David J. Serisier

Characterization of Bacterial Community Diversity in Cystic Fibrosis Lung Infections by Use of 16S Ribosomal DNA Terminal Restriction Fragment Length Polymorphism Profiling

ABSTRACT Progressive loss of lung function resulting from the inflammatory response to bacterial colonization is the leading cause of mortality in cystic fibrosis (CF) patients. A greater understanding of these bacterial infections is needed to improve lung disease management. As culture-based diagnoses are associated with fundamental drawbacks, we used terminal restriction fragment (T-RF) length polymorphism profiling and 16S rRNA clone data to characterize, without prior cultivation, the bacterial community in 71 sputa from 34 adult CF patients. Nineteen species from 15 genera were identified in 53 16S rRNA clones from three patients. Of these, 15 species have not previously been reported in CF lung infections and many were species requiring strict anaerobic conditions for growth. The species richness and evenness were determined from the T-RF length and volume for the 71 profiles. Species richness was on average 13.3 ± 7.9 per sample and 13.4 ± 6.7 per patient. On average, the T-RF bands of the lowest and highest volumes represented 0.6 and 59.2% of the total volume in each profile, respectively. The second through fifth most dominant T-RF bands represented 15.3, 7.5, 4.7, and 2.8% of the total profile volume, respectively. On average, the remaining T-RF bands represented 10.2% of the total profile volume. The T-RF band corresponding to Pseudomonas aeruginosa had the highest volume in 61.1% of the samples. However, 18 other T-RF band lengths were dominant in at least one sample. In conclusion, this reveals the enormous complexity of bacteria within the CF lung. Although their significance is yet to be determined, these findings alter our perception of CF lung infections.

Effect of Long-term, Low-Dose Erythromycin on Pulmonary Exacerbations Among Patients With Non–Cystic Fibrosis Bronchiectasis: The BLESS Randomized Controlled Trial

IMPORTANCE Macrolide antibiotics such as erythromycin may improve clinical outcomes in non-cystic fibrosis (CF) bronchiectasis, although associated risks of macrolide resistance are poorly defined. OBJECTIVE To evaluate the clinical efficacy and antimicrobial resistance cost of low-dose erythromycin given for 12 months to patients with non-CF bronchiectasis with a history of frequent pulmonary exacerbations. DESIGN, SETTING, AND PARTICIPANTS Twelve-month, randomized (1:1), double-blind, placebo-controlled trial of erythromycin in currently nonsmoking, adult patients with non-CF bronchiectasis with a history of 2 or more infective exacerbations in the preceding year. This Australian study was undertaken between October 2008 and December 2011 in a university teaching hospital, with participants also recruited via respiratory physicians at other centers and from public radio advertisements. INTERVENTIONS Twice-daily erythromycin ethylsuccinate (400 mg) or matching placebo. MAIN OUTCOME MEASURES The primary outcome was the annualized mean rate of protocol-defined pulmonary exacerbations (PDPEs) per patient. Secondary outcomes included macrolide resistance in commensal oropharyngeal streptococci and lung function. RESULTS Six-hundred seventy-nine patients were screened, 117 were randomized (58 placebo, 59 erythromycin), and 107 (91.5%) completed the study. Erythromycin significantly reduced PDPEs both overall (mean, 1.29 [95% CI, 0.93-1.65] vs 1.97 [95% CI, 1.45-2.48] per patient per year; incidence rate ratio [IRR], 0.57 [95% CI, 0.42-0.77]; P = .003), and in the prespecified subgroup with baseline Pseudomonas aeruginosa airway infection (mean difference, 1.32 [95% CI, 0.19-2.46]; P = .02). Erythromycin reduced 24-hour sputum production (median difference, 4.3 g [interquartile range [IQR], 1 to 7.8], P = .01) and attenuated lung function decline (mean absolute difference for change in postbronchodilator forced expiratory volume in the first second of expiration, 2.2 percent predicted [95% CI, 0.1% to 4.3%]; P = .04) compared with placebo. Erythromycin increased the proportion of macrolide-resistant oropharyngeal streptococci (median change, 27.7% [IQR, 0.04% to 41.1%] vs 0.04% [IQR, -1.6% to 1.5%]; difference, 25.5% [IQR,15.0% to 33.7%]; P < .001). CONCLUSION AND RELEVANCE Among patients with non-CF bronchiectasis, the 12-month use of erythromycin compared with placebo resulted in a modest decrease in the rate of pulmonary exacerbations and an increased rate of macrolide resistance. TRIAL REGISTRATION anzctr.org.au Identifier: ACTRN12609000578202.

Inhaled, dual release liposomal ciprofloxacin in non-cystic fibrosis bronchiectasis (ORBIT-2): a randomised, double-blind, placebo-controlled trial

Background The delivery of antipseudomonal antibiotics by inhalation to Pseudomonas aeruginosa-infected subjects with non-cystic fibrosis (CF) bronchiectasis is a logical extension of treatment strategies successfully developed in CF bronchiectasis. Dual release ciprofloxacin for inhalation (DRCFI) contains liposomal ciprofloxacin, formulated to optimise airway antibiotic delivery. Methods Phase II, 24-week Australian/New Zealand multicentre, randomised, double-blind, placebo-controlled trial in 42 adult bronchiectasis subjects with ≥2 pulmonary exacerbations in the prior 12 months and ciprofloxacin-sensitive P aeruginosa at screening. Subjects received DRCFI or placebo in three treatment cycles of 28 days on/28 days off. The primary outcome was change in sputum P aeruginosa bacterial density to the end of treatment cycle 1 (day 28), analysed by modified intention to treat (mITT). Key secondary outcomes included safety and time to first pulmonary exacerbation—after reaching the pulmonary exacerbation endpoint subjects discontinued study drug although remained in the study. Results DRCFI resulted in a mean (SD) 4.2 (3.7) log10 CFU/g reduction in P aeruginosa bacterial density at day 28 (vs −0.08 (3.8) with placebo, p=0.002). DRCFI treatment delayed time to first pulmonary exacerbation (median 134 vs 58 days, p=0.057 mITT, p=0.046 per protocol). DRCFI was well tolerated with a similar incidence of systemic adverse events to the placebo group, but fewer pulmonary adverse events. Conclusions Once-daily inhaled DRCFI demonstrated potent antipseudomonal microbiological efficacy in adults with non-CF bronchiectasis and ciprofloxacin-sensitive P aeruginosa. In this modest-sized phase II study, DRCFI was also well tolerated and delayed time to first pulmonary exacerbation in the per protocol population.

Use of 16S rRNA Gene Profiling by Terminal Restriction Fragment Length Polymorphism Analysis To Compare Bacterial Communities in Sputum and Mouthwash Samples from Patients with Cystic Fibrosis

ABSTRACT The bacterial communities present in the oral cavity and the lungs of 19 adult cystic fibrosis (CF) patients were compared by using terminal restriction fragment length polymorphism analysis of 16S rRNA gene PCR products amplified from nucleic acids extracted directly from bacteria in clinical samples. Sputum samples were not found to be subject to profound contamination by oral cavity bacteria. Evidence of colonization of the CF lung by certain oral bacterial species was found.

Clinical measures of disease in adult non-CF bronchiectasis correlate with airway microbiota composition

Rationale Despite the potentially important roles for infection in adult non-cystic fibrosis (CF) bronchiectasis disease progression, the bacterial species present in the lower airways of these patients is poorly characterised. Objectives To provide a comprehensive cross-sectional analysis of bacterial content of lower airway samples from patients with non-CF bronchiectasis using culture-independent microbiology. Methods Paired induced sputum and bronchoalveolar lavage samples, obtained from 41 adult patients with non-CF bronchiectasis, were analysed by 16S ribosomal RNA gene pyrosequencing. Assessment of species distribution and dispersal allowed ‘core’ and ‘satellite’ bacterial populations to be defined for this patient group. Microbiota characteristics correlated with clinical markers of disease. Measurement and main results 140 bacterial species were identified, including those associated with respiratory tract infections and opportunistic infections more generally. A group of core species, consisting of species detected frequently and in high abundance, was defined. Core species included those currently associated with infection in bronchiectasis, such as Pseudomonas aeruginosa, Haemophilus influenzae and Streptococcus pneumoniae, and many species that would be unlikely to be reported through standard diagnostic surveillance. These included members of the genera Veillonella, Prevotella and Neisseria. The comparative contribution of core and satellite groups suggested a low level of random species acquisition. Bacterial diversity was significantly positively correlated with forced expiratory volume in 1 s (FEV1) and bacterial community composition similarity correlated significantly with FEV1, neutrophil count and Leicester cough score. Conclusions Characteristics of the lower airways microbiota of adult patients with non-CF bronchiectasis correlate significantly with clinical markers of disease severity.

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.

Bacterial activity in cystic fibrosis lung infections

BackgroundChronic lung infections are the primary cause of morbidity and mortality in Cystic Fibrosis (CF) patients. Recent molecular biological based studies have identified a surprisingly wide range of hitherto unreported bacterial species in the lungs of CF patients. The aim of this study was to determine whether the species present were active and, as such, worthy of further investigation as potential pathogens.MethodsTerminal Restriction Fragment Length Polymorphism (T-RFLP) profiles were generated from PCR products amplified from 16S rDNA and Reverse Transcription Terminal Restriction Fragment Length Polymorphism (RT-T-RFLP) profiles, a marker of metabolic activity, were generated from PCR products amplified from 16S rRNA, both extracted from the same CF sputum sample. To test the level of activity of these bacteria, T-RFLP profiles were compared to RT-T-RFLP profiles.ResultsSamples from 17 individuals were studied. Parallel analyses identified a total of 706 individual T-RF and RT-T-RF bands in this sample set. 323 bands were detected by T-RFLP and 383 bands were detected by RT-T-RFLP (statistically significant; P ≤ 0.001). For the group as a whole, 145 bands were detected in a T-RFLP profile alone, suggesting metabolically inactive bacteria. 205 bands were detected in an RT-T-RFLP profile alone and 178 bands were detected in both, suggesting a significant degree of metabolic activity. Although Pseudomonas aeruginosa was present and active in many patients, a low occurrence of other species traditionally considered to be key CF pathogens was detected. T-RFLP profiles obtained for induced sputum samples provided by healthy individuals without CF formed a separate cluster indicating a low level of similarity to those from CF patients.ConclusionThese results indicate that a high proportion of the bacterial species detected in the sputum from all of the CF patients in the study are active. The widespread activity of bacterial species in these samples emphasizes the potential importance of these previously unrecognized species within the CF lung.

Risks of population antimicrobial resistance associated with chronic macrolide use for inflammatory airway diseases

Macrolide antibiotics have established efficacy in the management of cystic fibrosis and diffuse panbronchiolitis-uncommon lung diseases with substantial morbidity and the potential for rapid progression to death. Emerging evidence suggests benefits of maintenance macrolide treatment in more indolent respiratory diseases including chronic obstructive pulmonary disease and non-cystic fibrosis bronchiectasis. In view of the greater patient population affected by these disorders (and potential for macrolide use to spread to disorders such as chronic cough), widespread use of macrolides, particularly azithromycin, has the potential to substantially influence antimicrobial resistance rates of a range of respiratory microbes. In this Personal View, I explore theories around population (rather than patient) macrolide resistance, appraise evidence linking macrolide use with development of resistance, and highlight the risks posed by injudicious broadening of their use, particularly of azithromycin. These risks are weighed against the potential benefits of macrolides in less aggressive inflammatory airway disorders. A far-sighted approach to maintenance macrolide use in non-cystic fibrosis inflammatory airway diseases is needed, which minimises risks of adversely affecting community macrolide resistance: combining preferential use of erythromycin and restriction of macrolide use to those patients at greatest risk represents an appropriately cautious management approach.

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.

Determining Cystic Fibrosis-Affected Lung Microbiology: Comparison of Spontaneous and Serially Induced Sputum Samples by Use of Terminal Restriction Fragment Length Polymorphism Profiling

ABSTRACT Sampling of the lower airways of the adult cystic fibrosis (CF) lung has received insufficient detailed consideration, with the importance of sampling strategies for bacteriological outcome not known. Although spontaneously expectorated sputum (SES) samples are often used for diagnostic bacteriological analysis, induced sputum (IS) methods have advantages. This study examined whether significant differences in bacterial content were detected when using a culture-independent, molecular profiling technique to analyze SES or IS samples. Moreover, this work examined what trends relating to bacterial species distributions and reproducibility were found in sequentially induced sputum samples and what implications this has for pathogen detection. Terminal restriction fragment length polymorphism (T-RFLP) analysis was performed on a SES sample and 4 subsequent IS samples taken at 5-min intervals from 10 clinically stable, adult CF patients. This was repeated over 3 sampling days, with variability between samples, induction periods, and sampling days determined. A diverse range of bacterial species, including potentially novel pathogens, was found. No significant difference in bacterial content was observed for either SES or serial IS samples. On average, the analysis of a single sample from any time point resolved ∼58% of total bacterial diversity achieved by analysis of an SES sample and 4 subsequent IS samples. The reliance on analysis of a single respiratory sample was not sufficient for the detection of recognized CF pathogens in all instances. Close correlation between T-RFLP and microbiological data in the detection of key species indicates the importance of these findings in routine diagnostics for the detection of recognized and novel CF pathogens.