Airways Of Cystic Fibrosis Patients Research Articles

Fungal Colonization of the Airways of Patients with Cystic Fibrosis: the Role of the Environmental Reservoirs.

Filamentous fungi frequently colonize the airways of patients with cystic fibrosis and may cause severe diseases, such as the allergic bronchopulmonary aspergillosis. The most common filamentous fungi capable to chronically colonize the respiratory tract of the patients are Aspergillus fumigatus and Scedosporium species. Defining the treatment strategy may be challenging, the number of available drugs being limited and some of the causative agents being multiresistant microorganisms. The knowledge of the fungal niches in the outdoor and indoor environment is needed for understanding the origin of the contamination of the patients. In light of the abundance of some of the causative molds in compost, agricultural and flower fields, occupational activities related to such environments should be discouraged for patients with cystic fibrosis (CF). In addition, the microbiological monitoring of their indoor environment, including analysis of air and dust on surfaces, is essential to propose preventive measures aiming to reduce the exposure to environmental molds. Nevertheless, some specific niches were also identified in the indoor environment, in relation with humidity which favors the growth of thermotolerant molds. Potted plants were reported as indoor reservoirs for Scedosporium species. Likewise, Exophiala dermatitidis may be spread in the kitchen via dishwashers. However, genotype studies are still required to establish the link between dishwashers and colonization of the airways of CF patients by this black yeast. Moreover, as nothing is known regarding the other filamentous fungi associated with CF, further studies should be conducted to identify other potential specific niches in the habitat.

Pseudomonas aeruginosa heme metabolites biliverdin IXβ and IXδ are integral to lifestyle adaptations associated with chronic infection.

The opportunistic pathogen Pseudomonas aeruginosa causes long-term chronic infection in the airways of cystic fibrosis patients. The ability to scavenge iron and to establish chronic infection within this environment coincides with a switch to utilize heme as the primary iron source. Herein, we show the heme metabolites biliverdin beta and delta are themselves important signaling molecules integrating the switch in iron acquisition systems with cooperative behaviors such as motility and biofilm formation that are essential for long-term chronic infection. These significant findings will enhance the development of viable multi-targeted therapeutics effective against both heme utilization and cooperative behaviors essential for survival and persistence within the host.

Comparison of culture and PCR-DGGE methods to evaluate the airways of cystic fibrosis patients and determination of their antibiotic resistance profile.

Respiratory infections are the most serious condition in cystic fibrosis (CF) patients; therefore, a thorough comprehension of the diversity and dominant microbial species in CF airways has a crucial role in treatment. Our objective was to determine the antibiotic resistance profile of CF airways microbiota and compare culture methods and PCR-DGGE to evaluate bacterial diversity. Pharyngeal swabs from 121 CF patients were collected. The samples were then cultured, identified and antibiotic resistance testing was performed. Thirty samples were subjected to further molecular surveys. DNA contents of these samples were extracted and amplified using nested-PCR technique and their bacterial diversity was assessed by DGGE. The DGGE patterns were visualized and certain bands were excised and purified. Next, the DNA was amplified by another round of PCR and sent out for sequencing. Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae were the most prevalent species isolated using culture methods. S. aureus was the most common bacteria among 6 years and younger patients; while, P. aeruginosa had more prevalence among older ones. The PCR-DGGE results showed more diversity than culture methods, particularly in younger patients who exhibited more bacterial diversity than the older groups. Sequencing results unveiled the presence of certain bacterial species including Haemophilus parainfluenzae and Stenotrophomonas maltophilia which were completely missed in culture. Even though culture-dependent methods are cost-effective, PCR-DGGE appeared to be more efficient to determine bacterial diversity. PCR-DGGE detects less abundant species, though their viability could not be determined using this method.

Ceragenins exhibit bactericidal properties that are independent of the ionic strength in the environment mimicking cystic fibrosis sputum.

The purpose of the work was to investigate the impact of sodium chloride (NaCl) on the antimicrobial efficacy of ceragenins (CSAs) and antimicrobial peptides (AMPs) against bacterial and fungal pathogens associated with cystic fibrosis (CF) lung infections. CF-associated bacterial (Pseudomonas aeruginosa, Ochrobactrum spp., and Staphylococcus aureus), and fungal pathogens (Candida albicans, and Candida tropicalis) were used as target organisms for ceragenins (CSA-13 and CSA-131) and AMPs (LL-37 and omiganan). Susceptibility to the tested compounds was assessed using minimal inhibitory concentrations (MICs) and bactericidal concentrations (MBCs), as well as by colony counting assays in CF sputum samples supplemented with various concentrations of NaCl. Our results demonstrated that ceragenins exhibit potent antimicrobial activity in CF sputum regardless of the NaCl concentration when compared to LL-37 and omiganan. Given the broad-spectrum antimicrobial activity of ceragenins in the microenvironments mimicking the airways of CF patients, ceragenins might be promising agents in managing CF disease.

Genomic and Functional Characterization of Longitudinal Pseudomonas aeruginosa Isolates from Young Patients with Cystic Fibrosis.

Individuals with cystic fibrosis (CF) suffer from frequent and recurring microbial airway infections. The Gram-negative bacterium Pseudomonas aeruginosa is one of the most common organisms isolated from CF patient airways. P. aeruginosa establishes chronic infections that persist throughout a patient's lifetime and is a major cause of morbidity and mortality. Throughout the course of infection, P. aeruginosa must evolve and adapt from an initial state of early, transient colonization to chronic colonization of the airways. Here, we examined isolates of P. aeruginosa from children under the age of 3 years old with CF to determine genetic adaptations the bacterium undergoes during this early stage of colonization and infection. These isolates were collected when early aggressive antimicrobial therapy was not the standard of care and therefore highlight strain evolution under limited antibiotic pressure. Examination of specific phenotypic adaptations, such as lipid A palmitoylation, antibiotic resistance, and loss of quorum sensing, did not reveal a clear genetic basis for such changes. Additionally, we demonstrate that the geography of patient origin, within the United States or among other countries, does not appear to significantly influence genetic adaptation. In summary, our results support the long-standing model that patients acquire individual isolates of P. aeruginosa that subsequently become hyperadapted to the patient-specific airway environment. This study provides a multipatient genomic analysis of isolates from young CF patients in the United States and contributes data regarding early colonization and adaptation to the growing body of research about P. aeruginosa evolution in the context of CF airway disease. IMPORTANCE Chronic lung infection with Pseudomonas aeruginosa is of major concern for patients with cystic fibrosis (CF). During infection, P. aeruginosa undergoes genomic and functional adaptation to the hyperinflammatory CF airway, resulting in worsening lung function and pulmonary decline. All studies that describe these adaptations use P. aeruginosa obtained from older children or adults during late chronic lung infection; however, children with CF can be infected with P. aeruginosa as early as 3 months of age. Therefore, it is unclear when these genomic and functional adaptations occur over the course of CF lung infection, as access to P. aeruginosa isolates in children during early infection is limited. Here, we present a unique cohort of CF patients who were identified as being infected with P. aeruginosa at an early age prior to aggressive antibiotic therapy. Furthermore, we performed genomic and functional characterization of these isolates to address whether chronic CF P. aeruginosa phenotypes are present during early infection.

WS14.06 Single cell RNA sequencing of lung biopsies reveals altered immune-structural cell crosstalk in cystic fibrosis

Background: Cystic Fibrosis (CF) is a life-limiting genetic disease that involves chronic airway inflammation and progressive lung tissue remodeling, which are the main causes of mortality. A continuous crosstalk between immune cells and structural cells within the lung is essential for mounting effective immune responses and maintaining homeostasis. Many studies have investigated how individual cell types or classes are deregulated in CF, but few have studied the complete spectrum of lung resident cells. Therefore, we lack a complete overview of immune-structural cell crosstalk in CF lungs, which may be relevant for developing therapies aimed at restoring homeostasis in the airways of CF patients.

Mycobacterium abscessus DosRS two-component system controls a species-specific regulon required for adaptation to hypoxia.

Mycobacterium abscessus (Mab), an emerging opportunistic pathogen, predominantly infects individuals with underlying pulmonary diseases such as cystic fibrosis (CF). Current treatment outcomes for Mab infections are poor due to Mab's inherent antibiotic resistance and unique host interactions that promote phenotypic tolerance and hinder drug access. The hypoxic, mucus-laden airways in the CF lung and antimicrobial phagosome within macrophages represent hostile niches Mab must overcome via alterations in gene expression for survival. Regulatory mechanisms important for the adaptation and long-term persistence of Mab within the host are poorly understood, warranting further genetic and transcriptomics study of this emerging pathogen. DosRS Mab , a two-component signaling system (TCS), is one proposed mechanism utilized to subvert host defenses and counteract environmental stress such as hypoxia. The homologous TCS of Mycobacterium tuberculosis (Mtb), DosRS Mtb , is known to induce a ~50 gene regulon in response to hypoxia, carbon monoxide (CO) and nitric oxide (NO) in vitro and in vivo. Previously, a small DosR Mab regulon was predicted using bioinformatics based on DosR Mtb motifs however, the role and regulon of DosRS Mab in Mab pathogenesis have yet to be characterized in depth. To address this knowledge gap, our lab generated a Mab dosRS knockout strain (MabΔdosRS) to investigate differential gene expression, and phenotype in an in vitro hypoxia model of dormancy. qRT-PCR and lux reporter assays demonstrate Mab_dosR and 6 predicted downstream genes are induced in hypoxia. In addition, RNAseq revealed induction of a much larger hypoxia response comprised of >1000 genes, including 127 differentially expressed genes in a dosRS mutant strain. Deletion of DosRS Mab led to attenuated growth under low oxygen conditions, a shift in morphotype from smooth to rough, and down-regulation of 216 genes. This study provides the first look at the global transcriptomic response of Mab to low oxygen conditions encountered in the airways of CF patients and within macrophage phagosomes. Our data also demonstrate the importance of DosRS Mab for adaptation of Mab to hypoxia, highlighting a distinct regulon (compared to Mtb) that is significantly larger than previously described, including both genes conserved across mycobacteria as well as Mab-specific genes.

The Cystic Fibrosis Upper and Lower Airway Metagenome.

The microbial metagenome in cystic fibrosis (CF) airways was investigated by whole-genome shotgun sequencing of total DNA isolated from nasal lavage samples, oropharyngeal swabs, and induced sputum samples collected from 65 individuals with CF aged 7 to 50 years. Each patient harbored a personalized microbial metagenome unique in microbial load and composition, the exception being monocultures of the most common CF pathogens Staphylococcus aureus and Pseudomonas aeruginosa from patients with advanced lung disease. The sampling of the upper airways by nasal lavage uncovered the fungus Malassezia restricta and the bacterium Staphylococcus epidermidis as prominent species. Healthy and CF donors harbored qualitatively and quantitatively different spectra of commensal bacteria in their sputa, even in the absence of any typical CF pathogen. If P. aeruginosa, S. aureus, or Stenotrophomonas maltophilia belonged to the trio of the most abundant species in the CF sputum metagenome, common inhabitants of the respiratory tract of healthy subjects, i.e., Eubacterium sulci, Fusobacterium periodonticum, and Neisseria subflava, were present only in low numbers or not detectable. Random forest analysis identified the numerical ecological parameters of the bacterial community, such as Shannon and Simpson diversity, as the key parameters that globally distinguish sputum samples from CF and healthy donors. IMPORTANCE Cystic fibrosis (CF) is the most common life-limiting monogenetic disease in European populations and is caused by mutations in the CFTR gene. Chronic airway infections with opportunistic pathogens are the major morbidity that determines prognosis and quality of life in most people with CF. We examined the composition of the microbial communities of the oral cavity and upper and lower airways in CF patients across all age groups. From early on, the spectrum of commensals is different in health and CF. Later on, when the common CF pathogens take up residence in the lungs, we observed differential modes of depletion of the commensal microbiota in the presence of S. aureus, P. aeruginosa, S. maltophilia, or combinations thereof. It remains to be seen whether the implementation of lifelong CFTR (cystic fibrosis transmembrane conductance regulator) modulation will change the temporal evolution of the CF airway metagenome.

Multifaceted Interplay between Hfq and the Small RNA GssA in Pseudomonas aeruginosa.

Behind the pathogenic lifestyle of Pseudomonas aeruginosa exists a complex regulatory network of intertwined switches at both the transcriptional and posttranscriptional levels. Major players that mediate translation regulation of several genes involved in host-P. aeruginosa interaction are small RNAs (sRNAs) and the Hfq protein. The canonical role of Hfq in sRNA-driven regulation is to act as a matchmaker between sRNAs and target mRNAs. Besides, the sRNA CrcZ is known to sequester Hfq and abrogate its function of translation repression of target mRNAs. In this study, we describe the novel sRNA GssA in the strain PA14 and its multifaceted interplay with Hfq. We show that GssA is multiresponsive to environmental and physiological signals and acts as an apical repressor of key bacterial functions in the human host such as the production of pyocyanin, utilization of glucose, and secretion of exotoxin A. We suggest that the main role of Hfq is not to directly assist GssA in its regulatory role but to repress GssA expression. In the case of pyocyanin production, we suggest that Hfq interplays with GssA also by converging a positive effect on this pathway. Furthermore, our results indicate that both Hfq and GssA play a positive role in anaerobic growth, possibly by regulating the respiratory chain. On the other hand, we show that GssA can modulate not only Hfq expression at both transcriptional and posttranscriptional levels but also that of CrcZ, thus potentially influencing the pleiotropic role of Hfq. IMPORTANCE The pathogenic lifestyle of the bacterium Pseudomonas aeruginosa, a leading cause of life-threatening infections in the airways of cystic fibrosis patients, is based on the fine regulation of virulence-associated factors. Regulatory small RNAs (sRNAs) and the RNA-binding protein Hfq are recognized key components within the P. aeruginosa regulatory networks involved in host-pathogen interaction. In this study, we characterized in the highly virulent P. aeruginosa strain PA14 the novel sRNA GssA. We found that it can establish a many-sided reciprocal interplay with Hfq which goes beyond the canonical mechanism of direct physical interaction that had previously been characterized for other sRNAs. Given that the Hfq-driven regulatory network of virulence factors is very broad and important for the progression of infection, we consider GssA as a new RNA target that can potentially be used to develop new antibacterial drugs.

S2.3d Mixed biofilm of Aspergillus fumigatus and Stenotrophomonas maltophilia: Microscopic visualization of galactosaminogalactan and galactomannan polysaccharides in the extracellular matrix

S2.3 Novel diagnostic tools for invasive mold infection, September 21, 2022, 3:00 PM - 4:30 PM Introduction Aspergillus fumigatus (Af) and Stenotrophomonas maltophilia (Sm) are commonly co-isolated from the airways of cystic fibrosis patients, especially in mixed biofilms. A mixed Af-Sm biofilm model, developed by our lab, demonstrated that Sm exhibits an antibiosis effect on Af by (1) inhibiting fungal growth, (2) rendering the fungal mycelia highly branched, and (3) increasing the fungal cell wall thickness.1 The presence of three Af cell wall polysaccharides, galactomannan (GM), galactosaminogalactan (GAG), and α-1,3-glucan, have been described in the extracellular matrix (ECM) of Af biofilms.2 GM and GAG are known to be released out from the cell wall of Af.3 GAG contains N-acetylgalactosamines (GalNAc) which undergo partial deacetylation extracellularly, to be converted to GalN, and thus transform this polysaccharide into a polycation with strong surface binding properties.4Aim of the studyAnalyze the structure of fungal cell wall polysaccharides in the formation of Af biofilm alone or mixed with Sm.MethodsThe Af-Sm co-culture was prepared on an 8-chambers Lab-Tek slide as described previously1. Anti-GAG and anti-GM monoclonal antibodies produced at the Aspergillus Unit at Pasteur Institute of Paris, were used to focus on the 3D structure of GAG and GM in the ECM of the biofilm. These polysaccharides were analyzed by fluorescence confocal microscopy at 24 h.Results and ConclusionGM was found, as expected, in the hyphal Af cell wall but was very little in the ECM. GAG was also found in the cell wall but mainly formed a beautiful fibrillary network between the hyphae, showing the importance of this polysaccharide in cell-cell interaction and in the structuration of Af biofilm. GAG could be the surface receptor for Sm, which would promote strong adhesion between Sm and Af in the biofilm.Sources1. Melloul E et al. Front Microbiol. 2018;2. Beauvais A et al. Microbiol Spectr. 2015;3. Fontaine T et al. J Fungi. 2020;4. Briard B et al. Mycologia 2016.

Ruthenium(II) Polypyridyl Complexes for Antimicrobial Photodynamic Therapy: Prospects for Application in Cystic Fibrosis Lung Airways.

Antimicrobial photodynamic therapy (aPDT) depends on a variety of parameters notably related to the photosensitizers used, the pathogens to target and the environment to operate. In a previous study using a series of Ruthenium(II) polypyridyl ([Ru(II)]) complexes, we reported the importance of the chemical structure on both their photo-physical/physico-chemical properties and their efficacy for aPDT. By employing standard in vitro conditions, effective [Ru(II)]-mediated aPDT was demonstrated against planktonic cultures of Pseudomonas aeruginosa and Staphylococcus aureus strains notably isolated from the airways of Cystic Fibrosis (CF) patients. CF lung disease is characterized with many pathophysiological disorders that can compromise the effectiveness of antimicrobials. Taking this into account, the present study is an extension of our previous work, with the aim of further investigating [Ru(II)]-mediated aPDT under in vitro experimental settings approaching the conditions of infected airways in CF patients. Thus, we herein studied the isolated influence of a series of parameters (including increased osmotic strength, acidic pH, lower oxygen availability, artificial sputum medium and biofilm formation) on the properties of two selected [Ru(II)] complexes. Furthermore, these compounds were used to evaluate the possibility to photoinactivate P. aeruginosa while preserving an underlying epithelium of human bronchial epithelial cells. Altogether, our results provide substantial evidence for the relevance of [Ru(II)]-based aPDT in CF lung airways. Besides optimized nano-complexes, this study also highlights the various needs for translating such a challenging perspective into clinical practice.

A Shaving Proteomic Approach to Unveil Surface Proteins Modulation of Multi-Drug Resistant Pseudomonas aeruginosa Strains Isolated From Cystic Fibrosis Patients.

Cystic fibrosis (CF) is the most common rare disease caused by a mutation of the CF transmembrane conductance regulator gene encoding a channel protein of the apical membrane of epithelial cells leading to alteration of Na+ and K+ transport, hence inducing accumulation of dense and sticky mucus and promoting recurrent airway infections. The most detected bacterium in CF patients is Pseudomonas aeruginosa (PA) which causes chronic colonization, requiring stringent antibiotic therapies that, in turn induces multi-drug resistance. Despite eradication attempts at the first infection, the bacterium is able to utilize several adaptation mechanisms to survive in hostile environments such as the CF lung. Its adaptive machinery includes modulation of surface molecules such as efflux pumps, flagellum, pili and other virulence factors. In the present study we compared surface protein expression of PA multi- and pan-drug resistant strains to wild-type antibiotic-sensitive strains, isolated from the airways of CF patients with chronic colonization and recent infection, respectively. After shaving with trypsin, microbial peptides were analyzed by tandem-mass spectrometry on a high-resolution platform that allowed the identification of 174 differentially modulated proteins localized in the region from extracellular space to cytoplasmic membrane. Biofilm assay was performed to characterize all 26 PA strains in term of biofilm production. Among the differentially expressed proteins, 17 were associated to the virulome (e.g., Tse2, Tse5, Tsi1, PilF, FliY, B-type flagellin, FliM, PyoS5), six to the resistome (e.g., OprJ, LptD) and five to the biofilm reservoir (e.g., AlgF, PlsD). The biofilm assay characterized chronic antibiotic-resistant isolates as weaker biofilm producers than wild-type strains. Our results suggest the loss of PA early virulence factors (e.g., pili and flagella) and later expression of virulence traits (e.g., secretion systems proteins) as an indicator of PA adaptation and persistence in the CF lung environment. To our knowledge, this is the first study that, applying a shaving proteomic approach, describes adaptation processes of a large collection of PA clinical strains isolated from CF patients in early and chronic infection phases.

N-acetylcysteine (NAC) and Its Role in Clinical Practice Management of Cystic Fibrosis (CF): A Review.

N-acetylcysteine is the acetylated form of the amino acid L-cysteine and a precursor to glutathione (GSH). It has been known for a long time as a powerful antioxidant and as an antidote for paracetamol overdose. However, other activities related to this molecule have been discovered over the years, making it a promising drug for diseases such as cystic fibrosis (CF). Its antioxidant activity plays a key role in CF airway inflammation and redox imbalance. Furthermore, this molecule appears to play an important role in the prevention and eradication of biofilms resulting from CF airway infections, in particular that of Pseudomonas aeruginosa. The aim of this review is to provide an overview of CF and the role that NAC could play in preventing and eliminating biofilms, as a modulator of inflammation and as an antioxidant, restoring the redox balance within the airways in CF patients. To do this, NAC can act alone, but it can also be used as an adjuvant molecule to known drugs (antibiotics/anti-inflammatories) to increase their activity.

Mixed Populations and Co-Infection: Pseudomonas aeruginosa and Staphylococcus aureus.

The human pathogens Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated from chronic wounds or cystic fibrosis patient airways. Clinical studies analysing the impact of co-infection on patient clinical outcomes lead to contradictory results. However, laboratory approaches suggest that the two pathogens co-colonize the same infection niches and form a mixed-species biofilm, therefore favouring their resistance to antibiotics and immune response. In parallel, many recent studies have focused on the different interactions between the two bacterial species. It has long been recognized that P. aeruginosa usually outcompetes S. aureus, and the molecular mechanisms involved in this state of bacterial competition are now well understood. However, several recent studies show that interactions between P. aeruginosa and S. aureus can be diverse and evolve over time. Thus, many CF isolates of P. aeruginosa and S. aureus can coexist and develop cooperative behaviours. In this chapter, we will provide an overview of the current knowledge on the mixed populations of P. aeruginosa and S. aureus, from their mechanisms of establishment to their impacts on bacterial physiology and clinical outcomes.

Defective Glyoxalase 1 Contributes to Pathogenic Inflammation in Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disorder that affects multiple organs, although a decline in respiratory function represents the major cause of morbidity and mortality. The airways of CF patients are characterized by a chronic inflammatory state to which the receptor for advanced glycation end-products greatly contributes. Glyoxalase 1 (GLO1) is the major enzyme metabolizing methylglyoxal, a potent precursor of advanced glycation end-products. Its role in CF has never been investigated. We herein resorted to murine and human preclinical models of CF to define the contribution of GLO1 to inflammatory pathology. We found that the expression and activity of GLO1, measured by real-time PCR and Western blot or a specific spectrophotometric assay, respectively, are defective in mice and human bronchial cells from CF patients exposed to Aspergillus fumigatus, a common pathogen in CF, but could be restored upon blockade of interleukin-1 receptor signaling by anakinra in mice. This study suggests that GLO1 contributes to pathology in CF and may be potentially targeted to mitigate inflammation.

Genome features and antibiotic resistance of Pseudomonas aeruginosa strains isolated in patients with cystic fibrosis in the Russian Federation.

Cystic fibrosis (CF) is a common genetic disease, manifested by airway obstruction and chronic respiratory infection. The most prevalent infectious agent in airways of CF patients is Pseudomonas aeruginosa. This study aimed to determine sequence-types, antimicrobial resistance phenotypes and genes defining adaptive antibiotic resistance in P. aeruginosa isolates recovered from CF patients in Russia. In total, 84 P. aeruginosa strains from 64 CF patients were analyzed. Susceptibility to antibiotics was determined by disk diffusion test. Whole-genome sequencing (WGS) was performed on MGISEQ-2000 platform. SPAdes software, Galaxy, ResFinder, PubMLST were used for analysis of WGS data. Examined P. aeruginosa isolates belonged to 53 different sequence-types (STs), including 6 new STs. High-risk epidemic clone ST235 (10%) and clonal CF P. aeruginosa strains ST17, ST242, ST274 (7%) were detected. Non-susceptibility to ticarcillin-clavulanate, cefepime, imipenem was observed in 63%, 12% and 25% of isolates, respectively; to tobramycin - in 24%, to amikacin - in 35%; to ciprofloxacin, levofloxacin - in 35% and 57% of strains, respectively. Multidrug-resistant phenotype was detected in 18% of isolates. In examined strains, genes of beta-lactamases VIM-2 (5 ST235 strains), VEB-1 (two ST2592 strains), GES-1 (1 ST235 strain), PER-1 (1 ST235 strain) were found. Ciprofloxacin-modifying enzyme CrpP gene was detected in 67% of isolates, aminoglycoside-modifying enzymes AAD, ANT, AAC genes - in 7%, 4%, 12% of strains, respectively. P. aeruginosa isolates from CF patients in Russia demonstrate a high clonal diversity, which is similar to other P. aeruginosa infections. The isolates of high-risk clone and clonal CF P. aeruginosa strains are detected.

Cystic Fibrosis Sputum Impairs the Ability of Neutrophils to Kill Staphylococcus aureus.

Cystic fibrosis (CF) airway disease is characterized by chronic microbial infections and infiltration of inflammatory polymorphonuclear (PMN) granulocytes. Staphylococcus aureus (S. aureus) is a major lung pathogen in CF that persists despite the presence of PMNs and has been associated with CF lung function decline. While PMNs represent the main mechanism of the immune system to kill S. aureus, it remains largely unknown why PMNs fail to eliminate S. aureus in CF. The goal of this study was to observe how the CF airway environment affects S. aureus killing by PMNs. PMNs were isolated from the blood of healthy volunteers and CF patients. Clinical isolates of S. aureus were obtained from the airways of CF patients. The results show that PMNs from healthy volunteers were able to kill all CF isolates and laboratory strains of S. aureus tested in vitro. The extent of killing varied among strains. When PMNs were pretreated with supernatants of CF sputum, S. aureus killing was significantly inhibited suggesting that the CF airway environment compromises PMN antibacterial functions. CF blood PMNs were capable of killing S. aureus. Although bacterial killing was inhibited with CF sputum, PMN binding and phagocytosis of S. aureus was not diminished. The S. aureus-induced respiratory burst and neutrophil extracellular trap release from PMNs also remained uninhibited by CF sputum. In summary, our data demonstrate that the CF airway environment limits killing of S. aureus by PMNs and provides a new in vitro experimental model to study this phenomenon and its mechanism.

Compensatory evolution of Pseudomonas aeruginosa\u2019s slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.

Inhibition of Pseudomonas aeruginosa Alginate Synthesis by Ebselen Oxide and Its Analogues.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that is frequently found in the airways of cystic fibrosis (CF) patients due to the dehydrated mucus that collapses the underlying cilia and prevents mucociliary clearance. During this life-long chronic infection, P.aeruginosa cell accumulates mutations that lead to inactivation of the mucA gene that results in the constitutive expression of algD-algA operon and the production of alginate exopolysaccharide. The viscous alginate polysaccharide further occludes the airways of CF patients and serves as a protective matrix to shield P.aeruginosa from host immune cells and antibiotic therapy. Development of inhibitors of alginate production by P.aeruginosa would reduce the negative impact from this viscous polysaccharide. In addition to transcriptional regulation, alginate biosynthesis requires allosteric activation by bis (3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) binding to an Alg44 protein. Previously, we found that ebselen (Eb) and ebselen oxide (EbO) inhibited diguanylate cyclase from synthesizing c-di-GMP. In this study, we show that EbO, Eb, ebsulfur (EbS), and their analogues inhibit alginate production. Eb and EbS can covalently modify the cysteine 98 (C98) residue of Alg44 and prevent its ability to bind c-di-GMP. However, P.aeruginosa with Alg44 C98 substituted with alanine or serine was still inhibited for alginate production by Eb and EbS. Our results indicate that EbO, Eb, and EbS are lead compounds for reducing alginate production by P.aeruginosa. Future development of these inhibitors could provide a potential treatment for CF patients infected with mucoid P.aeruginosa.

Airways glutathione S-transferase omega-1 and its A140D polymorphism are associated with severity of inflammation and respiratory dysfunction in cystic fibrosis

BackgroundGlutathione S-transferase omega-1 (GSTO1-1) is a cytosolic enzyme that modulates the S-thiolation status of intracellular factors involved in cancer cell survival or in the inflammatory response. Studies focusing on chronic obstructive pulmonary disease (COPD) have demonstrated that GSTO1-1 is detectable in alveolar macrophages, airway epithelium and in the extracellular compartment, where its functions have not been completely understood. Moreover GSTO1-1 polymorphisms have been associated with an increased risk to develop COPD. Against this background, the aim of this study was to evaluate GSTO1-1 levels and its polymorphisms in cystic fibrosis (CF) patients. MethodsClinical samples from a previous study published by our groups were analyzed for GSTO1-1 levels and polymorphisms. For comparison, a model of lung inflammation in CFTR-knock out mice was also used. ResultsOur data document that soluble GSTO1-1 can be found in the airways of CF patients and correlates with inflammatory parameters such as neutrophilic elastase and the chemokine IL-8. A negative correlation was found between GSTO1-1 levels and the spirometric parameter FEV1 and the FEV1/FVC ratio. Additionally, the A140D polymorphism of GSTO1-1 was associated with lower levels of the antiinflammatory mediators PGE2 and 15(S)-HETE, and with lower values of the FEV1/FVC ratio in CF subjects with the homozygous CFTR ΔF508 mutation. ConclusionsOur data suggest that extracellular GSTO1-1 and its polymorphysms could have a biological and clinical significance in CF. Pathophysiological functions of GSTOs are far from being completely understood, and more studies are required to understand the role(s) of extracellular GSTO1-1 in inflamed tissues.