Although nearly half of acute COPD exacerbations are associated with bacterial infection, our knowledge of the microbial species associated with these events is limited to a handful of organisms detected primarily by culture-based methods (Papi et al., 2006
; Rosell et al., 2005
; Soler et al., 2007
). The overall aim of this study was to begin to address the overarching question whether previously undetected bacterial species exist in the airways of COPD patients during acute exacerbations. High-resolution, culture-independent analysis using the 16S rRNA PhyloChip revealed a much greater diversity of bacteria than has previously been appreciated in the airways of COPD patients being managed for severe exacerbations, including members of the Pseudomonadaceae, Enterobacteriaceae, and Helicobacteraceae, among others. The potential for a diverse airway bacterial community to play a role in chronic airway colonization and inflammation, a feature of COPD, has not been previously considered.
The identification of a diversity of bacterial communities in respiratory samples from COPD patients experiencing severe exacerbations suggests that the pathogenesis of these events could involve a polymicrobial process. Future studies in a larger cohort of patients, including nonintubated COPD patients with a greater range of exacerbation severity, will be necessary to determine relationships between community composition, structure, and pulmonary health. Only a handful of bacterial species, such as H. influenzae
and P. aeruginosa
, have previously been associated with COPD exacerbations. The possible role of other bacterial community members with pathogenic potential identified in this study, e.g. A. cryaerophilus
, B. diminuta
, and L. interrogans
, may merit further investigation. Many of these species have been implicated in other pathogenic processes such as endocarditis (Han and Andrade, 2005
; Marques da Silva et al., 2006
; Paster et al., 2002
) and bacteremia (Hsueh et al., 1997
; Woo et al., 2001
). L. interrogans
, the causative agent of human leptospirosis (Gaudie et al., 2008
), has recently been shown to induce pulmonary lesions in an experimental animal model of airway infection (Marinho et al., 2009
) and pulmonary hemorrhage in severe cases (Dall'Antonia et al., 2008
). Their potential for pathogenesis suggests the possibility of a role for these organisms in COPD chronic airway disease. Future studies involving functional and mechanistic analyses will be necessary to further assess this.
Multiple oropharyngeal and gut-associated bacterial species were also identified by PhyloChip analysis, suggesting a potential role in COPD exacerbations. Although contamination by oropharyngeal secretions is always a possibility, samples were collected through an endotracheal tube, diminishing the degree of direct contamination. Ongoing microaspiration during intubation, however, cannot be completely prevented, and it has been suggested that the oral cavity and gastrointestinal tract act as a microbial reservoir for seeding the airways in vulnerable patient populations (Garrouste-Orgeas et al., 1997
; Heyland and Mandell, 1992
; Orozco-Levi et al., 2003
). In our study the relative abundance of targeted gastrointestinal-associated species, H. cetorum
(Figura et al., 1993
; Garcia-Amado et al., 2007
), were confirmed by independent Q-PCR in airway samples from these patients. The presence of oropharyngeal or gut-associated bacteria in the lower airways may have significant implications for a disease population with greater risks from pulmonary infections. For example, Duan et al. (2003
) demonstrated in a rat model that coinfection of P. aeruginosa
with oropharyngeal bacterial species (isolated from cystic fibrosis patient sputa) resulted in enhanced lung damage and upregulation of P. aeruginosa
virulence gene expression. In our study, P. aeruginosa
and oropharyngeal and gut-associated species were present in the airways of all patients studied, suggesting the potential for enhanced pathogenesis in this patient population.
We recognize that our study numbers are small and represent a severely ill group of COPD patients. Therefore, caution must be exercised in extrapolating these findings to a broader group of COPD patients, especially nonintubated individuals experiencing less severe exacerbations. Although facilitating access to lower respiratory specimens that are otherwise challenging to obtain during severe exacerbations, the intubation status of these patients is an important consideration in weighing these findings. Although protracted intubation was associated with decreased bacterial community richness, the possibility for a more diverse bacterial community to play a role at least at the onset of exacerbation-associated respiratory failure remains. Control samples from nonintubated COPD and non-COPD patients were not available for this study, nor were longitudinal samples from these patients. In a previous study, however, we have found that endotracheal samples obtained from patients briefly intubated for elective surgery produced no detectable 16S rRNA PCR product (Flanagan et al., 2007
). The lack of an association between duration of active antimicrobial therapy and bacterial community structure is most likely due to both the small study numbers as well as the administration of previous antibiotic courses up to one month prior to sample collection in the study (e.g., patient 6).
Results of this study highlight the advantages of complementing culture-based methods with higher resolution approaches for bacterial detection to provide a more comprehensive assessment of the airway microbiota present in COPD patients. Culture-independent methods are particularly relevant to identify viable but nonculturable species that produce and exist in biofilms (Rayner et al., 1998
), which are characteristic of chronic airway infections (Costerton et al., 1999
; Singh et al., 2000
) and have recently been implicated in COPD (Martinez-Solano et al., 2008
). Because our PhyloChip analysis was based on DNA extracted from airway samples, it does not provide information on the viability of the species detected. However, we have previously noted that within 24
h of antimicrobial administration to cystic fibrosis patients, bacterial richness decreased approximately 10-fold as detected by the PhyloChip (Lynch, unpublished data). This suggests that DNA turnover is relatively rapid in the airways of chronic pulmonary disease patients, and that the taxa detected during antimicrobial administration in our COPD patients largely represent the viable portion of the community. This is supported by the finding that the PhyloChip detected all species that were isolated by concurrent clinical laboratory culture. Independent Q-PCR analysis of selected taxa also demonstrated strong concordance between calculated 16S rRNA copy number and PhyloChip-based fluorescence intensities, validating the abundance of individual species detected by the array. Although both cultures and the microarray demonstrated low detection rates for H. influenzae and M. catarrhalis,
two species commonly associated with COPD exacerbation (potentially due to antibiotic-mediated clearance), only the PhyloChip identified other Haemophilus
species across the majority of patients in this study, as well as other members of the Moraxellaceae family. Although these molecular methods may identify potentially relevant species undetected by culture, they provide no information on the viability or activity of these species. Hence, the significance of detecting species phylogenetically related to known pathogens in COPD airways is unclear, but may merit further study.