Cystic Fibrosis Lung Environment Research Articles

A gain-of-function mutation in zinc cluster transcription factor Rob1 drives Candida albicans adaptive growth in the cystic fibrosis lung environment.

Candida albicans chronically colonizes the respiratory tract of patients with Cystic Fibrosis (CF). It competes with CF-associated pathogens (e.g. Pseudomonas aeruginosa) and contributes to disease severity. We hypothesize that C. albicans undergoes specific adaptation mechanisms that explain its persistence in the CF lung environment. To identify the underlying genetic and phenotypic determinants, we serially recovered 146 C. albicans clinical isolates over a period of 30 months from the sputum of 25 antifungal-naive CF patients. Multilocus sequence typing analyses revealed that most patients were individually colonized with genetically close strains, facilitating comparative analyses between serial isolates. We strikingly observed differential ability to filament and form monospecies and dual-species biofilms with P. aeruginosa among 18 serial isolates sharing the same diploid sequence type, recovered within one year from a pediatric patient. Whole genome sequencing revealed that their genomes were highly heterozygous and similar to each other, displaying a highly clonal subpopulation structure. Data mining identified 34 non-synonymous heterozygous SNPs in 19 open reading frames differentiating the hyperfilamentous and strong biofilm-former strains from the remaining isolates. Among these, we detected a glycine-to-glutamate substitution at position 299 (G299E) in the deduced amino acid sequence of the zinc cluster transcription factor ROB1 (ROB1G299E), encoding a major regulator of filamentous growth and biofilm formation. Introduction of the G299E heterozygous mutation in a co-isolated weak biofilm-former CF strain was sufficient to confer hyperfilamentous growth, increased expression of hyphal-specific genes, increased monospecies biofilm formation and increased survival in dual-species biofilms formed with P. aeruginosa, indicating that ROB1G299E is a gain-of-function mutation. Disruption of ROB1 in a hyperfilamentous isolate carrying the ROB1G299E allele abolished hyperfilamentation and biofilm formation. Our study links a single heterozygous mutation to the ability of C. albicans to better survive during the interaction with other CF-associated microbes and illuminates how adaptive traits emerge in microbial pathogens to persistently colonize and/or infect the CF-patient airways.

Pseudomonas aeruginosa transcriptome analysis of metal restriction in ex vivo cystic fibrosis sputum.

Chronic Pseudomonas aeruginosa lung infections are a feature of cystic fibrosis (CF) that many patients experience even with the advent of highly effective modulator therapies. Identifying factors that impact P. aeruginosa in the CF lung could yield novel strategies to eradicate infection or otherwise improve outcomes. To complement published P. aeruginosa studies using laboratory models or RNA isolated from sputum, we analyzed transcripts of strain PAO1 after incubation in sputum from different CF donors prior to RNA extraction. We compared PAO1 gene expression in this "spike-in" sputum model to that for P. aeruginosa grown in synthetic cystic fibrosis sputum medium to determine key genes, which are among the most differentially expressed or most highly expressed. Using the key genes, gene sets with correlated expression were determined using the gene expression analysis tool eADAGE. Gene sets were used to analyze the activity of specific pathways in P. aeruginosa grown in sputum from different individuals. Gene sets that we found to be more active in sputum showed similar activation in published data that included P. aeruginosa RNA isolated from sputum relative to corresponding in vitro reference cultures. In the ex vivo samples, P. aeruginosa had increased levels of genes related to zinc and iron acquisition which were suppressed by metal amendment of sputum. We also found a significant correlation between expression of the H1-type VI secretion system and CFTR corrector use by the sputum donor. An ex vivo sputum model or synthetic sputum medium formulation that imposes metal restriction may enhance future CF-related studies.IMPORTANCEIdentifying the gene expression programs used by Pseudomonas aeruginosa to colonize the lungs of people with cystic fibrosis (CF) will illuminate new therapeutic strategies. To capture these transcriptional programs, we cultured the common P. aeruginosa laboratory strain PAO1 in expectorated sputum from CF patient donors. Through bioinformatic analysis, we defined sets of genes that are more transcriptionally active in real CF sputum compared to a synthetic cystic fibrosis sputum medium. Many of the most differentially active gene sets contained genes related to metal acquisition, suggesting that these gene sets play an active role in scavenging for metals in the CF lung environment which may be inadequately represented in some models. Future studies of P. aeruginosa transcript abundance in CF may benefit from the use of an expectorated sputum model or media supplemented with factors that induce metal restriction.

Experimental evolution of Pseudomonas aeruginosa to colistin in spatially confined microdroplets identifies evolutionary trajectories consistent with adaptation in microaerobic lung environments.

Antibiotic resistance is a continuing global health crisis. Identifying the evolutionary trajectories leading to increased antimicrobial resistance can be critical to the discovery of biomarkers for clinical diagnostics and new targets for drug discovery. While the combination of patient data and in vitro experimental evolution has been remarkably successful in extending our understanding of antimicrobial resistance, it can be difficult for in vitro methods to recapitulate the spatial structure and consequent microenvironments that characterize in vivo infection. Notably, in cystic fibrosis (CF) patients, changes to either the PmrA/PmrB or PhoP/PhoQ two-component systems have been identified as critical drivers for high levels of colistin and polymyxin resistance. When using microfluidic emulsions to provide spatially structured, low-competition environments, we found that adaptive mutations to phoQ were more successful than pmrB in increasing colistin resistance. Conversely, mutations to pmrB were readily identified using well-mixed unstructured cultures. We found that oxygen concentration gradients within the microdroplet emulsions favored adaptive changes to the PhoP/PhoQ pathway consistent with microaerobic conditions that can be found in the lungs of CF patients. We also observed mutations linked to hallmark adaptations to the CF lung environment, such as loss of motility and loss of O antigen biosynthesis (wbpL). Mutation to wbpL, in addition to causing loss of O antigen, was additionally shown to confer moderately increased colistin resistance. Taken together, our data suggest that distinct evolutionary trajectories to colistin resistance may be shaped by the microaerobic partitioning and spatial separation imposed within the CF lung.IMPORTANCEAntibiotic resistance remains one of the great challenges confronting public health in the world today. Individuals with compromised immune systems or underlying health conditions are often at an increased for bacterial infections. Patients with cystic fibrosis (CF) produce thick mucus that clogs airways and provides a very favorable environment for infection by bacteria that further decrease lung function and, ultimately, mortality. CF patients are often infected by bacteria such as Pseudomonas aeruginosa early in life and experience a series of chronic infections that, over time, become increasingly difficult to treat due to increased antibiotic resistance. Colistin is a major antibiotic used to treat CF patients. Clinical and laboratory studies have identified PmrA/PmrB and PhoP/PhoQ as responsible for increased resistance to colistin. Both have been identified in CF patient lungs, but why, in some cases, is it one and not the other? In this study, we show that distinct evolutionary trajectories to colistin resistance may be favored by the microaerobic partitioning found within the damaged CF lung.

Pseudomonas aeruginosa utilizes the host-derived polyamine spermidine to facilitate antimicrobial tolerance.

Pseudomonas aeruginosa undergoes diversification during infection of the cystic fibrosis (CF) lung. Understanding these changes requires model systems that capture the complexity of the CF lung environment. We previously identified loss-of-function mutations in the 2-component regulatory system sensor kinase gene pmrB in P. aeruginosa from CF lung infections and from experimental infection of mice. Here, we demonstrate that, while such mutations lowered in vitro minimum inhibitory concentrations for multiple antimicrobial classes, this was not reflected in increased antibiotic susceptibility in vivo. Loss of PmrB impaired aminoarabinose modification of LPS, increasing the negative charge of the outer membrane and promoting uptake of cationic antimicrobials. However, in vivo, this could be offset by increased membrane binding of other positively charged molecules present in lungs. The polyamine spermidine readily coated the surface of PmrB-deficient P. aeruginosa, reducing susceptibility to antibiotics that rely on charge differences to bind the outer membrane and increasing biofilm formation. Spermidine was elevated in lungs during P. aeruginosa infection in mice and during episodes of antimicrobial treatment in people with CF. These findings highlight the need to study antimicrobial resistance under clinically relevant environmental conditions. Microbial mutations carrying fitness costs in vitro may be advantageous during infection, where host resources can be utilized.

436 Characterizing lactate as a key metabolite in the cystic fibrosis lung environment

436 Characterizing lactate as a key metabolite in the cystic fibrosis lung environment

Staphylococcus aureus and Pseudomonas aeruginosa Isolates from the Same Cystic Fibrosis Respiratory Sample Coexist in Coculture.

ABSTRACTRespiratory infections with bacterial pathogens remain the major cause of morbidity in individuals with the genetic disease cystic fibrosis (CF). Some studies have shown that CF patients that harbor both Staphylococcus aureus and Pseudomonas aeruginosa in their lungs are at even greater risk for more severe and complicated respiratory infections and earlier death. However, the drivers for this worse clinical condition are not well understood. To investigate the interactions between these two microbes that might be responsible for their increased pathogenic potential, we obtained 28 pairs of S. aureus and P. aeruginosa from the same respiratory samples from 18 individuals with CF. We compared the survival of each S. aureus CF isolate cocultured with its corresponding coinfecting CF P. aeruginosa to when it was cocultured with non-CF laboratory strains of P. aeruginosa. We found that the S. aureus survival was significantly higher in the presence of the coinfecting P. aeruginosa compared to laboratory P. aeruginosa strains, regardless of whether the coinfecting isolate was mucoid or nonmucoid. We also tested how a non-CF S. aureus strain, JE2, behaved with each P. aeruginosa CF isolate and found that its interaction was similar to how the CF S. aureus isolate interacted with its coinfecting P. aeruginosa. Altogether, our work suggests that interactions between S. aureus and P. aeruginosa that promote coexistence in the CF lung are isolate-dependent and that this interaction appears to be driven mainly by P. aeruginosa.IMPORTANCE Previous studies have shown that in laboratory settings, Pseudomonas aeruginosa generally kills Staphylococcus aureus. However, these bacteria are often found coinfecting the lungs of cystic fibrosis (CF) patients, which has been associated with worse patient outcomes. To investigate the interactions between these two bacteria, we competed 28 coinfection pairs obtained from the same lung samples of 18 different CF patients. We compared these results to those we previously reported of each CF S. aureus isolate against a non-CF laboratory strain of P. aeruginosa. We found that S. aureus survival against its corresponding coinfection P. aeruginosa was higher than its survival against the laboratory strain of P. aeruginosa. These results suggest that there may be selection for coexistence of these microbes in the CF lung environment. Further understanding of the interactions between P. aeruginosa and S. aureus will provide insights into the drivers of coexistence and their impact on the host.

Essential Oils Biofilm Modulation Activity and Machine Learning Analysis on Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients.

The opportunistic pathogen Pseudomonas aeruginosa is often involved in airway infections of cystic fibrosis (CF) patients. It persists in the hostile CF lung environment, inducing chronic infections due to the production of several virulence factors. In this regard, the ability to form a biofilm plays a pivotal role in CF airway colonization by P. aeruginosa. Bacterial virulence mitigation and bacterial cell adhesion hampering and/or biofilm reduced formation could represent a major target for the development of new therapeutic treatments for infection control. Essential oils (EOs) are being considered as a potential alternative in clinical settings for the prevention, treatment, and control of infections sustained by microbial biofilms. EOs are complex mixtures of different classes of organic compounds, usually used for the treatment of upper respiratory tract infections in traditional medicine. Recently, a wide series of EOs were investigated for their ability to modulate biofilm production by different pathogens comprising S. aureus, S. epidermidis, and P. aeruginosa strains. Machine learning (ML) algorithms were applied to develop classification models in order to suggest a possible antibiofilm action for each chemical component of the studied EOs. In the present study, we assessed the biofilm growth modulation exerted by 61 commercial EOs on a selected number of P. aeruginosa strains isolated from CF patients. Furthermore, ML has been used to shed light on the EO chemical components likely responsible for the positive or negative modulation of bacterial biofilm formation.

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.

Biofilm Formation in Methicillin-Resistant Staphylococcus aureus Isolated in Cystic Fibrosis Patients Is Strain-Dependent and Differentially Influenced by Antibiotics.

Cystic fibrosis (CF) is a genetic disease with lung abnormalities making patients particularly predisposed to pulmonary infections. Staphylococcus aureus is the most frequently identified pathogen, and multidrug-resistant strains (MRSA, methicillin-resistant S. aureus) have been associated with more severe lung dysfunction leading to eradication recommendations. Diverse bacterial traits and adaptive skills, including biofilm formation, may, however, make antimicrobial therapy challenging. In this context, we compared the ability of a collection of genotyped MRSA isolates from CF patients to form biofilm with and without antibiotics (ceftaroline, ceftobiprole, linezolid, trimethoprim, and rifampicin). Our study used standardized approaches not previously applied to CF MRSA, the BioFilm Ring test® (BRT®), the Antibiofilmogram®, and the BioFlux™ 200 system which were adapted for use with the artificial sputum medium (ASM) mimicking conditions more relevant to the CF lung. We included 63 strains of 10 multilocus sequence types (STs) isolated from 35 CF patients, 16 of whom had chronic colonization. The BRT® showed that 27% of the strains isolated in 37% of the patients were strong biofilm producers. The Antibiofilmogram® performed on these strains showed that broad-spectrum cephalosporins had the lowest minimum biofilm inhibitory concentrations (bMIC) on a majority of strains. A focus on four chronically colonized patients with inclusion of successively isolated strains showed that ceftaroline, ceftobiprole, and/or linezolid bMICs may remain below the resistance thresholds over time. Studying the dynamics of biofilm formation by strains isolated 3years apart in one of these patients using BioFlux™ 200 showed that inhibition of biofilm formation was observed for up to 36h of exposure to bMIC and ceftaroline and ceftobiprole had a significantly greater effect than linezolid. This study has brought new insights into the behavior of CF MRSA which has been little studied for its ability to form biofilm. Biofilm formation is a common characteristic of prevalent MRSA clones in CF. Early biofilm formation was strain-dependent, even within a sample, and not only observed during chronic colonization. Ceftaroline and ceftobiprole showed a remarkable activity with a long-lasting inhibitory effect on biofilm formation and a conserved activity on certain strains adapted to the CF lung environment after years of colonization.

Calprotectin-Mediated Zinc Chelation Inhibits Pseudomonas aeruginosa Protease Activity in Cystic Fibrosis Sputum.

Pseudomonas aeruginosa induces pathways indicative of low zinc availability in the cystic fibrosis (CF) lung environment. To learn more about P. aeruginosa zinc access in CF, we grew P. aeruginosa strain PAO1 directly in expectorated CF sputum. The P. aeruginosa Zur transcriptional repressor controls the response to low intracellular zinc, and we used the NanoString methodology to monitor levels of Zur-regulated transcripts, including those encoding a zincophore system, a zinc importer, and paralogs of zinc containing proteins that do not require zinc for activity. Zur-controlled transcripts were induced in sputum-grown P. aeruginosa compared to those grown in control cultures but not if the sputum was amended with zinc. Amendment of sputum with ferrous iron did not reduce expression of Zur-regulated genes. A reporter fusion to a Zur-regulated promoter had variable activity in P. aeruginosa grown in sputum from different donors, and this variation inversely correlated with sputum zinc concentrations. Recombinant human calprotectin (CP), a divalent-metal binding protein released by neutrophils, was sufficient to induce a zinc starvation response in P. aeruginosa grown in laboratory medium or zinc-amended CF sputum, indicating that CP is functional in the sputum environment. Zinc metalloproteases comprise a large fraction of secreted zinc-binding P. aeruginosa proteins. Here, we show that recombinant CP inhibited both LasB-mediated casein degradation and LasA-mediated lysis of Staphylococcus aureus, which was reversible with added zinc. These studies reveal the potential for CP-mediated zinc chelation to posttranslationally inhibit zinc metalloprotease activity and thereby affect the protease-dependent physiology and/or virulence of P. aeruginosa in the CF lung environment. IMPORTANCE The factors that contribute to worse outcomes in individuals with cystic fibrosis (CF) with chronic Pseudomonas aeruginosa infections are not well understood. Therefore, there is a need to understand environmental factors within the CF airway that contribute to P. aeruginosa colonization and infection. We demonstrate that growing bacteria in CF sputum induces a zinc starvation response that inversely correlates with sputum zinc levels. Additionally, both calprotectin and a chemical zinc chelator inhibit the proteolytic activities of LasA and LasB proteases, suggesting that extracellular zinc chelators can influence proteolytic activity and thus P. aeruginosa virulence and nutrient acquisition in vivo.

Interspecies Metabolic Complementation in Cystic Fibrosis Pathogens via Purine Exchange

Cystic fibrosis (CF) is a genetic disease frequently associated with chronic lung infections caused by a consortium of pathogens. It is common for auxotrophy (the inability to biosynthesize certain essential metabolites) to develop in clinical isolates of the dominant CF pathogen Pseudomonas aeruginosa, indicating that the CF lung environment is replete in various nutrients. Many of these nutrients are likely to come from the host tissues, but some may come from the surrounding polymicrobial community within the lungs of CF patients as well. To assess the feasibility of nutrient exchange within the polymicrobial community of the CF lung, we selected P. aeruginosa and Staphylococcus aureus, two of the most prevalent species found in the CF lung environment. By comparing the polymicrobial culture of wild-type strains relative to their purine auxotrophic counterparts, we were able to observe metabolic complementation occurring in both P. aeruginosa and S. aureus when grown with a purine-producing cross-species pair. While our data indicate that some of this complementation is likely derived from extracellular DNA freed by lysis of S. aureus by the highly competitive P. aeruginosa, the partial complementation of S. aureus purine deficiency by P. aeruginosa demonstrates that bidirectional nutrient exchange between these classic competitors is possible.

Bacterial persisters in long-term infection: Emergence and fitness in a complex host environment.

Despite intensive antibiotic treatment, Pseudomonas aeruginosa often persists in the airways of cystic fibrosis (CF) patients for decades, and can do so without antibiotic resistance development. Using high-throughput screening assays of bacterial survival after treatment with high concentrations of ciprofloxacin, we have determined the prevalence of persisters in a large patient cohort using 460 longitudinal isolates of P. aeruginosa from 39 CF patients. Isolates were classed as high persister variants (Hip) if they regrew following antibiotic treatment in at least 75% of the experimental replicates. Strain genomic data, isolate phenotyping, and patient treatment records were integrated in a lineage-based analysis of persister formation and clinical impact. In total, 19% of the isolates were classified as Hip and Hip emergence increased over lineage colonization time within 22 Hip+ patients. Most Hip+ lineages produced multiple Hip isolates, but few Hip+ lineages were dominated by Hip. While we observed no strong signal of adaptive genetic convergence within Hip isolates, they generally emerged in parallel or following the development of ciprofloxacin resistance and slowed growth. Transient lineages were majority Hip-, while strains that persisted over a clinically diagnosed ‘eradication’ period were majority Hip+. Patients received indistinguishable treatment regimens before Hip emergence, but Hip+ patients overall were treated significantly more than Hip- patients, signaling repeated treatment failure. When subjected to in vivo-similar antibiotic dosing, a Hip isolate survived better than a non-Hip in a structured biofilm environment. In sum, the Hip phenotype appears to substantially contribute to long-term establishment of a lineage in the CF lung environment. Our results argue against the existence of a single dominant molecular mechanism underlying bacterial antibiotic persistence. We instead show that many routes, both phenotypic and genetic, are available for persister formation and consequent increases in strain fitness and treatment failure in CF airways.

Comparative Evolutionary Patterns of Burkholderia cenocepacia and B. multivorans During Chronic Co-infection of a Cystic Fibrosis Patient Lung.

During chronic respiratory infections of cystic fibrosis (CF) patients, bacteria adaptively evolve in response to the nutritional and immune environment as well as influence other infecting microbes. The present study was designed to gain insights into the genetic mechanisms underlying adaptation and diversification by the two most prevalent pathogenic species of the Burkholderia cepacia complex (Bcc), B. cenocepacia and B. multivorans. Herein, we study the evolution of both of these species during coinfection of a CF patient for 4.4 years using genome sequences of 9 B. multivorans and 11 B. cenocepacia. This co-infection spanned at least 3 years following initial infection by B. multivorans and ultimately ended in the patient’s death by cepacia syndrome. Both species acquired several mutations with accumulation rates of 2.08 (B. cenocepacia) and 2.27 (B. multivorans) SNPs/year. Many of the mutated genes are associated with oxidative stress response, transition metal metabolism, defense mechanisms against antibiotics, and other metabolic alterations consistent with the idea that positive selection might be driven by the action of the host immune system, antibiotic therapy and low oxygen and iron concentrations. Two orthologous genes shared by B. cenocepacia and B. multivorans were found to be under strong selection and accumulated mutations associated with lineage diversification. One gene encodes a nucleotide sugar dehydratase involved in lipopolysaccharide O-antigen (OAg) biosynthesis (wbiI). The other gene encodes a putative two-component regulatory sensor kinase protein required to sense and adapt to oxidative- and heavy metal- inducing stresses. This study contributes to understanding of shared and species-specific evolutionary patterns of B. cenocepacia and B. multivorans evolving in the same CF lung environment.

An in vitro model for assessing drug transport in cystic fibrosis treatment: Characterisation of the CuFi-1 cell line

Cystic fibrosis (CF) is a disease that most commonly affects the lungs and is characterized by mucus retention and a continuous cycle of bacterial infection and inflammation. Current CF treatment strategies are focused on targeted drug delivery to the lungs. Novel inhalable drug therapies require an in vitro CF model that appropriately mimics the in vivo CF lung environment to better understand drug delivery and transport across the CF epithelium, and predict drug therapeutic efficacy. Therefore, the aim of this research was to determine the appropriate air–liquid interface (ALI) culture method of the CuFi-1 (CF cell line) compared to the NuLi-1 (healthy cell line) cells to be used as in vitro models of CF airway epithelia. Furthermore, drug transport on both CuFi-1 and NuLi-1 was investigated to determine whether these cell lines could be used to study transport of drugs used in CF treatment using Ibuprofen (the only anti-inflammatory drug currently approved for CF) as a model drug. Differentiating characteristics specific to airway epithelia such as mucus production, inflammatory response and tight junction formation at two seeding densities (Low and High) were assessed throughout an 8-week ALI culture period. This study demonstrated that both the NuLi-1 and CuFi-1 cell lines fully differentiate in ALI culture with significant mucus secretion, IL-6 and IL-8 production, and functional tight junctions at week 8. Additionally, the High seeding density was found to alter the phenotype of the NuLi-1 cell line. For the first time, this study identifies that ibuprofen is transported via the paracellular pathway in ALI models of NuLi-1 and CuFi-1 cell lines. Overall, these findings highlight that NuLi-1 and CuFi-1 as promising in vitro ALI models to investigate the transport properties of novel inhalable drug therapies for CF treatment.

Trophic cooperation promotes bacterial survival of Staphylococcus aureus and Pseudomonas aeruginosa.

In the context of infection, Pseudomonas aeruginosa and Staphylococcus aureus are frequently co-isolated, particularly in cystic fibrosis (CF) patients. Within lungs, the two pathogens exhibit a range of competitive and coexisting interactions. In the present study, we explored the impact of S. aureus on the physiology of P. aeruginosa in the context of coexistence. Transcriptomic analyses showed that S. aureus significantly and specifically affects the expression of numerous genes involved in P. aeruginosa carbon and amino acid metabolism. In particular, 65% of the strains presented considerable overexpression of the genes involved in the acetoin catabolic (aco) pathway. We demonstrated that acetoin is (i) produced by clinical S. aureus strains, (ii) detected in sputa from CF patients and (iii) involved in P. aeruginosa's aco system induction. Furthermore, acetoin is catabolized by P. aeruginosa, a metabolic process that improves the survival of both pathogens by providing a new carbon source for P. aeruginosa and avoiding the toxic accumulation of acetoin on S. aureus. Due to its beneficial effects on both bacteria, acetoin catabolism could testify to the establishment of trophic cooperation between S. aureus and P. aeruginosa in the CF lung environment, thus promoting their persistence.

Investigation of role of anaerobic bacteria in cystic fibrosis patients

Repetitive pulmonary infections are the main cause of morbidity and mortality in cystic fibrosis (CF) patients. In recent years, non-culture dependent metagenomic studies showed complex dynamics of the pulmonary environment of CF patients and pointed out the importance of anaerobic bacteria. Molecular-based studies indicate that anaerobic bacteria can be found more than aerobic or facultative anaerobic bacteria in CF lung environment. However, limited number of studies are far away to clarify the importance of anaerobic bacteria in CF pulmonary disease. The aim of this study was to evaluate the role of anaerobic bacteria in CF patients admitted to Hacettepe University, Pediatric Respiratory Diseases Department, by using quantitative culture method for both aerobic and anaerobic bacteria. Anaerobic bacteria were identified by conventional and semi-automated methods. Antibiotic susceptibilities were performed by agar dilution method. Seventy-seven anaerobic bacteria were isolated from 35 (81.4%) of 43 patients. The total count of anaerobes and facultative bacteria (mean 16 x 106), was higher than aerobes and facultative bacteria (mean 14.1 x 106). If anaerobe culture were not performed merely 63.65% of all species could be obtained. In patients whose samples yielded intermediate or high numbers of PMNLs, significantly more obligate anaerobic bacteria were isolated (p= 0.046). Patients older than 18 years were colonized with higher number of anaerobic bacteria. Susceptibilities of 72 isolates out of 77, against ampicillin, sulbactam-ampicillin, piperacillin, piperacillin-tazobactam, moxifloxacin, metronidazole, imipenem, and clindamycin were also evaluated. Clindamycin was found to be the least effective antibiotic among all. None of the isolates was resistant to imipenem. This is the first study to show the role and importance of anaerobic bacteria in CF patients in our country. The resistance rates in anaerobic bacteria isolated from CF patients is concerning. Therefore, intermittent anaerobic culture and follow-up of resistance rates will be helpful in the follow-up of these patients.

New insights into the immunoproteome of B. cenocepacia J2315 using serum samples from cystic fibrosis patients

Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are currently available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to the majority of clinically available antimicrobials. An immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host, using bacterial cells grown under conditions mimicking the CF lung environment and serum samples from CF patients with a clinical record of Bcc infection. 24 proteins of the Bcc strain B. cenocepacia J2315 were identified as immunoreactive, 19 here reported as immunogenic for the first time. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.

Genetically diverse Pseudomonas aeruginosa populations display similar transcriptomic profiles in a cystic fibrosis explanted lung

Previous studies have demonstrated substantial genetic diversification of Pseudomonas aeruginosa across sub-compartments in cystic fibrosis (CF) lungs. Here, we isolate P. aeruginosa from five different sampling areas in the upper and lower airways of an explanted CF lung, analyze ex vivo transcriptional profiles by RNA-seq, and use colony re-sequencing and deep population sequencing to determine the genetic diversity within and across the various sub-compartments. We find that, despite genetic variation, the ex vivo transcriptional profiles of P. aeruginosa populations inhabiting different regions of the CF lung are similar. Although we cannot estimate the extent to which the transcriptional response recorded here actually reflects the in vivo transcriptomes, our results indicate that there may be a common in vivo transcriptional profile in the CF lung environment.

Reconciling Antimicrobial Susceptibility Testing and Clinical Response in Antimicrobial Treatment of Chronic Cystic Fibrosis Lung Infections.

Median cystic fibrosis (CF) survival has increased dramatically over time due to several factors, including greater availability and use of antimicrobial therapies. During the progression of CF lung disease, however, the emergence of multidrug antimicrobial resistance can limit treatment effectiveness, threatening patient longevity. Current planktonic-based antimicrobial susceptibility testing lacks the ability to predict clinical response to antimicrobial treatment of chronic CF lung infections. There are numerous reasons for these limitations including bacterial phenotypic and genotypic diversity, polymicrobial interactions, and impaired antibiotic efficacy within the CF lung environment. The parallels to other chronic diseases such as non-CF bronchiectasis are discussed as well as research priorities for moving forward.

Mucus accumulation in the lungs precedes structural changes and infection in children with cystic fibrosis.

Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography-defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared "permanent" because they did not dissolve in dilute BALF matrix, they could be solubilized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.