Abstract
Introduction: The most common pathogen in the lower airway of patients with COPD is Haemophilus influenzae. Emerging evidence has shown that microbes can be present in cell-associated biofilms or adopt a free-living existence. Whether free-living H. influenzae, that which is living independently & not attached to another organism or substrate, in COPD exists, or exhibit a greater degree of virulence associated with disease severity is unknown.
Methods: DNA was extracted using a commercial assay from cell free sputum supernatant in 53 COPD patients (43 male) attending a research project investigating biomarkers of COPD exacerbations. A H. influenzae plasmid, targeting the ompP6 gene, was designed for quantification of H. influenzae by Real-Time PCR (qPCR) utilising Taqman hydrolysis probes.
Results: H. influenzae was detected in free-living form in 87% of stable samples and 80% of exacerbation samples. The coefficient of variation for H. influenzae qPCR was 0.5%. The geometric mean (95%CI) of H. influenzae free-living bacteria in stable state was 8.3x102(1.5x102to 4.7x103) copies/ml. There was no association of H. influenzae and FEV1% predicted (r = 0.11, p=0.41). In 18 paired stable and exacerbation visits, there was no significant difference in cell free H. influenzae (mean fold difference 0.88, 95% fold difference 0.01 to 114.8, p=0.96). Cell free H. influenzae was associated with total sputum H. influenzae qPCR and neutrophil counts (r=0.36 and 0.37 respectively, p<0.01).
Conclusion: H. influenzae exists in a free-living phenotype in patients with COPD. Further investigation is needed to see if this is associated with disease severity and airway inflammation.
- Copyright ©ERS 2015