Upper Airways Microbiota in Antibiotic-Naive Wheezing and Healthy Infants from the Tropics of Rural Ecuador

Background: Observations that the airway microbiome is disturbed in asthma may be confounded by the widespread use of antibiotics and inhaled steroids. We have therefore examined the oropharyngeal microbiome in early onset wheezinginfants from a rural area of tropical Ecuador where antibiotic usage is minimal and glucocorticoid usage is absent. Materials and Methods: We performed pyrosequencing of amplicons of the polymorphic bacterial 16S rRNA gene from oropharyngeal samples from 24 infants with non-infectious early onset wheezing and 24 healthy controls (average age 10.2 months). We analyzed microbial community structure and differences between cases and controls by QIIME software. Results: We obtained 76,627 high quality sequences classified into 182 operational taxonomic units (OTUs). Firmicutes was the most common and diverse phylum (71.22% of sequences) with Streptococcus being the most common genus (49.72%). Known pathogens were found significantly more often in cases of infantile wheeze compared to controls, exemplified by Haemophilus spp. (OR = 2.12, 95% Confidence Interval (CI) 1.82–2.47; P = 5.46610223) and Staphylococcus spp. (OR = 124.1, 95%CI 59.0–261.2; P = 1.876102241). Other OTUs were less common in cases than controls, notably Veillonella spp. (OR = 0.59, 95%CI = 0.56–0.62; P = 8.06610286). Discussion: The airway microbiota appeared to contain many more Streptococci than found in Western Europe and the USA. Comparisons between healthy and wheezing infants revealed a significant difference in several bacterial phylotypes that were not confounded by antibiotics or use of inhaled steroids. The increased prevalence of pathogens such as Haemophilus and Staphylococcus spp. in cases may contribute to wheezing illnesses in this age group

The lung microbiome in health and disease

The Human Microbiome Project began 10 years ago, leading to a signifi cant growth in understanding of the role the human microbiome plays in health and disease. In this article, we explain with an emphasis on the lung, the origins of microbiome research. We discuss how 16S rRNA gene sequencing became the fi rst major molecular tool to examine the bacterial communities present within the human body. We highlight the pitfalls of molecular-based studies, such as false fi ndings resulting from contamination, and the limitations of 16S rRNA gene sequencing. Knowledge about the lung microbiome has evolved from initial scepticism to the realisation that it might have a signifi cant infl uence on many illnesses. We also discuss the lung microbiome in the context of disease by giving examples of important respiratory conditions. In addition, we draw attention to the challenges for metagenomic studies of respiratory samples and the importance of systematic bacterial isolation to enable host–microbiome interactions to be understood. We conclude by discussing how knowledge of the lung microbiome impacts current clinical diagnostics

Late-onset bloodstream infection and perturbed maturation of the gastrointestinal microbiota in premature infants

Late-onset bloodstream infection (LO-BSI) is a common complication of prematurity, and lack of timely diagnosis and treatment can have life-threatening consequences. We sought to identify clinical characteristics and microbial signatures in the gastrointestinal microbiota preceding diagnosis of LO-BSI in premature infants.Daily faecal samples and clinical data were collected over two years from 369 premature neonates (<32 weeks gestation). We analysed samples from 22 neonates who developed LO-BSI and 44 matched control infants. Next-generation sequencing of 16S rRNA gene regions amplified by PCR from total faecal DNA was used to characterise the microbiota of faecal samples preceding diagnosis from infants with LO-BSI and controls. Culture of selected samples was undertaken, and bacterial isolates identified using MALDI-TOF. Antibiograms from bloodstream and faecal isolates were compared to explore strain similarity.From the week prior to diagnosis, infants with LO-BSI had higher proportions of faecal aerobes/facultative anaerobes compared to controls. Risk factors for LO-BSI were identified by multivariate analysis. Enterobacteriaceal sepsis was associated with antecedent multiple lines, low birth weight and a faecal microbiota with prominent Enterobacteriaceae. Staphylococcal sepsis was associated with Staphylococcus OTU faecal over-abundance, and the number of days prior to diagnosis of mechanical ventilation and of the presence of centrally-placed lines. In 12 cases, the antibiogram of the bloodstream isolate matched that of a component of the faecal microbiota in the sample collected closest to diagnosis.The gastrointestinal tract is an important reservoir for LO-BSI organisms, pathogens translocating across the epithelial barrier. LO-BSI is associated with an aberrant microbiota, with abundant staphylococci and Enterobacteriaceae and a failure to mature towards predominance of obligate anaerobes

Pulmonary ORMDL3 is critical for induction of Alternaria -induced allergic airways disease

Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. Objective We sought to investigate in vivo the functional role of ORMDL3 in disease inception. Methods An Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. Results Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6–mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum–associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen–induced allergic airways disease. Conclusions This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses

Dynamic and physical clustering of gene expression during epidermal barrier formation in differentiating keratinocytes.

The mammalian epidermis is a continually renewing structure that provides the interface between the organism and an innately hostile environment. The keratinocyte is its principal cell. Keratinocyte proteins form a physical epithelial barrier, protect against microbial damage, and prepare immune responses to danger. Epithelial immunity is disordered in many common diseases and disordered epithelial differentiation underlies many cancers. In order to identify the genes that mediate epithelial development we used a tissue model of the skin derived from primary human keratinocytes. We measured global gene expression in triplicate at five times over the ten days that the keratinocytes took to fully differentiate. We identified 1282 gene transcripts that significantly changed during differentiation (false discovery rate <0.01%). We robustly grouped these transcripts by K-means clustering into modules with distinct temporal expression patterns, shared regulatory motifs, and biological functions. We found a striking cluster of late expressed genes that form the structural and innate immune defences of the epithelial barrier. Gene Ontology analyses showed that undifferentiated keratinocytes were characterised by genes for motility and the adaptive immune response. We systematically identified calcium-binding genes, which may operate with the epidermal calcium gradient to control keratinocyte division during skin repair. The results provide multiple novel insights into keratinocyte biology, in particular providing a comprehensive list of known and previously unrecognised major components of the epidermal barrier. The findings provide a reference for subsequent understanding of how the barrier functions in health and disease

Delayed acquisition of airway commensals in antibiotic naïve children and its relationship with wheezing in rural Ecuador.

INTRODUCTION: The hygiene hypothesis identified a relationship between living in rural areas and acquiring protective environmental factors against the development of asthma and atopy. In our previous study, we found a correlation between particular bacterial species and early-onset wheezing in infants from the rural tropics of Ecuador who were corticosteroid-naïve and had limited antibiotic exposure. We now describe a longitudinal study of infants conducted to determine the age-related changes of the microbiome and its relationship with wheezing. METHODS: We performed an amplicon sequencing of the 16S rRNA bacterial gene from the oropharyngeal samples obtained from 110 infants who had a history of recurrent episodic wheezing sampled at different ages (7, 12, and 24 months) and compared it to the sequencing of the oropharyngeal samples from 150 healthy infants sampled at the same time points. Bioinformatic analyses were conducted using QIIME and R. RESULTS: As expected, the microbiota diversity consistently increased as the infants grew older. Considering age-based microbiota changes, we found that infants with wheeze had significantly lower species richness than the healthy infants at 7 months, but not at 12 or 24 months. Most of the core and accessory organisms increased in abundance and prevalence with age, except for a few which decreased. At 7 months of age, infants with wheeze had notably higher levels of a single Streptococcus operational taxonomic unit and core microbiota member than controls. CONCLUSIONS: In a cohort with limited antibiotic and corticosteroid use, a progressively more complex and diverse respiratory microbial community develops with age. The respiratory microbiota in early life is altered in infants with wheeze, but this does not hold true in older infants

The role of bacteria in the pathogenesis and progression of idiopathic pulmonary fibrosis

Rationale:Idiopathic pulmonaryfibrosis (IPF)isa progressivelung disease of unknown cause that leads to respiratory failure and death within 5 years of diagnosis. Overt respiratory infection and immunosuppression carry a high morbidity and mortality, and polymorphisms in genes related to epithelial integrity and host defense predispose to IPF. Objectives: To investigate the role of bacteria in the pathogenesis and progression of IPF. Methods: We prospectively enrolled patients diagnosed with IPF according to international criteria together with healthy smokers, nonsmokers, and subjectswithmoderate chronic obstructive pulmonary disease as control subjects. Subjects underwent bronchoalveolar lavage (BAL), from which genomic DNA was isolated. The V3–V5 region of the bacterial 16S rRNA gene was amplified, allowing quantification of bacterial load and identification of communities by 16S rRNA quantitative polymerase chain reaction and pyrosequencing. Measurements and Main Results: Sixty-five patients with IPF had double the burden of bacteria in BAL fluid compared with 44 control subjects. Baseline bacterial burden predicted the rate of decline in lung volume and risk of death and associated independently with the rs35705950 polymorphism of the MUC5B mucin gene, a proven host susceptibilityfactorfor IPF. Sequencing yielded912,883 high-quality reads from all subjects.WeidentifiedHaemophilus, Streptococcus,Neisseria, and Veillonella spp. to be more abundant in cases than control subjects. Regression analyses indicated that these specific operational taxonomic units as well as bacterial burden associated independently with IPF. Conclusions: IPF is characterized by an increased bacterial burden in BAL that predicts decline in lung function and death. Trials of antimicrobial therapy are needed to determine if microbial burden is pathogenic in the disease

Predicting cell types and genetic variations contributing to disease by combining GWAS and epigenetic data

Genome-wide association studies (GWASs) identify single nucleotide polymorphisms (SNPs) that are enriched in individuals suffering from a given disease. Most disease-associated SNPs fall into non-coding regions, so that it is not straightforward to infer phenotype or function; moreover, many SNPs are in tight genetic linkage, so that a SNP identified as associated with a particular disease may not itself be causal, but rather signify the presence of a linked SNP that is functionally relevant to disease pathogenesis. Here, we present an analysis method that takes advantage of the recent rapid accumulation of epigenomics data to address these problems for some SNPs. Using asthma as a prototypic example; we show that non-coding disease-associated SNPs are enriched in genomic regions that function as regulators of transcription, such as enhancers and promoters. Identifying enhancers based on the presence of the histone modification marks such as H3K4me1 in different cell types, we show that the location of enhancers is highly cell-type specific. We use these findings to predict which SNPs are likely to be directly contributing to disease based on their presence in regulatory regions, and in which cell types their effect is expected to be detectable. Moreover, we can also predict which cell types contribute to a disease based on overlap of the disease-associated SNPs with the locations of enhancers present in a given cell type. Finally, we suggest that it will be possible to re-analyze GWAS studies with much higher power by limiting the SNPs considered to those in coding or regulatory regions of cell types relevant to a given disease

Author Correction: Progression of whole-blood transcriptional signatures from interferon-induced to neutrophil-associated patterns in severe influenza.

In the version of this article initially published, a source of funding was not included in the Acknowledgements section. That section should include the following: P.J.M.O. was supported by EU FP7 PREPARE project 602525. The error has been corrected in the HTML and PDF version of the article

Effects of different antibiotic classes on airway bacteria in stable COPD using culture and molecular techniques: a randomised controlled trial

Long-term antibiotic therapy is used to prevent exacerbations of COPD but there is uncertainty over whether this reduces airway bacteria. The optimum antibiotic choice remains unknown. We conducted an exploratory trial in stable patients with COPD comparing three antibiotic regimens against placebo