THOMAS A. NEFF LECTURE Chronic Obstructive Pulmonary Disease and Infection Disruption of the Microbiome? Sanjay Sethi1 1 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, University at Buffalo, State University of New York, Buffalo, New York; and Department of Veterans Affairs Western New York Healthcare System, Buffalo, New York
Abstract The dynamics of infection in chronic obstructive pulmonary disease (COPD) are complex, and microbiome technology has provided us with a new research tool for its better understanding. There is compartmentalization of the microbiota in the various parts of the lung. Studies of the lower airway lumen microbiota in COPD have yielded confusing results, and additional studies with scrupulous attention to prevent and account for upper airway contamination of bronchoalveolar lavage samples are required. Lung tissue microbiota has been examined in three studies, which also demonstrate varied
results based on the site of sampling (bronchial mucosa, lung parenchyma), and this variation extends to sampling sites within a lobe of the lung. The Vicious Circle Hypothesis embodies how an altered lung microbiome could contribute to COPD progression. Relating microbiota composition to airway and systemic inflammation and clinical outcomes are important research questions. Although various obstacles need to be surmounted, ultimately lung microbiome studies will provide new insights into how infection contributes to COPD. Keywords: chronic obstructive pulmonary disease; microbiome; bronchoalveolar lavage; exacerbation
(Received in original form July 1, 2013; accepted in final form November 20, 2013 ) Correspondence and requests for reprints should be addressed to Sanjay Sethi, M.D., VA WNY Healthcare System (151), 3495 Bailey Avenue, Buffalo NY 14215. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S43–S47, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201307-212MG Internet address: www.atsjournals.org
Before the realization that exposure to noxious particles or gases, such as tobacco or biomass smoke, is central to pathogenesis of chronic obstructive pulmonary disease (COPD), infective exacerbations and mucus hypersecretion were believed to be central to the development of COPD. However, studies done in the 1960s and 1970s did not support this hypothesis, and the role of infection was relegated to an epiphenomenon in COPD (1). The contribution of infection to progression of disease, through causing exacerbations and inflammation even during “stable” disease, is being increasingly recognized again (2). Much of this rediscovery of the importance of infection in COPD has been based on culture techniques, molecular epidemiology, and immunological and inflammation studies. The development of microbiome technology has provided us with a new research tool to further Sethi: Microbiome in COPD
understand the complex dynamics of infection in COPD (3, 4).
The “Lung” Microbiome Definition of the lung microbiome is complex. As with many other processes in COPD, there is compartmentalization of the microbiome in the various anatomic locations of the lung. In a study by Cabrera-Rubio and colleagues, sputum, bronchoalveolar lavage (BAL), bronchial biopsies, and bronchial aspirates were obtained in six patients with moderate COPD (5). Microbiota of the BAL and bronchial biopsies clustered together, but those of bronchial aspirates and sputum were different and distinct from each other (Figure 1). Erb-Downward and colleagues examined lung tissue samples that were centered around large and medium-sized
airways from lung explants of patients undergoing transplant for very severe COPD (6). In these samples, an abundant microbiome was seen, dominated by Pseudomonas spp., likely reflecting the site of sampling and the very severe COPD in these patients. Interestingly, they also found heterogeneity in the microbiota within and between lobes of the lung in a single subject. In contrast to the abundant microbiomes in the upper and lower tracheobronchial tree, when Sze and colleagues sampled lung parenchymal tissue from patients with very severe COPD, they demonstrated only a sparse microbiome. Although not more abundant, the composition of this tissue microbiome is distinct from that seen in control lung tissue (7). This compartmentalization of the lung microbiome implies that the compartment being sampled should be carefully selected on the basis of the S43
THOMAS A. NEFF LECTURE research question. Feasibility considerations could also limit which compartment is sampled. Obtaining repeated samples or samples from patients with underlying severe COPD could limit the choice to noninvasive methods, such as sputum collection.
Microbiome Alterations in COPD
Figure 1. Top panel: Taxonomic assignments of the 24 respiratory tract samples obtained from six patients at the level of bacterial classes. BAL = bronchoalveolar lavage; Bas = bronchial aspirate; Sp = sputum; Ts = tissue. Bottom panel: Principal component analysis (PCoA) of the microbiomes of the four respiratory sample types and oral samples (supragingival dental plaques). Bronchial mucosa and lavage samples cluster together. Sputum and bronchial aspirate samples differ from the bronchial mucosa and lavage samples and from each other. Reprinted by permission from Reference 5.
S44
Changes in the microbial milieu in the lower airways in COPD have been investigated by conventional microbiology and microbiome techniques in samples obtained by BAL or protected specimen brushing (PSB). Soler and colleagues sampled patients with mild, moderate, and severe COPD as well as control smokers without airflow obstruction by PSB and BAL and determined the presence of potential pathogenic bacteria (PPB) by quantitative cultures (8). Significant concentrations of PPB were found in one-third of the patients with COPD. However, among the “healthy” smokers, 50% had PPB in the lower airways. This study demonstrated that the microbial flora of the lower airways of patients with COPD is often colonized with PPB. Furthermore, this process of colonization appears to start early in the disease, with even the healthy smokers demonstrating significant PPB presence. The absence of a nonsmoking control group to verify the significance of their observation was a limitation of this study. To address this issue, we undertook a bronchoscopic sampling study of the distal airways in ex-smokers with COPD (9). Two sets of control subjects were included: one was healthy nonsmokers and the other was ex-smokers with normal lung function and no respiratory symptoms. BAL for the right middle lobe was obtained in all the participants for quantitative cultures, with greater than 100 cfu/ml of PPB regarded as significant growth. As contamination by the upper airway flora of BAL samples can be a major confounder, a two-scope technique was used. The first scope was used to instill lidocaine for local anesthesia up to the glottis. The second scope was then introduced; its channel was not used for suctioning or instillation of lidocaine, and BAL was obtained as described above. In this study, 35% of the 19 patients with COPD had significant PPB growth from their BAL, and none (0%) of the 20
AnnalsATS Volume 11 Supplement 1 | January 2014
THOMAS A. NEFF LECTURE ex-smokers without COPD and 1 (7%) of the 15 healthy nonsmokers had significant PPB in their BAL. This study clearly demonstrated that in COPD, despite the discontinuation of smoking, the microbiome is changed. Recent studies have used microbiome techniques to determine if smoking and the development of COPD alter the microbiota of distal airways using BAL samples. Morris and colleagues compared 45 healthy nonsmokers with 19 smokers with normal spirometry (10). Surprisingly, they did not find differences in total lower airway flora estimated by 16s quantification and found very minor differences in the operational taxonomic units between the two groups. Erb-Downward did a similar but smaller study comparing patients with COPD, healthy smokers, and nonsmokers and found no differences in the microbial flora of the three groups (6). Pragman and colleagues used stored BAL samples from control subjects and subjects with moderate and severe COPD to compare microbiota. An intriguing observation in that study was that microbiota of patients who were on inhaled corticosteroids and bronchodilators appeared to differ from patients not on those medications in the principal components analysis (11). What can explain these contradictory findings of the studies with conventional microbiology and with microbiome techniques? It maybe that the microbiota of the COPD airway is indeed not different between healthy individuals, smokers, and patients with COPD. This is unlikely given the findings of the studies using conventional microbiology and the relatively sparse microbiome described in other studies by Charlson and colleagues (12). Another possibility is that the differences in PPB are difficult to discern when the whole microbiome is explored (i.e., the trees are being lost when we look at the forest). The most likely explanation for the contradictory findings in the studies is the measures taken to avoid upper airway contamination of BAL samples. Although in the conventional microbiology studies PSB and/or the two-scope technique were used, the two microbiome studies did not use such measures. Charlson and colleagues in an elegant study have shown that the upper airway contains several logs of magnitude more operational taxonomic units than the lower airway in healthy individuals (12). Therefore, even a minimal Sethi: Microbiome in COPD
amount of upper airway contamination of the lower airway BAL samples would significantly obscure differences in lower airway microbiota. Additional studies of the lower airway microbiome in COPD with scrupulous attention to prevent and account for upper airway contamination of BAL samples are required. The lung parenchymal microbiome in COPD has been examined in a small study by Sze and colleagues (7). Lung tissue from patients with Global Initiative for Chronic Obstructive Lung Disease stage 4 COPD was compared with samples obtained from healthy control subjects and subjects with cystic fibrosis. Quantitatively, the microbiome was quite sparse in these tissue core samples and was in fact not different between the healthy control subjects and those with COPD, whereas samples from patients with cystic fibrosis demonstrated a much greater microbial abundance. However, when the community composition was compared, differences were found between the groups. The pathogenic significance of this changed but sparse microbiome in COPD is undetermined. The tissue cores studied contained much more alveolar tissue than small airways; therefore, these data predominantly represent the alveolar microbiome rather than the small airway microbiome. Whether the small airway microbiome differs in COPD will need additional studies, which will be challenging.
Disruption of the Lung Microbiome and Progression of COPD Although it is clear that noxious fume and particle exposure is the predominant driving force for disease progression in COPD, other factors undoubtedly contribute, including genetic predisposition, nutritional status, and respiratory infections. The possible contribution of an altered microbiome to COPD progression is embodied in the Vicious Circle Hypothesis (Figure 2) (2). Once innate lung defense mechanisms are perturbed by smoke exposure, the microbiome in the airway is disrupted, likely becoming much more abundant (9, 13). This unhealthy microbiome is able to drive the inflammatory process in COPD, which by worsening the protease–antiprotease balance in the lung causes progressive airflow obstruction through airway and
possibly parenchymal damage. Acute exacerbations of COPD represent abrupt major changes in the microbiome that result in large increases in airway and systemic inflammation, which leads to an increase in respiratory symptoms. Direct evidence to support this hypothesis comes from studies with conventional microbiology, which have found increased inflammatory cells, cytokines/chemokines, and proteases, with colonization in airway samples obtained by bronchoscopy or in sputum (8, 9, 14). Indirect evidence to support this hypothesis comes from pathological studies that have shown development of inflammatory mucus exudates and lymphoid follicles in the small airways in COPD lungs (15). These pathological changes are consistent with chronic infection. Advances in lung imaging have also revealed that bronchiectatic changes in the airways are prevalent in COPD (16). Furthermore, the presence of bronchiectasis is associated with higher incidence of airway bacterial colonization by sputum culture and worse clinical outcomes, including mortality (17). Microbiome studies could make significant advances in furthering our understanding of microbial contribution to the pathogenesis and progression of COPD. Whether differences in microbiota in the airway in COPD determine clinical expression of the disease (e.g., the presence of chronic bronchitis) could be explored. Microbiota composition relatedness to airway and systemic inflammation and, in longitudinal studies, to outcomes such as changes in lung function and health status, are important research questions.
Exacerbations of COPD and Lung Microbiome Exacerbations of COPD are major contributors to the morbidity and mortality of this disease. Because bacterial pathogens are often recovered from airway secretions in COPD during stable disease, it was unclear if they played a role in exacerbations or were innocent bystanders. Application of molecular differentiation among strains of bacteria was required to discover that exacerbations of COPD were linked to acquisition of new strains of PPB from the environment (18). When these infecting strains of bacteria were used as antigens, specific host responses could be discerned S45
THOMAS A. NEFF LECTURE
Figure 2. The Vicious Circle Hypothesis. COPD = chronic obstructive pulmonary disease. Reprinted by permission from Reference 2.
(19). Although much has been learned by conventional microbiology about microbial causation of exacerbations, the limits of that technology are becoming clear. When a molecular detection technique such as polymerase chain reaction is applied simultaneously with culture to sputum samples from COPD, the yield of PPB is doubled (20). Even in well-characterized clinically bacterial exacerbations based on sputum purulence, only half yield a PPB (21–23). Microbiome studies could provide exciting new observations in this scenario, which could lead to discovery of new pathogens that have been difficult to obtain on culture. Furthermore, it appears that bacterial species often associate with each other, and whether and how that happens during exacerbations of COPD could be described.
Challenges in Studying the Lung Microbiome Although the technology is here, and several studies have been published, there are several challenges that need to be tackled to fully use the benefits of microbiome research in COPD. Paramount among those is the issue of upper airway and environmental contamination that was alluded to earlier. This has been a challenge for conventional microbiology, but is magnified several fold because of the sensitivity of microbiome techniques to detect minute amounts of S46
bacteria. Furthermore, because the very nature of the microbiome approach is to avoid observer bias and be minimally biased in its selection of data, confounding of data by contamination becomes an even bigger problem. With conventional microbiology, upper airway and environmental contamination were dealt with by scrupulous techniques, identifying certain bacteria as pathogens, and using quantitative thresholds to define significant infection (8, 9, 21). Scrupulous techniques should be applied to all microbiome studies. However, identifying certain bacteria as pathogens and ignoring the rest defeats the very purpose of the microbiome approach. Most microbiome data provide relative rather than absolute quantities of pathogens in the lung. Therefore, defining significant thresholds in microbiome data is currently difficult. Clearly, further refinement of analysis of microbiome data is required to deal with these challenges. Adjusting BAL data for a standard oral microbiome has been done, but as many of the microbiota are actually shared between the two compartments, significant information from the BAL would be lost with that adjustment (10). Obtaining reliable microbiome data is only the first step, as interpretation of the findings is crucial. Which of the various microbes identified are contributing to the disease? The identification of possible pathogens in the microbiome data is often only to the level of the genus at best.
However, this may not provide enough granularity to distinguish pathogens from nonpathogens. As an example, identification of Haemophilus spp. is commonplace in respiratory tract secretions. Within this genus, H. influenzae has been documented as a pathogen, whereas H. haemolyticus and H. parainfluenzae behave as commensals (24). Furthermore, within a pathogenic species, strains can differ widely in their virulence, and that can translate to differences in clinical behavior (25).
Conclusions Traditional microbiology has clear limitations when it comes to uncultivable bacteria and its insensitivity to lowabundance bacteria. Despite the various obstacles to be surmounted, study of the lung microbiome in COPD could answer several important questions. It would be fascinating to find out whether different microbiomes exist in the different phenotypes of this complex disease. Are exacerbations that are clinically bacterial but do not have a predominant pathogen on culture induced by microbiome alterations? Does a virus infection alter the microbiome in the lung, and what are the clinical consequences? Therapeutics that address the microbiome could be developed. n Author disclosures are available with the text of this article at www.atsjournals.org.
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References 1 Tager I, Speizer FE. Role of infection in chronic bronchitis. N Engl J Med 1975;292:563–571. 2 Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359: 2355–2365. 3 Han MK, Huang YJ, Lipuma JJ, Boushey HA, Boucher RC, Cookson WO, Curtis JL, Erb-Downward J, Lynch SV, Sethi S, et al. Significance of the microbiome in obstructive lung disease. Thorax 2012;67:456–463. 4 Huang YJ, Charlson ES, Collman RG, Colombini-Hatch S, Martinez FD, Senior RM. The role of the lung microbiome in health and disease. A National Heart, Lung, and Blood Institute workshop report. Am J Respir Crit Care Med 2013;187:1382–1387. 5 Cabrera-Rubio R, Garcia-Nuñez ´ M, Seto´ L, Anto´ JM, Moya A, Monso´ E, Mira A. Microbiome diversity in the bronchial tracts of patients with chronic obstructive pulmonary disease. J Clin Microbiol 2012;50:3562–3568. 6 Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Schmidt LA, Young VB, Toews GB, Curtis JL, Sundaram B, et al. Analysis of the lung microbiome in the “healthy” smoker and in COPD. PLoS ONE 2011;6:e16384. 7 Sze MA, Dimitriu PA, Hayashi S, Elliott WM, McDonough JE, Gosselink JV, Cooper J, Sin DD, Mohn WW, Hogg JC. The lung tissue microbiome in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2012;185:1073–1080. 8 Soler N, Ewig S, Torres A, Filella X, Gonzalez J, Zaubet A. Airway inflammation and bronchial microbial patterns in patients with stable chronic obstructive pulmonary disease. Eur Respir J 1999;14:1015–1022. 9 Sethi S, Maloney J, Grove L, Wrona C, Berenson CS. Airway inflammation and bronchial bacterial colonization in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2006;173:991–998. 10 Morris A, Beck JM, Schloss PD, Campbell TB, Crothers K, Curtis JL, Flores SC, Fontenot AP, Ghedin E, Huang L, et al.; Lung HIV Microbiome Project. Comparison of the respiratory microbiome in healthy nonsmokers and smokers. Am J Respir Crit Care Med 2013; 187:1067–1075. 11 Pragman AA, Kim HB, Reilly CS, Wendt C, Isaacson RE. The lung microbiome in moderate and severe chronic obstructive pulmonary disease. PLoS ONE 2012;7:e47305. 12 Charlson ES, Bittinger K, Haas AR, Fitzgerald AS, Frank I, Yadav A, Bushman FD, Collman RG. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am J Respir Crit Care Med 2011;184:957–963. 13 Soler N, Ewig S, Ferrer M, Filella X, Xaubet A, Torres A. Airway inflammation and bronchial colonisation in stable COPD patients [abstract]. Am J Respir Crit Care Med 1999;159:A799.
Sethi: Microbiome in COPD
14 Banerjee D, Khair OA, Honeybourne D. Impact of sputum bacteria on airway inflammation and health status in clinical stable COPD. Eur Respir J 2004;23:685–691. 15 Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, Cherniack RM, Rogers RM, Sciurba FC, Coxson HO, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 2004;350:2645–2653. 16 Martinez-Garcia MA, Soler-Cataluna JJ, Donat-Sanz Y, Catalan-Serra P, Agramunt-Lerma M, Ballestin-Vicente J, Perpina-Tordera M. Factors associated with bronchiectasis in chronic obstructive pulmonary disease patients. Chest 2011;40:1130–1137. 17 Mart´ınez-Garc´ıa MA, de la Rosa Carrillo D, Soler-Cataluña JJ, DonatSanz Y, Serra PC, Lerma MA, Ballest´ın J, Sanchez ´ IV, Selma Ferrer MJ, Dalfo AR, et al. Prognostic value of bronchiectasis in patients with moderate-to-severe chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2013;187:823–831. 18 Sethi S, Evans N, Grant BJ, Murphy TF. New strains of bacteria and exacerbations of chronic obstructive pulmonary disease. N Engl J Med 2002;347:465–471. 19 Sethi S, Wrona C, Grant BJB, Murphy TF. Strain-specific immune response to Haemophilus influenzae in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2004;169:448–453. 20 Garcha DS, Thurston SJ, Patel AR, Mackay AJ, Goldring JJ, Donaldson GC, McHugh TD, Wedzicha JA. Changes in prevalence and load of airway bacteria using quantitative PCR in stable and exacerbated COPD. Thorax 2012;67:1075–1080. 21 Soler N, Agust´ı C, Angrill J, Puig De la Bellacasa J, Torres A. Bronchoscopic validation of the significance of sputum purulence in severe exacerbations of chronic obstructive pulmonary disease. Thorax 2007;62:29–35. 22 Wilson R, Anzueto A, Miravitlles M, Arvis P, Alder J, Haverstock D, Trajanovic M, Sethi S. Moxifloxacin vs amoxicillin/clavulanic acid in outpatient acute exacerbations of COPD: MAESTRAL results. Eur Respir J 2012;40:17–27. 23 Stockley RA, O’Brien C, Pye A, Hill SL. Relationship of sputum color to nature and outpatient management of acute exacerbations of COPD. Chest 2000;117:1638–1645. 24 Murphy TF, Brauer AL, Sethi S, Kilian M, Cai X, Lesse AJ. Haemophilus haemolyticus: a human respiratory tract commensal to be distinguished from Haemophilus influenzae. J Infect Dis 2007;195: 81–89. 25 Chin CL, Manzel LJ, Lehman EE, Humlicek AL, Shi L, Starner TD, Denning GM, Murphy TF, Sethi S, Look DC. Haemophilus influenzae from patients with chronic obstructive pulmonary disease exacerbation induce more inflammation than colonizers. Am J Respir Crit Care Med 2005;172:85–91.
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Abstract The dynamics of infection in chronic obstructive pulmonary disease (COPD) are complex, and microbiome technology has provided us with a new research tool for its better understanding. There is compartmentalization of the microbiota in the various parts of the lung. Studies of the lower airway lumen microbiota in COPD have yielded confusing results, and additional studies with scrupulous attention to prevent and account for upper airway contamination of bronchoalveolar lavage samples are required. Lung tissue microbiota has been examined in three studies, which also demonstrate varied
results based on the site of sampling (bronchial mucosa, lung parenchyma), and this variation extends to sampling sites within a lobe of the lung. The Vicious Circle Hypothesis embodies how an altered lung microbiome could contribute to COPD progression. Relating microbiota composition to airway and systemic inflammation and clinical outcomes are important research questions. Although various obstacles need to be surmounted, ultimately lung microbiome studies will provide new insights into how infection contributes to COPD. Keywords: chronic obstructive pulmonary disease; microbiome; bronchoalveolar lavage; exacerbation
(Received in original form July 1, 2013; accepted in final form November 20, 2013 ) Correspondence and requests for reprints should be addressed to Sanjay Sethi, M.D., VA WNY Healthcare System (151), 3495 Bailey Avenue, Buffalo NY 14215. E-mail: [email protected] Ann Am Thorac Soc Vol 11, Supplement 1, pp S43–S47, Jan 2014 Copyright © 2014 by the American Thoracic Society DOI: 10.1513/AnnalsATS.201307-212MG Internet address: www.atsjournals.org
Before the realization that exposure to noxious particles or gases, such as tobacco or biomass smoke, is central to pathogenesis of chronic obstructive pulmonary disease (COPD), infective exacerbations and mucus hypersecretion were believed to be central to the development of COPD. However, studies done in the 1960s and 1970s did not support this hypothesis, and the role of infection was relegated to an epiphenomenon in COPD (1). The contribution of infection to progression of disease, through causing exacerbations and inflammation even during “stable” disease, is being increasingly recognized again (2). Much of this rediscovery of the importance of infection in COPD has been based on culture techniques, molecular epidemiology, and immunological and inflammation studies. The development of microbiome technology has provided us with a new research tool to further Sethi: Microbiome in COPD
understand the complex dynamics of infection in COPD (3, 4).
The “Lung” Microbiome Definition of the lung microbiome is complex. As with many other processes in COPD, there is compartmentalization of the microbiome in the various anatomic locations of the lung. In a study by Cabrera-Rubio and colleagues, sputum, bronchoalveolar lavage (BAL), bronchial biopsies, and bronchial aspirates were obtained in six patients with moderate COPD (5). Microbiota of the BAL and bronchial biopsies clustered together, but those of bronchial aspirates and sputum were different and distinct from each other (Figure 1). Erb-Downward and colleagues examined lung tissue samples that were centered around large and medium-sized
airways from lung explants of patients undergoing transplant for very severe COPD (6). In these samples, an abundant microbiome was seen, dominated by Pseudomonas spp., likely reflecting the site of sampling and the very severe COPD in these patients. Interestingly, they also found heterogeneity in the microbiota within and between lobes of the lung in a single subject. In contrast to the abundant microbiomes in the upper and lower tracheobronchial tree, when Sze and colleagues sampled lung parenchymal tissue from patients with very severe COPD, they demonstrated only a sparse microbiome. Although not more abundant, the composition of this tissue microbiome is distinct from that seen in control lung tissue (7). This compartmentalization of the lung microbiome implies that the compartment being sampled should be carefully selected on the basis of the S43
THOMAS A. NEFF LECTURE research question. Feasibility considerations could also limit which compartment is sampled. Obtaining repeated samples or samples from patients with underlying severe COPD could limit the choice to noninvasive methods, such as sputum collection.
Microbiome Alterations in COPD
Figure 1. Top panel: Taxonomic assignments of the 24 respiratory tract samples obtained from six patients at the level of bacterial classes. BAL = bronchoalveolar lavage; Bas = bronchial aspirate; Sp = sputum; Ts = tissue. Bottom panel: Principal component analysis (PCoA) of the microbiomes of the four respiratory sample types and oral samples (supragingival dental plaques). Bronchial mucosa and lavage samples cluster together. Sputum and bronchial aspirate samples differ from the bronchial mucosa and lavage samples and from each other. Reprinted by permission from Reference 5.
S44
Changes in the microbial milieu in the lower airways in COPD have been investigated by conventional microbiology and microbiome techniques in samples obtained by BAL or protected specimen brushing (PSB). Soler and colleagues sampled patients with mild, moderate, and severe COPD as well as control smokers without airflow obstruction by PSB and BAL and determined the presence of potential pathogenic bacteria (PPB) by quantitative cultures (8). Significant concentrations of PPB were found in one-third of the patients with COPD. However, among the “healthy” smokers, 50% had PPB in the lower airways. This study demonstrated that the microbial flora of the lower airways of patients with COPD is often colonized with PPB. Furthermore, this process of colonization appears to start early in the disease, with even the healthy smokers demonstrating significant PPB presence. The absence of a nonsmoking control group to verify the significance of their observation was a limitation of this study. To address this issue, we undertook a bronchoscopic sampling study of the distal airways in ex-smokers with COPD (9). Two sets of control subjects were included: one was healthy nonsmokers and the other was ex-smokers with normal lung function and no respiratory symptoms. BAL for the right middle lobe was obtained in all the participants for quantitative cultures, with greater than 100 cfu/ml of PPB regarded as significant growth. As contamination by the upper airway flora of BAL samples can be a major confounder, a two-scope technique was used. The first scope was used to instill lidocaine for local anesthesia up to the glottis. The second scope was then introduced; its channel was not used for suctioning or instillation of lidocaine, and BAL was obtained as described above. In this study, 35% of the 19 patients with COPD had significant PPB growth from their BAL, and none (0%) of the 20
AnnalsATS Volume 11 Supplement 1 | January 2014
THOMAS A. NEFF LECTURE ex-smokers without COPD and 1 (7%) of the 15 healthy nonsmokers had significant PPB in their BAL. This study clearly demonstrated that in COPD, despite the discontinuation of smoking, the microbiome is changed. Recent studies have used microbiome techniques to determine if smoking and the development of COPD alter the microbiota of distal airways using BAL samples. Morris and colleagues compared 45 healthy nonsmokers with 19 smokers with normal spirometry (10). Surprisingly, they did not find differences in total lower airway flora estimated by 16s quantification and found very minor differences in the operational taxonomic units between the two groups. Erb-Downward did a similar but smaller study comparing patients with COPD, healthy smokers, and nonsmokers and found no differences in the microbial flora of the three groups (6). Pragman and colleagues used stored BAL samples from control subjects and subjects with moderate and severe COPD to compare microbiota. An intriguing observation in that study was that microbiota of patients who were on inhaled corticosteroids and bronchodilators appeared to differ from patients not on those medications in the principal components analysis (11). What can explain these contradictory findings of the studies with conventional microbiology and with microbiome techniques? It maybe that the microbiota of the COPD airway is indeed not different between healthy individuals, smokers, and patients with COPD. This is unlikely given the findings of the studies using conventional microbiology and the relatively sparse microbiome described in other studies by Charlson and colleagues (12). Another possibility is that the differences in PPB are difficult to discern when the whole microbiome is explored (i.e., the trees are being lost when we look at the forest). The most likely explanation for the contradictory findings in the studies is the measures taken to avoid upper airway contamination of BAL samples. Although in the conventional microbiology studies PSB and/or the two-scope technique were used, the two microbiome studies did not use such measures. Charlson and colleagues in an elegant study have shown that the upper airway contains several logs of magnitude more operational taxonomic units than the lower airway in healthy individuals (12). Therefore, even a minimal Sethi: Microbiome in COPD
amount of upper airway contamination of the lower airway BAL samples would significantly obscure differences in lower airway microbiota. Additional studies of the lower airway microbiome in COPD with scrupulous attention to prevent and account for upper airway contamination of BAL samples are required. The lung parenchymal microbiome in COPD has been examined in a small study by Sze and colleagues (7). Lung tissue from patients with Global Initiative for Chronic Obstructive Lung Disease stage 4 COPD was compared with samples obtained from healthy control subjects and subjects with cystic fibrosis. Quantitatively, the microbiome was quite sparse in these tissue core samples and was in fact not different between the healthy control subjects and those with COPD, whereas samples from patients with cystic fibrosis demonstrated a much greater microbial abundance. However, when the community composition was compared, differences were found between the groups. The pathogenic significance of this changed but sparse microbiome in COPD is undetermined. The tissue cores studied contained much more alveolar tissue than small airways; therefore, these data predominantly represent the alveolar microbiome rather than the small airway microbiome. Whether the small airway microbiome differs in COPD will need additional studies, which will be challenging.
Disruption of the Lung Microbiome and Progression of COPD Although it is clear that noxious fume and particle exposure is the predominant driving force for disease progression in COPD, other factors undoubtedly contribute, including genetic predisposition, nutritional status, and respiratory infections. The possible contribution of an altered microbiome to COPD progression is embodied in the Vicious Circle Hypothesis (Figure 2) (2). Once innate lung defense mechanisms are perturbed by smoke exposure, the microbiome in the airway is disrupted, likely becoming much more abundant (9, 13). This unhealthy microbiome is able to drive the inflammatory process in COPD, which by worsening the protease–antiprotease balance in the lung causes progressive airflow obstruction through airway and
possibly parenchymal damage. Acute exacerbations of COPD represent abrupt major changes in the microbiome that result in large increases in airway and systemic inflammation, which leads to an increase in respiratory symptoms. Direct evidence to support this hypothesis comes from studies with conventional microbiology, which have found increased inflammatory cells, cytokines/chemokines, and proteases, with colonization in airway samples obtained by bronchoscopy or in sputum (8, 9, 14). Indirect evidence to support this hypothesis comes from pathological studies that have shown development of inflammatory mucus exudates and lymphoid follicles in the small airways in COPD lungs (15). These pathological changes are consistent with chronic infection. Advances in lung imaging have also revealed that bronchiectatic changes in the airways are prevalent in COPD (16). Furthermore, the presence of bronchiectasis is associated with higher incidence of airway bacterial colonization by sputum culture and worse clinical outcomes, including mortality (17). Microbiome studies could make significant advances in furthering our understanding of microbial contribution to the pathogenesis and progression of COPD. Whether differences in microbiota in the airway in COPD determine clinical expression of the disease (e.g., the presence of chronic bronchitis) could be explored. Microbiota composition relatedness to airway and systemic inflammation and, in longitudinal studies, to outcomes such as changes in lung function and health status, are important research questions.
Exacerbations of COPD and Lung Microbiome Exacerbations of COPD are major contributors to the morbidity and mortality of this disease. Because bacterial pathogens are often recovered from airway secretions in COPD during stable disease, it was unclear if they played a role in exacerbations or were innocent bystanders. Application of molecular differentiation among strains of bacteria was required to discover that exacerbations of COPD were linked to acquisition of new strains of PPB from the environment (18). When these infecting strains of bacteria were used as antigens, specific host responses could be discerned S45
THOMAS A. NEFF LECTURE
Figure 2. The Vicious Circle Hypothesis. COPD = chronic obstructive pulmonary disease. Reprinted by permission from Reference 2.
(19). Although much has been learned by conventional microbiology about microbial causation of exacerbations, the limits of that technology are becoming clear. When a molecular detection technique such as polymerase chain reaction is applied simultaneously with culture to sputum samples from COPD, the yield of PPB is doubled (20). Even in well-characterized clinically bacterial exacerbations based on sputum purulence, only half yield a PPB (21–23). Microbiome studies could provide exciting new observations in this scenario, which could lead to discovery of new pathogens that have been difficult to obtain on culture. Furthermore, it appears that bacterial species often associate with each other, and whether and how that happens during exacerbations of COPD could be described.
Challenges in Studying the Lung Microbiome Although the technology is here, and several studies have been published, there are several challenges that need to be tackled to fully use the benefits of microbiome research in COPD. Paramount among those is the issue of upper airway and environmental contamination that was alluded to earlier. This has been a challenge for conventional microbiology, but is magnified several fold because of the sensitivity of microbiome techniques to detect minute amounts of S46
bacteria. Furthermore, because the very nature of the microbiome approach is to avoid observer bias and be minimally biased in its selection of data, confounding of data by contamination becomes an even bigger problem. With conventional microbiology, upper airway and environmental contamination were dealt with by scrupulous techniques, identifying certain bacteria as pathogens, and using quantitative thresholds to define significant infection (8, 9, 21). Scrupulous techniques should be applied to all microbiome studies. However, identifying certain bacteria as pathogens and ignoring the rest defeats the very purpose of the microbiome approach. Most microbiome data provide relative rather than absolute quantities of pathogens in the lung. Therefore, defining significant thresholds in microbiome data is currently difficult. Clearly, further refinement of analysis of microbiome data is required to deal with these challenges. Adjusting BAL data for a standard oral microbiome has been done, but as many of the microbiota are actually shared between the two compartments, significant information from the BAL would be lost with that adjustment (10). Obtaining reliable microbiome data is only the first step, as interpretation of the findings is crucial. Which of the various microbes identified are contributing to the disease? The identification of possible pathogens in the microbiome data is often only to the level of the genus at best.
However, this may not provide enough granularity to distinguish pathogens from nonpathogens. As an example, identification of Haemophilus spp. is commonplace in respiratory tract secretions. Within this genus, H. influenzae has been documented as a pathogen, whereas H. haemolyticus and H. parainfluenzae behave as commensals (24). Furthermore, within a pathogenic species, strains can differ widely in their virulence, and that can translate to differences in clinical behavior (25).
Conclusions Traditional microbiology has clear limitations when it comes to uncultivable bacteria and its insensitivity to lowabundance bacteria. Despite the various obstacles to be surmounted, study of the lung microbiome in COPD could answer several important questions. It would be fascinating to find out whether different microbiomes exist in the different phenotypes of this complex disease. Are exacerbations that are clinically bacterial but do not have a predominant pathogen on culture induced by microbiome alterations? Does a virus infection alter the microbiome in the lung, and what are the clinical consequences? Therapeutics that address the microbiome could be developed. n Author disclosures are available with the text of this article at www.atsjournals.org.
AnnalsATS Volume 11 Supplement 1 | January 2014
THOMAS A. NEFF LECTURE
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