Articles
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 Geraint B Rogers, Kenneth D Bruce, Megan L Martin, Lucy D Burr, David J Serisier
Summary 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.
Lancet Respir Med 2014 Published Online October 14, 2014 http://dx.doi.org/10.1016/ S2213-2600(14)70213-9 See Online/Comment http://dx.doi.org/10.1016/ S2213-2600(14)70223-1 Infection and Immunity Theme, South Australia Health and Medical Research Institute, North Terrace, Adelaide, SA, Australia (G B Rogers PhD); School of Medicine, Flinders University, Bedford Park, Adelaide, SA, Australia (G B Rogers); Institute of Pharmaceutical Science, King’s College London, London, UK (K D Bruce PhD); Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, QLD, Australia (M L Martin BN, D J Serisier DM); and Immunity, Infection, and Inflammation Program, Mater Research Institute–University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia (L D Burr MBBS, D J Serisier) Correspondence to: Dr David J Serisier, Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, QLD 4101, Australia [email protected]
Funding Mater Adult Respiratory Research Trust Fund.
Introduction Macrolide antibiotics have been shown to be clinically effective in several chronic respiratory diseases, most recently in non-cystic fibrosis bronchiectasis,1–3 although the mechanisms by which they work are unclear. Researchers believe that the effect is probably antiinflammatory or immunomodulatory,4 but clinical studies have not provided convincing evidence to support this belief. Conversely, clinical data do not support the alternative hypothesis of a traditional antibacterial mechanism for clinical efficacy, and the inherent in-vitro
resistance of Pseudomonas aeruginosa (a common pathogen in chronic airway disease) to macrolide antibiotics5 argues against this explanation. Indeed, data suggest that patients with airway infection by P aeruginosa derive the greatest clinical benefit from macrolide treatment.1,6 However, macrolide antibiotics also represent a substantial selective pressure on microbial communities, evidenced most simply by the emergence of bacterial resistance that results from their use.7,8 The increasingly widespread use of these drugs, particularly azithromycin, therefore poses a risk of inducing substantial
www.thelancet.com/respiratory Published online October 14, 2014 http://dx.doi.org/10.1016/S2213-2600(14)70213-9
1
Articles
See Online for appendix
population-level antibiotic resistance in a range of microorganisms.8,9 Furthermore, exposure to long-term macrolide antibiotics is likely to change the composition of bacterial communities in the airways (and other niches); such shifts in the composition of respiratory microbiota are likely to involve the displacement of macrolidesusceptible organisms by those with inherent tolerance. Such an effect would provide further important direct evidence to link macrolide use with adverse microbiological consequences in individuals with chronic airway disease, strengthening the argument for limiting the long-term prescription of macrolides.10 Furthermore, such an effect will be greatest in respiratory bacterial communities dominated by macrolide-sensitive organisms, which could be relevant to the relatively smaller clinical benefit of macrolides seen in people without P aeruginosa infection.1,6 The results of previous non-randomised studies that have used next-generation sequencing methods have shown profound effects of antibiotic consumption on the composition of human faecal microbiota.11,12 Furthermore, antibiotic-induced shifts in the composition of gut microbiota promote both body fat acquisition13 and severity of asthma14 in animal models. Although results from studies of the respiratory tract have shown links between microbiota composition and disease severity in chronic airway infections,15–20 no randomised studies have been done to investigate the effect of any antibiotic, including macrolides, on the composition of respiratory microbiota, as assessed by deep sequencing. In this analysis from the Bronchiectasis and Low-dose Erythromycin Study (BLESS) study, we aimed to assess whether long-term, low-dose erythromycin treatment changes the composition of respiratory microbiota in people with non-cystic fibrosis bronchiectasis. Additionally, we sought to assess whether this effect was associated with changes in the key clinical outcome measure of pulmonary exacerbations, and whether it might result in changes in airway microbiology that promote the ascendancy of pathogenic species.
Methods Study design and participants BLESS was a 12-month, double-blind, randomised, placebo-controlled trial to compare low-dose erythromycin ethylsuccinate (400 mg twice daily; equivalent to 250 mg twice daily of erythromycin base) in patients with noncystic fibrosis bronchiectasis.1 Adult patients aged 20–85 years with high-resolution CT scan-proven bronchiectasis, at least two separate pulmonary exacerbations requiring supplemental systemic antibiotic treatment in the preceding 12 months, and daily sputum production were eligible. Participants were required to have been clinically stable (defined as no symptoms of exacerbation, no requirement for supplemental antibiotic therapy, and forced expiratory volume in 1 s within 10% of best recently recorded value, if available) for at least 4 weeks before enrolment. Exclusion criteria1 included 2
cystic fibrosis, existing mycobacterial disease or bronchopulmonary aspergillosis, any reversible cause for exacerbations, maintenance oral antibiotic prophylaxis, previous macrolide use apart from short-term use, changes to drug treatment in the preceding 4 weeks, cigarette smoking within 6 months, and drug treatments or comorbidities with the potential for important interactions with erythromycin. All participants were required to have negative sputum mycobacterial cultures before randomisation. Further details of the study design and methods1 are provided in the appendix. The study was approved by the Mater Human Research Ethics Committee and all participants provided written informed consent. The present analysis is based on participants enrolled in BLESS who had adequate, paired sputum samples available from both the first and final visits (ie, baseline and visit 8 [week 48]).
Randomisation and masking Assignment was done with computer-generated randomisation sequences, blocked in random groups of two, four, and eight, and stratified by the presence of sputum P aeruginosa at screening. All participants and study personnel were masked to treatment assignment, including all investigators involved in sample processing and data entry.
Procedures Details of the main BLESS trial methods have been reported elsewhere.1 Spontaneously expectorated sputum used in the present analysis was frozen rapidly and stored at –80°C until molecular analysis. Nucleic acid extractions were done from 200 μL portions of sputum by a combination of physical disruption and a phenol–chloroform-based method, as described previously.16 A detailed extraction protocol is provided in the appendix. We did 16S rRNA gene sequencing to determine the composition of the respiratory microbiota. We used bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) of the 16S V1–V3 region with 16S rRNA gene universal bacterial primers 27F–519R (27F 5ʹ-AGRGTTTGATCMTGGCTCAG, 519R 5ʹ-GTNTTACNGCGGCKGCTG) in a single-step PCR using a HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA). Amplification was done under the following conditions: 94°C for 5 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min. Amplification was followed by a final elongation step at 72°C for 5 min. After PCR, all amplicon products from different samples were mixed in equal concentrations and purified with Agencourt Ampure beads (Agencourt Bioscience, Beverly, MA, USA). We sequenced samples using Roche 454 FLX titanium instruments and reagents (Roche, Branford, CT, USA) in accordance with the manufacturer’s guidelines. Sequences were depleted of barcodes and primers, then short sequences (
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 Geraint B Rogers, Kenneth D Bruce, Megan L Martin, Lucy D Burr, David J Serisier
Summary 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.
Lancet Respir Med 2014 Published Online October 14, 2014 http://dx.doi.org/10.1016/ S2213-2600(14)70213-9 See Online/Comment http://dx.doi.org/10.1016/ S2213-2600(14)70223-1 Infection and Immunity Theme, South Australia Health and Medical Research Institute, North Terrace, Adelaide, SA, Australia (G B Rogers PhD); School of Medicine, Flinders University, Bedford Park, Adelaide, SA, Australia (G B Rogers); Institute of Pharmaceutical Science, King’s College London, London, UK (K D Bruce PhD); Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, QLD, Australia (M L Martin BN, D J Serisier DM); and Immunity, Infection, and Inflammation Program, Mater Research Institute–University of Queensland, Translational Research Institute, Woolloongabba, QLD, Australia (L D Burr MBBS, D J Serisier) Correspondence to: Dr David J Serisier, Department of Respiratory Medicine, Mater Adult Hospital, South Brisbane, QLD 4101, Australia [email protected]
Funding Mater Adult Respiratory Research Trust Fund.
Introduction Macrolide antibiotics have been shown to be clinically effective in several chronic respiratory diseases, most recently in non-cystic fibrosis bronchiectasis,1–3 although the mechanisms by which they work are unclear. Researchers believe that the effect is probably antiinflammatory or immunomodulatory,4 but clinical studies have not provided convincing evidence to support this belief. Conversely, clinical data do not support the alternative hypothesis of a traditional antibacterial mechanism for clinical efficacy, and the inherent in-vitro
resistance of Pseudomonas aeruginosa (a common pathogen in chronic airway disease) to macrolide antibiotics5 argues against this explanation. Indeed, data suggest that patients with airway infection by P aeruginosa derive the greatest clinical benefit from macrolide treatment.1,6 However, macrolide antibiotics also represent a substantial selective pressure on microbial communities, evidenced most simply by the emergence of bacterial resistance that results from their use.7,8 The increasingly widespread use of these drugs, particularly azithromycin, therefore poses a risk of inducing substantial
www.thelancet.com/respiratory Published online October 14, 2014 http://dx.doi.org/10.1016/S2213-2600(14)70213-9
1
Articles
See Online for appendix
population-level antibiotic resistance in a range of microorganisms.8,9 Furthermore, exposure to long-term macrolide antibiotics is likely to change the composition of bacterial communities in the airways (and other niches); such shifts in the composition of respiratory microbiota are likely to involve the displacement of macrolidesusceptible organisms by those with inherent tolerance. Such an effect would provide further important direct evidence to link macrolide use with adverse microbiological consequences in individuals with chronic airway disease, strengthening the argument for limiting the long-term prescription of macrolides.10 Furthermore, such an effect will be greatest in respiratory bacterial communities dominated by macrolide-sensitive organisms, which could be relevant to the relatively smaller clinical benefit of macrolides seen in people without P aeruginosa infection.1,6 The results of previous non-randomised studies that have used next-generation sequencing methods have shown profound effects of antibiotic consumption on the composition of human faecal microbiota.11,12 Furthermore, antibiotic-induced shifts in the composition of gut microbiota promote both body fat acquisition13 and severity of asthma14 in animal models. Although results from studies of the respiratory tract have shown links between microbiota composition and disease severity in chronic airway infections,15–20 no randomised studies have been done to investigate the effect of any antibiotic, including macrolides, on the composition of respiratory microbiota, as assessed by deep sequencing. In this analysis from the Bronchiectasis and Low-dose Erythromycin Study (BLESS) study, we aimed to assess whether long-term, low-dose erythromycin treatment changes the composition of respiratory microbiota in people with non-cystic fibrosis bronchiectasis. Additionally, we sought to assess whether this effect was associated with changes in the key clinical outcome measure of pulmonary exacerbations, and whether it might result in changes in airway microbiology that promote the ascendancy of pathogenic species.
Methods Study design and participants BLESS was a 12-month, double-blind, randomised, placebo-controlled trial to compare low-dose erythromycin ethylsuccinate (400 mg twice daily; equivalent to 250 mg twice daily of erythromycin base) in patients with noncystic fibrosis bronchiectasis.1 Adult patients aged 20–85 years with high-resolution CT scan-proven bronchiectasis, at least two separate pulmonary exacerbations requiring supplemental systemic antibiotic treatment in the preceding 12 months, and daily sputum production were eligible. Participants were required to have been clinically stable (defined as no symptoms of exacerbation, no requirement for supplemental antibiotic therapy, and forced expiratory volume in 1 s within 10% of best recently recorded value, if available) for at least 4 weeks before enrolment. Exclusion criteria1 included 2
cystic fibrosis, existing mycobacterial disease or bronchopulmonary aspergillosis, any reversible cause for exacerbations, maintenance oral antibiotic prophylaxis, previous macrolide use apart from short-term use, changes to drug treatment in the preceding 4 weeks, cigarette smoking within 6 months, and drug treatments or comorbidities with the potential for important interactions with erythromycin. All participants were required to have negative sputum mycobacterial cultures before randomisation. Further details of the study design and methods1 are provided in the appendix. The study was approved by the Mater Human Research Ethics Committee and all participants provided written informed consent. The present analysis is based on participants enrolled in BLESS who had adequate, paired sputum samples available from both the first and final visits (ie, baseline and visit 8 [week 48]).
Randomisation and masking Assignment was done with computer-generated randomisation sequences, blocked in random groups of two, four, and eight, and stratified by the presence of sputum P aeruginosa at screening. All participants and study personnel were masked to treatment assignment, including all investigators involved in sample processing and data entry.
Procedures Details of the main BLESS trial methods have been reported elsewhere.1 Spontaneously expectorated sputum used in the present analysis was frozen rapidly and stored at –80°C until molecular analysis. Nucleic acid extractions were done from 200 μL portions of sputum by a combination of physical disruption and a phenol–chloroform-based method, as described previously.16 A detailed extraction protocol is provided in the appendix. We did 16S rRNA gene sequencing to determine the composition of the respiratory microbiota. We used bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) of the 16S V1–V3 region with 16S rRNA gene universal bacterial primers 27F–519R (27F 5ʹ-AGRGTTTGATCMTGGCTCAG, 519R 5ʹ-GTNTTACNGCGGCKGCTG) in a single-step PCR using a HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA). Amplification was done under the following conditions: 94°C for 5 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min. Amplification was followed by a final elongation step at 72°C for 5 min. After PCR, all amplicon products from different samples were mixed in equal concentrations and purified with Agencourt Ampure beads (Agencourt Bioscience, Beverly, MA, USA). We sequenced samples using Roche 454 FLX titanium instruments and reagents (Roche, Branford, CT, USA) in accordance with the manufacturer’s guidelines. Sequences were depleted of barcodes and primers, then short sequences (
