Nasopharyngeal microbiota in children is associated with severe asthma exacerbations.

BACKGROUND: The respiratory microbiome has been associated with the etiology and disease course of asthma. OBJECTIVE: We sought to assess the nasopharyngeal microbiota in children with a severe asthma exacerbation and their associations with medication, air quality, and viral infection. METHODS: A cross-sectional study was performed among children aged 2 to 18 years admitted to the medium care unit (MCU; n = 84) or intensive care unit (ICU; n = 78) with an asthma exacerbation. For case-control analyses, we matched all cases aged 2 to 6 years (n = 87) to controls in a 1:2 ratio. Controls were participants of either a prospective case-control study or a longitudinal birth cohort (n = 182). The nasopharyngeal microbiota was characterized by 16S-rRNA-gene sequencing. RESULTS: Cases showed higher Shannon diversity index (ICU and MCU combined; P = .002) and a distinct microbial community composition when compared with controls (permutational multivariate ANOVA R2 = 1.9%; P < .001). We observed significantly higher abundance of Staphylococcus and "oral" taxa, including Neisseria, Veillonella, and Streptococcus spp. and a lower abundance of Dolosigranulum pigrum, Corynebacterium, and Moraxella spp. (MaAsLin2; q < 0.25) in cases versus controls. Furthermore, Neisseria abundance was associated with more severe disease (ICU vs MCU MaAslin2, P = .03; q = 0.30). Neisseria spp. abundance was also related with fine particulate matter exposure, whereas Haemophilus and Streptococcus abundances were related with recent inhaled corticosteroid use. We observed no correlations with viral infection. CONCLUSIONS: Our results demonstrate that children admitted with asthma exacerbations harbor a microbiome characterized by overgrowth of Staphylococcus and "oral" microbes and an underrepresentation of beneficial niche-appropriate commensals. Several of these associations may be explained by (environmental or medical) exposures, although cause-consequence relationships remain unclear and require further investigations.

The respiratory microbiome in childhood asthma.

Asthma is the most prevalent noncommunicable disease in childhood, characterized by reversible airway constriction and inflammation of the lower airways. The respiratory tract consists of the upper and lower airways, which are lined with a diverse community of microbes. The composition and density of the respiratory microbiome differs across the respiratory tract, with microbes adapting to the gradually changing physiology of the environment. Over the past decade, both the upper and lower respiratory microbiomes have been implicated in the etiology and disease course of asthma, as well as in its severity and phenotype. We have reviewed the literature on the role of the respiratory microbiome in asthma, making a careful distinction between the relationship of the microbiome with development of childhood asthma and its relationship with the disease course, while accounting for age and the microbial niches studied. Furthermore, we have assessed the literature regarding the underlying asthma endotypes and the impact of the microbiome on the host immune response. We have identified distinct microbial signatures across the respiratory tract associated with asthma development, stability, and severity. These data suggest that the respiratory microbiome may be important for asthma development and severity and may therefore be a potential target for future microbiome-based preventive and treatment strategies.

Relationship between Respiratory Microbiome and Systemic Inflammatory Markers in COPD: A Pilot Study.

The respiratory microbiome may influence the development and progression of COPD by modulating local immune and inflammatory events. We aimed to investigate whether relative changes in respiratory bacterial abundance are also associated with systemic inflammation, and explore their relationship with the main clinical COPD phenotypes. Multiplex analysis of inflammatory markers and transcript eosinophil-related markers were analyzed on peripheral blood in a cohort of stable COPD patients (n = 72). Respiratory microbiome composition was analyzed by 16S rRNA microbial sequencing on spontaneous sputum. Spearman correlations were applied to test the relationship between the microbiome composition and systemic inflammation. The concentration of the plasma IL-8 showed an inverted correlation with the relative abundance of 17 bacterial genera in the whole COPD cohort. COPD patients categorized as eosinophilic showed positive relationships with blood eosinophil markers and inversely correlated with the degree of airway obstruction and the number of exacerbations during the previous year. COPD patients categorized as frequent exacerbators were enriched with the bacterial genera Pseudomonas which, in turn, was positively associated with the severity of airflow limitation and the prior year's exacerbation history. The associative relationships of the sputum microbiome with the severity of the disease emphasize the relevance of the interaction between the respiratory microbiota and systemic inflammation.

The mycobiomes and bacteriomes of sputum, saliva, and home dust.

Respiratory microbiome is an understudied area of research compared to other microbiomes of the human body. The respiratory tract is exposed to an array of environmental pollutants, including microbes. Yet, we know very little about the relationship between environmental and respiratory microbiome. The primary aim of our study was to compare the mycobiomes and bacteriomes between three sample types from the same participants, including home dust, saliva, and sputum. Samples were collected from 40 adolescents in a longitudinal cohort. We analyzed the samples using 16s bacterial rDNA and ITS fungal rDNA gene sequencing, as well as quantitative PCR with universal fungal and bacterial primers. Results showed that home dust had the greatest alpha diversity between the three sample types for both bacteria and fungi. Dust had the highest total fungal load and the lowest total bacterial load. Sputum had greater bacterial diversity than saliva, but saliva had greater fungal diversity than sputum. The distribution of major bacterial phyla differed between all sample types. However, the distribution of major fungal classes differed only between sputum and saliva. Future research should examine the biological significance of the taxa found in each sample type based on microbial ecology and associations with health effects.

Respiratory microbiome and epithelial interactions shape immunity in the lungs.

The airway epithelium represents a physical barrier to the external environment acting as the first line of defence against potentially harmful environmental stimuli including microbes and allergens. However, lung epithelial cells are increasingly recognized as active effectors of microbial defence, contributing to both innate and adaptive immune function in the lower respiratory tract. These cells express an ample repertoire of pattern recognition receptors with specificity for conserved microbial and host motifs. Modern molecular techniques have uncovered the complexity of the lower respiratory tract microbiome. The interaction between the microbiota and the airway epithelium is key to understanding how stable immune homeostasis is maintained. Loss of epithelial integrity following exposure to infection can result in the onset of inflammation in susceptible individuals and may culminate in lung disease. Here we discuss the current knowledge regarding the molecular and cellular mechanisms by which the pulmonary epithelium interacts with the lung microbiome in shaping immunity in the lung. Specifically, we focus on the interactions between the lung microbiome and the cells of the conducting airways in modulating immune cell regulation, and how defects in barrier structure and function may culminate in lung disease. Understanding these interactions is fundamental in the search for more effective therapies for respiratory diseases.

The characterization of bacterial communities of oropharynx microbiota in healthy children by combining culture techniques and sequencing of the 16S rRNA gene.

The high incidence of bacterial respiratory infections has led to a focus on evaluating the human respiratory microbiome. Studies based on culture-based and molecular methods have shown an increase in the bacterial community that includes the bacterial phyla Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria in the oropharynx of healthy individuals. Therefore, recognizing this microbial compound and subsequently identifying those carriers of specific pathogens can be of great help in predicting future infections and their control. In this prospective study, we sought to characterize the bacterial communities of the respiratory microbiome in healthy children aged between 3 and 6 years old by combining both cultural techniques and sequencing of the 16S rRNA gene. Seventy-seven oropharynx samples using Dacron swabs were collected from 77 healthy children in the kindergartens of Ilam, Iran. Bacterial identification was performed by phenotypic methods and in house developed PCR-based sequencing (the V1-V9 hypervariable region of the bacterial 16S ribosomal RNA gene). In total, 346 bacterial isolates were characterized based on phenotypic and sequencing-based molecular methods. The 3 most predominant phyla were Firmicutes (74%), Proteobacteria (22%), and Actinobacteria (4%). At the level of the genus, Staphylococci (coagulase-positive and coagulase-negative) and Streptococci were dominant. Also, the most commonly identified potentially pathogenic colonisers were S. aureus (75%), Enterobacteriaceae spp. (40.1%), and A. baumannii (15.6%). The present study identified 3 phyla and 9 family of bacteria in the oropharyngeal microbiome. Remarkably, the presence of potential pathogenic bacteria in the nasopharynx of healthy children can predispose them to infectious diseases, and also frequent exposure to human respiratory bacterial pathogens are further risk factors.

Airway microbiome, host immune response and recurrent wheezing in infants with severe respiratory syncytial virus bronchiolitis.

BACKGROUND: Early interactions between respiratory viruses and microbiota might modulate host immune responses and subsequently contribute to later development of recurrent wheezing and asthma in childhood. We aimed to study the possible association between respiratory microbiome, host immune response, and the development of recurrent wheezing in infants with severe respiratory syncytial virus (RSV) bronchiolitis. METHODS: Seventy-four infants who were hospitalized at Beijing Children's Hospital during an initial episode of severe RSV bronchiolitis at 6 months of age or less were included and followed up until the age of 3 years. Sputum samples were collected, and their microbiota profiles, LPS, and cytokines were analyzed by 16S rRNA-based sequencing, ELISA, and multiplex immunoassay, respectively. RESULTS: Twenty-six (35.1%) infants developed recurrent wheezing by the age of 3 years, and 48 (64.9%) did not. The relative abundance of Haemophilus, Moraxella, and Klebsiella was higher in infants who later developed recurrent wheezing than in those who did not (LDA score >3.5). Airway levels of LPS (P = .003), CXCL8 (P = .004), CCL5 (P = .029), IL-6 (P = .004), and IL-13 (P < .001) were significantly higher in infants who later developed recurrent wheezing than in those who did not. Moreover, high airway abundance of Haemophilus was associated with CXCL8 (r = 0.246, P = .037) level, and that of Moraxella was associated with IL-6 level (r = 0.236, P = .046) and IL-10 level (r = 0.266, P = .024). CONCLUSION: Our study suggests that higher abundance of Haemophilus and Moraxella in airway microbiome might modulate airway inflammation during severe RSV bronchiolitis in infancy, potentially contributing to the development of subsequent recurrent wheezing in later childhood.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

[Characterization and impact of respiratory microbiota on viral infections]. / Caractérisation et impact du microbiote bactérien respiratoire sur les maladies virales.

Considered for years as sterile, respiratory tract contains very variable bacterial microbiome. Driven by next generation sequencing techniques, microbiome analyses allow a better understanding of viral infection pathophysiology, in terms of incidence and lethality. After a short reminder of microbiome characterization methods, this review will describe its impact on adaptive and innate immunity of the infected host. Studied by viral challenges after antibiotic administration, microbiome depletion has an important impact on clinical outcome of the host. Patient's management and therapeutic choices could exploit results of this field of research, using microbiome characteristics as prognostic biomarkers. Numerous immunologic pathways are impacted by modification of the microbiome and its restoration, using probiotic or innovative molecules could confer better clinical prognostic of viral respiratory diseases.

Household air pollution and the lung microbiome of healthy adults in Malawi: a cross-sectional study.

BACKGROUND: Domestic combustion of biomass fuels, such as wood, charcoal, crop residue and dung causes Household Air Pollution (HAP). These inhaled particulates affect more than half of the world's population, causing respiratory problems such as infection and inflammatory lung disease. We examined whether the presence of black carbon in alveolar macrophages was associated with alterations in the lung microbiome in a Malawi population. METHODS: Bronchoalveolar lavage samples from 44 healthy adults were sequenced using 16S rDNA amplification to assess microbial diversity, richness and relative taxa abundance. Individuals were classified as high or low particulate exposure as determined by questionnaire and the percentage of black carbon within their alveolar macrophages. RESULTS: Subjects in the low and high particulate groups did not differ in terms of source of fuels used for cooking or lighting. There was no difference in alpha or beta diversity by particulate group. Neisseria and Streptococcus were significantly more abundant in samples from high particulate exposed individuals, and Tropheryma was found less abundant. Petrobacter abundance was higher in people using biomass fuel for household cooking and lighting, compared with exclusive use of electricity. CONCLUSIONS: Healthy adults in Malawi exposed to higher levels of particulates have higher abundances of potentially pathogenic bacteria (Streptococcus, Neisseria) within their lung microbiome. Domestic biomass fuel use was associated with an uncommon environmental bacterium (Petrobacter) associated with oil-rich niches.

Induced sputum in young healthy children with cystic fibrosis.

Young children with CF are often asymptomatic and non-productive, yet CF lung disease occurs early in life. Cough swabs are used routinely to sample bacteria from the CF respiratory tract in non-productive healthy children; bronchoscopy is used to definitively sample the lower airway, but is an invasive procedure. Induced sputum is a non-invasive approach to sampling the lower airway. The article concentrates on how well it is tolerated in children, how successful it is in identifying respiratory pathogens, and how it may be important in routine surveillance if 16S technology is to be used in the clinical forum.

Active herpesviruses are associated with intensive care unit (ICU) admission in patients pulmonary infection and alter the respiratory microbiome.

Background: The Herpesviridae family contains several human-related viruses, which are able to establish colonizing and latency in the human body, posing a significant threat to the prognosis of patients. Pulmonary infections represent one of the predominant infectious diseases globally, characterized by diverse and multifaceted clinical manifestations that have consistently attracted clinician's concern. However, the relationship of herpesviruses on the prognosis of pulmonary infections and the respiratory microbiota remains poorly understood. Methods: Here, we retrospectively analyzed respiratory samples from 100 patients with pulmonary infection detected by metagenomic next-generation sequencing (mNGS). Results: Employing mNGS, five herpesvirus species were detected: Human alphaherpesvirus 1 (HSV-1), Human gammaherpesvirus 4 (EBV), Human betaherpesvirus 5 (CMV), Human betaherpesvirus 7 (HHV-7), and Human betaherpesvirus 6B (HHV-6B). Regression analysis showed that the age and positivity of herpesviruses in patients were independently correlated with ICU admission rates. In addition, positivity of herpesvirus was related with increased ICU days and total hospital stay. The herpesvirus-positive group demonstrated markedly higher incidences of co-infections and fungi-positive, predominantly involving Pneumocystis jirovecii and Aspergillus fumigatus. Analysis of respiratory microbiota revealed a substantially altered community composition within the herpesvirus-positive group, and herpesviruses were significantly positively correlated with the diverse respiratory opportunistic pathogens. Conclusion: Overall results substantiate that the active herpesviruses in patients with pulmonary infections were significantly associated with high ICU admission rate. Moreover, the herpesviruses promotes the dysbiosis of the respiratory microbiota and an increased proportion of co-infections. These insights could contribute to unraveling the underlying mechanisms connecting active herpesviruses to the progression of severe illnesses.

Short-read full-length 16S rRNA amplicon sequencing for characterisation of the respiratory bacteriome of captive and free-ranging African elephants (Loxodonta africana).

Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.

Cross-domain microbiomes: the interaction of gut, lung and environmental microbiota in asthma pathogenesis.

Recent experimental and epidemiological studies underscore the vital interaction between the intestinal microbiota and the lungs, an interplay known as the "gut-lung axis". The significance of this axis has been further illuminated following the identification of intestinal microbial metabolites, such as short-chain fatty acids (SCFA), as key mediators in setting the tone of the immune system. Through the gut-lung axis, the gut microbiota and its metabolites, or allergens, are directly or indirectly involved in the immunomodulation of pulmonary diseases, thereby increasing susceptibility to allergic airway diseases such as asthma. Asthma is a complex outcome of the interplay between environmental factors and genetic predispositions. The concept of the gut-lung axis may offer new targets for the prevention and treatment of asthma. This review outlines the relationships between asthma and the respiratory microbiome, gut microbiome, and environmental microbiome. It also discusses the current advancements and applications of microbiomics, offering novel perspectives and strategies for the clinical management of chronic respiratory diseases like asthma.

The effects of different lung parts, age, and batches on the lung microbiota of healthy rats.

BACKGROUND: Rat models are valuable tools to study the lung microbiota in diseases. Yet the impacts of different lung parts, young and mature adult stages, and the different batches of the same conditions on the healthy rat lung microbiome have not been investigated. METHODS: The rat lung microbiome was analyzed to clarify the lung part-dependent and age-dependent differences and to evaluate the effects of several 'batch environmental factors' on normal rats, after eliminating potential contamination. RESULTS: The results showed that the contamination could be identified and excluded. The lung microbiome from left and right lung parts was very similar so one representative part could be used in the microbiome study. There were significantly different lung microbial communities between the young and mature adult groups, and also between the different feeding batches groups of the same repetitive feeding conditions, but a common lung microbiota characterized by Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria as the most dominant phyla were present in all adult rats. It indicated that the experiment under the same condition of the same rats batch was needed to compare the difference in the lung microbiota and repeated experiments were necessary to confirm the results. CONCLUSION: These data represented that the lung bacterial communities were dynamic and rapidly susceptible to environmental influence, clustered strongly by age or different feeding batches but similar in the different lung tissue parts. This study improved the basic understanding of the potential effects on the lung microbiome of healthy rats.

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.

The application of multi-omics in the respiratory microbiome: Progresses, challenges and promises.

The study of the respiratory microbiome has entered a multi-omic era. Through integrating different omic data types such as metagenome, metatranscriptome, metaproteome, metabolome, culturome and radiome surveyed from respiratory specimens, holistic insights can be gained on the lung microbiome and its interaction with host immunity and inflammation in respiratory diseases. The power of multi-omics have moved the field forward from associative assessment of microbiome alterations to causative understanding of the lung microbiome in the pathogenesis of chronic, acute and other types of respiratory diseases. However, the application of multi-omics in respiratory microbiome remains with unique challenges from sample processing, data integration, and downstream validation. In this review, we first introduce the respiratory sample types and omic data types applicable to studying the respiratory microbiome. We next describe approaches for multi-omic integration, focusing on dimensionality reduction, multi-omic association and prediction. We then summarize progresses in the application of multi-omics to studying the microbiome in respiratory diseases. We finally discuss current challenges and share our thoughts on future promises in the field.

Narrative review: respiratory tract microbiome and never smoking lung cancer.

Background and Objective: The lung microbiome was previously thought to be a sterile environment where only gaseous exchange takes place, but recent studies have shown the presence of microbiota in the lung. This review investigates the current literature on the effects of an environmental driven dysbiosis on the healthy oral and respiratory microbiome and its relationship to lung cancer risk in never-smokers. Methods: An online electronic search was performed on PubMed of all English-language literature using combinations of the following keywords: "lung cancer", "dysbiosis", "non-smokers", "oral microbiome", and "respiratory microbiome". All population-based studies reporting results on oral and/or respiratory microbiome in adults were considered for our narrative review. Key Content and Findings: Metagenomic analyses have been performed on isolated samples from healthy participants and compared to samples from those with lung cancer. Research shows that a decrease in alpha diversity of microbes in the oral microbiome is associated with increased risk of lung cancer, along with differences in beta diversity in the sputum of lung cancer cases and healthy controls. Further, several studies have observed that significant changes in the abundance of genera such as increased abundance of Lactobacillales, Bacilli, and Firmicutes associated with an increased lung cancer risk among participants with exposure to certain household solid fuels. Conclusions: These findings suggest potential carcinogenic processes such as increased inflammation associated with changes in flora. Additionally, studies showed that increase in certain taxa such as Bacteroides and Spirochetes might have a protective effect on lung cancer risk. The review also provides insight into how understanding the microbial changes can be beneficial for lung cancer treatment and disease-free survival. Larger studies in different populations need to be performed to strengthen the current associations between microbial diversity and lung cancer risk.