Leah Cuthbertson

Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition

Rationale Despite the potentially important roles for infection in adult non-cystic fibrosis (CF) bronchiectasis disease progression, the bacterial species present in the lower airways of these patients is poorly characterised. Objectives To provide a comprehensive cross-sectional analysis of bacterial content of lower airway samples from patients with non-CF bronchiectasis using culture-independent microbiology. Methods Paired induced sputum and bronchoalveolar lavage samples, obtained from 41 adult patients with non-CF bronchiectasis, were analysed by 16S ribosomal RNA gene pyrosequencing. Assessment of species distribution and dispersal allowed ‘core’ and ‘satellite’ bacterial populations to be defined for this patient group. Microbiota characteristics correlated with clinical markers of disease. Measurement and main results 140 bacterial species were identified, including those associated with respiratory tract infections and opportunistic infections more generally. A group of core species, consisting of species detected frequently and in high abundance, was defined. Core species included those currently associated with infection in bronchiectasis, such as Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, and many species that would be unlikely to be reported through standard diagnostic surveillance. These included members of the genera Veillonella, Prevotella and Neisseria. The comparative contribution of core and satellite groups suggested a low level of random species acquisition. Bacterial diversity was significantly positively correlated with forced expiratory volume in 1 s (FEV1) and bacterial community composition similarity correlated significantly with FEV1, neutrophil count and Leicester cough score. Conclusions Characteristics of the lower airways microbiota of adult patients with non-CF bronchiectasis correlate significantly with clinical markers of disease severity.

Three Clinically Distinct Chronic Pediatric Airway Infections Share a Common Core Microbiota

RATIONALE DNA-based microbiological studies are moving beyond studying healthy human microbiota to investigate diverse infectious diseases, including chronic respiratory infections, such as those in the airways of people with cystic fibrosis (CF) and non-CF bronchiectasis. The species identified in the respiratory secretion microbiota from such patients can be classified into those that are common and abundant among similar subjects (core) versus those that are infrequent and rare (satellite). This categorization provides a vital foundation for investigating disease pathogenesis and improving therapy. However, whether the core microbiota of people with different respiratory diseases, which are traditionally associated with specific culturable pathogens, are unique or shared with other chronic infections of the lower airways is not well studied. Little is also known about how these chronic infection microbiota change from childhood to adulthood. OBJECTIVES We sought to compare the core microbiota in respiratory specimens from children and adults with different chronic lung infections. METHODS We used bacterial 16S rRNA gene pyrosequencing, phylogenetic analysis, and ecological statistical tools to compare the core microbiota in respiratory samples from three cohorts of symptomatic children with clinically distinct airway diseases (protracted bacterial bronchitis, bronchiectasis, CF), and from four healthy children. We then compared the core pediatric respiratory microbiota with those in samples from adults with bronchiectasis and CF. MEASUREMENTS AND MAIN RESULTS All three pediatric disease cohorts shared strikingly similar core respiratory microbiota that differed from adult CF and bronchiectasis microbiota. The most common species in pediatric disease cohort samples were also detected in those from healthy children. The adult CF and bronchiectasis microbiota also differed from each other, suggesting common early infection airway microbiota that diverge by adulthood. The shared core pediatric microbiota included both traditional pathogens and many species not routinely identified by standard culture. CONCLUSIONS Our results indicate that these clinically distinct chronic airway infections share common early core microbiota, which are likely shaped by natural aspiration and impaired clearance of the same airway microbes, but that disease-specific characteristics select for divergent microbiota by adulthood. Longitudinal and interventional studies will be required to define the relationships between microbiota, treatments, and disease progression.

Reducing bias in bacterial community analysis of lower respiratory infections

High-throughput pyrosequencing and quantitative PCR (Q-PCR) analysis offer greatly improved accuracy and depth of characterisation of lower respiratory infections. However, such approaches suffer from an inability to distinguish between DNA derived from viable and non-viable bacteria. This discrimination represents an important step in characterising microbial communities, particularly in contexts with poor clearance of material or high antimicrobial stress, as non-viable bacteria and extracellular DNA can contribute significantly to analyses. Pre-treatment of samples with propidium monoazide (PMA) is an effective approach to non-viable cell exclusion (NVCE). However, the impact of NVCE on microbial community characteristics (abundance, diversity, composition and structure) is not known. Here, adult cystic fibrosis (CF) sputum samples were used as a paradigm. The effects of PMA treatment on CF sputum bacterial community characteristics, as analysed by pyrosequencing and enumeration by species-specific (Pseudomonas aeruginosa) and total bacterial Q-PCR, were assessed. At the local community level, abundances of both total bacteria and of P. aeruginosa were significantly lower in PMA-treated sample portions. Meta-analysis indicated no overall significant differences in diversity; however, PMA treatment resulted in a significant alteration in local community membership in all cases. In contrast, at the metacommunity level, PMA treatment resulted in an increase in community evenness, driven by an increase in diversity, predominately representing rare community members. Importantly, PMA treatment facilitated the detection of both recognised and emerging CF pathogens, significantly influencing ‘core’ and ‘satellite’ taxa group membership. Our findings suggest failure to implement NVCE may result in skewed bacterial community analyses.

Respiratory microbiota resistance and resilience to pulmonary exacerbation and subsequent antimicrobial intervention.

Pulmonary symptoms in cystic fibrosis (CF) begin in early life with chronic lung infections and concomitant airway inflammation leading to progressive loss of lung function. Gradual pulmonary function decline is interspersed with periods of acute worsening of respiratory symptoms known as CF pulmonary exacerbations (CFPEs). Cumulatively, CFPEs are associated with more rapid disease progression. In this study multiple sputum samples were collected from adult CF patients over the course of CFPEs to better understand how changes in microbiota are associated with CFPE onset and management. Data were divided into five clinical periods: pre-CFPE baseline, CFPE, antibiotic treatment, recovery, and post-CFPE baseline. Samples were treated with propidium monoazide prior to DNA extraction, to remove the impact of bacterial cell death artefacts following antibiotic treatment, and then characterised by 16S rRNA gene-targeted high-throughput sequencing. Partitioning CF microbiota into core and rare groups revealed compositional resistance to CFPE and resilience to antibiotics interventions. Mixed effects modelling of core microbiota members revealed no significant negative impact on the relative abundance of Pseudomonas aeruginosa across the exacerbation cycle. Our findings have implications for current CFPE management strategies, supporting reassessment of existing antimicrobial treatment regimens, as antimicrobial resistance by pathogens and other members of the microbiota may be significant contributing factors.

Siblings of patients with Crohn’s disease exhibit a biologically relevant dysbiosis in mucosal microbial metacommunities

Objective To determine the existence of mucosal dysbiosis in siblings of patients with Crohns disease (CD) using 454 pyrosequencing and to comprehensively characterise and determine the influence of genotypical and phenotypical factors, on that dysbiosis. Siblings of patients with CD have elevated risk of developing CD and display aspects of disease phenotype, including faecal dysbiosis. Whether the mucosal microbiota is disrupted in these at-risk individuals is unknown. Design Rectal biopsy DNA was extracted from 21 patients with quiescent CD, 17 of their healthy siblings and 19 unrelated healthy controls. Mucosal microbiota was analysed by 16S rRNA gene pyrosequencing and were classified into core and rare species. Genotypical risk was determined using Illumina Immuno BeadChip, faecal calprotectin by ELISA and blood T-cell phenotype by flow cytometry. Results Core microbiota of both patients with CD and healthy siblings was significantly less diverse than controls. Metacommunity profiling (Bray–Curtis (SBC) index) showed the sibling core microbial composition to be more similar to CD (SBC=0.70) than to healthy controls, whereas the sibling rare microbiota was more similar to healthy controls (SBC=0.42). Faecalibacterium prausnitzii contributed most to core metacommunity dissimilarity both between siblings and controls, and between patients and controls. Phenotype/genotype markers of CD risk significantly influenced microbiota variation between and within groups, of which genotype had the largest effect. Conclusions Individuals with elevated CD-risk display mucosal dysbiosis characterised by reduced diversity of core microbiota and lower abundance of F. prausnitzii. This dysbiosis in healthy people at risk of CD implicates microbiological processes in CD pathogenesis.

Time between Collection and Storage Significantly Influences Bacterial Sequence Composition in Sputum Samples from Cystic Fibrosis Respiratory Infections

ABSTRACT Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at −80°C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at −80°C within 12 h of collection results in significant changes in the detected community composition.

Implications of multiple freeze-thawing on respiratory samples for culture-independent analyses

Background Best practice when performing culture-independent microbiological analysis of sputum samples involves their rapid freezing and storage at −80 °C. However, accessing biobanked collections can mean that material has been passed through repeated freeze–thaw cycles. The aim of this study was to determine the impact of these cycles on microbial community profiles. Methods Sputum was collected from eight adults with cystic fibrosis, and each sample was subjected to six freeze–thaw cycles. Following each cycle, an aliquot was removed and treated with propidium monoazide (PMA) prior to DNA extraction and 16S rRNA gene pyrosequencing. Results The impact of freeze–thaw cycles was greatest on rare members of the microbiota, with variation beyond that detected with within-sample repeat analysis observed after three cycles. Conclusion Four or more freeze thaw cycles result in a significant distortion of microbiota profiles from CF sputum.

Rearing and foraging affects bumblebee (Bombus terrestris) gut microbiota

Bumblebees are ecologically and economically important as pollinators of crop and wild plants, especially in temperate systems. Species, such as the buff-tailed bumblebee (Bombus terrestris), are reared commercially to pollinate high-value crops. Their highly specific gut microbiota, characterized by low diversity, may affect nutrition and immunity and are likely to be important for fitness and colony health. However, little is known about how environmental factors affect bacterial community structure. We analysed the gut microbiota from three groups of worker bumblebees (B. terrestris) from distinct colonies that varied in rearing and foraging characteristics: commercially reared with restricted foraging (RR); commercially reared with outside foraging (RF); and wild-caught workers (W). Contrary to previous studies, which indicate that bacterial communities are highly conserved across workers, we found that RF individuals had an intermediate community structure compared with RR and W types. Further, this was shaped by differences in the abundances of common operational taxonomic units (OTUs) and the diversity of rare OTUs present, which we propose results from an increase in the variety of carbohydrates obtained through foraging.

136 Impact of propidium monoazide treatment on CF bacterial community pyrosequencing analysis

136 Impact of propidium monoazide treatment on CF bacterial community pyrosequencing analysis L. Cuthbertson1,2, G. Rogers2, L. Hoffman3, A. Oliver1, P. Wing3, M. Carroll4, K. Bruce2, A. Walker5, C. van der Gast1. 1NERC Centre for Ecology and Hydrology, McLean Building, Wallingford, United Kingdom; 2King’s College London, Pharmaceutical Science Division, London, United Kingdom; 3University of Washington, Department of Pediatrics, Seattle, United States; 4Southampton University Hospitals NHS Trust, Cystic Fibrosis Unit, Southampton, United Kingdom; 5Wellcome Trust Sanger Institute, Cambridge, United Kingdom