieved by stabilizing the R domain in a "released" conformation permissive to channel gating. Furthermore, by comparing PKA-C variants with different posttranslational modification patterns, we find that direct membrane tethering of the kinase through its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic, CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify virulence factors that demyristoylate PKA-C in vitro, raising the possibility that during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute to the exacerbation of lung disease.

Therapeutic Interventions for Pseudomonas Infections in Cystic Fibrosis Patients: A Review of Phase IV Trials.

Pseudomonas aeruginosa (Pa) poses a significant threat to individuals with cystic fibrosis (CF), as this bacterium is highly adaptable and resistant to antibiotics. While early-stage Pa infections can often be eradicated with aggressive antibiotic therapy, chronic infections are nearly impossible to eliminate and require treatments that focus on long-term bacterial suppression. Without such suppression, these persistent infections can severely damage the lungs, leading to serious complications and a reduced life expectancy for CF patients. Evidence for a specific treatment regimen for managing Pa infections in CF patients remains limited. This narrative review provides a detailed analysis of antimicrobial therapies assessed in completed phase IV trials, focusing on their safety and efficacy, especially with prolonged use. Key antibiotics, including tobramycin, colistin, meropenem, aztreonam, ceftolozane/tazobactam, ciprofloxacin, and azithromycin, are discussed, emphasizing their use, side effects, and delivery methods. Inhaled antibiotics are preferred for their targeted action and minimal side effects, while systemic antibiotics offer potency but carry risks like nephrotoxicity. The review also explores emerging treatments, such as phage therapy and antibiofilm agents, which show promise in managing chronic infections. Nonetheless, further research is necessary to enhance the safety and effectiveness of existing therapies while investigating new approaches for better long-term outcomes.

A dTDP-L-rhamnose 4-epimerase required for glycopeptidolipid biosynthesis in Mycobacterium abscessus

Mycobacterium abscessus causes severe lung infections in cystic fibrosis patients and exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in the S-to-R transition but the underlying molecular mechanisms of this transition remain incompletely understood. Herein, we characterized MAB_4111c in relation to GPL synthesis and investigated the effects of MAB_4111c deletion in M. abscessus pathogenicity. An enzymatic assay indicated that MAB_4111c, also designated Tle for Talose epimerase, is converting dTDP-L-Rhamnose into dTDP-6-deoxy-L-Talose. A tle deletion mutant was constructed in the S variant of M. abscessus and relative areas of Rhamnose and 6-deoxy-Talose and their methylated forms expressed as ratios of total monosaccharides, showed an altered GPL profile lacking 6-deoxy-Talose. Thus, Tle provides dTDP-6-deoxy-L-Talose, subsequently used by the glycosyltransferase Gtf1 to transfer 6-deoxy-Talose to the GPL backbone. Strikingly, the tle mutant exhibited a R morphotype, showed impaired sliding motility and biofilm formation, and these phenotypes were rescued upon functional complementation. Moreover, deletion of tle in M. abscessus results in increased pathogenicity and killing in zebrafish embryos. Together, our results underscore the importance of the dTDP-L-Rhamnose 4-epimerase activity in GPL biosynthesis and in influencing M. abscessus virulence.

Highly effective inhalable voriconazole-loaded nanomicelles for fungal infections in cystic fibrosis patients: A promising therapeutic strategy for allergic bronchopulmonary aspergillosis

In patients with cystic fibrosis (CF), growth of A. fumigatus and other fungal hyphae within the bronchial lumen can trigger an immunoglobulin E-mediated hypersensitivity response known as allergic bronchopulmonary aspergillosis (ABPA) which can potentially advance to fibrosis.The management of ABPA involves the administration of systemic glucocorticoids alongside azole antifungals, such as voriconazole (VRC). This study evaluated the potential of Soluplus® polymeric micelles as an effective nanocarrier for pulmonary delivery of VRC to improve antifungal therapy in CF patients. The micellar nanoformulation prepared in this study could increase up to 51-fold the aqueous solubility of VRC. Their micellar size, evaluated by dynamic light scattering, was 102.2 ± 1.3 nm for loaded freeze-dried nanoformulations with a unimodal size distribution. Nanomicelles showed excellent colloidal stability during the in vitro nebulization process and under dilution in similar interstitial fluids for pulmonary VRC delivery. Nanoformulations demonstrated high in vitro cytocompatibility in A549 and Vero cells. Interestingly, in vitro antifungal activity of VRC-loaded micelles was at least 2-fold higher than the drug aqueous suspension against different fungal species isolated from CF sputum, regardless of the presence of artificial CF mucus layer. Following single intratracheal dose, significantly (p < 0.05) higher VRC concentrations were observed within 2 h in the lung tissue for the micellar formulations compared with the VRC suspension. We concluded that these findings will help to develop a potent and innovative inhalable VRC micellar nanoformulation that may be effective against different lung disorders caused by fungi, such as ABPA.

Rapid differentiation of cystic fibrosis-related bacteria via reagentless atmospheric pressure photoionisation mass spectrometry

Breath analysis is an area of significant interest in medical research as it allows for non-invasive sampling with exceptional potential for disease monitoring and diagnosis. Volatile organic compounds (VOCs) found in breath can offer critical insight into a person’s lifestyle and/or disease/health state. To this end, the development of a rapid, sensitive, cost-effective and potentially portable method for the detection of key compounds in breath would mark a significant advancement. Herein, we have designed, built and tested a novel reagent-less atmospheric pressure photoionisation (APPI) source, coupled with mass spectrometry (MS), utilising a bespoke bias electrode within a custom 3D printed sampling chamber for direct analysis of VOCs. Optimal APPI-MS conditions were identified, including bias voltage, cone voltage and vaporisation temperature. Calibration curves were produced for ethanol, acetone, 2-butanone, ethyl acetate and eucalyptol, yielding R2 > 0.99 and limits of detection < 10 pg. As a pre-clinical proof of concept, this method was applied to bacterial headspace samples of Escherichia coli (EC), Pseudomonas aeruginosa (PSA) and Staphylococcus aureus (SA) collected in 1 L Tedlar bags. In particular, PSA and SA are commonly associated with lung infection in cystic fibrosis patients. The headspace samples were classified using principal component analysis with 86.9% of the total variance across the first three components and yielding 100% classification in a blind-sample study. All experiments conducted with the novel APPI arrangement were carried out directly in real-time with low-resolution MS, which opens up exciting possibilities in the future for on-site (e.g., in the clinic) analysis with a portable system.

Insights into Aspergillus fumigatus Colonization in Cystic Fibrosis and Cross-Transmission between Patients and Hospital Environments.

Approximately 60% of individuals with cystic fibrosis (CF) are affected by Aspergillus fumigatus infection. This condition is correlated with a decline in lung function and is identified as an independent risk factor contributing to hospital admissions among CF patients. This study investigates the dynamic interplay of A. fumigatus within the context of CF patients, tracing its evolution over time, with a specific emphasis on colonization dynamics. An analysis was conducted on 83 sequential A. fumigatus isolates derived from sputum samples of six patients receiving care at a renowned CF hospital in Brazil. Employing microsatellite genotyping techniques, alongside an investigation into cyp51A gene mutations, this research sheds light on the genetic variations, colonization, and resistance of A. fumigatus within the CF respiratory environment. Our research findings indicate that CF patients can harbor A. fumigatus strains from the same clonal complexes for prolonged periods. Additionally, we identified that clinical isolates have the potential to spread among patients in the same healthcare facility, evidencing hospital contamination. Two patients who underwent long-term Itraconazole treatment did not show phenotypic resistance. However, one of these patients exhibited mutations in the cyp51A gene, indicating the need to monitor resistance to azoles in these patients colonized for long periods by A. fumigatus. We also observed co-colonization or co-infection involving multiple genotypes in all patients over time. This comprehensive examination offers valuable insights into the pathogenesis of A. fumigatus infections in CF patients, potentially shaping future therapeutic strategies and management approaches. This enhanced understanding contributes to our knowledge of A. fumigatus impact on disease progression in individuals with cystic fibrosis. Additionally, the study provides evidence of cross-contamination among patients undergoing treatment at the same hospital.

Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

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

P209 Mucus composition and interactions with Pseudomonas aeruginosa in chronically infected cystic fibrosis and primary ciliary dyskinesia patients

P209 Mucus composition and interactions with Pseudomonas aeruginosa in chronically infected cystic fibrosis and primary ciliary dyskinesia patients

Relationship Between Biophysical Properties of Antimicrobial Peptides (AMPs) and their Associated Drug Efficacies

Antibiotic resistance is a growing global health threat. One consequence is that patients with cystic fibrosis (CF) are prone to developing antibiotic resistant lung infections caused by multiple strains of bacteria, including Pseudomonas aeruginosa. Due to the limited number of treatment options for patients with chronic antibiotic resistant infections, there is a need for finding new antibiotics that allow for effective eradication of bacterial infections, such as those in the CF lung. Many antimicrobial peptides (AMPs) have been annotated in databases and are considered as potential alternatives for current antibiotics. However, in many instances, the suitability of AMPs as drug molecules has not been extensively explored. Here, we propose that certain molecular properties of AMPs favor high antibiotic efficacy. Using information from AMP databases, we combined statistical analyses and machine learning techniques to identify relationships between various biophysical properties of AMPs and their drug efficacies. Analyses from classification and regression trees (CART) and random forests suggest that net charge and maximum average hydrophobic moment are the most important properties in determining if a peptide is useful against P. aeruginosa infections in CF patients. Maximum average hydrophobic residue, average alpha helix propensity score, hydrophobic proportion, and peptide length still contribute to this determination but to lesser degrees. Cation-pi interactions, on the other hand, do not appear to factor into this decision at all. Based on these properties, our current work is focused on designing and experimentally testing new peptides that may have activity against P. aeruginosa infections.

Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Lsr2, a pleiotropic regulator at the core of the infectious strategy of Mycobacterium abscessus.

Mycobacterium abscessus is a non-tuberculous mycobacterium, causing lung infections in cystic fibrosis patients. During pulmonary infection, M. abscessus switches from smooth (Mabs-S) to rough (Mabs-R) morphotypes, the latter being hyper-virulent. Previously, we isolated the lsr2 gene as differentially expressed during S-to-R transition. lsr2 encodes a pleiotropic transcription factor that falls under the superfamily of nucleoid-associated proteins. Here, we used two functional genomic methods, RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), to elucidate the molecular role of Lsr2 in the pathobiology of M. abscessus. Transcriptomic analysis shows that Lsr2 differentially regulates gene expression across both morphotypes, most of which are involved in several key cellular processes of M. abscessus, including host adaptation and antibiotic resistance. These results were confirmed through quantitative real-time PCR, as well as by minimum inhibitory concentration tests and infection tests on macrophages in the presence of antibiotics. ChIP-seq analysis revealed that Lsr2 extensively binds the M. abscessus genome at AT-rich sequences and appears to form long domains that participate in the repression of its target genes. Unexpectedly, the genomic distribution of Lsr2 revealed no distinctions between Mabs-S and Mabs-R, implying more intricate mechanisms at play for achieving target selectivity.IMPORTANCELsr2 is a crucial transcription factor and chromosome organizer involved in intracellular growth and virulence in the smooth and rough morphotypes of Mycobacterium abscessus. Using RNA-seq and chromatin immunoprecipitation-sequencing (ChIP-seq), we investigated the molecular role of Lsr2 in gene expression regulation along with its distribution on M. abscessus genome. Our study demonstrates the pleiotropic regulatory role of Lsr2, regulating the expression of many genes coordinating essential cellular and molecular processes in both morphotypes. In addition, we have elucidated the role of Lsr2 in antibiotic resistance both in vitro and in vivo, where lsr2 mutant strains display heightened sensitivity to antibiotics. Through ChIP-seq, we reported the widespread distribution of Lsr2 on M. abscessus genome, revealing a direct repressive effect due to its extensive binding on promoters or coding sequences of its targets. This study unveils the significant regulatory role of Lsr2, intricately intertwined with its function in shaping the organization of the M. abscessus genome.

Fungal Respiratory Colonization and Infections in Cystic Fibrosis Patients in the Middle East

Fungal Respiratory Colonization and Infections in Cystic Fibrosis Patients in the Middle East

Phage Milagro: a platform for engineering a broad host range virulent phage for Burkholderia.

Burkholderia infections are a significant concern in people with CF and other immunocompromising disorders, and are difficult to treat with conventional antibiotics due to their inherent drug resistance. Bacteriophages, or bacterial viruses, are now seen as a potential alternative therapy for these infections, but most of the naturally occurring phages are temperate and have narrow host ranges, which limit their utility as therapeutics. Here we describe the temperate Burkholderia phage Milagro and our efforts to engineer this phage into a potential therapeutic by expanding the phage host range and selecting for phage mutants that are strictly virulent. This approach may be used to generate new therapeutic agents for treating intractable infections in CF patients.

Neutrophil bactericidal activity and host defenses in cystic fibrosis: a narrative review.

Cystic fibrosis (CF) is a disorder that affects the cystic fibrosis transmembrane conductance regulator (CFTR). Without properly functioning CFTR channels, chloride does not exit respiratory epithelial cells, and consequently the mucus lining the surface of the cells becomes thick. This viscous mucus accumulates and causes abnormal function of the mucociliary apparatus, which can lead to bacterial colonization, infections with Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa), and eventually lung damage. Recent studies have shown that the increased susceptibility to respiratory infections in CF patients may also be due to defects in neutrophil function, but the exact mechanism is uncertain. The PubMed database was searched on February 10, 2023 and again on July 23, 2023 to compile a comprehensive list of clinical and experimental studies to evaluate neutrophil function in CF. The first search included a combination of MeSH terms: "cystic fibrosis" and "neutrophils/physiology". A separate second search included a combination of the MeSH terms: "neutrophils" and "cystic fibrosis transmembrane conductance regulator". Neutrophils from patients with CF have decreased transfer of chloride into phagolysosomes after bacterial ingestion and have dysregulated degranulation. This reduces the production of toxic oxidative radicals, especially hypochlorous acid (HOCl), and reduces bactericidal activity. CFTR potentiators correct the dysregulated degranulation in patients with CF and increased neutrophil killing activity. A reduced concentration of chloride in in vitro assays also reduces neutrophil killing activity; these observations are relevant to the reduced chloride concentrations in respiratory secretions in patients with CF. This literature review summarizes studies that demonstrate that an important defect in CF neutrophils lies in the oxygen-dependent pathway in phagolysosomes and studies with ivacaftor demonstrate that this drug corrects CF neutrophil function. These studies demonstrate the potential utility of using easily available neutrophils to study drug effects in CF patients.

Development of Quinazolinone Derivatives as Modulators of Virulence Factors of Pseudomonas aeruginosa Cystic Fibrosis Strains.

Pseudomonas aeruginosa (PA), one of the ESKAPE pathogens, is an opportunistic Gram-negative bacterium responsible for nosocomial infections in humans but also for infections in patients affected by AIDS, cancer, or cystic fibrosis (CF). Treatment of PA infections in CF patients is a global healthcare problem due to the ability of PA to gain antibiotic tolerance through biofilm formation. Anti-virulence compounds represent a promising approach as adjuvant therapy, which could reduce or eliminate the pathogenicity of PA without impacting its growth. Pyocyanin is one of the virulence factors whose production is modulated by the Pseudomonas quinolone signal (PQS) through its receptor PqsR. Different PqsR modulators have been synthesized over the years, highlighting this new powerful therapeutic strategy. Based on the promising structure of quinazolin-4(3H)-one, we developed compounds 7a-d, 8a,b, 9, 10, and 11a-f able to reduce biofilm formation and the production of virulence factors (pyocyanin and pyoverdine) at 50 µM in two PA strains responsible for CF acute and chronic infections. The developed compounds did not reduce the cell viability of IB3-1 bronchial CF cells, and computational studies confirmed the potential ability of novel compounds to act as potential Pqs system modulators.

Transposon sequencing identifies genes impacting Staphylococcus aureus invasion in a human macrophage model.

Staphylococcus aureus is a facultative intracellular pathogen in many host cell types, facilitating its persistence in chronic infections. The genes contributing to intracellular pathogenesis have not yet been fully enumerated. Here, we cataloged genes influencing S. aureus invasion and survival within human THP-1 derived macrophages using two laboratory strains (ATCC2913 and JE2). We developed an in vitro transposition method to produce highly saturated transposon mutant libraries in S. aureus and performed transposon insertion sequencing (Tn-Seq) to identify candidate genes with significantly altered abundance following macrophage invasion. While some significant genes were strain-specific, 108 were identified as common across both S. aureus strains, with most (n = 106) being required for optimal macrophage infection. We used CRISPR interference (CRISPRi) to functionally validate phenotypic contributions for a subset of genes. Of the 20 genes passing validation, seven had previously identified roles in S. aureus virulence, and 13 were newly implicated. Validated genes frequently evidenced strain-specific effects, yielding opposing phenotypes when knocked down in the alternative strain. Genomic analysis of de novo mutations occurring in groups (n = 237) of clonally related S. aureus isolates from the airways of chronically infected individuals with cystic fibrosis (CF) revealed significantly greater in vivo purifying selection in conditionally essential candidate genes than those not associated with macrophage invasion. This study implicates a core set of genes necessary to support macrophage invasion by S. aureus, highlights strain-specific differences in phenotypic effects of effector genes, and provides evidence for selection of candidate genes identified by Tn-Seq analyses during chronic airway infection in CF patients in vivo.

Higher levels of Pseudomonas aeruginosa LasB elastase expression are associated with early-stage infection in cystic fibrosis patients

Pseudomonas aeruginosa is a common pathogen in cystic fibrosis (CF) patients and a major contributor to progressive lung damage. P. aeruginosa elastase (LasB), a key virulence factor, has been identified as a potential target for anti-virulence therapy. Here, we sought to differentiate the P. aeruginosa isolates from early versus established stages of infection in CF patients and to determine if LasB was associated with either stage. The lasB gene was amplified from 255 P. aeruginosa clinical isolates from 70 CF patients from the Toulouse region (France). Nine LasB variants were identified and 69% of the isolates produced detectable levels of LasB activity. Hierarchical clustering using experimental and clinical data distinguished two classes of isolates, designated as ‘Early’ and ‘Established’ infection. Multivariate analysis revealed that the isolates from the Early infection class show higher LasB activity, fast growth, tobramycin susceptibility, non-mucoid, pigmented colonies and wild-type lasR genotype. These traits were associated with younger patients with polymicrobial infections and high pFEV1. Our findings show a correlation between elevated LasB activity in P. aeruginosa isolates and early-stage infection in CF patients. Hence, it is this patient group, prior to the onset of chronic disease, that may benefit most from novel therapies targeting LasB.

Antibacterial and Antibiofilm Effects of Lactobacilli Strains against Clinical Isolates of Pseudomonas aeruginosa under Conditions Relevant to Cystic Fibrosis.

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

In Vitro Activity of Finafloxacin against Panels of Respiratory Pathogens.

This study determined the in vitro activity of finafloxacin against panels of bacterial strains, representative of those associated with infection in cystic fibrosis patients and predominately isolated from clinical cases of respiratory disease. Many of these isolates were resistant to various antimicrobials evaluated including the aminoglycosides, cephalosporins, carbapenems and fluoroquinolones. Broth microdilution assays were performed at neutral and acidic pH, to determine antimicrobial activity. Finafloxacin demonstrated superior activity at reduced pH for all of the bacterial species investigated, highlighting the requirement to determine the activity of antimicrobials in host-relevant conditions.

Repurposing High-Throughput Screening Identifies Unconventional Drugs with Antibacterial and Antibiofilm Activities against Pseudomonas aeruginosa under Experimental Conditions Relevant to Cystic Fibrosis.

Pseudomonas aeruginosa is the most common pathogen infecting cystic fibrosis (CF) lungs, causing acute and chronic infections. Intrinsic and acquired antibiotic resistance allow P. aeruginosa to colonize and persist despite antibiotic treatment, making new therapeutic approaches necessary. Combining high-throughput screening and drug repurposing is an effective way to develop new therapeutic uses for drugs. This study screened a drug library of 3,386 drugs, mostly FDA approved, to identify antimicrobials against P. aeruginosa under physicochemical conditions relevant to CF-infected lungs. Based on the antibacterial activity, assessed spectrophotometrically against the prototype RP73 strain and 10 other CF virulent strains, and the toxic potential evaluated toward CF IB3-1 bronchial epithelial cells, five potential hits were selected for further analysis: the anti-inflammatory and antioxidant ebselen, the anticancer drugs tirapazamine, carmofur, and 5-fluorouracil, and the antifungal tavaborole. A time-kill assay showed that ebselen has the potential to cause rapid and dose-dependent bactericidal activity. The antibiofilm activity was evaluated by viable cell count and crystal violet assays, revealing carmofur and 5-fluorouracil as the most active drugs in preventing biofilm formation regardless of the concentration. In contrast, tirapazamine and tavaborole were the only drugs actively dispersing preformed biofilms. Tavaborole was the most active drug against CF pathogens other than P. aeruginosa, especially against Burkholderia cepacia and Acinetobacter baumannii, while carmofur, ebselen, and tirapazamine were particularly active against Staphylococcus aureus and B. cepacia. Electron microscopy and propidium iodide uptake assay revealed that ebselen, carmofur, and tirapazamine significantly damage cell membranes, with leakage and cytoplasm loss, by increasing membrane permeability. IMPORTANCE Antibiotic resistance makes it urgent to design new strategies for treating pulmonary infections in CF patients. The repurposing approach accelerates drug discovery and development, as the drugs' general pharmacological, pharmacokinetic, and toxicological properties are already well known. In the present study, for the first time, a high-throughput compound library screening was performed under experimental conditions relevant to CF-infected lungs. Among 3,386 drugs screened, the clinically used drugs from outside infection treatment ebselen, tirapazamine, carmofur, 5-fluorouracil, and tavaborole showed, although to different extents, anti-P. aeruginosa activity against planktonic and biofilm cells and broad-spectrum activity against other CF pathogens at concentrations not toxic to bronchial epithelial cells. The mode-of-action studies revealed ebselen, carmofur, and tirapazamine targeted the cell membrane, increasing its permeability with subsequent cell lysis. These drugs are strong candidates for repurposing for treating CF lung P. aeruginosa infections.