Variability and effects of bronchial colonisation in patients with moderate COPD

The bronchial tree and the pulmonary parenchyma are sterile in healthy subjects, but in patients with chronic obstructive pulmonary disease (COPD) potentially pathogenic microorganisms (PPMs) are often recovered from bronchial secretions during periods of clinical stability and, particularly, during episodes of exacerbation, when PPM loads increase significantly 1, 2. Most studies on PPM colonisation in COPD have focused on patients with severe disease 3–5. In contrast, the relationships between the microbiology of bronchial colonisation and local inflammatory response in moderate COPD patients, when therapeutic interventions are expected to have maximal effects, have only been assessed in small selected population samples 6–8. With the hypothesis that the characteristics of bronchial colonisation have an effect on bronchial inflammation and lung function that may be identified in stable outpatients with moderate COPD, the present study sought: first, to determine the prevalence and load of bronchial colonisation in a cohort of COPD outpatients with moderate airflow limitation never admitted to a hospital for an exacerbation of the disease; and secondly, to investigate in these patients the relationships between colonisation characteristics, bronchial inflammation markers and post-bronchodilator changes in forced expiratory volume in 1 s (FEV₁) during follow-up.

METHODS

Molecular genotyping

Molecular PPM typing was performed using pulse field gel electrophoresis (PFGE) to determine whether a PPM recovered from consecutive samples corresponded to the persistence of the same strain or to the acquisition of a new one 26. In brief, chromosomal DNA from multiple (more than four) PPMs growing in chocolate agar was extracted with agarose and incubated at 37°C for 18 h. After enzyme digestion with Sma I (New England Biolabs, Ipswich, MA, USA), restriction fragments were separated in agarose gel with tris-borate-ethylenediaminetetraacetic acid (Sigma Chemical, St Louis, MI, USA) through PFGE using a homogeneous electric camp contour clamp (CHEF DR II system; BioRad, Ivey sur Seine, France), beginning with an initial 5.6-s pulse that was linearly increased until a 40.6-s pulse was reached and then maintained for 24 h at 5 V·cm⁻¹ and 14°C. Bacteriophage λ concatemer (New England Biolabs) was included as the molecular weight DNA marker. Obtained patterns were photographed with a 360-nm transilluminator after staining, and analysed using Diversity Database Software (BioRad). PPMs cultured from follow-up sputum samples obtained from patients who grew the same species at baseline and showed the same molecular profile in both samples analysed by PFGE were considered long-term persistent strains, whereas PPMs cultured at follow-up which were not present in the baseline sample were considered as strains acquired during follow-up (fig. 1⇓).

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Fig. 1—

Pulse-field electrophoresis of samples growing Haemophilus influenzae in baseline and follow-up samples. a) Persistent H. influenzae strain, and b) appearance of a strain newly acquired during follow-up.

RESULTS

40 consecutive, clinically stable COPD outpatients fulfilled the inclusion criteria and were enrolled. Participating patients were mostly male (39 (97.5%)), had a mean age±sd of 66.5±8.1 yrs and showed moderate lung function impairment (post-bronchodilator FEV₁ 57.9% of predicted (sd 19.1%)) (table 1⇓). In these patients, 79 baseline visits and consecutive follow-up examinations were performed after 8.0±3.2 months. Sputum was obtained with isotonic saline in 23 baseline and 34 follow-up visits, and was induced with hypertonic saline in the remaining examinations. PPMs were recovered from nearly three-quarters of these sputum samples, both at baseline and at the follow-up visits, and in half of them this recovery was associated with a neutrophilic response. H. influenzae was the PPM recovered most often (in 28 and 25 cases at baseline and follow-up visits, respectively), and samples growing that microorganism also had the highest microbial loads. Low loads of P. aeruginosa, enterobacteria and H. parainfluenzae, however, were also often cultivated from the sputum samples (table 2⇓).

59 samples showed bronchial colonisation at the follow-up examination, preceded by positive sputum cultures for PPMs in the baseline sputum sample in 42 (53.2%) cases. These successive positive cultures, however, were in most cases due to new PPMs acquired during follow-up (30 (38.0%) cases). Long-term persistence of the same strain was the cause in only 12 (15.2%) cases, a situation that was mainly attributable to colonisation by P. aeruginosa or enterobacteria (table 3⇓). No statistically significant differences in the measured bronchial inflammation parameters were found between sputum samples with long-term persistent and acquired colonisation (table 4⇓).

The identification of bronchial colonisation by H. influenzae in the follow-up sputum was associated with a bronchial inflammatory response identifiable through a higher prevalence of sputum neutrophilia in samples positive for this PPM (68.0 versus 42.6%; p = 0.04, Chi-squared test), and higher concentrations of IL-1β (median 1,636 (IQR 597–7,736) versus 601 (153–1,320) pg·mL⁻¹; p = 0.005, Mann–Whitney U-test) and IL-12 (14 (7–29) versus 2 (1–17) pg·mL⁻¹; p = 0.01, Mann–Whitney U-test) (fig. 2⇓; table 5⇓). This inflammatory response was not observed in samples colonised by P. aeruginosa/enterobacteria or H. parainfluenzae (table 5⇓). The recovered load of H. influenzae was also positively correlated with the sputum concentrations of IL-1β in patients with sputum cultures positive for this PPM (ρ = 0.64, p = 0.001, Spearman’s correlation coefficient) (fig. 3⇓). In samples colonised by P. aeruginosa or enterobacteria, a neutrophilic inflammatory response was also observed (p = 0.05, Chi-squared test) (table 5⇓). This neutrophilic inflammatory response, mainly found in patients colonised by H. influenzae, P. aeruginosa or enterobacteria (27 out of 40 neutrophilic samples, 67.5%), showed a statistically significant relationship with FEV₁ declines over the median decline during follow-up in our sample of moderate COPD patients (OR 2.67, 95% CI 1.07–6.62; p = 0.03). That relationship was not modified after adjustment for covariates and was not found for IL-1β or IL-12, nor were statistically significant relationships found between inhaled corticosteroid use or appearance of an exacerbation and follow-up inflammatory markers in sputum (data not shown).

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Fig. 2—

a) Interleukin (IL)-1β and b) IL-12 inflammatory markers in sputum according to the presence of bronchial colonisation by Haemophilus influenzae. Boxes represent median (horizontal dividing line) and interquartile range; whiskers represent sd.

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Fig. 3—

Scatterplot of total bacterial count (colony-forming units (cfu)·mL⁻¹) and sputum interleukin (IL)-1β (Spearman’s correlation coefficient ρ = 0.64, p = 0.001) in patients colonised by Haemophilus influenzae at the follow-up examination (n = 25). Bacterial count data have been logarithmically transformed.

DISCUSSION

In the present study, focusing on the effects of bronchial colonisation on inflammation and lung function in moderate COPD, we found that PPMs were recovered from bronchial secretions in nearly three-quarters of the patients. The recovery of H. influenzae, P. aeruginosa and enterobacteria from bronchial secretions was closely related to a neutrophilic response, suggesting that the cellular inflammatory effects of bronchial colonisation on moderate COPD depend mainly on the presence of these PPMs. H. influenzae colonisation was additionally associated with higher sputum concentrations of such inflammation markers as IL-1β and IL-12, a relationship that was shown to be load-mediated for IL-1β. Bronchial colonisation by H. parainfluenzae, however, was not associated with an inflammatory response, a finding suggesting that the effect of this microorganism on the bronchial mucosa must be considered as marginal. The repetition of the sputum cultures was recurrently positive for PPMs in more than half of the patients, in most cases due to colonisation during follow-up by a newly acquired PPM; thus, long-term persistence of the same strain was observed in fewer than a fifth of the cases, all, however, with an associated neutrophilic response. The finding of sputum neutrophilia during stability was associated with a significant lung function decline in these moderate COPD patients, a relationship that was independent of current smoking.

Previous studies of bronchial colonisation in stable COPD patients, in which the protected specimen brush was used to collect microbiology samples, have demonstrated cultures positive for PPMs in one-third of the patients, with H. influenzae, S. pneumoniae and M. catarrhalis the PPMs most often recovered 1. When sputum has been used for the identification of bronchial colonisation in COPD, the cultures positive for PPMs have been found in one-fifth 5 to three-quarters 4 of the samples. In patients unable to spontaneously produce appropriate sputum samples during their stable periods, the use of sputum induction has facilitated the study of bronchial colonisation by allowing most stable COPD patients to be sampled 6, 27. Study of the cytological characteristics of sputum in this context has shown cultures positive for PPMs with neutrophilia in half of the studied cases 3, 28, a rate similar to the prevalence found in the present study, in which H. influenzae was the most frequently isolated PPM.

Bronchial colonisation by H. influenzae was significantly related to sputum neutrophilia and higher sputum concentrations of the inflammatory mediators IL-1β and IL-12 in our sample of stable COPD patients with moderate lung function impairment. These observations suggest that colonisation by these PPMs is already able to induce an inflammatory response in the bronchial mucosa of such patients. This inflammatory response was seen to be species-specific because in our series, a similar effect was not found in COPD patients colonised by H. parainfluenzae, a PPM that occasionally causes COPD exacerbation and pneumonia 29–31, but has been shown to exhibit low mucosal adherence and a minimal potential to cause bronchial inflammation 32, 33. Our choice of H. influenzae load as a main study variable was justified by its close relationship to the inflammatory response of the bronchial mucosa. Most PPMs recovered from participating COPD patients grew low microbial loads (≤10⁵ cfu·mL⁻¹), with high microbial loads being found almost exclusively when H. influenzae was the colonising PPM in our study. High-load H. influenzae colonisation was significantly associated with neutrophilic sputum samples (Murray-Washington ≥3) and higher levels of IL-1β in sputum. When dose–response relationships between PPM load and bronchial inflammatory mediators in sputum have been assessed in previous studies not focusing on H. influenzae, they have supported a causal role for bronchial colonisation in the pathogenesis of bronchial inflammation in COPD 4, 34, 35, and loads >10⁵ cfu·mL⁻¹ have usually been associated with neutrophilic inflammation 3. Most series of patients with advanced disease, however, have shown positive correlations between microbial load and IL-8, myeloperoxidase, neutrophil elastase and leukotriene B₄ 3, 4, 28. Together, all of these findings point to a significant impact of bronchial colonisation load on the mucosal inflammatory response, which, according to our results, may already be found, though with a different pattern, in patients with moderate disease when H. influenzae is the colonising bacteria. IL-1β has been related to bronchial colonisation in animal models 36, and our observation of high concentrations of this inflammatory mediator and IL-12, a cytokine related to the mucosal response against bacteria 37, in moderate COPD patients with H. influenzae colonisation supports the hypothesis of differences in the inflammatory response to bronchial colonisation according to the severity of lung function impairment. Our finding of a different pattern may have clinical implications and requires further assessment.

We have found that samples showing bronchial colonisation were preceded by positive sputum cultures for PPMs in more than half the cases, and we have used molecular typing techniques to investigate whether recovered PPMs corresponded to newly acquired or long-term persistent strains. According to this analysis, bronchial colonisation was mostly attributable to new PPMs rather than to persistent ones. Persistent colonisation was observed in fewer than a fifth of the samples that were positive on follow-up and was most often attributable to long-term colonisation by P. aeruginosa or enterobacteria. Two previous studies reported that microbial persistence accounted for about a half of the cases of bronchial colonisation in stable COPD patients 3, 5, a prevalence higher than we have found. These studies, however, mostly investigated patients with severe COPD, and only one of them used molecular typing techniques to rule out colonisation by a new strain of the same PPM 5. Our observations suggest that, in spite of a high prevalence of bronchial colonisation in moderate COPD patients, long-term microbial persistence is less often the cause, probably due to a higher turnover of colonising strains in less advanced disease.

Our findings in moderate COPD patients colonised by H. influenzae, P. aeruginosa and enterobacteria confirm the close association between colonisation by these PPMs and neutrophilia reported in other studies 34. This association supports an early relationship between colonisation and the appearance of cellular inflammatory mediators in bronchial secretions, years before the disease causes severe impairment and at a stage when these relationships could be more easily identifiable 38, 39. The importance of the neutrophilic inflammatory response associated with bronchial colonisation is emphasised by our finding of a statistically significant relationship between the appearance of this inflammatory response and lung function impairment during follow-up. A similar functional impairment associated with bronchial colonisation has been documented by Wilkinson et al. 3, but was not found by Hill et al. 4. In our study, the decline in FEV₁ in COPD patients with sputum neutrophilia in some cases approached the magnitude previously seen in exacerbated COPD patients with more severe disease 40. This finding supports the hypothesis that bronchial colonisation may have a subclinical impact in moderate COPD patients, who may be less prone to report their daily symptoms as an exacerbation 41. Such patients may have functional impairment equivalent to the FEV₁ decline that is associated with exacerbation in patients with advanced disease, who suffer easily from dyspnoea with even slight impairments in their lung function.

We have not performed genomic analysis of the sputum samples that do not grow PPMs, and we cannot rule out underdiagnosis of microbial persistence due to bronchial colonisation at loads below the detection limit of the sputum culture. This situation must be considered as unusual, however, because when this approach has been used undiagnosed, bronchial colonisation has only been identified in one-tenth of the culture-negative sputum samples 42. The lack of virological data is also a limitation of our study, but respiratory viruses are seldom recovered from bronchial secretions in stable COPD patients, who only give positive samples in one-tenth of the cases 43, 44. The lowest prevalence of viral recovery has been found in infrequent exacerbators 44, such as those in our study.

In summary, we found that the bronchial tree of clinically stable outpatients with moderate COPD and without previous admissions due to respiratory diseases is often colonised by PPMs, but high sputum loads are only reached by H. influenzae. The presence of this PPM is associated with an inflammatory response of the bronchial mucosa, characterised by neutrophilic inflammation and high levels of IL-1β and IL-12, findings that are not characteristic of patients colonised by H. parainfluenzae. Colonising P. aeruginosa and enterobacteria are more often persistent and also associated with sputum neutrophilia. These observations suggest that the effect of bronchial colonisation on COPD patients is mainly mediated by H. influenzae, P. aeruginosa and enterobacteria, PPMs that are able to cause an identifiable inflammatory response in moderately ill COPD patients.

Support statement

This study was funded by Fondo de Investigación Sanitaria PI060684, Red Respira Instituto de Salud Carlos III (ISCIII)-RTIC-03/11, and Fundació Catalana de Pneumologia. CB06/06/1089. El CIBER de Enfermedades Respiratorias (CIBERES) is an initiative of ISCIII.

Statement of interest

None declared.