Abstract Although culture-independent techniques have shown that the lungs are not sterile, little is known about the lung microbiome in chronic obstructive pulmonary disease (COPD). We used pyrosequencing of 16S amplicons to analyze the lung microbiome in two ways: first, using bronchoalveolar lavage (BAL) to sample the distal bronchi and air-spaces; and second, by examining multiple discrete tissue sites in the lungs of six subjects removed at the time of transplantation. We performed BAL on three never-smokers (NS) with normal spirometry, seven smokers with normal spirometry (“heathy smokers”, HS), and four subjects with COPD (CS). Bacterial 16 s sequences were found in all subjects, without significant quantitative differences between groups. Both taxonomy-based and taxonomy-independent approaches disclosed heterogeneity in the bacterial communities between HS subjects that was similar to that seen in healthy NS and two mild COPD patients. The moderate and severe COPD patients had very limited community diversity, which was also noted in 28% of the healthy subjects. Both approaches revealed extensive membership overlap between the bacterial communities of the three study groups. No genera were common within a group but unique across groups. Our data suggests the existence of a core pulmonary bacterial microbiome that includes Pseudomonas, Streptococcus, Prevotella, Fusobacterium, Haemophilus, Veillonella, and Porphyromonas. Most strikingly, there were significant micro-anatomic differences in bacterial communities within the same lung of subjects with advanced COPD. These studies are further demonstration of the pulmonary microbiome and highlight global and micro-anatomic changes in these bacterial communities in severe COPD patients.

Citation: Erb-Downward JR, Thompson DL, Han MK, Freeman CM, McCloskey L, Schmidt LA, et al. (2011) Analysis of the Lung Microbiome in the “Healthy” Smoker and in COPD. PLoS ONE 6(2): e16384. https://doi.org/10.1371/journal.pone.0016384 Editor: Stefan Bereswill, Charité-University Medicine Berlin, Germany Received: October 7, 2010; Accepted: December 14, 2010; Published: February 22, 2011 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: These investigations were supported by Department of Internal Medicine Pilot Grant U020559; the Michigan Institute for Clinical and Health Research, Grant UL1 RR024986; National Heart, Lung and Blood Institute grant R01 HL082480; National Institute of Diabetes and Digestive and Kidney Diseases R01 grant R01 DK070875; T32 HL07749; and by a Research Enhancement Award Program and a Career Development Award (C.M.F.) from the Biomedical Laboratory Research and Development Service, Department of Veterans Affairs. Partial support also came from by the Tissue Procurement Core of the University of Michigan Comprehensive Cancer Center, Grant P30 CA46952, and by the Lung Tissue Research Consortium (Clinical Centers), Grant N01 HR046162. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Chronic obstructive pulmonary disease (COPD) is a progressive and potentially fatal lung disease that is projected to be responsible for the fifth largest burden of disease worldwide by 2020 [1], [2]. COPD is characterized by largely irreversible airflow limitation, mucus hypersecretion, small airway fibrosis, and destruction of the alveolar space (emphysema) [3]. In developed nations, the leading cause of COPD is tobacco smoke exposure, predominately direct, whereas in developing nations indoor air pollution from combustion of biomass fuel also contributes significantly [4]. Not all smokers develop COPD, but why some do is not currently known. Because no current treatments halt COPD progression, new insights into its pathogenesis are urgently needed. From the initial description of COPD as a distinct clinical condition responsible for productive cough and shortness of breath in patients without tuberculosis [5], there has been considerable controversy about the role of lower respiratory tract bacteria in its pathogenesis. This is the case both for its prolonged early asymptomatic phase and, until recently, for the acute exacerbations that punctuate its later stages [6], which can induce accelerated and sustained loss of lung function [7]. In part, this controversy arose because classical, culture-based studies suggested that the lungs of healthy individuals were sterile [8], [9], [10], while the lungs of COPD patients were believed to be colonized. More recently, culture-independent microbiological techniques demonstrated that the lungs are not sterile during health and documented changes in the lung microbiome in several lung diseases [11], [12], [13], [14], [15]. Nevertheless, the role of the lung bacterial microbiome in COPD pathogenesis and progression remains undefined. Our two objectives addressed two gaps in the understanding of the pulmonary microbiome as related to smoking and COPD. The first was to assess the lung microbiome in smokers with neither signs of disease nor decreased lung function (“healthy” smokers) through analysis of bronchoalveolar lavage (BAL) fluid, which samples a broad region of lungs, and compare this to healthy non-smokers. The second objective was to determine whether pulmonary microanatomic/microenvironmental disparities lead to differences in the structure of localized bacterial communities in COPD, through analysis of multiple sample sites from surgical explants. We analyzed both types of samples by massively parallel pyrosequencing of bacterial 16S amplicons, a technique that provides a culture-independent analysis of the resident pulmonary microbiome and offers a breadth of analysis not previously available for studies of pulmonary biology and disease.

Methods and Materials Ethics Statement All clinical investigations were conducted according to the principles expressed in the Declaration of Helsinki. The study protocol was approved by the institutional review boards of the University of Michigan Healthcare System and the Ann Arbor Veterans Affairs Healthcare System. All patients provided written informed consent. The institutional review boards have examined the protocols and certified that “The risks are reasonable in relation to benefits to subjects and the knowledge to be gained. The risks of the study have been minimized to the extent possible.” Subject Enrollment – Patient populations Specimens were obtained from subjects enrolled in an observational study registered with ClinicalTrials.gov as NCT00281229. Bronchoalveolar lavage (BAL) samples (n = 14) came from volunteers who underwent research bronchoscopy at the VA Ann Arbor Healthcare System. Surgical specimens (n = 8) were obtained from six patients undergoing clinically-indicated lung transplantation for COPD at the University of Michigan Health Care System. All subjects underwent pre-procedure spirometry, PA and lateral chest radiogram, electrocardiogram, complete blood count and automated chemistry analysis, prospectively collected medication history, and clinical evaluation; surgical participants also underwent computerized tomography (CT) of the chest and full pulmonary function testing. We excluded subjects who had mental incompetence or active psychiatric illness precluding informed consent; asthma as primary clinical pulmonary diagnosis; cystic fibrosis, clinically significant bronchiectasis or other inflammatory or fibrotic lung diseases; and those taking prednisone >20 mg daily. Spirometry was expressed as a function of appropriate predicted equations for the included population [16], [17]. We categorized subjects using the spirometric classification of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [18]. For the BAL cohort analyses, subjects were segregated into three groups: healthy smokers (HS) exhibiting no evidence of underlying lung disease, post-bronchodilator FEV1/FVC>0.70 and FEV1%>80% predicted; never smokers (NS) having no smoking history or evidence of lung disease, post-bronchodilator FEV1/FVC>0.70 and FEV1%>80% predicted; COPD subjects (CS) having a post-bronchodilator FEV1/FVC<0.70 and FEV1%<80% predicted. Samples Volunteer subjects underwent fiberoptic bronchoscopy under moderate conscious sedation according to published guidelines [19] using nebulized and instilled lidocaine, intravenous fentanyl, midazolam, and in some cases, diphenhydramine. Initially, we performed bronchoscopy via the nostril, which was anesthetized using viscous lidocaine (2%), but for the last 11 procedures, bronchoscopy was performed via the mouth to minimize contamination. The bronchoscope was successively wedged into a single subsegment of the right middle lobe and the lingula, each of which were lavaged with a total of 120 ml normal saline, heated to body temperature, which was removed using manual suction on a 30 ml syringe. The total recovered volume was pooled and transported to the laboratory immediately where 15 ml of unprocessed BAL fluid was reserved for microbiome analysis, before the remainder was processed for other research studies. BALs were divided evenly into 2 ml Eppendorf tubes, spun at 13,000 rpm for 30 minutes at 4°C in a microcentrifuge, the supernatants discarded, the pellets snap frozen in liquid nitrogen, and stored at -80°C. Lung explants were obtained immediately following removal and were sterilely dissected in the hospital pathology lab in a laminar flow hood, using appropriate biosafety precautions. The lung explant was cut either sagittally or coronally to expose the airways for sampling. Airways were isolated starting proximally, and 1 cm2 tissue samples were sequentially collected from the segmental and distal portions of the upper, middle, and lower lobes, and processed separately. The anatomic location of sampling was carefully recorded to allow radiographic correlation with airway structure. Computed tomography High resolution computed tomography (HRCT) images were acquired using 64-row detector CT scanners (GEMedical Imaging, Wisconsin, USA). After initial anterior-posterior and lateral planning images, supine end-inhalation axial images were obtained in a volumetric fashion. No intravenous contrast material was administered. The images obtained were 1.25 mm thick images obtained at every 1.25 mm interval with a pitch of1.375∶1, using 120 kvp, variable tube currents (ranged between 80-375 mA based on body habitus), 0.5-0.6 s tube rotation speed and with a noise index of 22. These images were reconstructed using bone algorithm and viewed using routine lung window settings (window width of 1300 HU and window level of -600 HU). DNA Isolation To extract DNA from the BAL samples, tubes containing sample pellets (corresponding to 5 ml BAL) were suspended in a total of 500 µl Bacterial Lysis Buffer (BLB) (Roche Diagnostics), then transferred to 2 ml bead beating tubes (Mo Bio). Samples were homogenized in a Mini Bead-Beater 16 (Biospec) for 1 minute followed by centrifugation for 1 minute at 13,000 rpm in a fixed angle microcentrifuge (Microfuge 18, Beckman Coulter, Indianapolis, IN). Subsequently, 40 µl of proteinase K was added to each sample, which was then incubated for 10 minutes at 65°, followed by a second bead-beating for 1 minute, then centrifugation at full speed for 1 minute. Finally, tubes were incubated for 10 minutes at 95°C. DNA was harvested from these lysates using the MagNA Pure Compact system and Nucleic Acid Isolation Kit I (Roche, Indianapolis, IN). DNA concentration was quantified using the NanoDrop® ND-1000 Spectrophotometer (Nanodrop Technologies). We isolated DNA from lung tissue samples by the same procedure, except that the duration of each bead beating step was increased to 2 minutes. 16S Quantitative PCR qPCR was used to quantify the 16S content of our samples. Reactions were performed on a Lightcycler 480 (Roche) using the following protocol: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of 95°C for 15 sec, and 60°C for 60 sec. Readings were taken in single acquisition mode. The primers and probes consisted of a forward-primer TCC TAC GGG AGG CAG CAG T, the reverse-primer GGA CTA CCA GGG TAT CTA ATC TT, and the 16S specific probe 5′-FAM/CGT ATT ACC GCG GCT GCT GGC AC/3′-TAMSp. A standard curve was constructed using 24 two-fold dilutions of Helicobacter hepaticus DNA (a bacterium known to have only a single copy of the 16S gene in its genome) beginning at 1000 ng. Samples were run in duplicate and at 1∶10 and 1∶100 fold dilutions. 454 Pyrosequencing The bacterial tag-encoded FLX-Titanium amplicon pyrosequencing (bTEFAP) method targeting the V1-V3 variable regions of 16S rRNA was used to create amplicon libraries [20]. V1-V3 primer sets corresponded to 27F (5′- GAGTTTGATCNTGGCTCAG-3′) and 519R (5′- GWNTTACNGCGGCKGCTG-3′), along with appropriate sample nucleotide bar codes and the Roche A & B primers. The pyrosequencing was performed following established protocols [21]. Data Analysis Taxonomy. A locally run version of RDP Classifier (http://rdp.cme.msu.edu) was used for phylotyping 16S rDNA sequences. Sequences containing fewer than 50 nucleotides, and sequences without a valid barcode or those that had the barcode in the wrong position, were removed as low-quality reads. A confidence cut-off of 50% was used to produce accurate taxonomic identifications [22]. Data tables were constructed from the Classifier output and analyzed using several custom R scripts and the vegan package for R (http://CRAN.R-project.org/package=vegan) [23]. Operational Taxonomic Units (OTUs). The open-source, platform-independent, community-supported software program, mothur (http://www.mothur.org; [24]), was used to process and analyze the sequence data. Sequence reads were cleaned and filtered using quality control procedures described above, pre-clustering, and chimera elimination. 16S rDNA analysis was performed using an OTU cutoff of 3% and followed the Costello Stool Analysis example (http://www.mothur.org/wiki/Costello_stool_analysis). Statistical analysis Statistical analyses were performed using Prism 5 (GraphPad Software) for One-way ANOVA and R (http://www.r-project.org).

Discussion This study using massively parallel pyrosequencing of bacterial 16S amplicons provides novel information on the microbiota of a range of subjects including healthy non-smokers, smokers with normal lung function, and stable COPD subjects with mild or severe spirometric disease. We have demonstrated three key findings. First, the lungs of healthy smokers contain a bacterial microbiome that is quantitatively significant, diverse (but of limited membership), and quite distinct from that reported for the oral cavity or nasopharynx [26]. Second, the diversity of the lung bacterial microbiome is often lower in subjects with decreased lung function, most commonly associated with dominance by Pseudomonas spp. Third, this is the first study to describe that the numerous microanatomic sites within the lung can give rise to significant differences in bacterial community structure. Our current study has examined the lung microbiome with an unprecedented depth, averaging ∼12,000 sequences per sample. This markedly greater sequencing depth increases confidence that we have sufficiently sampled the lungs to characterize the microbial lung community accurately. Our studies of the BAL from healthy smokers are consistent with the recent demonstration of a diverse bacterial lung microbiome in healthy individuals [12], a study which had ∼3,000 sequence reads total for the entire study. Importantly, our finding that some smokers had a less diverse lung microbiota relative to smokers with normal lung function indicates that alteration in lung microbiota can occur in subjects with no spirometric evidence of disease. Whether this relative reduction in diversity is persistent, is an effect of the inflammatory changes that characterize COPD, or could in part contribute to disease progression are all questions that will require longitudinal follow-up in larger groups of subjects. Our results are consistent with findings at another mucosal site, the gastrointestinal tract, where decreases in the diversity of the microbiota are associated with increased incidence of inflammatory bowel disease [30], [31]. Lung community dysbiosis could provide the constant inflammatory stimulus that has long been observed in COPD [32]. Thus, in the lungs, as with other sites on the mucosa, a diverse microbiota may be important for health, including colonization resistance, epithelial integrity, and immunoregulation [33], [34]. Collectively, our results provide a unifying framework for characterizing the role of Pseudomonas and Haemophilus in the development, progression and/or exacerbation of COPD. Prior to the use of culture-independent techniques, Haemophilus was the organism most frequently grown from samples of COPD lungs [35], with Pseudomonas oftentimes noted [36]. The current work suggests that these organisms are generally present even under healthy conditions. While larger studies must be performed, our data support a model where dominance of an organism within the lung's microbial community is associated with disease. We have also shown that because of local differences in lung airway microarchitecture, samples from different airways taken throughout the lungs can contain very different bacterial communities. This result was most pronounced in the airways of the left upper lobe in one transplant recipient (Figure 4) where the bacterial community was dominated by a bacterial genera (Haemophilus) that was not detected at high levels in the other airways. A previous study [12] found a significant correlation between COPD and the presence of Haemophilus spp. in sterile brushings of the left upper lobes. At first pass, this finding appears to conflict with the domination by Pseudomonas spp. of BAL samples in our study and of endotracheal aspirates in a study of severe acute exacerbations of COPD [13]. However, since COPD pathology may be anatomically heterogeneous, our observation that the lung microarchitecture allows for the development of distinct localized microbial communities during disease provides a unifying hypothesis for all the culture-based and -independent studies on the role of bacteria in COPD progression. The demonstration of spatially distinct bacterial communities in the lungs may be very important to our understanding of pulmonary health and disease because anatomic variation in the microbiome is becoming the focus for dissecting disease mechanisms at other body sites. For example, regions of skin prone to dermatitis have been shown to have different microbial communities when compared to adjacent areas that remain disease-free [37]. Similarly, differences between teeth in the structure of the microbial community predisposes to disease within the same mouth [38]. Mechanistically, the microbial communities in teeth with periodontal disease are locally enriched for methanogenic Archea when compared to those that are disease-free [38]. Although these organisms are not disease-causing, the evidence suggests that they alter the local environment such that bacteria known to cause disease can thrive. It is possible that similar pathogenic syntropic interactions also exist in the lungs. We observed the most striking microanatomic community differences in the upper lobes of the lung, which is consistent with clinical observations that emphysema in COPD most commonly begins in the upper lobes. Recognized limitations of our study include the absence of oropharyngeal samples and the relatively small sample size. While the latter is a matter for future studies, the former will be dealt with here. The oropharyngeal sample as a control for microbiologic studies of the lung is rooted in the belief that the lungs should be sterile; thus, the purpose of the oropharyngeal sample is to rule out contamination. However, despite the lack of this sample, we can demonstrate that the lung microbiota that we have identified is not the result of contamination. First, the levels of 16S detected in subject BAL are too high to be consistent with contamination (Figure 1), but high enough to be consistent with a low level colonization. Second, the domination of Proteobacteria, in particular Pseudomonas spp., in BAL samples is radically different from the nasal cavity, which is dominated by the phylum Actinobacteria [26], and the oropharynx, which is dominated by Firmicutes [26]. In some subjects, Proteobacteria have been shown to have a larger presence in the oropharynx, but Pseudomonads were never encountered [26]. Finally, when the airways from explanted lungs were sampled directly, the bacteria identified were the same as those identified in BAL samples. However, all of our advanced COPD explants lacked the diversity observed in the “healthy smoker” controls. Collectively, these data argue strongly that the airways of the lungs are an independent microbial habitat, and that our results are not simply a result of contamination. In summary, these results demonstrate the need to consider, in a systematic, anatomically-correlated fashion, both the lung microbiome and the host inflammatory response when studying COPD. We believe that CT imaging will be central to this endeavor. By demonstrating that one person's lungs can harbor both generalized areas of “healthy” microbiome and a single site containing a “pathogenic” community, our results suggest a mechanism by which the interaction of lung pathogens and host immunity might contribute to localized disease progression, even in the absence of overt exacerbation.

Supporting Information Table S1. Table S1 depicts the complete population breakdown of the bacterial genera present in the lung explant tissue samples shown in Figure 4. https://doi.org/10.1371/journal.pone.0016384.s001 (DOC)

Acknowledgments The authors thank Drs. James M. Beck, James C. Hogg, Homer Twigg, Carlos H. Martinez for helpful discussion; Dr. Catherine Spino and Glen Feak for database support; Liujian Zhao for assistance in tissue processing; Christi Getty and Catherine Meldrum for support in patient recruitment; and Mary Freer, Joyce O'Brien and Rebecca Weeks for administrative support. We'd also like to thank Dr. Scot Dowd for his assistance with pyrosequencing.

Author Contributions Conceived and designed the experiments: GBH JLC FJM GBT. Performed the experiments: JED DLT. Analyzed the data: JED BS CMF MKH. Contributed reagents/materials/analysis tools: JLC FJM GBH. Wrote the paper: JED GBH. Designed the software used in analysis: JED. Study Coordinators: DLT LM. Significant intellectual contribution: VBY LAS GBT.