Impact of extended Elexacaftor/Tezacaftor/Ivacaftor therapy on the gut microbiome in cystic fibrosis.

BACKGROUND: There is a paucity of knowledge on the longer-term effects of CF transmembrane conductance regulator (CFTR) modulator therapies upon the gut microbiome and associated outcomes. In a pilot study, we investigated longitudinal Elexacaftor/Tezacaftor/Ivacaftor (ETI) therapy on the gut microbiota, metabolomic functioning, and clinical outcomes in people with CF (pwCF). STUDY DESIGN: Faecal samples from 20 pwCF were acquired before and then following 3, 6, and 17+ months of ETI therapy. Samples were subjected to microbiota sequencing and targeted metabolomics to profile and quantify short-chain fatty acid composition. Ten healthy matched controls were included for comparison. Clinical data, including markers of intestinal function were integrated to investigate relationships. RESULTS: Extended ETI therapy increased core microbiota diversity and composition, which translated to gradual shifts in whole microbiota composition towards that observed in healthy controls. Despite becoming more similar over time, CF microbiota and functional metabolite compositions remained significantly different to healthy controls. Antibiotic treatment for pulmonary infection significantly explained a relatively large degree of variation within the whole microbiota and rarer satellite taxa. Clinical outcomes were not significantly different following ETI. CONCLUSIONS: Whilst differences persisted, a positive trajectory towards the microbiota observed in healthy controls was found. We posit that progression was predominately impeded by pulmonary antibiotics administration. We recommend future studies use integrated omics approaches within a combination of long-term longitudinal patient studies and model experimental systems. This will deepen our understanding of the impacts of CFTR modulator therapy and respiratory antibiotic interventions upon the gut microbiome and gastrointestinal pathophysiology in CF.

Ecological patterns and processes of temporal turnover within lung infection microbiota.

BACKGROUND: Chronic infection and consequent airway inflammation are the leading causes of morbidity and early mortality for people living with cystic fibrosis (CF). However, lower airway infections across a range of chronic respiratory diseases, including in CF, do not follow classical 'one microbe, one disease' concepts of infection pathogenesis. Instead, they are comprised of diverse and temporally dynamic lung infection microbiota. Consequently, temporal dynamics need to be considered when attempting to associate lung microbiota with changes in disease status. Set within an island biogeography framework, we aimed to determine the ecological patterns and processes of temporal turnover within the lung microbiota of 30 paediatric and adult CF patients prospectively sampled over a 3-year period. Moreover, we aimed to ascertain the contributions of constituent chronic and intermittent colonizers on turnover within the wider microbiota. RESULTS: The lung microbiota within individual patients was partitioned into constituent chronic and intermittent colonizing groups using the Leeds criteria and visualised with persistence-abundance relationships. This revealed bacteria chronically infecting a patient were both persistent and common through time, whereas intermittently infecting taxa were infrequent and rare; respectively representing the resident and transient portions of the wider microbiota. It also indicated that the extent of chronic colonization was far greater than could be appreciated with microbiological culture alone. Using species-time relationships to measure temporal turnover and Vellend's rationalized ecological processes demonstrated turnover in the resident chronic infecting groups was conserved and underpinned principally by the deterministic process of homogenizing dispersal. Conversely, intermittent colonizing groups, representing newly arrived immigrants and transient species, drove turnover in the wider microbiota and were predominately underpinned by the stochastic process of drift. For adult patients, homogenizing dispersal and drift were found to be significantly associated with lung function. Where a greater frequency of homogenizing dispersal was observed with worsening lung function and conversely drift increased with better lung function. CONCLUSIONS: Our work provides a novel ecological framework for understanding the temporal dynamics of polymicrobial infection in CF that has translational potential to guide and improve therapeutic targeting of lung microbiota in CF and across a range of chronic airway diseases. Video Abstract.

Tezacaftor/Ivacaftor therapy has negligible effects on the cystic fibrosis gut microbiome.

People with cystic fibrosis (pwCF) experience a range of persistent gastrointestinal symptoms throughout life. There is evidence indicating interaction between the microbiota and gut pathophysiology in CF. However, there is a paucity of knowledge on the potential effects of CF transmembrane conductance regulator (CFTR) modulator therapies on the gut microbiome. In a pilot study, we investigated the impact of Tezacaftor/Ivacaftor dual combination CFTR modulator therapy on the gut microbiota and metabolomic functioning in pwCF. Fecal samples from 12 pwCF taken at baseline and following placebo or Tezacaftor/Ivacaftor administration were subjected to microbiota sequencing and to targeted metabolomics to assess the short-chain fatty acid (SCFA) composition. Ten healthy matched controls were included as a comparison. Inflammatory calprotectin levels and patient symptoms were also investigated. No significant differences were observed in overall gut microbiota characteristics between any of the study stages, extended also across intestinal inflammation, gut symptoms, and SCFA-targeted metabolomics. However, microbiota and SCFA metabolomic compositions, in pwCF, were significantly different from controls in all study treatment stages. CFTR modulator therapy with Tezacaftor/Ivacaftor had negligible effects on both the gut microbiota and SCFA composition across the course of the study and did not alter toward compositions observed in healthy controls. Future longitudinal CFTR modulator studies will investigate more effective CFTR modulators and should use prolonged sampling periods, to determine whether longer-term changes occur in the CF gut microbiome. IMPORTANCE People with cystic fibrosis (pwCF) experience persistent gastrointestinal (GI) symptoms throughout life. The research question "how can we relieve gastrointestinal symptoms, such as stomach pain, bloating, and nausea?" remains a top priority for clinical research in CF. While CF transmembrane conductance regulator (CFTR) modulator therapies are understood to correct underlying issues of CF disease and increasing the numbers of pwCF are now receiving some form of CFTR modulator treatment. It is not known how these therapies affect the gut microbiome or GI system. In this pilot study, we investigated, for the first time, effects of the dual combination CFTR modulator medicine, Tezacaftor/Ivacaftor. We found it had negligible effects on patient GI symptoms, intestinal inflammation, or gut microbiome composition and functioning. Our findings are important as they fill important knowledge gaps on the relative effectiveness of these widely used treatments. We are now investigating triple combination CFTR modulators with prolonged sampling periods.

Prior exposure of microbial communities to seawater reduces resilience but increases compositional and functional resistance to flooding events.

Storm surges, flooding, and the encroachment of seawater onto agricultural land are predicted to increase with climate change. These flooding events fundamentally alter many soil properties and have knock-on effects on the microbial community composition and its functioning. The hypotheses tested in this study were (1) that the extent of change (resistance) of microbial community functioning and structure during seawater flooding is a factor of pre-adaptation to the stress, and (2) if structure and function are altered, the pre-adaptation will result in communities returning to previous state prior to flooding (resilience) faster than unexposed communities. We chose a naturally occurring saltmarsh-terrestrial pasture gradient from which three elevations were selected to create mesocosms. By selecting these sites, we were able to incorporate the legacy of differing levels of seawater ingress and exposure. Mesocosms were submerged in seawater for 0, 1, 96- and 192-h, with half of the mesocosms sacrificed immediately after flooding, and the other half taken after a 14 day "recovery" period. The following parameters were monitored: 1) changes in soil environmental parameters, 2) prokaryotic community composition, and 3) microbial functioning. Our results indicated that any length of seawater inundation significantly altered the physicochemical properties of all the soils, although a greater change is observed in the pasture site compared to the saltmarsh sites. These changes remained following a recovery period. Interestingly, our results indicated that for community composition, there was a high degree of resistance for the Saltmarsh mesocosms, with the Pasture mesocosm displaying higher resilience. Further, we observed a functional shift in the enzyme activities with labile hemicellulose being preferentially utilised over cellulose, with the effect increasing with longer floods. These results suggest that changing bacterial physiology is more critical to understanding the impact of storm surges on agricultural systems than bulk community change.

Effects of postage on recovery of pathogens from cystic fibrosis sputum samples.

BACKGROUND: Regular surveillance microbiology of sputum is used in cystic fibrosis (CF) to monitor for new pathogens and target treatments. A move to remote clinics has meant greater reliance on samples collected at home and posted back. The impact of delays and sample disruption caused by posting has not been systematically assessed but could have significant implications for CF microbiology. METHODS: Sputum samples collected from adult CF patients were mixed, split, and either processed immediately or posted back to laboratory. Processing involved a further split into aliquots for culture-dependant and-independent microbiology (quantitative PCR [QPCR] and microbiota sequencing). We calculated retrieval by both approaches for five typical CF pathogens: Pseudomonas aeruginosa, Burkholderia cepacia complex, Achromobacter xylosoxidans, Staphylococcus aureus and Stenotrophomonas maltophilia. RESULTS: 93 paired samples were collected from 73 CF patients. Median interval between sample posting and receipt was 5 days (range 1-10). For culture, overall concordance for posted and fresh samples was 86% across the five targeted pathogens (ranging from 57 to 100% for different organisms), with no bias towards either sample type. For QPCR, overall concordance was 62% (range 39-84%), again with no bias towards fresh or posted samples. There were no significant differences in culture or QPCR for samples with short (≤3days) versus extended (≥7days) postal delays. Posting had no significant impact on pathogen abundance nor on microbiota characteristics. CONCLUSIONS: Posted sputum samples reliably reproduced culture-based and molecular microbiology of freshly collected samples, even after prolonged delays at ambient conditions. This supports use of posted samples during remote monitoring.

Mineralogy affects prokaryotic community composition in an acidic metal mine.

The microbial ecology of acidic mine and sulfide cave ecosystems is well characterised with respect to aquatic communities, typically revealing low taxonomic complexity and dominance by a relatively limited number of cosmopolitan acidophilic bacterial and archaeal taxa. Whilst pH, temperature, and geochemistry are recognised drivers of diversity in these ecosystems, the specific question of a possible influence of substratum mineralogy on microbial community composition remains unanswered. Here we address this void, using 81 subterranean mineral samples from a low temperature abandoned, acidic, sulfide ore mine system at Mynydd Parys (Parys Mountain in English), Wales, UK. Four primary and 15 secondary minerals were identified via x-ray diffraction, each sample containing a maximum of five and an average of two minerals. The mineralogy of primary (e.g. pyrite and quartz) and secondary (e.g. melanterite and pisanite) minerals was significantly correlated with prokaryotic community structure at multiple taxonomic levels, implying that the mineralosphere effect reported in less extreme terrestrial environments is also implicated in driving prokaryotic community composition in extremely acidic, base metal-bearing sulfide mineralisation at Mynydd Parys. Twenty phyla were identified, nine of which were abundant (mean relative abundance >1%). While taxa characteristic of acidic mines were detected, for example Leptospirillum (phylum Nitrospirae), Acidithiobacillus (phylum Proteobacteria), Sulfobacillus (phylum Firmicutes) and Ferroplasma (phylum Euryarchaeota), their abundance in individual samples was highly variable. Indeed, in the majority of the 81 samples investigated the abundance of these and other typical acidic mine taxa was low, with 25% of samples devoid of sequences from recognised acidic mine taxa. Most notable amongst the bacterial taxa not previously reported in such environments were the recently cultivated Muribaculaceae family (phylum Bacteroidetes), which often dominated Mynydd Parys samples regardless of their mineralogical content. Our results pose further questions regarding the mechanisms by which taxa not previously reported in such extreme environments appear to survive in Mynydd Parys, opening up research pathways for exploring the biodiversity drivers underlying microbial community composition and function in extremely acidic mine environments.

Bacterial Culture Underestimates Lung Pathogen Detection and Infection Status in Cystic Fibrosis.

Microbiological surveillance of airway secretions is central to clinical care in cystic fibrosis (CF). However, the efficacy of microbiological culture, the diagnostic gold standard for pathogen detection, has been increasingly questioned. Here we compared culture with targeted quantitative PCR (QPCR) for longitudinal detection of 2 key pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Prospectively collected respiratory samples taken from 20 pediatric and 20 adult CF patients over a period of 3-years were analyzed. Patients were eligible if considered free of chronic Pseudomonas infection within 12-months prior to start of study. QPCR revealed high levels of infection with both pathogens not apparent from culture alone. Pseudomonas and Staphylococcus were detected by culture on at least one sampling occasion in 12 and 29 of the patients, respectively. Conversely, both pathogens were detected in all 40 patients by QPCR. Classification of infection status also significantly altered in both pediatric and adult patients, where the number of patients deemed chronically infected with Pseudomonas and Staphylococcus increased from 1 to 28 and 9 to 34, respectively. Overall, Pseudomonas and Staphylococcus infection status classification changed respectively for 36 and 27 of all patients. In no cases did molecular identification lead to a patient being in a less clinically serious infection category. Pathogen detection and infection status classification significantly increased when assessed by QPCR in comparison to culture. This could have implications for clinical care of CF patients, including accuracy of infection diagnosis, relevant and timely antibiotic selection, antimicrobial resistance development, establishment of chronic infection, and cross-infection control. IMPORTANCE Chronic lung infection is the leading cause of morbidity and early mortality for people with cystic fibrosis (pwCF). Microbiological surveillance to detect lung pathogens is recommended as best practise in CF patient care. Here we studied pathogen detection in 40 pwCF over several years. We found that microbiological culture, the diagnostic gold standard, was significantly disparate to targeted culture-independent approaches for detection and determination of chronic infection status of two important pathogens in CF. Pathogen detection was significantly lower by culture and consequently infection status was also misclassified in most cases. In particular, the extent of chronic infection by both P. aeruginosa and S. aureus not realized with culture was striking. Our findings have implications for the development of infection and clinical care of pwCF. Future longitudinal studies with greater patient numbers will be needed to establish the full extent of the clinical implications indicated from this study.

Reproducibility of Bacterial Cellulose Nanofibers Over Sub-Cultured Generations for the Development of Novel Textiles.

The textile industry is in crisis and under pressure to minimize the environmental impact on its practices. Bacterial cellulose (BC), a naturally occurring form of cellulose, displays properties superior to those of its cotton plant counterpart, such as enhanced purity, crystallinity, tensile strength, and water retention and is thus suitable for an array of textile applications. It is synthesized from a variety of microorganisms but is produced in most abundance by Komagataeibacter xylinus. K. xylinus is available as a type strain culture and exists in the microbial consortium commonly known as Kombucha. Whilst existing literature studies have described the effectiveness of both K. xylinus isolates and Kombucha in the production of BC, this study investigated the change in microbial communities across several generations of sub-culturing and the impact of these communities on BC yield. Using Kombucha and the single isolate strain K. xylinus as inocula in Hestrin and Schramm liquid growth media, BC pellicles were propagated. The resulting pellicles and residual liquid media were used to further inoculate fresh liquid media, and this process was repeated over three generations. For each generation, the thickness of the pellicles and their appearance under SEM were recorded. 16S rRNA sequencing was conducted on both pellicles and liquid media samples to assess changes in communities. The results indicated that the genus Komagataeibacter was the most abundant species in all samples. Cultures seeded with Kombucha yielded thicker cellulose pellicles than those seeded with K. xylinus, but all the pellicles had similar nanofibrillar structures, with a mix of liquid and pellicle inocula producing the best yield of BC after three generations of sub-culturing. Therefore, Kombucha starter cultures produce BC pellicles which are more reproducible across generations than those created from pure isolates of K. xylinus and could provide a reproducible sustainable model for generating textile materials.

Intestinal function and transit associate with gut microbiota dysbiosis in cystic fibrosis.

BACKGROUND: Most people with cystic fibrosis (pwCF) suffer from gastrointestinal symptoms and are at risk of gut complications. Gut microbiota dysbiosis is apparent within the CF population across all age groups, with evidence linking dysbiosis to intestinal inflammation and other markers of health. This pilot study aimed to investigate the potential relationships between the gut microbiota and gastrointestinal physiology, transit, and health. STUDY DESIGN: Faecal samples from 10 pwCF and matched controls were subject to 16S rRNA sequencing. Results were combined with clinical metadata and MRI metrics of gut function to investigate relationships. RESULTS: pwCF had significantly reduced microbiota diversity compared to controls. Microbiota compositions were significantly different, suggesting remodelling of core and rarer satellite taxa in CF. Dissimilarity between groups was driven by a variety of taxa, including Escherichia coli, Bacteroides spp., Clostridium spp., and Faecalibacterium prausnitzii. The core taxa were explained primarily by CF disease, whilst the satellite taxa were associated with pulmonary antibiotic usage, CF disease, and gut function metrics. Species-specific ordination biplots revealed relationships between taxa and the clinical or MRI-based variables observed. CONCLUSIONS: Alterations in gut function and transit resultant of CF disease are associated with the gut microbiota composition, notably the satellite taxa. Delayed transit in the small intestine might allow for the expansion of satellite taxa resulting in potential downstream consequences for core community function in the colon.

Relationships between community composition, productivity and invasion resistance in semi-natural bacterial microcosms.

Common garden experiments that inoculate a standardised growth medium with synthetic microbial communities (i.e. constructed from individual isolates or using dilution cultures) suggest that the ability of the community to resist invasions by additional microbial taxa can be predicted by the overall community productivity (broadly defined as cumulative cell density and/or growth rate). However, to the best of our knowledge, no common garden study has yet investigated the relationship between microbial community composition and invasion resistance in microcosms whose compositional differences reflect natural, rather than laboratory-designed, variation. We conducted experimental invasions of two bacterial strains (Pseudomonas fluorescens and Pseudomonas putida) into laboratory microcosms inoculated with 680 different mixtures of bacteria derived from naturally occurring microbial communities collected in the field. Using 16S rRNA gene amplicon sequencing to characterise microcosm starting composition, and high-throughput assays of community phenotypes including productivity and invader survival, we determined that productivity is a key predictor of invasion resistance in natural microbial communities, substantially mediating the effect of composition on invasion resistance. The results suggest that similar general principles govern invasion in artificial and natural communities, and that factors affecting resident community productivity should be a focal point for future microbial invasion experiments.

Much like animals and plants, microorganisms such as bacteria and fungi naturally live in communities, where different species exist together and share the same resources. These communities can be quite stable over time and resist the invasion of new species ­ for example, by collectively and rapidly consuming all the available resources before invaders arrive. The gut microbiome is one example of such a microbial community, but there are many others. There have been many studies of how artificial microbial communities created in the lab resist invasion, but it remains unclear how naturally-occurring microbial communities do so, because they are harder to study in the lab. A leading theory is that certain combinations of microbes (i.e. communities) grow and consume resources faster than other combinations ­ this is known as achieving high productivity. Jones et al. conducted invasion experiments across hundreds of naturally-occurring microbial communities collected from woodland puddles that form in the exposed roots of beech trees. Each community contained different combinations of bacteria, but they all largely survived by breaking down leaf litter, so Jones et al. created a tea from beech leaves in which to grow these natural communities in the lab. The relationships between community composition, productivity and invasion resistance were then assessed using a combination of DNA sequencing, measurements of community growth and measurements of invader survival. Jones et al. found that natural combinations of bacteria that grew well together drove invasion resistance in these communities, mirroring results seen in much more artificial communities grown in the lab. These results suggest that productivity is a key factor underpinning invasion resistance in naturally-occurring microbial communities. This is a useful insight that could shape thinking about how the long-term stability of beneficial microbial communities ­ such as healthy gut microbiomes ­ might be improved, and how harmful communities ­ such as dental plaques ­ could be destabilised. The next step will be to conduct similar experiments in other natural microbe communities to see how generally applicable these results are.

Model Systems to Study the Chronic, Polymicrobial Infections in Cystic Fibrosis: Current Approaches and Exploring Future Directions.

A recent workshop titled "Developing Models to Study Polymicrobial Infections," sponsored by the Dartmouth Cystic Fibrosis Center (DartCF), explored the development of new models to study the polymicrobial infections associated with the airways of persons with cystic fibrosis (CF). The workshop gathered 35+ investigators over two virtual sessions. Here, we present the findings of this workshop, summarize some of the challenges involved with developing such models, and suggest three frameworks to tackle this complex problem. The frameworks proposed here, we believe, could be generally useful in developing new model systems for other infectious diseases. Developing and validating new approaches to study the complex polymicrobial communities in the CF airway could open windows to new therapeutics to treat these recalcitrant infections, as well as uncovering organizing principles applicable to chronic polymicrobial infections more generally.

Bacterial communities in larger islands have reduced temporal turnover.

Patterns of species diversity provide fundamental insights into the underlying mechanisms and processes that regulate biodiversity. The species-time relationship (STR) has the potential to be one such pattern; in a comparable manner to its more extensively studied spatial analogue, the species-area relationship (SAR), which has been pivotal in the development of ecological models and theories. We sought to determine the mechanisms and processes that underpin STR patterns of temporal turnover by sampling bacterial communities within ten water-filled tree-holes on the same European beech tree through the course of a year. We took this natural model system to represent an archipelago of islands of varying sizes and with shared common immigration sources. We observed an inverse relationship between STR-derived turnover rates and island size. Further, turnover was related to island size and not island isolation within the study system as indicated by a low frequency of dispersal limitation and high homogenizing dispersal. Compared to SARs, STRs are understudied, as such, the findings from the current study should provide a renewed interest in STR-based patterns and processes.

Gut microbiota development during infancy: Impact of introducing allergenic foods.

BACKGROUND: The gut microbiota potentially plays an important role in the immunologic education of the host during early infancy. OBJECTIVE: We sought to determine how the infant gut microbiota evolve during infancy, particularly in relation to hygiene-related environmental factors, atopic disorders, and a randomized introduction of allergenic solids. METHODS: A total of 1303 exclusively breast-fed infants were enrolled in a dietary randomized controlled trial (Enquiring About Tolerance study) from 3 months of age. In this nested longitudinal study, fecal samples were collected at baseline, with additional sampling of selected cases and controls at 6 and 12 months to study the evolution of their gut microbiota, using 16S ribosomal RNA gene-targeted amplicon sequencing. RESULTS: In the 288 baseline samples from exclusively breast-fed infant at 3 months, the gut microbiota was highly heterogeneous, forming 3 distinct clusters: Bifidobacterium-rich, Bacteroides-rich, and Escherichia/Shigella-rich. Mode of delivery was the major discriminating factor. Increased Clostridium sensu stricto relative abundance at 3 months was associated with presence of atopic dermatitis on examination at age 3 and 12 months. From the selected cases and controls with longitudinal samples (n = 70), transition to Bacteroides-rich communities and influx of adult-specific microbes were observed during the first year of life. The introduction of allergenic solids promoted a significant increase in Shannon diversity and representation of specific microbes, such as genera belonging to Prevotellaceae and Proteobacteria (eg, Escherichia/Shigella), as compared with infants recommended to exclusively breast-feed. CONCLUSIONS: Specific gut microbiota characteristics of samples from 3-month-old breast-fed infants were associated with cesarean birth, and greater Clostridium sensu stricto abundance was associated with atopic dermatitis. The randomized introduction of allergenic solids from age 3 months alongside breast-feeding was associated with differential dynamics of maturation of the gut microbial communities.

Towards a microbial process-based understanding of the resilience of peatland ecosystem service provisioning - A research agenda.

Peatlands are wetland ecosystems with great significance as natural habitats and as major global carbon stores. They have been subject to widespread exploitation and degradation with resulting losses in characteristic biota and ecosystem functions such as climate regulation. More recently, large-scale programmes have been established to restore peatland ecosystems and the various services they provide to society. Despite significant progress in peatland science and restoration practice, we lack a process-based understanding of how soil microbiota influence peatland functioning and mediate the resilience and recovery of ecosystem services, to perturbations associated with land use and climate change. We argue that there is a need to: in the short-term, characterise peatland microbial communities across a range of spatial and temporal scales and develop an improved understanding of the links between peatland habitat, ecological functions and microbial processes; in the medium term, define what a successfully restored 'target' peatland microbiome looks like for key carbon cycle related ecosystem services and develop microbial-based monitoring tools for assessing restoration needs; and in the longer term, to use this knowledge to influence restoration practices and assess progress on the trajectory towards 'intact' peatland status. Rapid advances in genetic characterisation of the structure and functions of microbial communities offer the potential for transformative progress in these areas, but the scale and speed of methodological and conceptual advances in studying ecosystem functions is a challenge for peatland scientists. Advances in this area require multidisciplinary collaborations between peatland scientists, data scientists and microbiologists and ultimately, collaboration with the modelling community. Developing a process-based understanding of the resilience and recovery of peatlands to perturbations, such as climate extremes, fires, and drainage, will be key to meeting climate targets and delivering ecosystem services cost effectively.

The airways microbiome of individuals with asthma treated with high and low doses of inhaled corticosteroids.

BACKGROUND: Inhaled corticosteroids (ICS) are the mainstay of asthma treatment, but evidence suggests a link between ICS usage and increased rates of respiratory infections. We assessed the composition of the asthmatic airways microbiome in asthma patients taking low and high dose ICS and the stability of the microbiome over a 2 week period. METHODS: We prospectively recruited 55 individuals with asthma. Of these, 22 were on low-dose ICS and 33 on high-dose ICS (16 on budesonide, 17 on fluticasone propionate). Sputum from each subject underwent DNA extraction, amplification and 16S rRNA gene sequencing of the bacterial component of the microbiome. 19 subjects returned for further sputum induction after 24 h and 2 weeks. RESULTS: A total of 5,615,037 sequencing reads revealed 167 bacterial taxa in the asthmatic airway samples, with the most abundant being Streptococcus spp. No significant differences in sputum bacterial load or overall community composition were seen between the low- and high-dose ICS groups. However, Streptococcus spp. showed significantly higher relative abundance in subjects taking low-dose ICS (p = 0.002). Haemophilus parainfluenzae was significantly more abundant in subjects on high-dose fluticasone propionate than those on high-dose budesonide (p = 0.047). There were no statistically significant changes in microbiota composition over a 2-week period. DISCUSSION: Whilst no significant differences were observed between the low- and high-dose ICS groups, increased abundance of the potential pathogen H. parainfluenzae was observed in patients taking high-dose fluticasone propionate compared to those taking high-dose budesonide. The microbiota were stable over fourteen days, providing novel evidence of the established community of bacteria in the asthmatic airways. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT02671773.

Lung function and microbiota diversity in cystic fibrosis.

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. 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. Video Abstract.

Bacterial adaptation is constrained in complex communities.

A major unresolved question is how bacteria living in complex communities respond to environmental changes. In communities, biotic interactions may either facilitate or constrain evolution depending on whether the interactions expand or contract the range of ecological opportunities. A fundamental challenge is to understand how the surrounding biotic community modifies evolutionary trajectories as species adapt to novel environmental conditions. Here we show that community context can dramatically alter evolutionary dynamics using a novel approach that 'cages' individual focal strains within complex communities. We find that evolution of focal bacterial strains depends on properties both of the focal strain and of the surrounding community. In particular, there is a stronger evolutionary response in low-diversity communities, and when the focal species have a larger genome and are initially poorly adapted. We see how community context affects resource usage and detect genetic changes involved in carbon metabolism and inter-specific interaction. The findings demonstrate that adaptation to new environmental conditions should be investigated in the context of interspecific interactions.

Bacterial dominance is due to effective utilisation of secondary metabolites produced by competitors.

Interactions between bacteria govern the progression of respiratory infections; however, the mechanisms underpinning these interactions are still unclear. Understanding how a bacterial species comes to dominate infectious communities associated with respiratory infections has direct relevance to treatment. In this study, Burkholderia, Pseudomonas, and Staphylococcus species were isolated from the sputum of an individual with Cystic Fibrosis and assembled in a fully factorial design to create simple microcosms. Measurements of growth and habitat modification were recorded over time, the later using proton Nuclear Magnetic Resonance spectra. The results showed interactions between the bacteria became increasingly neutral over time. Concurrently, the bacteria significantly altered their ability to modify the environment, with Pseudomonas able to utilise secondary metabolites produced by the other two isolates, whereas the reverse was not observed. This study indicates the importance of including data about the habitat modification of a community, to better elucidate the mechanisms of bacterial interactions.

Selection and assembly of indigenous bacteria and methanogens from spent metalworking fluids and their potential as a starting culture in a fluidized bed reactor.

Waste metalworking fluids (MWFs) are highly biocidal resulting in real difficulties in the, otherwise favoured, bioremediation of these high chemical oxygen deman (COD) wastes anaerobically in bioreactors. We have shown, as a proof of concept, that it is possible to establish an anaerobic starter culture using strains isolated from spent MWFs which are capable of reducing COD or, most significantly, methanogenesis in this biocidal waste stream. Bacterial strains (n = 99) and archaeal methanogens (n = 28) were isolated from spent MWFs. The most common bacterial strains were Clostridium species (n = 45). All methanogens were identified as Methanosarcina mazei. Using a random partitions design (RPD) mesocosm experiment, we found that bacterial diversity and species-species interactions had significant effects on COD reduction but that bacterial composition did not. The RPD study showed similar effects on methanogenesis, except that composition was also significant. We identified bacterial species with positive and negative effects on methane production. A consortium of 16 bacterial species and three methanogens was used to initiate a fluidized bed bioreactor (FBR), in batch mode. COD reduction and methane production were variable, and the reactor was oscillated between continuous and batch feeds. In both microcosm and FBR experiments, periodic inconsistencies in bacterial reduction in fermentative products to formic and acetic acids were identified as a key issue.