Lucy D. Burr

A novel microbiota stratification system predicts future exacerbations in bronchiectasis.

RATIONALE Although airway microbiota composition correlates with clinical measures in non-cystic fibrosis bronchiectasis, these data are unlikely to provide useful prognostic information at the individual patient level. A system enabling microbiota data to be applied clinically would represent a substantial translational advance. OBJECTIVES This study aims to determine whether stratification of patients according to the predominant microbiota taxon can provide improved clinical insight compared with standard diagnostics. METHODS The presence of bacterial respiratory pathogens was assessed in induced sputum from 107 adult patients by culture, quantitative PCR, and, in 96 samples, by ribosomal gene pyrosequencing. Prospective analysis was performed on samples from 42 of these patients. Microbiological data were correlated with concurrent clinical measures and subsequent outcomes. MEASUREMENTS AND MAIN RESULTS Microbiota analysis defined three groups: Pseudomonas aeruginosa dominated (n = 26), Haemophilus influenzae dominated (n = 34), and other taxa dominated (n = 36). Patients with P. aeruginosa- and H. influenzae-dominated communities had significantly worse lung function, higher serum levels of C-reactive protein (CRP), and higher sputum levels of IL-8 and IL-1β. Predominance of P. aeruginosa, followed by Veillonella species, was the best predictor of future exacerbation frequency, with H. influenzae-dominated communities having significantly fewer episodes. Detection of P. aeruginosa was associated with poor lung function and exacerbation frequency, irrespective of analytical strategy. Quantitative PCR revealed significant correlations between H. influenzae levels and sputum IL-8, IL-1β, and serum CRP. Genus richness was negatively correlated with 24-hour sputum weight, age, serum CRP, sputum IL-1β, and IL-8. CONCLUSIONS Stratification of patients with non-cystic fibrosis bronchiectasis on the basis of predominant bacterial taxa is more clinically informative than either conventional culture or quantitative PCR-based analysis. Further investigation is now required to assess the mechanistic basis of these associations.

The effect of long-term macrolide treatment on respiratory microbiota composition in non-cystic fibrosis bronchiectasis: an analysis from the randomised, double-blind, placebo-controlled BLESS trial.

BACKGROUND Long-term macrolide treatment has proven benefit in inflammatory airways diseases, but whether it leads to changes in the composition of respiratory microbiota is unknown. We aimed to assess whether long-term, low-dose erythromycin treatment changes the composition of respiratory microbiota in people with non-cystic fibrosis bronchiectasis. METHODS Microbiota composition was determined by 16S rRNA gene sequencing of sputum samples from participants in the BLESS trial, a 12-month, double-blind, placebo-controlled trial of twice-daily erythromycin ethylsuccinate (400 mg) in adult patients with non-cystic fibrosis bronchiectasis and at least two infective exacerbations in the preceding year. The primary outcome was within-patient change in respiratory microbiota composition (assessed by Bray-Curtis index) between baseline and week 48, comparing erythromycin with placebo. The BLESS trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12608000460303. FINDINGS The BLESS trial took place between Oct 15, 2008, and Dec 14, 2011. Paired sputum samples were available from 86 randomly assigned patients, 42 in the placebo group and 44 in the erythromycin group. The change in microbiota composition between baseline and week 48 was significantly greater with erythromycin than with placebo (median Bray-Curtis score 0·52 [IQR 0·14-0·78] vs 0·68 [0·46-0·93]; median difference 0·16, 95% CI 0·01-0·33; p=0·03). In patients with baseline airway infection dominated by Pseudomonas aeruginosa, erythromycin did not change microbiota composition significantly. In those with infection dominated by organisms other than P. aeruginosa, erythromycin caused a significant change in microbiota composition (p=0·03 [by analysis of similarity]), representing a reduced relative abundance of Haemophilus influenzae (35·3% [5·5-91·6] vs 6·7% [0·8-74·8]; median difference 12·6%, 95% CI 0·4-28·3; p=0·04; interaction p=0·02) and an increased relative abundance of P aeruginosa (0·02% [0·00-0·33] vs 0·13% [0·01-39·58]; median difference 6·6%, 95% CI 0·1-37·1; p=0·002; interaction p=0·45). Compared with placebo, erythromycin reduced the rate of pulmonary exacerbations over the 48 weeks of the study in patients with P. aeruginosa-dominated infection (median 1 [IQR 0-3] vs 3 [2-5]; median difference -2, 95% CI -4 to -1; p=0·01), but not in those without P. aeruginosa-dominated infection (1 [0-2] vs 1 [0-3]; median difference 0, -1 to 0; p=0·41; interaction p=0·04). INTERPRETATION Long-term erythromycin treatment changes the composition of respiratory microbiota in patients with bronchiectasis. In patients without P. aeruginosa airway infection, erythromycin did not significantly reduce exacerbations and promoted displacement of H. influenzae by more macrolide-tolerant pathogens including P. aeruginosa. These findings argue for a cautious approach to chronic macrolide use in patients without P. aeruginosa airway infection. FUNDING Mater Adult Respiratory Research Trust Fund.

Adult Non-Cystic Fibrosis Bronchiectasis Is Characterised by Airway Luminal Th17 Pathway Activation

Background Non-cystic fibrosis (CF) bronchiectasis is characterised by chronic airway infection and neutrophilic inflammation, which we hypothesised would be associated with Th17 pathway activation. Methods Th17 pathway cytokines were quantified in bronchoalveolar lavage fluid (BALF), and gene expression of IL-17A, IL-1β, IL-8 and IL-23 determined from endobronchial biopsies (EBx) in 41 stable bronchiectasis subjects and 20 healthy controls. Relationships between IL-17A levels and infection status, important clinical measures and subsequent Pseudomonas aeruginosa infection were determined. Results BALF levels of all Th17 cytokines (median (IQR) pg/mL) were significantly higher in bronchiectasis than control subjects, including IL-17A (1.73 (1.19, 3.23) vs. 0.27 (0.24, 0.35), 95% CI 1.05 to 2.21, p<0.0001) and IL-23 (9.48 (4.79, 15.75) vs. 0.70 (0.43, 1.79), 95% CI 4.68 to 11.21, p<0.0001). However, BALF IL-17A levels were not associated with clinical measures or airway microbiology, nor predictive of subsequent P. aeruginosa infection. Furthermore, gene expression of IL-17A in bronchiectasis EBx did not differ from control. In contrast, gene expression (relative to medians of controls) in bronchiectasis EBx was significantly higher than control for IL1β (4.12 (1.24, 8.05) vs 1 (0.13, 2.95), 95% CI 0.05 to 4.07, p = 0.04) and IL-8 (3.75 (1.64, 11.27) vs 1 (0.54, 3.89), 95% CI 0.32 to 4.87, p = 0.02) and BALF IL-8 and IL-1α levels showed significant relationships with clinical measures and airway microbiology. P. aeruginosa infection was associated with increased levels of IL-8 while Haemophilus influenzae was associated with increased IL-1α. Conclusions and Clinical Relevance Established adult non-CF bronchiectasis is characterised by luminal Th17 pathway activation, however this pathway may be relatively less important than activation of non-antigen-specific innate neutrophilic immunity.

FUT2 genotype influences lung function, exacerbation frequency and airway microbiota in non-CF bronchiectasis

Objective To assess whether FUT2 (secretor) genotype affects disease severity and airway infection in patients with non-cystic fibrosis bronchiectasis. Participants Induced sputum samples were obtained from 112 adult patients with high-resolution CT scan-proven bronchiectasis and at least two exacerbations in the previous year, as part of an unrelated randomised control trial. Outcome measures Presence of null FUT2 polymorphisms were determined by gene sequencing and verified by endobronchial biopsy histochemical staining. Outcome measures were FEV1% predicted, exacerbation frequency, and bacterial, fungal and viral components of the microbiota (measured by culture independent approaches). Results Patients were grouped by FUT2 loss-of-function genotype; categorised as non-secretors (n=27, sese), heterozygous secretors (n=54, Sese) or homozygous secretors (n=31, SeSe). FEV1% was significantly lower in SeSe patients compared with sese patients (mean 61.6 (SD 20.0) vs 74.5 (18.0); p=0.023). Exacerbation frequency was significantly higher in SeSe (mean count 5.77) compared with sese (4.07; p=0.004) and Sese (4.63; p=0.026) genotypes. The time until first exacerbation was significantly shorter in SeSe compared with Sese (HR=0.571 (95% CI 0.343 to 0.950); p=0.031), with a similar trend for sese patients (HR=0.577 (0.311 to 1.07); p=0.081). sese had a significantly reduced frequency of Pseudomonas aeruginosa-dominated airway infection (8.7%) compared with Sese (31%; p=0.042) and SeSe (36%; p=0.035). In contrast, fungal, viral and non-dominant bacterial components of the microbiome were not significantly different between FUT2 genotypes. Conclusions FUT2 genotype in patients with non-cystic fibrosis bronchiectasis was significantly associated with disease outcomes, with homozygous secretors exhibiting lower lung function, higher exacerbation number and a higher frequency of P. aeruginosa-dominated infection. Trial registration number ACTRN12609000578202 (anzctr.org.au); Pre-results.

Oxidative and endoplasmic reticulum stress in respiratory disease

Oxidative stress and endoplasmic reticulum (ER) stress are related states that can occur in cells as part of normal physiology but occur frequently in diseases involving inflammation. In this article, we review recent findings relating to the role of oxidative and ER stress in the pathophysiology of acute and chronic nonmalignant diseases of the lung, including infections, cystic fibrosis, idiopathic pulmonary fibrosis and asthma. We also explore the potential of drugs targeting oxidative and ER stress pathways to alleviate disease.

Impact of Long-Term Erythromycin Therapy on the Oropharyngeal Microbiome and Resistance Gene Reservoir in Non-Cystic Fibrosis Bronchiectasis

Recent demonstrations that long-term macrolide therapy can prevent exacerbations in chronic airways diseases have led to a dramatic increase in their use. However, little is known about the wider, potentially adverse impacts of these treatments. Substantial disruption of the upper airway commensal microbiota might reduce its contribution to host defense and local immune regulation, while increases in macrolide resistance carriage would represent a serious public health concern. Using samples from a randomized controlled trial, we show that low-dose erythromycin given over 48 weeks influences the composition of the oropharyngeal commensal microbiota. We report that macrolide therapy is associated with significant changes in the relative abundances of members of the Actinomyces genus and with significant increases in the carriage of transmissible macrolide resistance. Determining the clinical significance of these changes, relative to treatment benefit, now represents a research priority. ABSTRACT Long-term macrolide therapy reduces rates of pulmonary exacerbation in bronchiectasis. However, little is known about the potential for macrolide therapy to alter the composition and function of the oropharyngeal commensal microbiota or to increase the carriage of transmissible antimicrobial resistance. We assessed the effect of long-term erythromycin on oropharyngeal microbiota composition and the carriage of transmissible macrolide resistance genes in 84 adults with bronchiectasis, enrolled in the Bronchiectasis and Low-dose Erythromycin Study (BLESS) 48-week placebo-controlled trial of twice-daily erythromycin ethylsuccinate (400 mg). Oropharyngeal microbiota composition and macrolide resistance gene carriage were determined by 16S rRNA gene amplicon sequencing and quantitative PCR, respectively. Long-term erythromycin treatment was associated with a significant increase in the relative abundance of oropharyngeal Haemophilus parainfluenzae (P = 0.041) and with significant decreases in the relative abundances of Streptococcus pseudopneumoniae (P = 0.024) and Actinomyces odontolyticus (P = 0.027). Validation of the sequencing results by quantitative PCR confirmed a significant decrease in the abundance of Actinomyces spp. (P = 0.046). Erythromycin treatment did not result in a significant increase in the number of subjects who carried erm(A), erm(B), erm(C), erm(F), mef(A/E), and msrA macrolide resistance genes. However, the abundance of erm(B) and mef(A/E) gene copies within carriers who had received erythromycin increased significantly (P < 0.05). Our findings indicate that changes in oropharyngeal microbiota composition resulting from long-term erythromycin treatment are modest and are limited to a discrete group of taxa. Associated increases in levels of transmissible antibiotic resistance genes within the oropharyngeal microbiota highlight the potential for this microbial system to act as a reservoir for resistance. IMPORTANCE Recent demonstrations that long-term macrolide therapy can prevent exacerbations in chronic airways diseases have led to a dramatic increase in their use. However, little is known about the wider, potentially adverse impacts of these treatments. Substantial disruption of the upper airway commensal microbiota might reduce its contribution to host defense and local immune regulation, while increases in macrolide resistance carriage would represent a serious public health concern. Using samples from a randomized controlled trial, we show that low-dose erythromycin given over 48 weeks influences the composition of the oropharyngeal commensal microbiota. We report that macrolide therapy is associated with significant changes in the relative abundances of members of the Actinomyces genus and with significant increases in the carriage of transmissible macrolide resistance. Determining the clinical significance of these changes, relative to treatment benefit, now represents a research priority.

Culture-independent detection of nontuberculous mycobacteria in clinical respiratory samples

ABSTRACT Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust.

PPARγ is reduced in the airways of non-CF bronchiectasis subjects and is inversely correlated with the presence of Pseudomonas aeruginosa

Background Chronic airway inflammation in conditions such as cystic fibrosis (CF) and non-CF bronchiectasis is characterised by a predominant neutrophilic inflammatory response, commonly due to the presence of pathogenic bacteria such as Pseudomonas aeruginosa. We hypothesised that down-regulation of the anti-inflammatory nuclear transcription regulator peroxisome proliferator-activated receptor gamma (PPARγ in non-CF bronchiectasis subjects may explain why this exuberant neutrophilic inflammation is able to persist unchecked in the inflamed airway. Methods PPARγ gene expression was assessed in bronchoalveolar lavage fluid (BAL) of 35 macrolide naïve non-CF bronchiectasis subjects and compared with that in 20 healthy controls. Human RNA was extracted from pelleted BAL and PPARγ expression was determined by reverse-transcription quantitative PCR. Bacterial DNA was extracted from paired induced sputum and total bacterial load was determined by 16S rRNA qPCR. Quantification of individual bacterial species was achieved by qPCR. Results PPARγ expression was lower in subjects with non-CF bronchiectasis compared with healthy control subjects (control: 1.00, IQR 0.55–1.44, n = 20 vs. Bronchiectasis: 0.49, IQR 0.12–0.89; n = 35; p<0.001, Mann-Whitney U test). This lower PPARγ expression correlated negatively with Pseudomonas aeruginosa (r = -0.53, n = 31; p = 0.002). No significant association was seen between PPARγ and total bacterial levels or levels Haemophilus influenzae. Conclusion PPARγ is expressed in low levels in the airways of non-CF bronchiectasis subjects, despite an aggressive inflammatory response. This low level PPARγ expression is particularly associated with the presence of high levels of P. aeruginosa, and may represent an intrinsic link with this bacterial pathogen.

Understanding the impact of antibiotic therapies on the respiratory tract resistome: a novel pooled-template metagenomic sequencing strategy

Determining the effects of antimicrobial therapies on airway microbiology at a population-level is essential. Such analysis allows, for example, surveillance of antibiotic-induced changes in pathogen prevalence, the emergence and spread of antibiotic resistance, and the transmission of multi-resistant organisms. However, current analytical strategies for understanding these processes are limited. Culture- and PCR-based assays for specific microbes require the a priori selection of targets, while antibiotic sensitivity testing typically provides no insight into either the molecular basis of resistance, or the carriage of resistance determinants by the wider commensal microbiota. Shotgun metagenomic sequencing provides an alternative approach that allows the microbial composition of clinical samples to be described in detail, including the prevalence of resistance genes and virulence traits. While highly informative, the application of metagenomics to large patient cohorts can be prohibitively expensive. Using sputum samples from a randomised placebo-controlled trial of erythromycin in adults with bronchiectasis, we describe a novel, cost-effective strategy for screening patient cohorts for changes in resistance gene prevalence. By combining metagenomic screening of pooled DNA extracts with validatory quantitative PCR-based analysis of candidate markers in individual samples, we identify population-level changes in the relative abundance of specific macrolide resistance genes. This approach has the potential to provide an important adjunct to current analytical strategies, particularly within the context of antimicrobial clinical trials.

Opportunistic bacteria confer the ability to ferment prebiotic starch in the adult cystic fibrosis gut

ABSTRACT Chronic disruption of the intestinal microbiota in adult cystic fibrosis (CF) patients is associated with local and systemic inflammation, and has been linked to the risk of serious comorbidities. Supplementation with high amylose maize starch (HAMS) might provide clinical benefit by promoting commensal bacteria and the biosynthesis of immunomodulatory metabolites. However, whether the disrupted CF gut microbiota has the capacity to utilise these substrates is not known. We combined metagenomic sequencing, in vitro fermentation, amplicon sequencing, and metabolomics to define the characteristics of the faecal microbiota in adult CF patients and assess HAMS fermentation capacity. Compared to healthy controls, the faecal metagenome of adult CF patients had reduced bacterial diversity and prevalence of commensal fermentative clades. In vitro fermentation models seeded with CF faecal slurries exhibited reduced acetate levels compared to healthy control reactions, but comparable levels of butyrate and propionate. While the commensal genus Faecalibacterium was strongly associated with short chain fatty acid (SCFA) production by healthy microbiota, it was displaced in this role by Clostridium sensu stricto 1 in the microbiota of CF patients. A subset of CF reactions exhibited enterococcal overgrowth, resulting in lactate accumulation and reduced SCFA biosynthesis. The addition of healthy microbiota to CF faecal slurries failed to displace predominant CF taxa, or substantially influence metabolite biosynthesis. Despite significant microbiota disruption, the adult CF gut microbiota retains the capacity to exploit HAMS. Our findings highlight the potential for taxa associated with the altered CF gut microbiotato mediate prebiotic effects in microbial systems subject to ongoing perturbation, irrespective of the depletion of common commensal clades.