Quantitative analysis of pathogens in the lower respiratory tract of patients with chronic obstructive pulmonary disease

Abstract

Background: Bacterial infection of the lower respiratory tract is believed to play a major role in the pathogenesis of chronic obstructive pulmonary disease (COPD) and acute exacerbations of COPD (AECOPD). This study investigates the potential relationship between AECOPD and the load of six common bacterial pathogens in the lower respiratory tract using real-time quantitative PCR (RT-qPCR) in COPD patients.

Methods: Protected specimen brush (PSB) and bronchoalveolar lavage fluid (BALF) samples from the lower respiratory tract of 66 COPD patients and 33 healthy subjects were collected by bronchoscopy. The load of Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonos aeruginosa, Haemophilus influenzeae, and Moraxella catarrhalis were detected by RT-qPCR.

Results: High Klebsiella pneumoniae, Pseudomonos aeruginosa, Haemophilus influenzeae and Moraxella catarrhalis burden were detected by RT-qPCR in both PSB and BALF samples obtained from stable COPD and AECOPD patients compared with healthy subjects. The load of the above four pathogenic strains in PSB and BALF samples obtained from AECOPD patients were significantly higher compared with stable COPD patients. Finally, positive correlations between bacterial loads and inflammatory mediators such as neutrophil count and cytokine levels of IL-1β, IL-6 and IL-8, as well as negative correlations between bacterial loads and the forced expiratory volume in one second (FEV1) % predicted, forced vital capacity (FVC) % predicted, and FEV1/FVC ratio, were detected.

Conclusions: These findings suggest that increased bacterial loads mediated inflammatory response in the lower respiratory tract and were associated with AECOPD. In addition, these results provide guidance for antibiotic therapy of AECOPD patients.

Fig. 1

Standard curves for Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonos aeruginosa, Haemophilus influenzeae, and Moraxella catarrhalis assays. Mean threshold cycle (CT) value ± SEM of three replicates (three per run) for each reaction is shown in relation to the log of CFU calculated per 20 μl of reaction mixture. Relative correlation coefficient (R ²) and linear regression equations are reported in the legend

Fig. 2

Bacterial load in PSB and BALF samples obtained from COPD patients and healthy subjects. The burden of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonos aeruginosa, Haemophilus influenzeae, and Moraxella catarrhalis in PSB samples (a), and of Klebsiella pneumoniae, Pseudomonos aeruginosa, Haemophilus influenzeae, and Moraxella catarrhalis in BALF samples (b) obtained from AECOPD patients compared with stable COPD patients. All data are expressed as means ± SEM. ^* P < 0.05 and ^** P < 0.01 vs. the HS group; ^# P < 0.05 and ^## P < 0.01 vs. the stable COPD group

Fig. 3

Infiltration of inflammatory cells in airways of COPD patients. The number of total inflammatory cells including neutrophils, lymphocytes, and macrophages in BALF samples obtained from patients in the HS, stable COPD and AECOPD groups were determined (a). The percentage of each cell type from total cells (b). Data are expressed as means ± SEM (n = 20 per group). ^* P < 0.05 and ^** P < 0.01 vs. the HS group; ^# P < 0.05 and ^## P < 0.01 vs. the stable COPD group