Cystic Fibrosis Airway Microbiome Research Articles

Insights into the Adolescent Cystic Fibrosis Airway Microbiome Using Shotgun Metagenomics.

Cystic fibrosis (CF) is an inherited genetic disorder which manifests primarily in airway disease. Recent advances in molecular technologies have unearthed the diverse polymicrobial nature of the CF airway. Numerous studies have characterised the genus-level composition of this airway community using targeted 16S rDNA sequencing. Here, we employed whole-genome shotgun metagenomics to provide a more comprehensive understanding of the early CF airway microbiome. We collected 48 sputum samples from 11 adolescents and children with CF over a 12-month period and performed shotgun metagenomics on the Illumina NextSeq platform. We carried out functional and taxonomic analysis of the lung microbiome at the species and strain levels. Correlations between microbial diversity measures and independent demographic and clinical variables were performed. Shotgun metagenomics detected a greater diversity of bacteria than culture-based methods. A large proportion of the top 25 most-dominant species were anaerobes. Samples dominated by Staphylococcus aureus and Prevotella melaninogenica had significantly higher microbiome diversity, while no CF pathogen was associated with reduced microbial diversity. There was a diverse resistome present in all samples in this study, with 57.8% agreement between shotgun metagenomics and culture-based methods for detection of resistance. Pathogenic sequence types (STs) of S. aureus, Pseudomonas aeruginosa, Haemophilus influenzae and Stenotrophomonas maltophilia were observed to persist in young CF patients, while STs of S. aureus were both persistent and shared between patients. This study provides new insight into the temporal changes in strain level composition of the microbiome and the landscape of the resistome in young people with CF. Shotgun metagenomics could provide a very useful one-stop assay for detecting pathogens, emergence of resistance and conversion to persistent colonisation in early CF disease.

Commensal Bacteria in the Cystic Fibrosis Airway Microbiome Reduce P. aeruginosa Induced Inflammation.

Chronic Pseudomonas aeruginosa infections play an important role in the progress of lung disease in patients suffering from cystic fibrosis (CF). Recent studies indicate that polymicrobial microbiome profiles in the airway are associated with less inflammation. Thus, the hypothesis was raised that certain commensal bacteria might protect the host from inflammation. We therefore performed a screening study with commensals isolated from CF airway microbiome samples to identify potential beneficial commensals. We isolated more than 80 aerobic or facultative anaerobic commensal strains, including strains from genera Streptococcus, Neisseria, Actinomyces, Corynebacterium, Dermabacter, Micrococcus and Rothia. Through a screening experiment of co-infection in human epithelial cell lines, we identified multiple commensal strains, especially strains belonging to Streptococcus mitis, that reduced P. aeruginosa triggered inflammatory responses. The results were confirmed by co-infection experiments in ex-vivo precision cut lung slices (PCLS) from mice. The underlying mechanisms of the complex host-pathogen-commensal crosstalk were investigated from both the host and the bacterial sides with a focus on S. mitis. Transcriptome changes in the host in response to co-infection and mono-infection were evaluated, and the results indicated that several signalling pathways mediating inflammatory responses were downregulated by co-infection with S. mitis and P. aeruginosa compared to P. aeruginosa mono-infection, such as neutrophil extracellular trap formation. The genomic differences among S. mitis strains with and without protective effects were investigated by whole genome sequencing, revealing genes only present in the S. mitis strains showing protective effects. In summary, through both in vitro and ex vivo studies, we could identify a variety of commensal strains that may reduce host inflammatory responses induced by P. aeruginosa infection. These findings support the hypothesis that CF airway commensals may protect the host from inflammation.

498: Bacteriophage and antibiotic resistance detected by metagenomic sequencing in the cystic fibrosis airway microbiome

498: Bacteriophage and antibiotic resistance detected by metagenomic sequencing in the cystic fibrosis airway microbiome

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities. Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial communities.

Changes in the Cystic Fibrosis Airway Microbiome in Response to CFTR Modulator Therapy.

The development of CFTR modulator therapies significantly changed the treatment scheme of people with cystic fibrosis. However, CFTR modulator therapy is still a life-long treatment, which is not able to correct the genetic defect and cure the disease. Therefore, it becomes crucial to understand the effects of such modulation of CFTR function on the airway physiology, especially on airway infections and inflammation that are currently the major life-limiting factors in people with cystic fibrosis. In this context, understanding the dynamics of airway microbiome changes in response to modulator therapy plays an essential role in developing strategies for managing airway infections. Whether and how the newly available therapies affect the airway microbiome is still at the beginning of being deciphered. We present here a brief review summarizing the latest information about microbiome alterations in light of modern cystic fibrosis modulator therapy.

P082 Patterns of acquisition and loss in the cystic fibrosis airway microbiome: competition among bacteria contrasts with reinforcement among fungi and mycobacteria

P082 Patterns of acquisition and loss in the cystic fibrosis airway microbiome: competition among bacteria contrasts with reinforcement among fungi and mycobacteria

Antibiotic multidrug resistance in the cystic fibrosis airway microbiome is associated with decreased diversity

BackgroundCystic fibrosis (CF) is associated with significant morbidity and early mortality due to recurrent acute and chronic lung infections. The chronic use of multiple antibiotics increases the possibility of multidrug resistance (MDR). Antibiotic susceptibility determined by culture-based techniques may not fully represent the resistance profile. The study objective was to detect additional antibiotic resistance using molecular methods and relate the presence of MDR to airway microbiome diversity and pulmonary function. MethodsBacterial DNA was extracted from sputum samples and amplified for the V4 region of the 16S rRNA gene. An qPCR array was used to detect antibiotic resistance genes. Clinical culture results and pulmonary function were also noted for each encounter. ResultsSix study participants contributed samples from 19 encounters. Those samples with MDR (n = 7) had significantly lower diversity measured by inverse Simpson's index than those without (n = 12) (2.193 ± 0.427 vs 6.023 ± 1.564, p = 0.035). Differential abundance showed that samples with MDR had more Streptococcus (p = 0.002) and Alcaligenaceae_unclassified (p = 0.002). Pulmonary function was also decreased when MDR was present (FEV1, 51 ± 22.9 vs 77 ± 26.7, p = 0.054; FVC, 64.5 ± 22.7 vs 91.6 ± 27.7, p = 0.047). ConclusionsThe presence of MDR within the CF airway microbiome was associated with decreased microbial diversity, the presence of Alcaligenes, and decreased pulmonary function.

Aspergillus fumigatus Inhibits Pseudomonas aeruginosa in Co-culture: Implications of a Mutually Antagonistic Relationship on Virulence and Inflammation in the CF Airway.

Many cystic fibrosis (CF) airway infections are considered to be polymicrobial and microbe–microbe interactions may play an important role in disease pathology. Pseudomonas aeruginosa and Aspergillus fumigatus are the most prevalent bacterial and fungal pathogens isolated from the CF airway, respectively. We have previously shown that patients co-colonized with these pathogens had comparable outcomes to those chronically colonized with P. aeruginosa. Our objective was to examine the interactions between A. fumigatus and P. aeruginosa, specifically the effects of co-colonization on biofilm formation, virulence and host pro-inflammatory responses. Our findings suggest that co-infections of A. fumigatus and P. aeruginosa in the Galleria mellonella acute infection model showed that pre-exposure of larvae to sub-lethal inocula of A. fumigatus increased the mortality caused by subsequent P. aeruginosa infection. Co-infection of human bronchial epithelial cells (CFBE41o-) with both pathogens did not enhance IL-6 and IL-8 production beyond the levels observed following single infections. In addition, both pathogens stimulated cytokine secretion via the same two mitogen-activated protein kinases (MAPKs) signaling pathways, ERK and p38. Mixed species biofilms showed overall reduced biofilm development with crystal violet staining. Quantification by species-specific qPCR revealed that both pathogens had mutually antagonistic effects on each other. A. fumigatus supernatants showed strong anti-Pseudomonal activity and gliotoxin was the main active agent. Gliotoxin resulted in varying levels of anti-biofilm activity toward other bacteria commonly found in the CF airways. Gliotoxin produced by A. fumigatus colonizing the CF airways may have a significant impact on the CF airway microbiome composition with potential clinical implications.

Benchmark Evaluation of True Single Molecular Sequencing to Determine Cystic Fibrosis Airway Microbiome Diversity.

Cystic fibrosis (CF) is an autosomal recessive disease associated with recurrent lung infections that can lead to morbidity and mortality. The impact of antibiotics for treatment of acute pulmonary exacerbations on the CF airway microbiome remains unclear with prior studies giving conflicting results and being limited by their use of 16S ribosomal RNA sequencing. Our primary objective was to validate the use of true single molecular sequencing (tSMS) and PathoScope in the analysis of the CF airway microbiome. Three control samples were created with differing amounts of Burkholderia cepacia, Pseudomonas aeruginosa, and Prevotella melaninogenica, three common bacteria found in cystic fibrosis lungs. Paired sputa were also obtained from three study participants with CF before and >6 days after initiation of antibiotics. Antibiotic resistant B. cepacia and P. aeruginosa were identified in concurrently obtained respiratory cultures. Direct sequencing was performed using tSMS, and filtered reads were aligned to reference genomes from NCBI using PathoScope and Kraken and unique clade-specific marker genes using MetaPhlAn. A total of 180–518 K of 6–12 million filtered reads were aligned for each sample. Detection of known pathogens in control samples was most successful using PathoScope. In the CF sputa, alpha diversity measures varied based on the alignment method used, but similar trends were found between pre- and post-antibiotic samples. PathoScope outperformed Kraken and MetaPhlAn in our validation study of artificial bacterial community controls and also has advantages over Kraken and MetaPhlAn of being able to determine bacterial strains and the presence of fungal organisms. PathoScope can be confidently used when evaluating metagenomic data to determine CF airway microbiome diversity.

Antibiotic susceptibility and molecular mechanisms of macrolide resistance in streptococci isolated from adult cystic fibrosis patients.

The cystic fibrosis (CF) airways are colonized by polymicrobial communities with high bacterial load and are influenced by frequent antibiotic exposures. This community includes diverse streptococci, some of which have been directly or indirectly associated with pulmonary exacerbations. As many streptococci are naturally competent, horizontal transfer of antibiotic-resistant determinants coupled with frequent and/or chronic antibiotic exposure may contribute to high resistance rates. In this study, we assessed antibiotic resistance in 413 streptococcal isolates from adult CF patients against nine antibiotics relevant in CF treatment. We observed very low rates of cephalosporin resistance [cefepime and ceftriaxone ( < 2%)], and higher rates of resistance to tetracycline (∼34%) and sulfamethoxazole/trimethoprim (∼45%). The highest rate of antibiotic resistance was to the macrolides [azithromycin (56.4%) and erythromycin (51.6%)]. We also investigated the molecular mechanisms of macrolide resistance and found that only half of our macrolide-resistant streptococci isolates contained the mef (efflux pump) or erm (methylation of 23S ribosomal target site) genes. The majority of isolates were, however, found to have point mutations at position 2058 or 2059 of the 23S ribosomal subunit - a molecular mechanism of resistance not commonly reported in the non-pyogenic and non-pneumococcal streptococci, and unique in comparison with previous studies. The high rates of resistance observed here may result in poor outcomes where specific streptococci are contributing to CF airway disease and serve as a reservoir of resistance genes within the CF airway microbiome.

WS21.6 Comparison of the CF airway microbiome obtained by bronchoscopy vs. sputum

WS21.5 A comparative study of motility and biofilm production in cystic fibrosis (CF) and environmental Pseudomonas aeruginosa (Pa) isolates K.A. Ramsay1, D.W. Reid2,3, S.C. Bell1,2, R.S. Ware1,4, R.E. Watts3, T.J. Kidd1. 1The University of Queensland, Queensland Children’s Medical Research Institute, Brisbane, Australia; 2The Prince Charles Hospital, Thoracic Medicine, Brisbane, Australia; 3QIMR Berghofer Medical Research Institute, Brisbane, Australia; 4The University of Queensland, School of Population Health, Brisbane, Australia

Metabolite transfer with the fermentation product 2,3-butanediol enhances virulence by Pseudomonas aeruginosa

The respiratory tract of cystic fibrosis (CF) patients harbor persistent microbial communities (CF airway microbiome) with Pseudomonas aeruginosa emerging as a dominant pathogen. Within a polymicrobial infection, interactions between co-habitant microbes can be important for pathogenesis, but even when considered, these interactions are not well understood. Here, we show with in vitro experiments that, compared with glucose, common fermentation products from co-habitant bacteria significantly increase virulence factor production, antimicrobial activity and biofilm formation of P. aeruginosa. The maximum stimulating effect was produced with the fermentation product 2,3-butanediol, which is a substrate for P. aeruginosa, resulting in a metabolic relationship between fermenters and this pathogen. The global transcription regulator LasI LasR, which controls quorum sensing, was upregulated threefold with 2,3-butanediol, resulting in higher phenazine and exotoxin concentrations and improved biofilm formation. This indicates that the success of P. aeruginosa in CF airway microbiomes could be governed by the location within the food web with fermenting bacteria. Our findings suggest that interbacterial metabolite transfer in polymicrobial infections stimulates virulence of P. aeruginosa and could have a considerable impact on disease progression.

125 Investigation of cystic fibrosis airway microbiome in patients showing a severe decline in lung function and not responding to conventional antimicrobial therapy

123 A shotgun metaproteomics approach to study the faecal microbiome of patients with cystic fibrosis reveals a reduction of butyrate-producing bacteria G. Debyser1, B. Mesuere2, L. Clement2, G. Duytschaever1, P. Van Hecke1, P. Dawyndt2, K. De Boeck3, P. Vandamme1, B. Devreese1. 1Ghent University, Department of Biochemistry and Microbiology, Ghent, Belgium; 2Ghent University, Department of Applied Mathematics and Computer Science, Ghent, Belgium; 3University Hospital of Leuven, Department of Pediatrics, Leuven, Belgium

The cystic fibrosis airway microbiome

Culture and molecular approaches have established that lower airway infections are polymicrobial. We consider how this new perspective in cystic fibrosis (CF) may affect treatment choices. Standard clinical microbiology of CF infection exacerbations often fails to provide indications of microbial causes that may drive the onset of exacerbation and the anticipated bacteriologic responses to the usual parenteral antibiotics prescribed as treatment. Antimicrobial responses by nonclassical members of the CF airway microbiome may explain why most patients clinically improve. These other organisms contribute to disease either directly as pathogens missed by conventional microbiology or through synergy with conventional pathogens. With these considerations, therapy may best be guided by directed antibiotic therapy to numerically significant isolates. An example is the Streptococcus milleri group, which we now believe to represent new pathogens that profile the exacerbations of infection in the CF lung and that necessitate specific antibiotic therapy to prevent loss of lung function and reduce frequency of exacerbations. A comprehensive understanding of airway infections offers the potential for improved disease management in CF patients. Accurate quantitative microbiology will be a prerequisite for routine intervention based on the polymicrobial perspective of CF infection exacerbations.

The polymicrobial nature of airway infections in cystic fibrosis: Cangene Gold Medal LectureThis article is based on a presentation by Dr. Christopher Sibley at the 60th Annual Meeting of the Canadian Society of Microbiologists in Hamilton, Ontario, on 15 June 2010. Dr. Sibley was the recipient of the 2010 Cangene Gold Medal as the Canadian Graduate Student Microbiologist of the

Microbial communities characterize the airways of cystic fibrosis (CF) patients. Members of these diverse and dynamic communities can be thought of as pathogens, benign commensals, or synergens--organisms not considered pathogens in the traditional sense but with the capacity to alter the pathogenesis of the community through microbe-microbe or polymicrobe-host interactions. Very few bacterial pathogens have been implicated as clinically relevant in CF; however, the CF airway microbiome can be a reservoir of previously unrecognized but clinically relevant organisms. A combination of culture-dependent and culture-independent approaches provides a more comprehensive perspective of CF microbiology than either approach alone. Here we review these concepts, highlight the future challenges for CF microbiology, and discuss the implications for the management of CF airway infections. We suggest that the success of treatment interventions for chronic CF lung disease will rely on the context of the microbes within microbial communities. The microbiology of CF airways may serve as a model to investigate the emergent properties of other clinically relevant microbial communities in the human body.