Lower airway microbiota in COPD and healthy controls.

BACKGROUND: The lower airway microbiota in patients with chronic obstructive pulmonary disease (COPD) are likely altered compared with the microbiota in healthy individuals. Information on how the microbiota is affected by smoking, use of inhaled corticosteroids (ICS) and COPD severity is still scarce. METHODS: In the MicroCOPD Study, participant characteristics were obtained through standardised questionnaires and clinical measurements at a single centre from 2012 to 2015. Protected bronchoalveolar lavage samples from 97 patients with COPD and 97 controls were paired-end sequenced with the Illumina MiSeq System. Data were analysed in QIIME 2 and R. RESULTS: Alpha-diversity was lower in patients with COPD than controls (Pielou evenness: COPD=0.76, control=0.80, p=0.004; Shannon entropy: COPD=3.98, control=4.34, p=0.01). Beta-diversity differed with smoking only in the COPD cohort (weighted UniFrac: permutational analysis of variance R2=0.04, p=0.03). Nine genera were differentially abundant between COPD and controls. Genera enriched in COPD belonged to the Firmicutes phylum. Pack years were linked to differential abundance of taxa in controls only (ANCOM-BC (Analysis of Compositions of Microbiomes with Bias Correction) log-fold difference/q-values: Haemophilus -0.05/0.048; Lachnoanaerobaculum -0.04/0.03). Oribacterium was absent in smoking patients with COPD compared with non-smoking patients (ANCOM-BC log-fold difference/q-values: -1.46/0.03). We found no associations between the microbiota and COPD severity or ICS. CONCLUSION: The lower airway microbiota is equal in richness in patients with COPD to controls, but less even. Genera from the Firmicutes phylum thrive particularly in COPD airways. Smoking has different effects on diversity and taxonomic abundance in patients with COPD compared with controls. COPD severity and ICS use were not linked to the lower airway microbiota.

Comparing microbiota profiles in induced and spontaneous sputum samples in COPD patients.

BACKGROUND: Induced and spontaneous sputum are used to evaluate the airways microbiota. Whether the sputum types can be used interchangeably in microbiota research is unknown. Our aim was to compare microbiota in induced and spontaneous sputum from COPD patients sampled during the same consultation. METHODS: COPD patients from Bergen, Norway, were followed between 2006/2010, examined during the stable state and exacerbations. 30 patients delivered 36 sample pairs. DNA was extracted by enzymatic and mechanical lysis methods. The V3-V4 region of the 16S rRNA gene was PCR-amplified and prepared for paired-end sequencing. Illumina Miseq System was used for sequencing, and Quantitative Insights Into Microbial Ecology (QIIME) and Stata were used for bioinformatics and statistical analyses. RESULTS: Approximately 4 million sequences were sorted into 1004 different OTUs and further assigned to 106 different taxa. Pair-wise comparison of both taxonomic composition and beta-diversity revealed significant differences in one or both parameters in 1/3 of sample pairs. Alpha-diversity did not differ. Comparing abundances for each taxa identified, showed statistically significant differences between the mean abundances in induced versus spontaneous samples for 15 taxa when disease state was considered. This included potential pathogens like Haemophilus and Moraxella. CONCLUSION: When studying microbiota in sputum samples one should take into consideration how samples are collected and avoid the usage of both induced and spontaneous sputum in the same study.

Comparison of inflammatory markers in induced and spontaneous sputum in a cohort of COPD patients.

BACKGROUND: Sputum induction is a non-invasive method for obtaining measurements of inflammation in the airways. Whether spontaneously sampled sputum can be a valid surrogate is unknown. The aim of this study was to compare levels of six inflammatory markers in sputum pairs consisting of induced and spontaneous sputum sampled on the same consultation either in a stable state or during exacerbations of chronic obstructive pulmonary disease (COPD). METHODS: 433 COPD patients aged 40-76, Global initiative for chronic Obstructive Lung Disease (GOLD) stage II-IV were enrolled in 2006/07 and followed every six months for three years. 356 patients were followed for potential exacerbations. Interleukin-6, interleukin-8, interleukin-18, interferon gamma-inducible protein-10, monokine induced by gamma interferon and tumor necrosis factor-alpha (IL-6, IL-8, IL-18, IP-10, MIG and TNF-α) were measured by bead based multiplex immunoassay in 60 paired sputum samples from 45 patients. Albumin was measured by enzyme immunoassay, for concentration correction. Culturing for bacterial growth was performed on 24 samples. Bland-Altman plots were used to assess agreement. The paired non-parametric Wilcoxon signed-rank test, the non-parametric Spearman's rank correlation test and Kruskal-Wallis test were used for statistical analyses. For all analyses, a p-value < 0.05 was considered significant. RESULTS: Agreement between the two measurements was generally low for all six markers. TNF-α was significantly higher in spontaneous sputum at exacerbations (p = 0.002) and trending higher at the steady state (p = 0.06). Correlation coefficients between the levels of markers in induced and spontaneous sputum varied between 0.58 (IL-18) to 0.83 (IP-10). In spontaneous sputum IL-18 and MIG were higher in ex-smokers (p < 0.05). The levels of all markers were higher in GOLD stage III & IV except for IL-6 in spontaneous sputum and IL-18 in induced sputum, compared with GOLD stage II, although not statistically significant. In spontaneous sputum the levels of IL-6 were significantly higher if Haemophilus influenzae (HI) was not cultured. CONCLUSION: We observed a low agreement and significant differences in inflammatory markers between induced and spontaneous sputum, both at steady state and exacerbations. We recommend considering sampling method when reporting on inflammatory markers in sputum.

The lower airways microbiome and antimicrobial peptides in idiopathic pulmonary fibrosis differ from chronic obstructive pulmonary disease.

BACKGROUND: The lower airways microbiome and host immune response in chronic pulmonary diseases are incompletely understood. We aimed to investigate possible microbiome characteristics and key antimicrobial peptides and proteins in idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). METHODS: 12 IPF patients, 12 COPD patients and 12 healthy controls were sampled with oral wash (OW), protected bronchoalveolar lavage (PBAL) and right lung protected sterile brushings (rPSB). The antimicrobial peptides and proteins (AMPs), secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 & hBD-2), were measured in PBAL by enzyme linked immunosorbent assay (ELISA). The V3V4 region of the bacterial 16S rDNA gene was sequenced. Bioinformatic analyses were performed with QIIME 2. RESULTS: hBD-1 levels in PBAL for IPF were lower compared with COPD. The predominant phyla in IPF were Firmicutes, Bacteroides and Actinobacteria; Proteobacteria were among top three in COPD. Differential abundance analysis at genus level showed significant differences between study groups for less abundant, mostly oropharyngeal, microbes. Alpha diversity was lower in IPF in PBAL compared to COPD (p = 0.03) and controls (p = 0.01), as well as in rPSB compared to COPD (p = 0.02) and controls (p = 0.04). Phylogenetic beta diversity showed significantly more similarity for IPF compared with COPD and controls. There were no significant correlations between alpha diversity and AMPs. CONCLUSIONS: IPF differed in microbial diversity from COPD and controls, accompanied by differences in antimicrobial peptides. Beta diversity similarity between OW and PBAL in IPF may indicate that microaspiration contributes to changes in its microbiome.

A longitudinal study of the pulmonary mycobiome in subjects with and without chronic obstructive pulmonary disease.

BACKGROUND: Few studies have examined the stability of the pulmonary mycobiome. We report longitudinal changes in the oral and pulmonary mycobiome of participants with and without COPD in a large-scale bronchoscopy study (MicroCOPD). METHODS: Repeated sampling was performed in 30 participants with and 21 without COPD. We collected an oral wash (OW) and a bronchoalveolar lavage (BAL) sample from each participant at two time points. The internal transcribed spacer 1 region of the ribosomal RNA gene cluster was PCR amplified and sequenced on an Illumina HiSeq sequencer. Differences in taxonomy, alpha diversity, and beta diversity between the two time points were compared, and we examined the effect of intercurrent antibiotic use. RESULTS: Sample pairs were dominated by Candida. We observed less stability in the pulmonary taxonomy compared to the oral taxonomy, additionally emphasised by a higher Yue-Clayton measure in BAL compared to OW (0.69 vs 0.22). No apparent effect was visually seen on taxonomy from intercurrent antibiotic use or participant category. We found no systematic variation in alpha diversity by time either in BAL (p-value 0.16) or in OW (p-value 0.97), and no obvious clusters on bronchoscopy number in PCoA plots. Pairwise distance analyses showed that OW samples from repeated sampling appeared more stable compared to BAL samples using the Bray-Curtis distance metric (p-value 0.0012), but not for Jaccard. CONCLUSION: Results from the current study propose that the pulmonary mycobiome is less stable than the oral mycobiome, and neither COPD diagnosis nor intercurrent antibiotic use seemed to influence the stability.

The lower airways microbiota and antimicrobial peptides indicate dysbiosis in sarcoidosis.

BACKGROUND: The role of the pulmonary microbiome in sarcoidosis is unknown. The objectives of this study were the following: (1) examine whether the pulmonary fungal and bacterial microbiota differed in patients with sarcoidosis compared with controls; (2) examine whether there was an association between the microbiota and levels of the antimicrobial peptides (AMPs) in protected bronchoalveolar lavage (PBAL). METHODS: Thirty-five sarcoidosis patients and 35 healthy controls underwent bronchoscopy and were sampled with oral wash (OW), protected BAL (PBAL), and left protected sterile brushes (LPSB). The fungal ITS1 region and the V3V4 region of the bacterial 16S rRNA gene were sequenced. Bioinformatic analyses were performed with QIIME 2. The AMPs secretory leucocyte protease inhibitor (SLPI) and human beta defensins 1 and 2 (hBD-1 and hBD-2), were measured in PBAL by enzyme-linked immunosorbent assay (ELISA). RESULTS: Aspergillus dominated the PBAL samples in sarcoidosis. Differences in bacterial taxonomy were minor. There was no significant difference in fungal alpha diversity between sarcoidosis and controls, but the bacterial alpha diversity in sarcoidosis was significantly lower in OW (p = 0.047) and PBAL (p = 0.03) compared with controls. The beta diversity for sarcoidosis compared with controls differed for both fungi and bacteria. AMP levels were significantly lower in sarcoidosis compared to controls (SLPI and hBD-1: p < 0.01). No significant correlations were found between alpha diversity and AMPs. CONCLUSIONS: The pulmonary fungal and bacterial microbiota in sarcoidosis differed from in controls. Lower antimicrobial peptides levels were seen in sarcoidosis, indicating an interaction between the microbiota and the innate immune system. Whether this dysbiosis represents a pathogenic mechanism in sarcoidosis needs to be confirmed in experimental studies. Video Abstract.

The airway microbiota and exacerbations of COPD.

AIM: The aim of this study was to investigate whether the compositionality of the lower airway microbiota predicts later exacerbation risk in persons with COPD in a cohort study. MATERIALS AND METHODS: We collected lower airways microbiota samples by bronchoalveolar lavage and protected specimen brushes, and oral wash samples from 122 participants with COPD. Bacterial DNA was extracted from all samples, before we sequenced the V3-V4 region of the 16S RNA gene. The frequency of moderate and severe COPD exacerbations was surveyed in telephone interviews and in a follow-up visit. Compositional taxonomy and α and ß diversity were compared between participants with and without later exacerbations. RESULTS: The four most abundant phyla were Firmicutes, Bacteroidetes, Proteobacteria and Fusobacteria in both groups, and the four most abundant genera were Streptococcus, Veillonella, Prevotella and Gemella. The relative abundances of different taxa showed a large variation between samples and individuals, and no statistically significant difference of either compositional taxonomy, or α or ß diversity could be found between participants with and without COPD exacerbations within follow-up. CONCLUSION: The findings from the current study indicate that individual differences in the lower airway microbiota in persons with COPD far outweigh group differences between frequent and nonfrequent COPD exacerbators, and that the compositionality of the microbiota is so complex as to present large challenges for use as a biomarker of later exacerbations.

Sputum microbiota and inflammation at stable state and during exacerbations in a cohort of chronic obstructive pulmonary disease (COPD) patients.

BACKGROUND: Exacerbations of chronic obstructive pulmonary disease (COPD) are debilitating events and spur disease progression. Infectious causes are frequent; however, it is unknown to what extent exacerbations are caused by larger shifts in the airways' microbiota. The aim of the current study was to analyse the changes in microbial composition between stable state and during exacerbations, and the corresponding immune response. METHODS: The study sample included 36 COPD patients examined at stable state and exacerbation from the Bergen COPD Cohort and Exacerbations studies, and one patient who delivered sputum on 13 different occasions during the three-year study period. A physician examined the patients at all time points, and sputum induction was performed by stringent protocol. Only induced sputum samples were used in the current study, not spontaneously expectorated sputum. Sputum inflammatory markers (IL-6, IL-8, IL-18, IP-10, MIG, TNF-α) and antimicrobial peptides (AMPs, i.e. LL-37/hCAP-18, SLPI) were measured in supernatants, whereas target gene sequencing (16S rRNA) was performed on corresponding cell pellets. The microbiome bioinformatics platform QIIME2TM and the statistics environment R were applied for bioinformatics analyses. RESULTS: Levels of IP-10, MIG, TNF-α and AMPs were significantly different between the two disease states. Of 36 sample pairs, 24 had significant differences in the 12 most abundant genera between disease states. The diversity was significantly different in several individuals, but not when data was analysed on a group level. The one patient case study showed longitudinal dynamics in microbiota unrelated to disease state. CONCLUSION: Changes in the sputum microbiota with changing COPD disease states are common, and are accompanied by changes in inflammatory markers. However, the changes are highly individual and heterogeneous events.

Protected sampling is preferable in bronchoscopic studies of the airway microbiome.

The aim was to evaluate susceptibility of oropharyngeal contamination with various bronchoscopic sampling techniques. 67 patients with obstructive lung disease and 58 control subjects underwent bronchoscopy with small-volume lavage (SVL) through the working channel, protected bronchoalveolar lavage (PBAL) and bilateral protected specimen brush (PSB) sampling. Subjects also provided an oral wash (OW) sample, and negative control samples were gathered for each bronchoscopy procedure. DNA encoding bacterial 16S ribosomal RNA was sequenced and bioinformatically processed to cluster into operational taxonomic units (OTU), assign taxonomy and obtain measures of diversity. The proportion of Proteobacteria increased, whereas Firmicutes diminished in the order OW, SVL, PBAL, PSB (p<0.01). The alpha-diversity decreased in the same order (p<0.01). Also, beta-diversity varied by sampling method (p<0.01), and visualisation of principal coordinates analyses indicated that differences in diversity were smaller between OW and SVL and OW and PBAL samples than for OW and the PSB samples. The order of sampling (left versus right first) did not influence alpha- or beta-diversity for PSB samples. Studies of the airway microbiota need to address the potential for oropharyngeal contamination, and protected sampling might represent an acceptable measure to minimise this problem.

Is elevated pre-ictal heart rate associated with secondary generalization in partial epilepsy?

BACKGROUND: People with epilepsy are at risk for sudden unexpected death. Cardiac arrhythmia is one possible mechanism. We have studied seizure-related changes in cardiac rhythm. METHODS: Video-EEG and ECG from 38 patients with epileptic seizures during long-term monitoring for investigation of partial epilepsy with ictal impairment of consciousness were obtained. Seizures were classified as either complex partial or secondarily generalized. Inter-ictal, pre-ictal, ictal and post-ictal heart rate was calculated for the first recorded seizure. RESULTS: Heart rate during the pre-ictal period was higher (p=0.016) in patients with secondarily generalized seizures (n=11) compared to patients with complex partial seizures (n=27). Heart rate was also elevated during and after generalized seizures (p<0.015). Inter-ictal heart rate was not different in patients with secondary generalization compared to patients with partial seizures. CONCLUSION: We report elevated heart rate prior to partial seizure onset in those attacks which become secondarily generalized compared to seizures which remain localized. The finding may be relevant for the understanding of sudden death in epilepsy.