Genomic attributes of airway commensal bacteria and mucosa.

Microbial communities at the airway mucosal barrier are conserved and highly ordered, in likelihood reflecting co-evolution with human host factors. Freed of selection to digest nutrients, the airway microbiome underpins cognate management of mucosal immunity and pathogen resistance. We show here the initial results of systematic culture and whole-genome sequencing of the thoracic airway bacteria, identifying 52 novel species amongst 126 organisms that constitute 75% of commensals typically present in heathy individuals. Clinically relevant genes encode antimicrobial synthesis, adhesion and biofilm formation, immune modulation, iron utilisation, nitrous oxide (NO) metabolism and sphingolipid signalling. Using whole-genome content we identify dysbiotic features that may influence asthma and chronic obstructive pulmonary disease. We match isolate gene content to transcripts and metabolites expressed late in airway epithelial differentiation, identifying pathways to sustain host interactions with microbiota. Our results provide a systematic basis for decrypting interactions between commensals, pathogens, and mucosa in lung diseases of global significance.

IL-1α is required for T cell-driven weight loss after respiratory viral infection.

Respiratory viral infections remain a major cause of hospitalization and death worldwide. Patients with respiratory infections often lose weight. While acute weight loss is speculated to be a tolerance mechanism to limit pathogen growth, severe weight loss following infection can cause quality of life deterioration. Despite the clinical relevance of respiratory infection-induced weight loss, its mechanism is not yet completely understood. We utilized a model of CD 8+ T cell-driven weight loss during respiratory syncytial virus (RSV) infection to dissect the immune regulation of post-infection weight loss. Supporting previous data, bulk RNA sequencing indicated significant enrichment of the interleukin (IL)-1 signaling pathway after RSV infection. Despite increased viral load, infection-associated weight loss was significantly reduced after IL-1α (but not IL-1ß) blockade. IL-1α depletion resulted in a reversal of the gut microbiota changes observed following RSV infection. Direct nasal instillation of IL-1α also caused weight loss. Of note, we detected IL-1α in the brain after either infection or nasal delivery. This was associated with changes in genes controlling appetite after RSV infection and corresponding changes in signaling molecules such as leptin and growth/differentiation factor 15. Together, these findings indicate a lung-brain-gut signaling axis for IL-1α in regulating weight loss after RSV infection.

Molecular assessment of mycobacterial burden in the treatment of nontuberculous mycobacterial disease.

Introduction: Nontuberculous pulmonary disease causes significant morbidity and mortality. Efforts to tackle infections are hampered by the lack of reliable biomarkers for diagnosis, assessment and prognostication. The aim of this study was to develop molecular assays capable of identifying and quantifying multiple nontuberculous mycobacterial (NTM) species and to examine their utility in following individual patients' clinical courses. Methods: DNA was extracted from 410 sputum samples obtained longitudinally from a cohort of 38 patients who were commencing treatment for either Mycobacterium abscessus or Mycobacterium avium complex or who were patients with bronchiectasis who had never had positive cultures for mycobacteria. NTM quantification was performed with quantitative PCR assays developed in-house. Results: The molecular assays had high in vitro sensitivity and specificity for the detection and accurate quantification of NTM species. The assays successfully identified NTM DNA from human sputum samples (in vivo sensitivity: 0.86-0.87%; specificity: 0.62-0.95%; area under the curve: 0.74-0.92). A notable association between NTM copy number and treatment (Friedman ANOVA (df)=22.8 (3), p≤0.01 for M. abscessus treatment group) was also demonstrated. Conclusion: The quantitative PCR assays developed in this study provide affordable, real-time and rapid measurement of NTM burden, with significant implications for prompt management decisions.

An invisible threat? Aspergillus positive cultures and co-infecting bacteria in airway samples.

BACKGROUND: Aspergillus fumigatus (Af) infection is associated with poor lung health in chronic suppurative lung diseases but often goes undetected. We hypothesised that inhibition of Af growth by Pseudomonas aeruginosa (Pa) increases the frequency of false-negative Af culture in co-infected people. Using a substantial group of cystic fibrosis (CF) airway samples, we assessed the relationship between Af and bacterial pathogens, additionally comparing fungal culture with next-generation sequencing. METHODS: Frequency of co-culture was assessed for 44,554 sputum/BAL cultures, from 1,367 CF patients between the years 2010-2020. In a subgroup, Internal Transcribed Spacer-2 (ITS2) fungal sequencing was used to determine sequencing-positive, culture-negative (S+/C-) rates. RESULTS: Pa+ samples were nearly 40% less likely (P<0.0001) than Pa- samples to culture Af, an effect that was also seen with some other Gram-negative isolates. This impact varied with Pa density and appeared to be moderated by Staphylococcus aureus co-infection. Sequencing identified Af-S+/C- for 40.1% of tested sputa. Samples with Pa had higher rates of Af-S+/C- (49.3%) than those without (35.7%; RR 1.38 [1.02-1.93], P<0.05). Af-S+/C- rate was not changed by other common bacterial infections. Pa did not affect the S+/C- rates of Candida, Exophiala or Scedosporium. CONCLUSIONS: Pa/ Af co-positive cultures are less common than expected in CF. Our findings suggest an Af-positive culture is less likely in the presence of Pa. Interpretation of negative cultures should be cautious, particularly in Pa-positive samples, and a companion molecular diagnostic could be useful. Further work investigating mechanisms, alternative detection techniques and other chronic suppurative lung diseases is needed.

Evidence of immunometabolic dysregulation and airway dysbiosis in athletes susceptible to respiratory illness.

BACKGROUND: Respiratory tract infection (RTI) is a leading cause of training and in-competition time-loss in athlete health. The immune factors associated with RTI susceptibility remain unclear. In this study, we prospectively characterise host immune factors in elite athletes exhibiting RTI susceptibility. METHODS: Peripheral blood lymphocyte flow cytometry phenotyping and 16S rRNA microbial sequencing of oropharyngeal swabs was performed in a prospective elite athlete cohort study (n = 121). Mass cytometry, peripheral blood mononuclear cell (PBMC) stimulation and plasma metabolic profiling was performed in age-matched highly-susceptible (HS) athletes (≥4RTI in last 18 months) (n = 22) compared to non-susceptible (NS) (≤1RTI in last 18 months) (n = 23) athletes. Findings were compared to non-athletic healthy controls (HC) (n = 19). FINDINGS: Athletes (n = 121) had a reduced peripheral blood memory T regulatory cell compartment compared to HC (p = 0.02 (95%CI:0.1,1.0)) and reduced upper airway bacterial biomass compared to HC (p = 0.032, effect size r = 0.19). HS athletes (n = 22) had lower circulating memory T regulatory cells compared to NS (n = 23) athletes (p = 0.005 (95%CI:-1.5,-0.15)) and HC (p = 0.002 (95%CI:-1.9,-0.3) with PBMC microbial stimulation assays revealing a T-helper 2 skewed immune response compared to HC. Plasma metabolomic profiling showed differences in sphingolipid pathway metabolites (a class of lipids important in infection and inflammation regulation) in HS compared to NS athletes and HC, with sphingomyelin predictive of RTI infection susceptibility (p = 0.005). INTERPRETATION: Athletes susceptible to RTI have reduced circulating memory T regulatory cells, metabolic dysregulation of the sphingolipid pathway and evidence of upper airway bacterial dysbiosis. FUNDING: This study was funded by the English Institute of Sport (UK).

Comparison of the airway microbiota in children with chronic suppurative lung disease.

RATIONALE: The airway microbiota is important in chronic suppurative lung diseases, such as primary ciliary dyskinesia (PCD) and cystic fibrosis (CF). This comparison has not previously been described but is important because difference between the two diseases may relate to the differing prognoses and lead to pathological insights and potentially, new treatments. OBJECTIVES: To compare the longitudinal development of the airway microbiota in children with PCD to that of CF and relate this to age and clinical status. METHODS: Sixty-two age-matched children (age range 0.5-17 years) with PCD or CF (n=31 in each group) were recruited prospectively and followed for 1.1 years. Throat swabs or sputum as well as clinical information were collected at routine clinical appointments. 16S rRNA gene sequencing was performed. MEASUREMENTS AND MAIN RESULTS: The microbiota was highly individual and more diverse in PCD and differed in community composition when compared with CF. While Streptococcus was the most abundant genus in both conditions, Pseudomonas was more abundant in CF with Haemophilus more abundant in PCD (Padj=0.0005). In PCD only, an inverse relationship was seen in the relative abundance of Streptococcus and Haemophilus with age. CONCLUSIONS: Bacterial community composition differs between children with PCD and those with CF. Pseudomonas is more prevalent in CF and Haemophilus in PCD, at least until infection with Pseudomonas supervenes. Interactions between organisms, particularly members of Haemophilus, Streptococcus and Pseudomonas genera appear important. Study of the interactions between these organisms may lead to new therapies or risk stratification.

Airway microbial communities, smoking and asthma in a general population sample.

BACKGROUND: Normal airway microbial communities play a central role in respiratory health but are poorly characterized. Cigarette smoking is the dominant global environmental influence on lung function, and asthma has become the most prevalent chronic respiratory disease worldwide. Both conditions have major microbial components that are incompletely defined. METHODS: We investigated airway bacterial communities in a general population sample of 529 Australian adults. Posterior oropharyngeal swabs were analyzed by sequencing of the 16S rRNA gene. The microbiota were characterized according to their prevalence, abundance and network memberships. FINDINGS: The microbiota were similar across the general population, and were strongly organized into co-abundance networks. Smoking was associated with diversity loss, negative effects on abundant taxa, profound alterations to network structure and expansion of Streptococcus spp. By contrast, the asthmatic microbiota were selectively affected by an increase in Neisseria spp. and by reduced numbers of low abundance but prevalent organisms. INTERPRETATION: Our study shows that the healthy airway microbiota in this population were contained within a highly structured ecosystem, suggesting balanced relationships between the microbiome and human host factors. The marked abnormalities in smokers may contribute to chronic obstructive pulmonary disease (COPD) and lung cancer. The narrow spectrum of abnormalities in asthmatics encourages investigation of damaging and protective effects of specific bacteria. FUNDING: The study was funded by the Asmarley Trust and a Wellcome Joint Senior Investigator Award to WOCC and MFM (WT096964MA and WT097117MA). The Busselton Healthy Ageing Study is supported by the Government of Western Australia (Office of Science, Department of Health) the City of Busselton, and private donations.

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

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

The fungal airway microbiome in cystic fibrosis and non-cystic fibrosis bronchiectasis.

BACKGROUND: The prevalence of fungal disease in cystic fibrosis (CF) and non-CF bronchiectasis is increasing and the clinical spectrum is widening. Poor sensitivity and a lack of standard diagnostic criteria renders interpretation of culture results challenging. In order to develop effective management strategies, a more accurate and comprehensive understanding of the airways fungal microbiome is required. The study aimed to use DNA sequences from sputum to assess the load and diversity of fungi in adults with CF and non-CF bronchiectasis. METHODS: Next generation sequencing of the ITS2 region was used to examine fungal community composition (n = 176) by disease and underlying clinical subgroups including allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, non-tuberculous mycobacteria, and fungal bronchitis. Patients with no known active fungal disease were included as disease controls. RESULTS: ITS2 sequencing greatly increased the detection of fungi from sputum. In patients with CF fungal diversity was lower, while burden was higher than those with non-CF bronchiectasis. The most common operational taxonomic unit (OTU) in patients with CF was Candida parapsilosis (20.4%), whereas in non-CF bronchiectasis sputum Candida albicans (21.8%) was most common. CF patients with overt fungal bronchitis were dominated by Aspergillus spp., Exophiala spp., Candida parapsilosis or Scedosporium spp. CONCLUSION: This study provides a framework to more accurately characterize the extended spectrum of fungal airways diseases in adult suppurative lung diseases.

Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection.

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

Lung function and microbiota diversity in cystic fibrosis.

BACKGROUND: Chronic infection and concomitant airway inflammation is the leading cause of morbidity and mortality for people living with cystic fibrosis (CF). Although chronic infection in CF is undeniably polymicrobial, involving a lung microbiota, infection surveillance and control approaches remain underpinned by classical aerobic culture-based microbiology. How to use microbiomics to direct clinical management of CF airway infections remains a crucial challenge. A pivotal step towards leveraging microbiome approaches in CF clinical care is to understand the ecology of the CF lung microbiome and identify ecological patterns of CF microbiota across a wide spectrum of lung disease. Assessing sputum samples from 299 patients attending 13 CF centres in Europe and the USA, we determined whether the emerging relationship of decreasing microbiota diversity with worsening lung function could be considered a generalised pattern of CF lung microbiota and explored its potential as an informative indicator of lung disease state in CF. RESULTS: We tested and found decreasing microbiota diversity with a reduction in lung function to be a significant ecological pattern. Moreover, the loss of diversity was accompanied by an increase in microbiota dominance. Subsequently, we stratified patients into lung disease categories of increasing disease severity to further investigate relationships between microbiota characteristics and lung function, and the factors contributing to microbiota variance. Core taxa group composition became highly conserved within the severe disease category, while the rarer satellite taxa underpinned the high variability observed in the microbiota diversity. Further, the lung microbiota of individual patient were increasingly dominated by recognised CF pathogens as lung function decreased. Conversely, other bacteria, especially obligate anaerobes, increasingly dominated in those with better lung function. Ordination analyses revealed lung function and antibiotics to be main explanators of compositional variance in the microbiota and the core and satellite taxa. Biogeography was found to influence acquisition of the rarer satellite taxa. CONCLUSIONS: Our findings demonstrate that microbiota diversity and dominance, as well as the identity of the dominant bacterial species, in combination with measures of lung function, can be used as informative indicators of disease state in CF. Video Abstract.

Viral respiratory infections and the oropharyngeal bacterial microbiota in acutely wheezing children.

Acute viral wheeze in children is a major cause of hospitalisation and a major risk factor for the development of asthma. However, the role of the respiratory tract microbiome in the development of acute wheeze is unclear. To investigate whether severe wheezing episodes in children are associated with bacterial dysbiosis in the respiratory tract, oropharyngeal swabs were collected from 109 children with acute wheezing attending the only tertiary paediatric hospital in Perth, Australia. The bacterial community from these samples was explored using next generation sequencing and compared to samples from 75 non-wheezing controls. No significant difference in bacterial diversity was observed between samples from those with wheeze and healthy controls. Within the wheezing group, attendance at kindergarten or preschool was however, associated with increased bacterial diversity. Rhinovirus (RV) infection did not have a significant effect on bacterial community composition. A significant difference in bacterial richness was observed between children with RV-A and RV-C infection, however this is likely due to the differences in age group between the patient cohorts. The bacterial community within the oropharynx was found to be diverse and heterogeneous. Age and attendance at day care or kindergarten were important factors in driving bacterial diversity. However, wheeze and viral infection were not found to significantly relate to the bacterial community. Bacterial airway microbiome is highly variable in early life and its role in wheeze remains less clear than viral influences.

Inhaled corticosteroid suppression of cathelicidin drives dysbiosis and bacterial infection in chronic obstructive pulmonary disease.

Bacterial infection commonly complicates inflammatory airway diseases such as chronic obstructive pulmonary disease (COPD). The mechanisms of increased infection susceptibility and how use of the commonly prescribed therapy inhaled corticosteroids (ICS) accentuates pneumonia risk in COPD are poorly understood. Here, using analysis of samples from patients with COPD, we show that ICS use is associated with lung microbiota disruption leading to proliferation of streptococcal genera, an effect that could be recapitulated in ICS-treated mice. To study mechanisms underlying this effect, we used cellular and mouse models of streptococcal expansion with Streptococcus pneumoniae, an important pathogen in COPD, to demonstrate that ICS impairs pulmonary clearance of bacteria through suppression of the antimicrobial peptide cathelicidin. ICS impairment of pulmonary immunity was dependent on suppression of cathelicidin because ICS had no effect on bacterial loads in mice lacking cathelicidin (Camp -/-) and exogenous cathelicidin prevented ICS-mediated expansion of streptococci within the microbiota and improved bacterial clearance. Suppression of pulmonary immunity by ICS was mediated by augmentation of the protease cathepsin D. Collectively, these data suggest a central role for cathepsin D/cathelicidin in the suppression of antibacterial host defense by ICS in COPD. Therapeutic restoration of cathelicidin to boost antibacterial immunity and beneficially modulate the lung microbiota might be an effective strategy in COPD.

Longitudinal development of the airway microbiota in infants with cystic fibrosis.

The pathogenesis of airway infection in cystic fibrosis (CF) is poorly understood. We performed a longitudinal study coupling clinical information with frequent sampling of the microbiota to identify changes in the airway microbiota in infancy that could underpin deterioration and potentially be targeted therapeutically. Thirty infants with CF diagnosed on newborn screening (NBS) were followed for up to two years. Two hundred and forty one throat swabs were collected as a surrogate for lower airway microbiota (median 35 days between study visits) in the largest longitudinal study of the CF oropharyngeal microbiota. Quantitative PCR and Illumina sequencing of the 16S rRNA bacterial gene were performed. Data analyses were conducted in QIIME and Phyloseq in R. Streptococcus spp. and Haemophilus spp. were the most common genera (55% and 12.5% of reads respectively) and were inversely related. Only beta (between sample) diversity changed with age (Bray Curtis r2 = 0.15, P = 0.03). Staphylococcus and Pseudomonas were rarely detected. These results suggest that Streptococcus spp. and Haemophilus spp., may play an important role in early CF. Whether they are protective against infection with more typical CF micro-organisms, or pathogenic and thus meriting treatment needs to be determined.

The lung and gut microbiome: what has to be taken into consideration for cystic fibrosis?

The 15th European Cystic Fibrosis Society (ECFS) Basic Science pre-conference Symposium focused on the topic of the microbiome, asking the question "The lung and gut microbiome: what has to be considered for cystic fibrosis (CF)?" This review gives an overview of the main points raised during the symposium, which dealt with the technical considerations, pathophysiology and clinical implications of the microbiome in CF.

Comparison of the upper and lower airway microbiota in children with chronic lung diseases.

RATIONALE: The lower airway microbiota is important in normal immunological development and chronic lung diseases (CLDs). Young children cannot expectorate and because of the uncertainty whether upper airway samples reflect the lower airway microbiota, there have been few longitudinal paediatric studies to date. OBJECTIVES: To assess whether throat swabs (TS) and cough swabs (CS) are representative of the lower airway microbiota. METHODS: TS, CS, bronchoalveolar lavage and bronchial brushings were prospectively collected from 49 children undergoing fibreoptic bronchoscopy for CLDs. Bacterial DNA was extracted and the 16S rRNA gene V4 region sequenced using the Illumina MiSeq. RESULTS: 5.97 million high quality reads were obtained from 168 samples (47 TS, 37 CS, 42 BALF and 42 bronchial brushings). CS sequenced poorly. At a community level, no difference in alpha diversity (richness, evenness or Shannon Diversity Index) was seen between lower airway samples and TS (P > 0.05). Less than 6.31% of beta diversity variation related to sampling method for TS (P = 0.001). Variation between pathologies and individual patients was greater (20%, 54% respectively P ≤ 0.001) than between TS and lower airway samples. There was strong correlation in the relative abundance of genera between samples (r = 0.78, P < 0.001). Similarity between upper and lower airway samples was observed to be less for individuals where one sample type was dominated by a single organism. CONCLUSIONS: At the community structure level, TS correlate with lower airway samples and distinguish between different CLDs. TS may be a useful sample for the study of the differences in longitudinal changes in the respiratory microbiota between different CLDs. Differences are too great however for TS to be used for clinical decision making.

Respiratory Disease following Viral Lung Infection Alters the Murine Gut Microbiota.

Alterations in the composition of the gut microbiota have profound effects on human health. Consequently, there is great interest in identifying, characterizing, and understanding factors that initiate these changes. Despite their high prevalence, studies have only recently begun to investigate how viral lung infections have an impact on the gut microbiota. There is also considerable interest in whether the gut microbiota could be manipulated during vaccination to improve efficacy. In this highly controlled study, we aimed to establish the effect of viral lung infection on gut microbiota composition and the gut environment using mouse models of common respiratory pathogens respiratory syncytial virus (RSV) and influenza virus. This was then compared to the effect of live attenuated influenza virus (LAIV) vaccination. Both RSV and influenza virus infection resulted in significantly altered gut microbiota diversity, with an increase in Bacteroidetes and a concomitant decrease in Firmicutes phyla abundance. Although the increase in the Bacteroidetes phylum was consistent across several experiments, differences were observed at the family and operational taxonomic unit level. This suggests a change in gut conditions after viral lung infection that favors Bacteroidetes outgrowth but not individual families. No change in gut microbiota composition was observed after LAIV vaccination, suggesting that the driver of gut microbiota change is specific to live viral infection. Viral lung infections also resulted in an increase in fecal lipocalin-2, suggesting low-grade gut inflammation, and colonic Muc5ac levels. Owing to the important role that mucus plays in the gut environment, this may explain the changes in microbiota composition observed. This study demonstrates that the gut microbiota and the gut environment are altered following viral lung infections and that these changes are not observed during vaccination. Whether increased mucin levels and gut inflammation drive, or are a result of, these changes is still to be determined.

The impact of persistent bacterial bronchitis on the pulmonary microbiome of children.

INTRODUCTION: Persistent bacterial bronchitis (PBB) is a leading cause of chronic wet cough in young children. This study aimed to characterise the respiratory bacterial microbiota of healthy children and to assess the impact of the changes associated with the development of PBB. Blind, protected brushings were obtained from 20 healthy controls and 24 children with PBB, with an additional directed sample obtained from PBB patients. DNA was extracted, quantified using a 16S rRNA gene quantitative PCR assay prior to microbial community analysis by 16S rRNA gene sequencing. RESULTS: No significant difference in bacterial diversity or community composition (R2 = 0.01, P = 0.36) was observed between paired blind and non-blind brushes, showing that blind brushings are a valid means of accessing the airway microbiota. This has important implications for collecting lower respiratory samples from healthy children. A significant decrease in bacterial diversity (P < 0.001) and change in community composition (R2 = 0.08, P = 0.004) was observed among controls, in comparison with patients. Bacterial communities within patients with PBB were dominated by Proteobacteria, and indicator species analysis showed that Haemophilus and Neisseria were significantly associated with the patient group. In 15 (52.9%) cases the dominant organism by sequencing was not identified by standard routine clinical culture. CONCLUSION: The bacteria present in the lungs of patients with PBB were less diverse in terms of richness and evenness. The results validate the clinical diagnosis, and suggest that more attention to bacterial communities in children with chronic cough may lead to more rapid recognition of this condition with earlier treatment and reduction in disease burden.