Chronic obstructive pulmonary disease and the airway microbiome: A review for clinicians.

Chronic obstructive pulmonary disease (COPD) is a complex heterogeneous disease characterized by progressive airflow limitation and chronic inflammation. The progressive development and long-term repeated acute exacerbation of COPD make many patients still unable to control the deterioration of the disease after active treatment, and even eventually lead to death. An increasing number of studies have shown that the occurrence and development of COPD are closely related to the composition and changes of airway microbiome. This article reviews the interaction between COPD and airway microbiome, the potential mechanisms of interaction, and the treatment methods related to microbiome. We elaborated the internal correlation between airway microbiome and different stages of COPD, inflammatory endotypes, glucocorticoid and antibiotic treatment, analyze the pathophysiological mechanisms such as the "vicious cycle" hypothesis, abnormal inflammation-immune response of the host and the "natural selection" of COPD to airway microbiome, introduce the treatment of COPD related to microbiome and emphasize the predictive value of airway microbiome for the progression, exacerbation and prognosis of COPD, as well as the guiding role for clinical management of patients, in order to provide a new perspective for exploring the pathogenesis of COPD, and also provide clues and guidance for finding new treatment targets.

Characterization of Pulmonary Bacteriobiota in Critically Ill Patients in Southern Peru through Next-Generation Sequencing Technology.

Sequence variation in the 16S gene is widely used to characterize diverse microbial communities. This was the first pilot study carried out in our region where the pulmonary microbiota of critically ill patients was investigated and analyzed, with the aim of finding a specific profile for these patients that can be used as a diagnostic marker. An study of critical patients mechanically ventilated for non-respiratory indications, in a polyvalent intensive care unit, was carried out; samplee were extracted by endotracheal aspiration and subsequently the microbiota was characterized through Next-Generation Sequencing Technology (NGS). The predominant phyla among the critically ill patients were Proteobacteria, Firmicutes and Bacteroidata. In the surviving patients group, the predominant phyla were Proteobacteria, Bacteroidata and Firmicutes, in the group of deceased patients thy were Firmicutes, Proteobacteria, and Bacteroidata. We found a decrease in commensal bacteria in deceased patients and a progressive increase in in-hospital germs.

Respiratory syncytial virus infection disrupts pulmonary microbiota to induce microglia phenotype shift.

The lung-brain axis is an emerging biological pathway that is being investigated in relation to microbiome medicine. Increasing evidence suggests that pulmonary viral infections can lead to distinct pathological imprints in the brain, so there is a need to explore and understand this mechanism and find possible interventions. This study used respiratory syncytial virus (RSV) infection in mice as a model to establish the potential lung-brain axis phenomenon. We hypothesized that RSV infection could disrupt the lung microbiota, compromise immune barriers, and induce a significant shift in microglia phenotype. One week old mice were randomized into the control, Ampicillin, RSV, and RSV+Ampicillin treated groups (n = 6 each). Seven days after the respective treatments, the mice were anaesthetized. Immunofluorescence and real-time qRT-PCR was used to detect virus. Hematoxylin-eosin staining was used to detect histopathology. Malondialdehyde and superoxide dismutase were used to determine oxidative stress and antioxidant capacity. Real-time qRT-PCR and enzyme-linked immunosorbent assay (ELISA) were used to measure Th differentiation in the lung. Real-time qRT-PCR, ELISA, and confocal immunofluorescence were used to determine the microglia phenotype. 16S DNA technology was used to detect lung microflora. RSV infection induces elevated oxidative stress, reduced antioxidant, and significant dysbacteriosis in the lungs of mice. Pulmonary microbes were found to enhance Th1-type immunoreactivity induced by RSV infection and eventually induced M1-type dominant microglia in the brains of mice. This study was able to establish a correlation between the pulmonary microbiome and brain function. Therefore, we recommend a large sample size study with robust data analysis for the long-term effects of antibiotics and RSV infection on brain physiology.

Clinical identification and microbiota analysis of Chlamydia psittaci- and Chlamydia abortus- pneumonia by metagenomic next-generation sequencing.

Introduction: Recently, the incidence of chlamydial pneumonia caused by rare pathogens such as C. psittaci or C. abortus has shown a significant upward trend. The non-specific clinical manifestations and the limitations of traditional pathogen identification methods determine that chlamydial pneumonia is likely to be poorly diagnosed or even misdiagnosed, and may further result in delayed treatment or unnecessary antibiotic use. mNGS's non-preference and high sensitivity give us the opportunity to obtain more sensitive detection results than traditional methods for rare pathogens such as C. psittaci or C. abortus. Methods: In the present study, we investigated both the pathogenic profile characteristics and the lower respiratory tract microbiota of pneumonia patients with different chlamydial infection patterns using mNGS. Results: More co-infecting pathogens were found to be detectable in clinical samples from patients infected with C. psittaci compared to C. abortus, suggesting that patients infected with C. psittaci may have a higher risk of mixed infection, which in turn leads to more severe clinical symptoms and a longer disease course cycle. Further, we also used mNGS data to analyze for the first time the characteristic differences in the lower respiratory tract microbiota of patients with and without chlamydial pneumonia, the impact of the pattern of Chlamydia infection on the lower respiratory tract microbiota, and the clinical relevance of these characteristics. Significantly different profiles of lower respiratory tract microbiota and microecological diversity were found among different clinical subgroups, and in particular, mixed infections with C. psittaci and C. abortus resulted in lower lung microbiota diversity, suggesting that chlamydial infections shape the unique lung microbiota pathology, while mixed infections with different Chlamydia may have important effects on the composition and diversity of the lung microbiota. Discussion: The present study provides possible evidences supporting the close correlation between chlamydial infection, altered microbial diversity in patients' lungs and clinical parameters associated with infection or inflammation in patients, which also provides a new research direction to better understand the pathogenic mechanisms of pulmonary infections caused by Chlamydia.

Pulmonary Microbial Composition in Sepsis-Induced Acute Respiratory Distress Syndrome.

Background: Acute respiratory distress syndrome (ARDS) is an unresolved challenge in the field of respiratory and critical care, and the changes in the lung microbiome during the development of ARDS and their clinical diagnostic value remain unclear. This study aimed to explore the role of the lung microbiome in disease progression in patients with sepsis-induced ARDS and potential therapeutic targets. Methods: Patients with ARDS were divided into two groups according to the initial site of infection, intrapulmonary infection (ARDSp, 111 cases) and extrapulmonary infection (ARDSexp, 45 cases), and a total of 28 patients with mild pulmonary infections were enrolled as the control group. In this study, we sequenced the DNA in the bronchoalveolar lavage fluid collected from patients using metagenomic next-generation sequencing (mNGS) to analyze the changes in the lung microbiome in patients with different infectious site and prognosis and before and after antibiotic treatment. Results: The Shannon-Wiener index indicated a statistically significant reduction in microbial diversity in the ARDSp group compared with the ARDSexp and control groups. The ARDSp group was characterized by a reduction in microbiome diversity, mainly in the normal microbes of the lung, whereas the ARDSexp group was characterized by an increase in microbiome diversity, mainly in conditionally pathogenic bacteria and intestinal microbes. Further analysis showed that an increase in Bilophila is a potential risk factor for death in ARDSexp. An increase in Escherichia coli, Staphylococcus aureus, Candida albicans, enteric microbes, or conditional pathogens may be risk factors for death in ARDSp. In contrast, Hydrobacter may be a protective factor in ARDSp. Conclusion: Different initial sites of infection and prognoses are likely to affect the composition and diversity of the pulmonary microbiome in patients with septic ARDS. This study provides insights into disease development and exploration of potential therapeutic targets.

Role of pulmonary microorganisms in the development of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a chronic obstructive respiratory disease characterized by irreversible airway limitation and persistent respiratory symptoms. The main clinical symptoms of COPD are dyspnoea, chronic cough, and sputum. COPD is often accompanied by other respiratory diseases, which can cause worsening of the disease. COPD patients with dyspnoea and aggravation of cough and sputum symptoms represent acute exacerbations of COPD (AECOPD). There is mounting evidence suggesting that dysbiosis of pulmonary microbiota participates in the disease. However, investigations of dysbiosis of pulmonary microbiota and the disease are still in initial phases. To screen, diagnose, and treat this respiratory disease, integrating data from different studies can improve our understanding of the occurrence and development of COPD and AECOPD. In this review, COPD epidemiology and the primary triggering mechanism are explored. Emerging knowledge regarding the association of inflammation, caused by pulmonary microbiome imbalance, and changes in lung microbiome flora species involved in the development of the disease are also highlighted. These data will further our understanding of the pathogenesis of COPD and AECOPD and may yield novel strategies for the use of pulmonary microbiota as a potential therapeutic intervention.

Persistent Legionnaires' Disease and Associated Antibiotic Treatment Engender a Highly Disturbed Pulmonary Microbiome Enriched in Opportunistic Microorganisms.

Despite the importance of pneumonia to public health, little is known about the composition of the lung microbiome during infectious diseases, such as pneumonia, and how it evolves during antibiotic therapy. To study the possible relation of the pulmonary microbiome to the severity and outcome of this respiratory disease, we analyzed the dynamics of the pathogen and the human lung microbiome during persistent infections caused by the bacterium Legionella pneumophila and their evolution during antimicrobial treatment. We collected 10 bronchoalveolar lavage fluid samples from three patients during long-term hospitalization due to pneumonia and performed a unique longitudinal study of the interkingdom microbiome, analyzing the samples for presence of bacteria, archaea, fungi, and protozoa by high-throughput Illumina sequencing of marker genes. The lung microbiome of the patients was characterized by a strong predominance of the pathogen, a low diversity of the bacterial fraction, and an increased presence of opportunistic microorganisms. The fungal fraction was more stable than the bacterial fraction. During long-term treatment, no genomic changes or antibiotic resistance-associated mutations that could explain the persistent infection occurred, according to whole-genome sequencing analyses of the pathogen. After antibiotic treatment, the microbiome did not recover rapidly but was mainly constituted of antibiotic-resistant species and enriched in bacteria, archaea, fungi, or protozoa associated with pathogenicity. The lung microbiome seems to contribute to nonresolving Legionella pneumonia, as it is strongly disturbed during infection and enriched in opportunistic and/or antibiotic-resistant bacteria and microorganisms, including fungi, archaea, and protozoa that are often associated with infections.IMPORTANCE The composition and dynamics of the lung microbiome during pneumonia are not known, although the lung microbiome might influence the severity and outcome of this infectious disease, similar to what was shown for the microbiome at other body sites. Here we report the findings of a comprehensive analysis of the lung microbiome composition of three patients with long-term pneumonia due to L. pneumophila and its evolution during antibiotic treatment. This work adds to our understanding of how the microbiome changes during disease and antibiotic treatment and points to microorganisms and their interactions that might be beneficial. In addition to bacteria and fungi, our analyses included archaea and eukaryotes (protozoa), showing that both are present in the pulmonary microbiota and that they might also play a role in the response to the microbiome disturbance.

High-throughput 16S rDNA sequencing of the pulmonary microbiome of rats with allergic asthma.

A decrease in microbial infection in adolescents is implicated with an increase in the incidence of asthma and allergic diseases in adulthood, indicating that the microbiome plays a critical role in asthma. However, the microbial composition of the lower respiratory tract remains unclear, hindering the further exploration of the pathogenesis of asthma. This study aims to explore the microbial distribution and composition in the lungs of normal rats and rats with allergic asthma via 16S rDNA sequencing. The DNA of the pulmonary microbiome was extracted from the left lungs collected from normal control group (NC), saline control group (SC), and allergic asthma group (AA) under aseptic conditions. After the 16s rDNA V4-V5 region was amplified, the products were sequenced using Illumina high-throughput technology and subjected to operational taxonomic unit (OTU) cluster and taxonomy analysis. The OTU values of AA increased significantly compared with those of NC and SC. Microbiome structure analysis showed that the dominant phylum of the pulmonary microbiome changed from Proteobacteria in NC to Firmicutes in AA. Linear discriminant analysis indicated that the key microbiomes involved in the three groups varied. Numerous microbiomes stably settled in the lungs of the rats in NC and AA. The structure and diversity of the pulmonary microbiome in AA differed from those in NC.

Microbial Evidence in Congenital Pulmonary Airway Malformations of Young Asymptomatic Infants.

Introduction: Experts of pediatric endosurgery recommend early thoracoscopic resection of congenital pulmonary airway malformations (CPAM) even in asymptomatic infants due to the risk of later infections. However, neither the rate of lower airway inflammation nor the underlying microbes and their pathogenic potential have been revealed in CPAMs yet. Using latest gene-sequencing techniques, we present the first study analyzing the pulmonary microbiome in young asymptomatic infants with CPAM. Materials and Methods: Seven asymptomatic infants (age 2-3 months) underwent thoracoscopic lobectomy for CPAM. Preoperatively, blood test was taken to rule out systemic infections. Surgical specimen was screened and graded for local inflammation by hematoxylin and eosin (HE) histology. The pulmonary microbiome and mycobiome were assessed using next-generation sequencing. Results: Preoperatively, all infants had normal white blood cell counts and normal C-reactive protein (CRP) levels. Microbial evidence was found in 4/7 patients, 3 being bacteria (Pseudomonas twice, Streptococcus sp. once) and 1 fungal species (Pneumocystis jirovecii, mostly known from immunosuppressed patients, and Preussia funiculata). Histologically, both Pseudomonas and fungi were associated with low and intermediate pulmonary inflammation, whereas Streptococcus was negative for leucocyte infiltration. Conclusion: For the first time ever this study revealed genetic evidence of pathogenic microbes in 4/7 CPAMs causing lower airway inflammation in 3/7 asymptomatic young infants. Especially pseudomonas and fungi represent considerable pathogenic potential and call for a broader, age-dependent, multicenter study of CPAMs. Such data could be important for pediatric surgeons counseling parents about the necessity and timing of thoracoscopic resections.

Analysis of the Pulmonary Microbiome Composition of Legionella pneumophila-Infected Patients.

The analysis of the lung microbiome composition is a field of research that recently emerged. It gained great interest in pulmonary diseases such as pneumonia since the microbiome seems to be involved in host immune responses, inflammation, and protection against pathogens. Thus, it is possible that the microbial communities living in the lungs play a role in the outcome and severity of lung infections such as Legionella-caused pneumonia and in the response to antibiotic therapy. In this chapter, all steps necessary for the characterization of the bacterial and fungal fraction of the lung microbiome using high-throughput sequencing approaches are explained, starting from the selection of clinical samples to the analysis of the taxonomic composition, diversity, and ecology of the microbiome.

Exact variance component tests for longitudinal microbiome studies.

In metagenomic studies, testing the association between microbiome composition and clinical outcomes translates to testing the nullity of variance components. Motivated by a lung human immunodeficiency virus (HIV) microbiome project, we study longitudinal microbiome data by using variance component models with more than two variance components. Current testing strategies only apply to models with exactly two variance components and when sample sizes are large. Therefore, they are not applicable to longitudinal microbiome studies. In this paper, we propose exact tests (score test, likelihood ratio test, and restricted likelihood ratio test) to (a) test the association of the overall microbiome composition in a longitudinal design and (b) detect the association of one specific microbiome cluster while adjusting for the effects from related clusters. Our approach combines the exact tests for null hypothesis with a single variance component with a strategy of reducing multiple variance components to a single one. Simulation studies demonstrate that our method has a correct type I error rate and superior power compared to existing methods at small sample sizes and weak signals. Finally, we apply our method to a longitudinal pulmonary microbiome study of HIV-infected patients and reveal two interesting genera Prevotella and Veillonella associated with forced vital capacity. Our findings shed light on the impact of the lung microbiome on HIV complexities. The method is implemented in the open-source, high-performance computing language Julia and is freely available at https://github.com/JingZhai63/VCmicrobiome.

Lung inflammation and disease: A perspective on microbial homeostasis and metabolism.

It is now well appreciated that the human microbiome plays a significant role in a number of processes in the body, significantly affecting its metabolic, inflammatory, and immune homeostasis. Recent research has revealed that almost every mucosal surface in the human body is associated with a resident commensal microbiome of its own. While the gut microbiome and its role in regulation of host metabolism along with its alteration in a disease state has been well studied, there is a lacuna in understanding the resident microbiota of other mucosal surfaces. Among these, the scientific information on the role of lung microbiota in pulmonary diseases is currently severely limited. Historically, lungs have been considered to be sterile and lung diseases have only been studied in the context of bacterial pathogenesis. Recently however, studies have revealed a resilient microbiome in the upper and lower respiratory tracts and there is increased evidence on its central role in respiratory diseases. Knowledge of lung microbiome and its metabolic fallout (local and systemic) is still in its nascent stages and attracting immense interest in recent times. In this review, we will provide a perspective on lung-associated metabolic disorders defined for lung diseases (e.g., chronic obstructive pulmonary disease, asthma, and respiratory depression due to infection) and correlate it with lung microbial perturbation. Such perturbations may be due to altered biochemical or metabolic stress as well. Finally, we will draw evidence from microbiome and classical microbiology literature to demonstrate how specific lung morbidities associate with specific metabolic characteristics of the disease, and with the role of microbiome in this context. © 2018 IUBMB Life, 71(1):152-165, 2019.

Intestinal Microbiota Disruption Reduces Regulatory T Cells and Increases Respiratory Viral Infection Mortality Through Increased IFNγ Production.

Alterations in gastrointestinal microbiota indirectly modulate the risk of atopic disease, but effects on respiratory viral infections are less clear. Using the murine paramyxoviral virus type 1, Sendai virus (SeV), we examined the effect of altering gastrointestinal microbiota on the pulmonary antiviral immune response. C57BL6 mice were treated with streptomycin before or during infection with SeV and resulting immune response studied. Ingestion of the non-absorbable antibiotic streptomycin led to a marked reduction in intestinal microbial diversity without a significant effect on lung microbiota. Reduction in diversity in the gastrointestinal tract was followed by greatly increased mortality to respiratory viral infection (p < 0.0001). This increase in mortality was associated with a dysregulated immune response characterized by decreased lung (p = 0.01) and intestinal (p = 0.03) regulatory T cells (Tregs), and increased lung IFNγ (p = 0.049), IL-6 (p = 0.015), and CCL2 (p = 0.037). Adoptive transfer of Treg cells or neutralization of IFNγ prevented increased mortality. Furthermore, Lin-CD4+ cells appeared to be a potential source of the increased IFNγ. Together, these results demonstrate gastrointestinal microbiota modulate immune responses at distant mucosal sites and have the ability to significantly impact mortality in response to a respiratory viral infection.

The lung microbiome in patients with pneumocystosis.

BACKROUND: Pneumocystis jirovecii pneumonia (PCP) is an opportunistic fungal infection that is associated with a high morbidity and mortality in immunocompromised individuals. In this study, we analysed the microbiome of the lower respiratory tract from critically ill intensive care unit patients with and without pneumocystosis. METHODS: Broncho-alveolar fluids from 65 intubated and mechanically ventilated intensive care unit patients (34 PCP+ and 31 PCP- patients) were collected. Sequence analysis of bacterial 16S rRNA gene V3/V4 regions was performed to study the composition of the respiratory microbiome using the Illumina MiSeq platform. RESULTS: Differences in the microbial composition detected between PCP+ and PCP- patients were not statistically significant on class, order, family and genus level. In addition, alpha and beta diversity metrics did not reveal significant differences between PCP+ and PCP- patients. The composition of the lung microbiota was highly variable between PCP+ patients and comparable in its variety with the microbiota composition of the heterogeneous collective of PCP- patients. CONCLUSIONS: The lower respiratory tract microbiome in patients with pneumocystosis does not appear to be determined by a specific microbial composition or to be dominated by a single bacterial species.