Background: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. Results:We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. (Continued on next page)Conclusions: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF.

SignificanceDrug-resistant subpopulations of microbes or tumor cells are difficult to detect but can confound disease treatment. In this deep characterization of a chronic fungal infection, we report unprecedented heterogeneity in the drug resistance-related gene MRR1 among Clavispora (Candida) lusitaniae isolates from a single individual. Cells expressing Mrr1 variants that led to drug resistance, by elevated expression of the MDR1-encoded efflux protein, were present at low levels in each sample and thus were undetected in standard assays. We provide evidence that these drug-resistant fungi may arise indirectly in response to other factors present in the infection. Our work suggests that alternative methods may be able to identify drug-resistant subpopulations and thus positively impact patient care.

BackgroundHuman-associated microbial communities include fungi, but we understand little about which fungal species are present, their relative and absolute abundances, and how antimicrobial therapy impacts fungal communities. The disease cystic fibrosis (CF) often involves chronic airway colonization by bacteria and fungi, and these infections cause irreversible lung damage. Fungi are detected more frequently in CF sputum samples upon initiation of antimicrobial therapy, and several studies have implicated the detection of fungi in sputum with worse outcomes. Thus, a more complete understanding of fungi in CF is required.ResultsWe characterized the fungi and bacteria in expectorated sputa from six CF subjects. Samples were collected upon admission for systemic antibacterial therapy and upon the completion of treatment and analyzed using a pyrosequencing-based analysis of fungal internal transcribed spacer 1 (ITS1) and bacterial 16S rDNA sequences. A mixture of Candida species and Malassezia dominated the mycobiome in all samples (74%–99% of fungal reads). There was not a striking trend correlating fungal and bacterial richness, and richness showed a decline after antibiotic therapy particularly for the bacteria. The fungal communities within a sputum sample resembled other samples from that subject despite the aggressive antibacterial therapy. Quantitative PCR analysis of fungal 18S rDNA sequences to assess fungal burden showed variation in fungal density in sputum before and after antibacterial therapy but no consistent directional trend. Analysis of Candida ITS1 sequences amplified from sputum or pure culture-derived genomic DNA from individual Candida species found little (<0.5%) or no variation in ITS1 sequences within or between strains, thereby validating this locus for the purpose of Candida species identification. We also report the enhancement of the publically available Visualization and Analysis of Microbial Population Structures (VAMPS) tool for the analysis of fungal communities in clinical samples.ConclusionsFungi are present in CF respiratory sputum. In CF, the use of intravenous antibiotic therapy often does not profoundly impact bacterial community structure, and we observed a similar stability in fungal species composition. Further studies are required to predict the effects of antibacterials on fungal burden in CF and fungal community stability in non-CF populations.

Individuals with cystic fibrosis (CF) often acquire chronic lung infections that lead to irreversible damage. We sought to examine regional variation in the microbial communities in the lungs of individuals with mild-to-moderate CF lung disease, to examine the relationship between the local microbiota and local damage, and to determine the relationships between microbiota in samples taken directly from the lung and the microbiota in spontaneously expectorated sputum. In this initial study, nine stable, adult CF patients with an FEV1>50% underwent regional sampling of different lobes of the right lung by bronchoalveolar lavage (BAL) and protected brush (PB) sampling of mucus plugs. Sputum samples were obtained from six of the nine subjects immediately prior to the procedure. Microbial community analysis was performed on DNA extracted from these samples and the extent of damage in each lobe was quantified from a recent CT scan. The extent of damage observed in regions of the right lung did not correlate with specific microbial genera, levels of community diversity or composition, or bacterial genome copies per ml of BAL fluid. In all subjects, BAL fluid from different regions of the lung contained similar microbial communities. In eight out of nine subjects, PB samples from different regions of the lung were also similar in microbial community composition, and were similar to microbial communities in BAL fluid from the same lobe. Microbial communities in PB samples were more diverse than those in BAL samples, suggesting enrichment of some taxa in mucus plugs. To our knowledge, this study is the first to examine the microbiota in different regions of the CF lung in clinically stable individuals with mild-to-moderate CF-related lung disease.