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.

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.

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.

Mild Cystic Fibrosis Lung Disease Is Associated with Bacterial Community Stability.

Microbial communities in the airways of persons with CF (pwCF) are variable, may include genera that are not typically associated with CF, and their composition can be difficult to correlate with long-term disease outcomes. Leveraging two large data sets characterizing sputum communities of 167 pwCF and associated metadata, we identified five bacterial community types. These communities explain 24% of the variability in lung function in this cohort, far more than single factors like Simpson diversity, which explains only 4%. Subjects with Pseudomonas-dominated communities tended to be older and have reduced percent predicted FEV1 (ppFEV1) compared to subjects with Streptococcus-dominated communities, consistent with previous findings. To assess the predictive power of these five communities in a longitudinal setting, we used random forests to classify 346 additional samples from 24 subjects observed 8 years on average in a range of clinical states. Subjects with mild disease were more likely to be observed at baseline, that is, not in the context of a pulmonary exacerbation, and community structure in these subjects was more self-similar over time, as measured by Bray-Curtis distance. Interestingly, we found that subjects with mild disease were more likely to remain in a mixed Pseudomonas community, providing some support for the climax-attack model of the CF airway. In contrast, patients with worse outcomes were more likely to show shifts among community types. Our results suggest that bacterial community instability may be a risk factor for lung function decline and indicates the need to understand factors that drive shifts in community composition. IMPORTANCE While much research supports a polymicrobial view of the CF airway, one in which the community is seen as the pathogenic unit, only controlled experiments using model bacterial communities can unravel the mechanistic role played by different communities. This report uses a large data set to identify a small number of communities as a starting point in the development of tractable model systems. We describe a set of five communities that explain 24% of the variability in lung function in our data set, far more than single factors like Simpson diversity, which explained only 4%. In addition, we report that patients with severe disease experienced more shifts among community types, suggesting that bacterial community instability may be a risk factor for lung function decline. Together, these findings provide a proof of principle for selecting bacterial community model systems.

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.

Identification of microbial signatures linked to oilseed rape yield decline at the landscape scale.

BACKGROUND: The plant microbiome plays a vital role in determining host health and productivity. However, we lack real-world comparative understanding of the factors which shape assembly of its diverse biota, and crucially relationships between microbiota composition and plant health. Here we investigated landscape scale rhizosphere microbial assembly processes in oilseed rape (OSR), the UK's third most cultivated crop by area and the world's third largest source of vegetable oil, which suffers from yield decline associated with the frequency it is grown in rotations. By including 37 conventional farmers' fields with varying OSR rotation frequencies, we present an innovative approach to identify microbial signatures characteristic of microbiomes which are beneficial and harmful to the host. RESULTS: We show that OSR yield decline is linked to rotation frequency in real-world agricultural systems. We demonstrate fundamental differences in the environmental and agronomic drivers of protist, bacterial and fungal communities between root, rhizosphere soil and bulk soil compartments. We further discovered that the assembly of fungi, but neither bacteria nor protists, was influenced by OSR rotation frequency. However, there were individual abundant bacterial OTUs that correlated with either yield or rotation frequency. A variety of fungal and protist pathogens were detected in roots and rhizosphere soil of OSR, and several increased relative abundance in root or rhizosphere compartments as OSR rotation frequency increased. Importantly, the relative abundance of the fungal pathogen Olpidium brassicae both increased with short rotations and was significantly associated with low yield. In contrast, the root endophyte Tetracladium spp. showed the reverse associations with both rotation frequency and yield to O. brassicae, suggesting that they are signatures of a microbiome which benefits the host. We also identified a variety of novel protist and fungal clades which are highly connected within the microbiome and could play a role in determining microbiome composition. CONCLUSIONS: We show that at the landscape scale, OSR crop yield is governed by interplay between complex communities of both pathogens and beneficial biota which is modulated by rotation frequency. Our comprehensive study has identified signatures of dysbiosis within the OSR microbiome, grown in real-world agricultural systems, which could be used in strategies to promote crop yield. Video abstract.

Bacterial Signatures of Paediatric Respiratory Disease: An Individual Participant Data Meta-Analysis.

Introduction: The airway microbiota has been linked to specific paediatric respiratory diseases, but studies are often small. It remains unclear whether particular bacteria are associated with a given disease, or if a more general, non-specific microbiota association with disease exists, as suggested for the gut. We investigated overarching patterns of bacterial association with acute and chronic paediatric respiratory disease in an individual participant data (IPD) meta-analysis of 16S rRNA gene sequences from published respiratory microbiota studies. Methods: We obtained raw microbiota data from public repositories or via communication with corresponding authors. Cross-sectional analyses of the paediatric (<18 years) microbiota in acute and chronic respiratory conditions, with >10 case subjects were included. Sequence data were processed using a uniform bioinformatics pipeline, removing a potentially substantial source of variation. Microbiota differences across diagnoses were assessed using alpha- and beta-diversity approaches, machine learning, and biomarker analyses. Results: We ultimately included 20 studies containing individual data from 2624 children. Disease was associated with lower bacterial diversity in nasal and lower airway samples and higher relative abundances of specific nasal taxa including Streptococcus and Haemophilus. Machine learning success in assigning samples to diagnostic groupings varied with anatomical site, with positive predictive value and sensitivity ranging from 43 to 100 and 8 to 99%, respectively. Conclusion: IPD meta-analysis of the respiratory microbiota across multiple diseases allowed identification of a non-specific disease association which cannot be recognised by studying a single disease. Whilst imperfect, machine learning offers promise as a potential additional tool to aid clinical diagnosis.

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.

Exploring the putative interactions between chronic kidney disease and chronic periodontitis.

Chronic kidney disease (CKD) and chronic periodontitis (CP) are both common diseases, which are found disproportionately comorbid with each other and have been reported to have a detrimental effect on the progression of each respective disease. They have an overlap in risk factors and both are a source of systemic inflammation along with a wide selection of immunological and non-specific effects that can affect the body over the lifespan of the conditions. Previous studies have investigated the directionality of the relationship between these two diseases; however, there is a lack of literature that has examined how these diseases may be interacting at the localized and systemic level. This review discusses how oral microorganisms have the ability to translocate and have distal effects and provides evidence for microbial involvement in a systemic disease. Furthermore, it summarizes the reported local and systemic effects of CKD and CP and discusses how the interaction of these effects may be responsible for directionality associations reported.

Acquisition and Development of the Extremely Preterm Infant Microbiota Across Multiple Anatomical Sites.

OBJECTIVES: Microbial communities influencing health and disease are being increasingly studied in preterm neonates. There exists little data, however, detailing longitudinal microbial acquisition, especially in the most extremely preterm (<26 weeks' gestation). This study aims to characterize the development of the microbiota in this previously under-represented cohort. METHODS: Seven extremely preterm infant-mother dyads (mean gestation 23.6 weeks) were recruited from a single neonatal intensive care unit. Oral and endotracheal secretions, stool, and breast milk (n = 157 total), were collected over the first 60 days of life. Targeted 16S rRNA gene sequencing identified bacterial communities present. RESULTS: Microbiota of all body sites were most similar immediately following birth and diverged longitudinally. Throughout the sampling period Escherichia, Enterococcus, Staphylococcus, and an Enterobacteriaceae were dominant and well dispersed across all sites. Temporal divergence of the stool from other microbiota was driven by decreasing diversity and significantly greater proportional abundance of Bifidobacteriaceae compared to other sites. CONCLUSIONS: Four taxa dominated all anatomical sampling sites. Rare taxa promoted dissimilarity. Cross-seeding between upstream communities and the stool was demonstrated, possibly relating to buccal colostrum/breast milk exposure and indwelling tubes. Given the importance of dysbiosis in health and disease of extremely preterm infants, better understanding of microbial acquisition within this context may be of clinical benefit.

Plant Rhizosphere Selection of Plasmodiophorid Lineages from Bulk Soil: The Importance of "Hidden" Diversity.

Microbial communities closely associated with the rhizosphere can have strong positive and negative impacts on plant health and growth. We used a group-specific amplicon approach to investigate local scale drivers in the diversity and distribution of plasmodiophorids in rhizosphere/root and bulk soil samples from oilseed rape (OSR) and wheat agri-systems. Plasmodiophorids are plant- and stramenopile-associated protists including well known plant pathogens as well as symptomless endobiotic species. We detected 28 plasmodiophorid lineages (OTUs), many of them novel, and showed that plasmodiophorid communities were highly dissimilar and significantly divergent between wheat and OSR rhizospheres and between rhizosphere and bulk soil samples. Bulk soil communities were not significantly different between OSR and wheat systems. Wheat and OSR rhizospheres selected for different plasmodiophorid lineages. An OTU corresponding to Spongospora nasturtii was positively selected in the OSR rhizosphere, as were two genetically distinct OTUs. Two novel lineages related to Sorosphaerula veronicae were significantly associated with wheat rhizosphere samples, indicating unknown plant-protist relationships. We show that group-targeted eDNA approaches to microbial symbiont-host ecology reveal significant novel diversity and enable inference of differential activity and potential interactions between sequence types, as well as their presence.

Extreme rainfall affects assembly of the root-associated fungal community.

Global warming is resulting in increased frequency of weather extremes. Root-associated fungi play important roles in terrestrial biogeochemical cycling processes, but the way in which they are affected by extreme weather is unclear. Here, we performed long-term field monitoring of the root-associated fungus community of a short rotation coppice willow plantation, and compared community dynamics before and after a once in 100 yr rainfall event that occurred in the UK in 2012. Monitoring of the root-associated fungi was performed over a 3-yr period by metabarcoding the fungal internal transcribed spacer (ITS) region. Repeated soil testing and continuous climatic monitoring supplemented community data, and the relative effects of environmental and temporal variation were determined on the root-associated fungal community. Soil saturation and surface water were recorded throughout the early growing season of 2012, following extreme rainfall. This was associated with a crash in the richness and relative abundance of ectomycorrhizal fungi, with each declining by over 50%. Richness and relative abundance of saprophytes and pathogens increased. We conclude that extreme rainfall events may be important yet overlooked determinants of root-associated fungal community assembly. Given the integral role of ectomycorrhizal fungi in biogeochemical cycles, these events may have considerable impacts upon the functioning of terrestrial ecosystems.

Converting highly productive arable cropland in Europe to grassland: -a poor candidate for carbon sequestration.

Sequestration of atmospheric CO2 as organic carbon by agricultural soils (SOC) is promoted as a climate change mitigation option. IPCC provides guidelines for determining carbon stocks and sequestration potential, incentivising policy changes towards management of farmland for carbon sequestration. However, the basis of the assumption that agricultural soils can sequester significant atmospheric CO2 has been questioned. We sought to determine the potential for conversion of arable cropland to grassland to sequester carbon in the short to medium term and potential limiting factors. There were no differences in SOC stocks in the top 30 cm between grassland up to 17 years old and arable cropland at 14 sites across the UK. However, SOC showed different distribution patterns, being concentrated in the top 10 cm under grassland. Soil microbial communities were significantly different between arable and grassland, with higher biomass and lesser dominance by bacteria in grassland soils. A land use conversion experiment showed these changes occurred within one year of land use change. Failure of grassland soils to accumulate SOC was attributed to reduced available soil nitrogen, resulting in low productivity. The implications of these results for carbon sequestration in soils as a climate change mitigation strategy are discussed.

Reducing Viability Bias in Analysis of Gut Microbiota in Preterm Infants at Risk of NEC and Sepsis.

Necrotising enterocolitis (NEC) and sepsis are serious diseases of preterm infants that can result in feeding intolerance, the need for bowel resection, impaired physiological and neurological development, and high mortality rates. Neonatal healthcare improvements have allowed greater survival rates in preterm infants leading to increased numbers at risk of developing NEC and sepsis. Gut bacteria play a role in protection from or propensity to these conditions and have therefore, been studied extensively using targeted 16S rRNA gene sequencing methods. However, exact epidemiology of these conditions remain unknown and the role of the gut microbiota in NEC remains enigmatic. Many studies have confounding variables such as differing clinical intervention strategies or major methodological issues such as the inability of 16S rRNA gene sequencing methods to determine viable from non-viable taxa. Identification of viable community members is important to identify links between the microbiota and disease in the highly unstable preterm infant gut. This is especially important as remnant DNA is robust and persists in the sampling environment following cell death. Chelation of such DNA prevents downstream amplification and inclusion in microbiota characterisation. This study validates use of propidium monoazide (PMA), a DNA chelating agent that is excluded by an undamaged bacterial membrane, to reduce bias associated with 16S rRNA gene analysis of clinical stool samples. We aim to improve identification of the viable microbiota in order to increase the accuracy of clinical inferences made regarding the impact of the preterm gut microbiota on health and disease. Gut microbiota analysis was completed on stools from matched twins (n = 16) that received probiotics. Samples were treated with PMA, prior to bacterial DNA extraction. Meta-analysis highlighted a significant reduction in bacterial diversity in 68.8% of PMA treated samples as well as significantly reduced overall rare taxa abundance. Importantly, overall abundances of genera associated with protection from and propensity to NEC and sepsis such as: Bifidobacterium; Clostridium, and Staphylococcus sp. were significantly different following PMA-treatment. These results suggest non-viable cell exclusion by PMA-treatment reduces bias in gut microbiota analysis from which clinical inferences regarding patient susceptibility to NEC and sepsis are made.

The gut microbiota of siblings offers insights into microbial pathogenesis of inflammatory bowel disease.

Siblings of patients with Crohn's disease (CD) have elevated risk of developing CD and display aspects of disease phenotype, including faecal dysbiosis. In our recent article we have used 16S rRNA gene targeted high-throughput sequencing to comprehensively characterize the mucosal microbiota in healthy siblings of CD patients, and determine the influence of genotypic and phenotypic factors on the gut microbiota (dysbiosis). We have demonstrated that the core microbiota of both patients with CD and healthy siblings is significantly less diverse than controls. Faecalibacterium prausnitzii contributed most to core metacommunity dissimilarity between both patients and controls and between siblings and controls. Phenotype/genotype markers of CD risk significantly influenced microbiota variation between and within groups, of which genotype had the largest effect. Individuals with elevated CD-risk display mucosal dysbiosis characterized by reduced diversity of core microbiota and lower abundance of F. prausnitzii. The presence of this dysbiosis in healthy people at-risk of CD implicates microbiological processes in CD pathogenesis.