Early-life nasal microbiota dynamics relate to longitudinal respiratory phenotypes in urban children.

BACKGROUND: Five distinct respiratory phenotypes based on latent classes of longitudinal patterns of wheezing, allergic sensitization. and pulmonary function measured in urban children from ages from 0 to 7 years have previously been described. OBJECTIVE: Our aim was to determine whether distinct respiratory phenotypes are associated with early-life upper respiratory microbiota development and environmental microbial exposures. METHODS: Microbiota profiling was performed using 16S ribosomal RNA-based sequencing of nasal samples collected at age 12 months (n = 120) or age 36 months (n = 142) and paired house dust samples collected at 3 months (12-month, n = 73; 36-month, n = 90) from all 4 centers in the Urban Environment and Childhood Asthma (URECA) cohort. RESULTS: In these high-risk urban children, nasal microbiota increased in diversity between ages 12 and 36 months (ß = 2.04; P = .006). Age-related changes in microbiota evenness differed significantly by respiratory phenotypes (interaction P = .0007), increasing most in the transient wheeze group. At age 12 months, respiratory illness (R2 = 0.055; P = .0001) and dominant bacterial genus (R2 = 0.59; P = .0001) explained variance in nasal microbiota composition, and enrichment of Moraxella and Haemophilus members was associated with both transient and high-wheeze respiratory phenotypes. By age 36 months, nasal microbiota was significantly associated with respiratory phenotypes (R2 = 0.019; P = .0376), and Moraxella-dominated microbiota was associated specifically with atopy-associated phenotypes. Analysis of paired house dust and nasal samples indicated that 12 month olds with low wheeze and atopy incidence exhibited the largest number of shared bacterial taxa with their environment. CONCLUSION: Nasal microbiota development over the course of early childhood and composition at age 3 years are associated with longitudinal respiratory phenotypes. These data provide evidence supporting an early-life window of airway microbiota development that is influenced by environmental microbial exposures in infancy and associates with wheeze- and atopy-associated respiratory phenotypes through age 7 years.

Microscopic Colitis Patients Possess a Perturbed and Inflammatory Gut Microbiota.

BACKGROUND: Microscopic colitis (MC), an inflammatory disease of the colon, is characterized by chronic non-bloody diarrhea with characteristic inflammation and for some, collagen deposits in mucosal biopsies. The etiology of MC is unclear, although previous findings implicate luminal factors and thus the gut microbiome. However, the relationships between fecal microbiota and MC are relatively unexplored. METHODS: Stool microbiota of MC (n = 15) and healthy controls (HC; n = 21) were assessed by 16S rRNA V4 amplicon sequencing and analysis performed in QIIME. Gut microbiota functions were predicted using Piphillin and inflammatory potential assessed using an in vitro HT29 colonocyte cell assay. RESULTS: MC patient fecal microbiota were less diverse (Faiths index; p < 0.01) and compositionally distinct (PERMANOVA, weighted UniFrac, R2 = 0.08, p = 0.02) compared with HC subjects. MC microbiota were significantly depleted of members of the Clostridiales, enriched for Prevotella and more likely to be dominated by this genus (Chi2 = 0.03). Predicted pathways enriched in MC microbiota included those related to biosynthesis of antimicrobials, and sphingolipids, to glycan degradation, host defense evasion, and Th17 cell differentiation and activation. In vitro, exposure of cultured colonocytes to cell-free products of MC patient feces indicates reduced gene expression of IL-1B and occludin and increased GPR119 and the lymphocyte chemoattractant CCL20. CONCLUSION: MC gut microbiota are distinct from HC and characterized by lower bacterial diversity and Prevotella enrichment and distinct predicted functional pathways. Limited in vitro experiments indicate that compared with cell-free products from healthy fecal microbiota, MC microbiota induce distinct responses when co-cultured with epithelial cells, implicating microbiota perturbation in MC-associated mucosal dysfunction.

Distinct associations of sputum and oral microbiota with atopic, immunologic, and clinical features in mild asthma.

BACKGROUND: Whether microbiome characteristics of induced sputum or oral samples demonstrate unique relationships to features of atopy or mild asthma in adults is unknown. OBJECTIVE: We sought to determine sputum and oral microbiota relationships to clinical or immunologic features in mild atopic asthma and the impact on the microbiota of inhaled corticosteroid (ICS) treatment administered to ICS-naive subjects with asthma. METHODS: Bacterial microbiota profiles were analyzed in induced sputum and oral wash samples from 32 subjects with mild atopic asthma before and after inhaled fluticasone treatment, 18 atopic subjects without asthma, and 16 nonatopic healthy subjects in a multicenter study (NCT01537133). Associations with clinical and immunologic features were examined, including markers of atopy, type 2 inflammation, immune cell populations, and cytokines. RESULTS: Sputum bacterial burden inversely associated with bronchial expression of type 2 (T2)-related genes. Differences in specific sputum microbiota also associated with T2-low asthma phenotype, a subgroup of whom displayed elevations in lung inflammatory mediators and reduced sputum bacterial diversity. Differences in specific oral microbiota were more reflective of atopic status. After ICS treatment of patients with asthma, the compositional structure of sputum microbiota showed greater deviation from baseline in ICS nonresponders than in ICS responders. CONCLUSIONS: Novel associations of sputum and oral microbiota to immunologic features were observed in this cohort of subjects with or without ICS-naive mild asthma. These findings confirm and extend our previous report of reduced bronchial bacterial burden and compositional complexity in subjects with T2-high asthma, with additional identification of a T2-low subgroup with a distinct microbiota-immunologic relationship.

Distinct nasal airway bacterial microbiotas differentially relate to exacerbation in pediatric patients with asthma.

BACKGROUND: In infants, distinct nasopharyngeal bacterial microbiotas differentially associate with the incidence and severity of acute respiratory tract infection and childhood asthma development. OBJECTIVE: We hypothesized that distinct nasal airway microbiota structures also exist in children with asthma and relate to clinical outcomes. METHODS: Nasal secretion samples (n = 3122) collected after randomization during the fall season from children with asthma (6-17 years, n = 413) enrolled in a trial of omalizumab (anti-IgE) underwent 16S rRNA profiling. Statistical analyses with exacerbation as the primary outcome and rhinovirus infection and respiratory illnesses as secondary outcomes were performed. Using A549 epithelial cells, we assessed nasal isolates of Moraxella, Staphylococcus, and Corynebacterium species for their capacity to induce epithelial damage and inflammatory responses. RESULTS: Six nasal airway microbiota assemblages, each dominated by Moraxella, Staphylococcus, Corynebacterium, Streptococcus, Alloiococcus, or Haemophilus species, were observed. Moraxella and Staphylococcus species-dominated microbiotas were most frequently detected and exhibited temporal stability. Nasal microbiotas dominated by Moraxella species were associated with increased exacerbation risk and eosinophil activation. Staphylococcus or Corynebacterium species-dominated microbiotas were associated with reduced respiratory illness and exacerbation events, whereas Streptococcus species-dominated assemblages increased the risk of rhinovirus infection. Nasal microbiota composition remained relatively stable despite viral infection or exacerbation; only a few taxa belonging to the dominant genera exhibited relative abundance fluctuations during these events. In vitro, Moraxella catarrhalis induced significantly greater epithelial damage and inflammatory cytokine expression (IL-33 and IL-8) compared with other dominant nasal bacterial isolates tested. CONCLUSION: Distinct nasal airway microbiotas of children with asthma relate to the likelihood of exacerbation, rhinovirus infection, and respiratory illnesses during the fall season.

Features of the bronchial bacterial microbiome associated with atopy, asthma, and responsiveness to inhaled corticosteroid treatment.

BACKGROUND: Compositional differences in the bronchial bacterial microbiota have been associated with asthma, but it remains unclear whether the findings are attributable to asthma, to aeroallergen sensitization, or to inhaled corticosteroid treatment. OBJECTIVES: We sought to compare the bronchial bacterial microbiota in adults with steroid-naive atopic asthma, subjects with atopy but no asthma, and nonatopic healthy control subjects and to determine relationships of the bronchial microbiota to phenotypic features of asthma. METHODS: Bacterial communities in protected bronchial brushings from 42 atopic asthmatic subjects, 21 subjects with atopy but no asthma, and 21 healthy control subjects were profiled by using 16S rRNA gene sequencing. Bacterial composition and community-level functions inferred from sequence profiles were analyzed for between-group differences. Associations with clinical and inflammatory variables were examined, including markers of type 2-related inflammation and change in airway hyperresponsiveness after 6 weeks of fluticasone treatment. RESULTS: The bronchial microbiome differed significantly among the 3 groups. Asthmatic subjects were uniquely enriched in members of the Haemophilus, Neisseria, Fusobacterium, and Porphyromonas species and the Sphingomonodaceae family and depleted in members of the Mogibacteriaceae family and Lactobacillales order. Asthma-associated differences in predicted bacterial functions included involvement of amino acid and short-chain fatty acid metabolism pathways. Subjects with type 2-high asthma harbored significantly lower bronchial bacterial burden. Distinct changes in specific microbiota members were seen after fluticasone treatment. Steroid responsiveness was linked to differences in baseline compositional and functional features of the bacterial microbiome. CONCLUSION: Even in subjects with mild steroid-naive asthma, differences in the bronchial microbiome are associated with immunologic and clinical features of the disease. The specific differences identified suggest possible microbiome targets for future approaches to asthma treatment or prevention.

STING-dependent type I IFN production inhibits cell-mediated immunity to Listeria monocytogenes.

Infection with Listeria monocytogenes strains that enter the host cell cytosol leads to a robust cytotoxic T cell response resulting in long-lived cell-mediated immunity (CMI). Upon entry into the cytosol, L. monocytogenes secretes cyclic diadenosine monophosphate (c-di-AMP) which activates the innate immune sensor STING leading to the expression of IFN-ß and co-regulated genes. In this study, we examined the role of STING in the development of protective CMI to L. monocytogenes. Mice deficient for STING or its downstream effector IRF3 restricted a secondary lethal challenge with L. monocytogenes and exhibited enhanced immunity that was MyD88-independent. Conversely, enhancing STING activation during immunization by co-administration of c-di-AMP or by infection with a L. monocytogenes mutant that secretes elevated levels of c-di-AMP resulted in decreased protective immunity that was largely dependent on the type I interferon receptor. These data suggest that L. monocytogenes activation of STING downregulates CMI by induction of type I interferon.

Airway Microbiota and the Implications of Dysbiosis in Asthma.

The mucosal surfaces of the human body are typically colonized by polymicrobial communities seeded in infancy and are continuously shaped by environmental exposures. These communities interact with the mucosal immune system to maintain homeostasis in health, but perturbations in their composition and function are associated with lower airway diseases, including asthma, a developmental and heterogeneous chronic disease with various degrees and types of airway inflammation. This review will summarize recent studies examining airway microbiota dysbioses associated with asthma and their relationship with the pathophysiology of this disease.

A prl mutation in SecY suppresses secretion and virulence defects of Listeria monocytogenes secA2 mutants.

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.

The Fungal Microbiome of the Upper Airway Is Associated With Future Loss of Asthma Control and Exacerbation Among Children With Asthma.

BACKGROUND: Accumulating evidence suggests that the upper airway bacterial microbiota is implicated in asthma inception, severity, and exacerbation. Unlike bacterial microbiota, the role of the upper airway fungal microbiome (mycobiome) in asthma control is poorly understood. RESEARCH QUESTION: What are the upper airway fungal colonization patterns among children with asthma and their relationship with subsequent loss of asthma control and exacerbation of asthma? STUDY DESIGN AND METHODS: The study was coupled with the Step Up Yellow Zone Inhaled Corticosteroids to Prevent Exacerbations (ClinicalTrials.gov Identifier: NCT02066129) clinical trial. The upper airway mycobiome was investigated using Internal transcribed spacer 1 (ITS1) sequencing of nasal blow samples collected from children with asthma when asthma was well controlled (baseline, n = 194) and during early signs of loss of asthma control (yellow zone [YZ], n = 107). RESULTS: At baseline, 499 fungal genera were detected in the upper airway samples, with two commensal fungal species, Malassezia globosa and Malassezia restricta, being most dominant. The relative abundance of Malassezia species varies by age, BMI, and race. Higher relative abundance of M globosa at baseline was associated with lower risk of future YZ episodes (P = .038) and longer time to development of first YZ episode (P = .022). Higher relative abundance of M globosa at YZ episode was associated with lower risk of progression from YZ episode to severe asthma exacerbation (P = .04). The upper airway mycobiome underwent significant changes from baseline to YZ episode, and increased fungal diversity was correlated highly with increased bacterial diversity (ρ = 0.41). INTERPRETATION: The upper airway commensal mycobiome is associated with future asthma control. This work highlights the importance of the mycobiota in asthma control and may contribute to the development of fungi-based markers to predict asthma exacerbation.

Gluten Degradation by the Gut Microbiota of Ulcerative Colitis Patients.

Several studies have reported improved disease symptomatology in ulcerative colitis (UC) patients consuming a gluten free diet. This observation coupled with diversity depletion in the gut microbiota of UC patients led us to hypothesize that UC-associated enteric microbes differentially metabolize dietary gluten to produce immunogenic products that promote inflammation. Gluten concentration in stool was determined using gluten-specific ELISA, and gluten intake was assessed by food frequency questionnaire (FFQ) in UC (n = 12) and healthy controls (HC; n = 13). Gluten-metabolizing bacteria were isolated on minimal media supplemented with 1% gluten from UC and HC and identified by 16S rRNA profiling. Cell-free culture media from gluten metabolizing gut bacterial isolates was assessed for immunogenicity in vitro using HT29 colonocytes. Compared to HC, UC patients did not consume gluten differently (Mann−Whitney; p > 0.10) and exhibited equivalent levels of gluten in their feces (Mann−Whitney; p = 0.163). The profile of gluten-degrading bacteria isolated from UC stool was distinct (Chi-square; p ≤ 0.0001). Compared with Enterococcus isolates, products of gluten degradation by Bacillus strains induced higher IL8 and lower occludin (Mann−Whitney; p = 0.002 and p = 0.059, respectively) gene expression in colonocytes irrespective of whether they originated from UC or healthy gut. Members of HC and UC microbiota exhibit gluten-degrading ability, metabolites of which influence genes involved in inflammation and barrier function in enteric colonocyte cultures. Preliminary findings of this study warrant further investigations into the mechanisms by which gut microbiota contribute to UC pathogenesis through gluten degradation.

Human Respiratory and Gut Microbiomes-Do They Really Contribute to Respiratory Health?

Human gastrointestinal and respiratory tracts are colonized by diverse polymicrobial communities shortly after birth, which are continuously molded by environmental exposure. The development of the resident microbiota in early life is a critical factor in the maturation of a healthy immune system. Disturbances to the intricate relationship between environmental exposure and maturation of the infant microbiome have been increasingly identified as a potential contributor to a range of childhood diseases. This review details recent evidence that implicates the contribution of gut and airway microbiome to pediatric respiratory health.

The gut microbiome: Relationships with disease and opportunities for therapy.

Over the past decade, our view of human-associated microbes has expanded beyond that of a few species toward an appreciation of the diverse and niche-specialized microbial communities that develop in the human host with chronological age. The largest reservoir of microbes exists in the distal gastrointestinal tract, both in the lumen, where microbes facilitate primary and secondary metabolism, and on mucosal surfaces, where they interact with host immune cell populations. While local microbial-driven immunomodulation in the gut is well described, more recent studies have demonstrated a role for the gut microbiome in influencing remote organs and mucosal and hematopoietic immune function. Unsurprisingly, therefore, perturbation to the composition and function of the gut microbiota has been associated with chronic diseases ranging from gastrointestinal inflammatory and metabolic conditions to neurological, cardiovascular, and respiratory illnesses. Considerable effort is currently focused on understanding the natural history of microbiome development in humans in the context of health outcomes, in parallel with improving our knowledge of microbiome-host molecular interactions. These efforts ultimately aim to develop effective approaches to rehabilitate perturbed human microbial ecosystems as a means to restore health or prevent disease. This review details the role of the gut microbiome in modulating host health with a focus on immunomodulation and discusses strategies for manipulating the gut microbiome for the management or prevention of chronic inflammatory conditions.

Motility and biofilm formation of the emerging gastrointestinal pathogen Campylobacter concisus differs under microaerophilic and anaerobic environments.

Campylobacter concisus has been isolated from patients with gastroenteritis and inflammatory bowel disease (IBD), as well as healthy subjects. While strain differences may plausibly explain virulence differentials, an alternative hypothesis posits that the pathogenic potential of this species may depend on altered ecosystem conditions in the inflamed gut. One potential difference is oxygen availability, which is frequently increased under conditions of inflammation and is known to regulate bacterial virulence. Hence, we hypothesized that oxygen influences C. concisus physiology. We therefore characterized the effect of microaerophilic or anaerobic environments on C. concisus motility and biofilm formation, two important determinants of host colonization and dissemination. C. concisus isolates (n = 46) sourced from saliva, gut mucosal biopsies and feces of patients with IBD (n = 23), gastroenteritis (n = 8) and healthy subjects (n = 13), were used for this study. Capacity to form biofilms was determined using crystal violet assay, while assessment of dispersion through soft agar permitted motility to be assessed. No association existed between GI disease and either motility or biofilm forming capacity. Oral isolates exhibited significantly greater capacity for biofilm formation compared to fecal isolates (p<0.03), and showed a strong negative correlation between motility and biofilm formation (r = -0.7; p = 0.01). Motility significantly increased when strains were cultured under microaerophilic compared to anaerobic conditions (p<0.001). Increased biofilm formation under microaerophillic conditions was also observed for a subset of isolates. Hence, differences in oxygen availability appear to influence key physiological aspects of the opportunistic gastrointestinal pathogen C. concisus.

The upper-airway microbiota and loss of asthma control among asthmatic children.

The airway microbiome has an important role in asthma pathophysiology. However, little is known on the relationships between the airway microbiome of asthmatic children, loss of asthma control, and severe exacerbations. Here we report that the microbiota's dynamic patterns and compositions are related to asthma exacerbations. We collected nasal blow samples (n = 319) longitudinally during a clinical trial at 2 time-points within one year: randomization when asthma is under control, and at time of early loss of asthma control (yellow zone (YZ)). We report that participants whose microbiota was dominated by the commensal Corynebacterium + Dolosigranulum cluster at RD experience the lowest rates of YZs (p = 0.005) and have longer time to develop at least 2 episodes of YZ (p = 0.03). The airway microbiota have changed from randomization to YZ. A switch from the Corynebacterium + Dolosigranulum cluster at randomization to the Moraxella- cluster at YZ poses the highest risk of severe asthma exacerbation (p = 0.04). Corynebacterium's relative abundance at YZ is inversely associated with severe exacerbation (p = 0.002).

Delayed gut microbiota development in high-risk for asthma infants is temporarily modifiable by Lactobacillus supplementation.

Gut microbiota dysbiosis and metabolic dysfunction in infancy precedes childhood atopy and asthma development. Here we examined gut microbiota maturation over the first year of life in infants at high risk for asthma (HR), and whether it is modifiable by early-life Lactobacillus supplementation. We performed a longitudinal comparison of stool samples collected from HR infants randomized to daily oral Lactobacillus rhamnosus GG (HRLGG) or placebo (HRP) for 6 months, and healthy (HC) infants. Meconium microbiota of HRP participants is distinct, follows a delayed developmental trajectory, and is primarily glycolytic and depleted of a range of anti-inflammatory lipids at 6 months of age. These deficits are partly rescued in HRLGG infants, but this effect was lost at 12 months of age, 6 months after cessation of supplementation. Thus we show that early-life gut microbial development is distinct, but plastic, in HR infants. Our findings offer a novel strategy for early-life preventative interventions.

Bacterial biogeography of adult airways in atopic asthma.

BACKGROUND: Perturbations to the composition and function of bronchial bacterial communities appear to contribute to the pathophysiology of asthma. Unraveling the nature and mechanisms of these complex associations will require large longitudinal studies, for which bronchoscopy is poorly suited. Studies of samples obtained by sputum induction and nasopharyngeal brushing or lavage have also reported asthma-associated microbiota characteristics. It remains unknown, however, whether the microbiota detected in these less-invasive sample types reflect the composition of bronchial microbiota in asthma. RESULTS: Bacterial microbiota in paired protected bronchial brushings (BB; n = 45), induced sputum (IS; n = 45), oral wash (OW; n = 45), and nasal brushings (NB; n = 27) from adults with mild atopic asthma (AA), atopy without asthma (ANA), and healthy controls (HC) were profiled using 16S rRNA gene sequencing. Though microbiota composition varied with sample type (p < 0.001), compositional similarity was greatest for BB-IS, particularly in AAs and ANAs. The abundance of genera detected in BB correlated with those detected in IS and OW (r median [IQR] 0.869 [0.748-0.942] and 0.822 [0.687-0.909] respectively), but not with those in NB (r = 0.004 [- 0.003-0.011]). The number of taxa shared between IS-BB and NB-BB was greater in AAs than in HCs (p < 0.05) and included taxa previously associated with asthma. Of the genera abundant in NB, only Moraxella correlated positively with abundance in BB; specific members of this genus were shared between the two compartments only in AAs. Relative abundance of Moraxella in NB of AAs correlated negatively with that of Corynebacterium but positively with markers of eosinophilic inflammation in the blood and BAL fluid. The genus, Corynebacterium, trended to dominate all NB samples of HCs but only half of AAs (p = 0.07), in whom abundance of this genus was negatively associated with markers of eosinophilic inflammation. CONCLUSIONS: Induced sputum is superior to nasal brush or oral wash for assessing bronchial microbiota composition in asthmatic adults. Although compositionally similar to the bronchial microbiota, the microbiota in induced sputum are distinct, reflecting enrichment of oral bacteria. Specific bacterial genera are shared between the nasal and the bronchial mucosa which are associated with markers of systemic and bronchial inflammation.

Characterisation of the transcriptomes of genetically diverse Listeria monocytogenes exposed to hyperosmotic and low temperature conditions reveal global stress-adaptation mechanisms.

The ability of Listeria monocytogenes to adapt to various food and food- processing environments has been attributed to its robustness, persistence and prevalence in the food supply chain. To improve the present understanding of molecular mechanisms involved in hyperosmotic and low-temperature stress adaptation of L. monocytogenes, we undertook transcriptomics analysis on three strains adapted to sub-lethal levels of these stress stimuli and assessed functional gene response. Adaptation to hyperosmotic and cold-temperature stress has revealed many parallels in terms of gene expression profiles in strains possessing different levels of stress tolerance. Gene sets associated with ribosomes and translation, transcription, cell division as well as fatty acid biosynthesis and peptide transport showed activation in cells adapted to either cold or hyperosmotic stress. Repression of genes associated with carbohydrate metabolism and transport as well as flagella was evident in stressed cells, likely linked to activation of CodY regulon and consequential cellular energy conservation.