Efficacy of biologically-directed daylight therapy on sleep and circadian rhythm in Parkinson's disease: a randomised, double-blind, parallel-group, active-controlled, phase 2 clinical trial.

Background: New non-pharmacological treatments for improving non-motor symptoms in Parkinson's disease (PD) are urgently needed. Previous light therapies for modifying sleep behaviour lacked standardised protocols and were not personalised for an individual patient chronotype. We aimed to assess the efficacy of a biologically-directed light therapy in PD that targets retinal inputs to the circadian system on sleep, as well as other non-motor and motor functions. Methods: In this randomised, double-blind, parallel-group, active-controlled trial at the Queensland University of Technology, Australia, participants with mild to moderate PD were computer randomised (1:1) to receive one of two light therapies that had the same photometric luminance and visual appearance to allow blinding of investigators and participants to the intervention. One of these biologically-directed lights matched natural daylight (Day Mel), which is known to stimulate melanopsin cells. The light therapy of the other treatment arm of the study, specifically supplemented the stimulation of retinal melanopsin cells (Enhanced Mel), targeting deficits to the circadian system. Both lights were administered 30 min per day over 4-weeks and personalised to an individual patient's chronotype, while monitoring environmental light exposure with actigraphy. Co-primary endpoints were a change from baseline in mean sleep macrostructure (polysomnography, PSG) and an endocrine biomarker of circadian phase (dim light melatonin secretion onset, DLMO) at weeks 4 and 6. Participants data were analysed using an intention to treat principle. All endpoints were evaluated by applying a mixed model analysis. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12621000077864. Findings: Between February 4, 2021 and August 8, 2022, 144 participants with PD were consecutively screened, 60 enrolled and randomly assigned to a light intervention. There was no significant difference in co-primary outcomes between randomised groups overall or at any individual timepoint during follow-up. The mean (95% CI) for PSG, N3% was 24.15 (19.82-28.48) for Day Mel (n = 23) and 19.34 (15.20-23.47) for the Enhanced Mel group (n = 25) in week 4 (p = 0.12); and 21.13 (16.99-25.28) for Day Mel (n = 26) and 18.48 (14.34-22.62) for the Enhanced Mel group (n = 25) in week 6, (p = 0.37). The mean (95% CI) DLMO (decimal time) was 19.82 (19.20-20.44) for Day Mel (n = 22) and 19.44 (18.85-20.04) for the Enhanced Mel group (n = 24) in week 4 (p = 0.38); and 19.90 (19.27-20.53) for Day Mel (n = 23) and 19.04 (18.44-19.64) for the Enhanced Mel group (n = 25) in week 6 (p = 0.05). However, both the controlled daylight (Day Mel) and the enhanced melanopsin (Enhanced Mel) interventions demonstrated significant improvement in primary PSG sleep macrostructure. The restorative deep sleep phase (PSG, N3) significantly improved at week 6 in both groups [model-based mean difference to baseline (95% CI): -3.87 (-6.91 to -0.83), p = 0.04]. There was a phase-advance in DLMO in both groups which did not reach statistical significance between groups at any time-point. There were no safety concerns or severe adverse events related to the intervention. Interpretation: Both the controlled daylight and melanopsin booster light showed efficacy in improving measures of restorative deep sleep in people with mild to moderate PD. That there was no significant difference between the two intervention groups may be due to the early disease stage. The findings suggest that controlled indoor daylight that is personalised to the individuals' chronotype could be effective for improving sleep in early to moderate PD, and further studies evaluating controlled daylight interventions are now required utilising this standardised approach, including in advanced PD. Funding: The Michael J Fox Foundation for Parkinson's Research, Shake IT Up Australia, National Health and Medical Research Council, and Australian Research Council.

Interleukin-22 suppresses major histocompatibility complex II in mucosal epithelial cells.

Major histocompatibility complex (MHC) II is dynamically expressed on mucosal epithelial cells and is induced in response to inflammation and parasitic infections, upon exposure to microbiota, and is increased in chronic inflammatory diseases. However, the regulation of epithelial cell-specific MHC II during homeostasis is yet to be explored. We discovered a novel role for IL-22 in suppressing epithelial cell MHC II partially via the regulation of endoplasmic reticulum (ER) stress, using animals lacking the interleukin-22-receptor (IL-22RA1), primary human and murine intestinal and respiratory organoids, and murine models of respiratory virus infection or with intestinal epithelial cell defects. IL-22 directly downregulated interferon-γ-induced MHC II on primary epithelial cells by modulating the expression of MHC II antigen A α (H2-Aα) and Class II transactivator (Ciita), a master regulator of MHC II gene expression. IL-22RA1-knockouts have significantly higher MHC II expression on mucosal epithelial cells. Thus, while IL-22-based therapeutics improve pathology in chronic disease, their use may increase susceptibility to viral infections.

The Impact of Long-Term Macrolide Exposure on the Gut Microbiome and Its Implications for Metabolic Control.

Long-term low-dose macrolide therapy is now widely used in the treatment of chronic respiratory diseases for its immune-modulating effects, although the antimicrobial properties of macrolides can also have collateral impacts on the gut microbiome. We investigated whether such treatment altered intestinal commensal microbiology and whether any such changes affected systemic immune and metabolic regulation. In healthy adults exposed to 4 weeks of low-dose erythromycin or azithromycin, as used clinically, we observed consistent shifts in gut microbiome composition, with a reduction in microbial capacity related to carbohydrate metabolism and short-chain fatty acid biosynthesis. These changes were accompanied by alterations in systemic biomarkers relating to immune (interleukin 5 [IL-5], IL-10, monocyte chemoattractant protein 1 [MCP-1]) and metabolic (serotonin [5-HT], C-peptide) homeostasis. Transplantation of erythromycin-exposed murine microbiota into germ-free mice demonstrated that changes in metabolic homeostasis and gastrointestinal motility, but not systemic immune regulation, resulted from changes in intestinal microbiology caused by macrolide treatment. Our findings highlight the potential for long-term low-dose macrolide therapy to influence host physiology via alteration of the gut microbiome. IMPORTANCE Long-term macrolide therapy is widely used in chronic respiratory diseases although its antibacterial activity can also affect the gut microbiota, a key regulator of host physiology. Macrolide-associated studies on the gut microbiota have been limited to short antibiotic courses and have not examined its consequences for host immune and metabolic regulation. This study revealed that long-term macrolides depleted keystone bacteria and impacted host regulation, mediated directly by macrolide activity or indirectly by alterations to the gut microbiota. Understanding these macrolide-associated mechanisms will contribute to identifying the risk of long-term exposure and highlights the importance of targeted therapy for maintenance of the gut microbiota.

Assessment of Long-Term Macrolide Exposure on the Oropharyngeal Microbiome and Macrolide Resistance in Healthy Adults and Consequences for Onward Transmission of Resistance.

While the use of long-term macrolide therapy to prevent exacerbations in chronic respiratory diseases is widespread, its impact on the oropharyngeal microbiota and macrolide resistance, and the potential for onward transmission of resistance to close contacts are poorly understood. We determined the effects of long-term exposure to azithromycin or erythromycin on phenotypic and genotypic macrolide resistance within the oropharyngeal microbiome of healthy adults and their close contacts in a randomized, single-blinded, parallel-group trial of 4 weeks of twice-daily oral 400 mg erythromycin ethylsuccinate or twice-daily oral 125 mg azithromycin. Using oropharyngeal swabs collected from 20 index healthy adults and 20 paired close contacts, the oropharyngeal microbial composition and macrolide resistance in streptococci were assessed by 16S rRNA sequencing and antibiotic susceptibility testing of oropharyngeal cultures, respectively, at baseline and weeks 4 and 8 (washout). Targeted quantitative PCR of antibiotic resistance genes was performed to evaluate paired changes in resistance gene levels in index patients and close contacts and to relate the potential transmission of antibiotic resistance. Neither azithromycin nor erythromycin altered oropharyngeal microbiota characteristics significantly. Proportional macrolide resistance in oropharyngeal streptococci increased with both erythromycin and azithromycin, remaining above baseline levels for the azithromycin group at washout. Levels of resistance genes increased significantly with azithromycin[erm(B) and mef] and erythromycin (mef), returning to baseline levels at washout only for the erythromycin group. We found no evidence of onward transmission of resistance to close contacts, as indicated by the lack of concomitant changes in resistance gene levels detected in close contacts. (This study has been registered with the Australian and New Zealand Clinical Trials Registry under identifier ACTRN12617000278336.).

Carriage and Transmission of Macrolide Resistance Genes in Patients With Chronic Respiratory Conditions and Their Close Contacts.

BACKGROUND: Long-term macrolide therapy has been shown to provide benefit to those with a range of chronic respiratory conditions. However, concerns remain about the impact of macrolide exposure on the carriage and abundance of antibiotic resistance genes within the oropharynx. The potential for onward transmission of resistance from macrolide recipients to their close contacts also is poorly understood. RESEARCH QUESTION: Does long-term macrolide use impact carriage of resistance within the oropharyngeal microbiota in people with chronic respiratory conditions and risk of onward transmission to their close contacts? STUDY DESIGN AND METHODS: Oropharyngeal swabs were collected from 93 individuals with chronic respiratory conditions, 53 of whom were receiving long-term macrolide therapy. An oropharyngeal swab also was collected from a close cohabiting contact of each patient. Detection and abundance of 10 macrolide-associated resistance genes with the potential to disseminate via horizontal gene transfer were assessed by quantitative polymerase chain reaction analysis. RESULTS: Detection of resistance genes in macrolide recipients was comparable with that in nonrecipients. However, the normalized gene abundance of erm(B) was significantly higher in the macrolide recipient group (P = .045). Among the close contacts, no between-group differences in resistance gene detection or abundance were identified. Within-group analysis showed that the detection of erm(F) and mef in macrolide recipients, but not nonrecipients, was associated significantly with detection in close contacts (P = .003 and P = .004, respectively). However, between-group analysis showed that treatment group did not predict cocarriage between patients and their close contacts (P > .05 for each gene). INTERPRETATION: Although levels of erm(B) were higher in those receiving long-term macrolide therapy and evidence of gene cocarriage with close contacts was found, no evidence was found that macrolide use increased the onward transmission risk to their close contacts. This study therefore addresses concerns that long-term macrolide therapy could promote the dissemination of transmissible macrolide resistance.

Targeting the P2Y13 Receptor Suppresses IL-33 and HMGB1 Release and Ameliorates Experimental Asthma.

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma.

The cystic fibrosis gut as a potential source of multidrug resistant pathogens.

BACKGROUND: The emergence of multidrug resistant (MDR) pathogens represents a profound threat to global health. Individuals with CF have amongst the highest cumulative antibiotic exposure of any patient group, including to critically-important last-line agents. While there is little evidence that antibiotic resistance in airway pathogens results in worse clinical outcomes for CF patients, the potential emergence of MDR pathogens in non-respiratory systems, as a consequence of CF care, represents a potential health threat to the wider population, including family and carers. METHODS: Stool from 19 adults with CF and 16 healthy adult controls was subjected to metagenomic sequencing, to assess faecal resistome, and culture-based analysis. Resistant isolates were identified phenotypically, and genetic determinants of resistance characterised by whole genome sequencing. RESULTS: CF and control faecal resistomes differed significantly (P = 0.0003). The proportion of reads that mapped to mobile genetic elements was significantly higher in CF (P = 0.014) and the composition was significantly different (P = 0.0001). Notably, CF patients displayed higher carriage of plasmid-mediated aminoglycoside-modifying genes ant(6)-Ib, aac(6')-Ip, and aph(3')-IIIa (P < 0.01). Culture-based analysis supported higher aminoglycoside resistance, with a higher proportion of aminoglycoside-resistant, Gram-negative bacteria (P < 0.0001). Isolated extended spectrum beta lactamase (ESBL)-positive Escherichia coli from CF stool exhibited phenotypic resistance to tobramycin and gentamicin. Genomic analysis showed co-localisation of both aminoglycoside resistance and ESBL genes, consistent with MDR emergence through horizontal gene transfer. CONCLUSIONS: The carriage of potentially transmissible resistance within the adult CF gut microbiome is considerably greater than in healthy individuals and could contribute to the emergence and dissemination of MDR pathogens.

The impact of CFTR modulator therapies on CF airway microbiology.

Major historical advances in cystic fibrosis (CF) respiratory clinical care, including mechanical airway clearance and inhaled medications, have aimed to address the consequences of cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction. In contrast, CFTR modulator therapies instead target the underlying protein defect that leads to CF lung disease. The extent to which these therapies might reduce susceptibility to chronic lung infections remains to be seen. However, by improving airway clearance, reducing the requirement for antibiotics, and in some cases, through direct antimicrobial effects, CFTR modulators are likely to result in substantial changes in CF airway microbiology. These changes could contribute substantially to the clinical benefit associated with modulator therapies, as well as providing an important indicator of treatment efficacy and residual pathophysiology. Indeed, the widespread introduction of modulator therapies might require us to re-consider our models of CF airway microbiology.

Management of Australian Adults with Bronchiectasis in Tertiary Care: Evidence-Based or Access-Driven?

PURPOSE: Australian data regarding the management of patients with bronchiectasis is scarce. We sought to compare the management of adults with bronchiectasis attending tertiary Australian centres with recent national and international guidelines. METHODS: The Australian Bronchiectasis Registry is a centralised database of patients with radiologically confirmed bronchiectasis unrelated to cystic fibrosis recruited from 14 tertiary Australian hospitals. We excluded children (<18 years) and those with incomplete data, leaving 589 adults for cross-sectional analyses. We compared the proportion of patients receiving certain therapies, as compared to the proportion eligible for those treatments according to the current guidelines and baseline clinical information available from the registry. RESULTS: Pulmonary rehabilitation was attended by 22%, although it was indicated in 67% of the cohort. Airway clearance was undertaken in 52% of patients, although 71% reported chronic productive cough. Sputum bacterial culture results were available for 59%, and mycobacterial culture results were available for 29% of the cohort. Inhaled antibiotics were used in half of potentially eligible patients. Despite guideline recommendations against routine use, inhaled corticosteroids were used in 48% of patients. Long-term macrolides were used in 28% of participants. CONCLUSIONS: Discrepancies exist between guideline recommendations and real-world treatment of bronchiectasis in Australia, even in tertiary centres. These findings suggest the need for increased patient referral to pulmonary rehabilitation, increased attention to airway clearance, increased collection of sputum samples (especially for mycobacterial culture) and rationalisation of inhaled corticosteroid use. These findings encourage a review of treatment access and will inform ongoing education to promote evidence-based care for people living with bronchiectasis.

Long-term macrolide antibiotics for the treatment of bronchiectasis in adults: an individual participant data meta-analysis.

BACKGROUND: Bronchiectasis guidelines recommend long-term macrolide treatment for patients with three or more exacerbations per year without Pseudomonas aeruginosa infection. Randomised controlled trials suggest that long-term macrolide treatment can prevent exacerbations in adult patients with bronchiectasis, but these individual studies have been too small to do meaningful subgroup analyses. We did a systematic review and individual patient data (IPD) meta-analysis to explore macrolide benefit in subpopulations, including those in which macrolide therapy is not currently recommended. METHODS: We searched MEDLINE, Embase, the Cochrane Central Register of Controlled Trials, and Web of Science from Jan 1, 2000, to Sept 30, 2018, to identify double blind, randomised, placebo-controlled trials of macrolide antibiotics in adult patients with bronchiectasis. We applied no language restrictions. Randomised controlled trials were eligible if treatment was defined a priori as long term and had a primary or secondary outcome of bronchiectasis exacerbations. Studies in patients with cystic fibrosis bronchiectasis were excluded. The primary outcome of the meta-analysis was frequency of exacerbations requiring treatment with antibiotics. Secondary endpoints were time to first exacerbation, change in quality of life according to the St George's Respiratory Questionnaire (SGRQ), and change in FEV1. IPD meta-analysis was done using fixed effects models adjusting for age, sex, FEV1, and trial. We did prespecified subgroup analyses for each of the primary and secondary endpoints using one-step meta-analysis only. Subgroups were defined by age, sex, previous exacerbation frequency, smoking status, inhaled corticosteroid use at baseline, body-mass index at baseline, cause, C-reactive protein at baseline, baseline FEV1 percentage of predicted, SGRQ total score, and Pseudomonas aeruginosa in sputum culture at baseline. The meta-analysis is registered with the PROSPERO international register of systematic reviews, number CRD42018102908. FINDINGS: Of 234 identified studies, we included three randomised controlled trials, and IPD was obtained for 341 participants. Macrolide antibiotics reduced the frequency of exacerbations (adjusted incidence rate ratio [IRR] 0·49, 95% CI 0·36 to 0·66; p<0·0001). We also found that macrolide treatment improved the time to first exacerbation (adjusted hazard ratio 0·46, 0·34 to 0·61; p<0·0001) and was associated with improved quality of life measured by the SGRQ (mean improvement 2·93 points, 0·03 to 5·83; p=0·048). Macrolides were not associated with a significant improvement in FEV1 (67 mL at 1 year, -22 to 112; p=0·14). Effect estimates in prespecified subgroup analyses revealed a reduced frequency of exacerbations in all prespecified subgroups, including a high level of benefit in patients with P aeruginosa infection (IRR 0·36, 0·18-0·72; p=0·0044) and in patients with one to two exacerbations per year (0·37, 0·16-0·88; p=0·025). Studies were rated as low risk of bias across all domains. INTERPRETATION: Long-term macrolide treatment significantly reduces the frequency of exacerbations in patients with bronchiectasis, with similar benefits observed in all subgroups based on patient characteristics. This finding suggests that macrolides might be considered in patients in whom macrolides are not indicated according to the current guidelines, particularly if alternative approaches to reduce exacerbations have been unsuccessful. However, downsides of long-term macrolide treatment must also be taken into account. FUNDING: European Respiratory Society.

Australian adults with bronchiectasis: The first report from the Australian Bronchiectasis Registry.

BACKGROUND: /objective: There are no large, multi-centre studies of Australians with bronchiectasis. The Australian Bronchiectasis Registry (ABR) was established in 2015 to create a longitudinal research platform. We aimed to describe the baseline characteristics of adult ABR participants and assess the impact of disease severity and exacerbation phenotype on quality of life (QoL). METHODS: The ABR is a centralised database of patients with radiologically confirmed bronchiectasis unrelated to cystic fibrosis. We analysed the baseline data of adult patients (≥18 years). RESULTS: From March 2016-August 2018, 799 adults were enrolled from 14 Australian sites. Baseline data were available for 589 adults predominantly from six tertiary centres (420 female, median age 71 years (interquartile range 64-77), 14% with chronic Pseudomonas aeruginosa infection). Most patients had moderate or severe disease based on the Bronchiectasis Severity Index (BSI) (84%) and FACED (59%) composite scores. Using Global Lung function Initiative-2012 reference equations, the majority of patients (48%) had normal spirometry; only 34% had airflow obstruction (FEV1/FVC < LLN). Disease severity scores (BSI and FACED) were negatively correlated with QoL-Bronchiectasis domain scores (rs between -0.09 and -0.58). The frequent exacerbator phenotype (≥3 in the preceding year) was identified in 23%; this group had lower scores in all QoL-B domains (p ≤ 0.001) and more hospitalisations (p < 0.001) than those with <3 exacerbations. CONCLUSIONS: The largest cohort of Australian adults with bronchiectasis has been described. Using contemporary criteria, most patients with bronchiectasis did not have airflow obstruction. The frequent exacerbation trait connotes poorer QoL and greater health-care utilisation.

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

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.

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.

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

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.

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.

Culture-Independent Detection of Nontuberculous Mycobacteria in Clinical Respiratory Samples.

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.

Macrolide Treatment Inhibits Pseudomonas aeruginosa Quorum Sensing in Non-Cystic Fibrosis Bronchiectasis. An Analysis from the Bronchiectasis and Low-Dose Erythromycin Study Trial.

RATIONALE: The mechanism by which low-dose macrolide therapy reduces exacerbations in non-cystic fibrosis bronchiectasis is not known. Pseudomonas aeruginosa quorum sensing controls the expression of a range of pathogenicity traits and is inhibited by macrolide in vitro. Quorum sensing inhibition renders P. aeruginosa less pathogenic, potentially reducing its contribution to airway damage. OBJECTIVES: The aim of this study was to determine whether long-term low-dose erythromycin inhibits P. aeruginosa quorum sensing within the airways of patients with non-cystic fibrosis bronchiectasis. METHODS: Analysis was performed on induced sputum from P. aeruginosa-positive subjects at recruitment to the BLESS (Bronchiectasis and Low-Dose Erythromycin Study) trial and after 48 weeks of treatment with erythromycin or placebo. To avoid changes in gene expression during culture, bacterial mRNA was extracted directly from sputum, and the relative expression of functionally critical quorum sensing genes was determined by quantitative polymerase chain reaction. MEASUREMENTS AND MAIN RESULTS: In keeping with the BLESS study, a significant reduction in total exacerbations was seen in this subgroup (placebo: 6, [interquartile range (IQR), 4-8]; erythromycin: 3, [IQR, 3-4]; P = 0.008; Mann-Whitney test). Erythromycin therapy did not change P. aeruginosa bacterial load determined by polymerase chain reaction. A significant reduction was observed in the expression of the quorum sensing genes, lasR (erythromycin: fold change, 0.065 [IQR, 0.01-0.85], n = 11; placebo: fold change, 1.000 [IQR, 0.05-3.05]; P = 0.047, Mann-Whitney U test) and pqsA (erythromycin: fold change, 0.07 [IQR, 0.02-0.25]; placebo: fold change, 1.000 [IQR, 0.21-4.31], P = 0.017, Mann-Whitney U test), after 48 weeks of erythromycin, compared with placebo. CONCLUSIONS: We demonstrate inhibition of P. aeruginosa quorum sensing within the airways of patients with non-cystic fibrosis bronchiectasis receiving long-term, low-dose erythromycin, without a reduction in bacterial load, representing a potential mechanism of therapeutic impact beyond a classical antimicrobial or antiinflammatory pathway.

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-1ß, 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 IL1ß (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.

Matrix metalloproteinases vary with airway microbiota composition and lung function in non-cystic fibrosis bronchiectasis.

RATIONALE: Despite growing evidence for the roles of airway remodeling and bacterial infection in the progression of non-cystic fibrosis bronchiectasis, relationships between collagen-degrading proteases and chronic airway infection are poorly understood. OBJECTIVES: The aim of this study was to determine which matrix metalloproteinases (MMPs) are elevated in bronchiectasis, whether these MMP levels vary based on patients' dominant infective microbe, and how these levels correlate with clinical measures of disease severity. METHODS: We determined concentrations of nine MMPs and four tissue inhibitors of metalloproteinases (TIMPs) in induced sputum from 86 patients with bronchiectasis and 8 healthy control subjects by Luminex protein assay. Concentrations were then assessed in relation to lung function, inflammatory markers, and airway microbiota composition, determined by 16S rRNA gene amplicon sequencing. Airway microbiota composition was classified as Pseudomonas aeruginosa-dominated, Haemophilus influenzae-dominated, or dominated by another species. MMP-8 and MMP-9 activity levels were also measured in a subset of patients. MEASUREMENTS AND MAIN RESULTS: MMP-1, -3, -7, -8, and -9 and TIMP-2 and -4 levels, as well as MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios, were significantly higher in patients with bronchiectasis than in healthy control subjects (all: P < 0.001, except MMP-7: P < 0.05). Patients with bronchiectasis with H. influenzae-dominated airway infections demonstrated higher MMP-2 levels (P < 0.01) and MMP-8 activity (P < 0.05) than those with P. aeruginosa-dominated airway infections. Among patients with bronchiectasis, there were significant inverse correlations between FEV1 as a percentage of predicted value, MMP-8 and MMP-1 levels, and MMP-8/TIMP-1 and MMP-9/TIMP-1 ratios (P < 0.01). CONCLUSIONS: Increased MMP levels (particularly MMP-8 and MMP-1) and MMP/TIMP ratios in patients with bronchiectasis compared with healthy control subjects correlated with lower lung function and higher levels of inflammatory markers. Further, MMP profiles differed in patients with bronchiectasis according to the dominant pathogen determined by gene sequencing, raising the possibility of differential airway remodeling according to airway microbiology.