Analysis of the bacterial community in chronic obstructive pulmonary disease sputum samples by denaturing gradient gel electrophoresis and real-time PCR

Abstract

BackgroundThe Global Initiative defines COPD for chronic obstructive lung disease as an entirely preventable and treatable disease characterized by sputum production, bacterial colonisation, neutrophilic bronchial airway inflammation and poor health status. The World Health Organization (WHO) estimates that COPD will become the fourth-most common cause of death worldwide, just behind ischemic heart disease, cerebrovascular disease and HIV/AIDS, by 2030. The aim of this study was to determine the main structure feature of sputum potentially pathogenic microorganisms in subjects with COPD during the clinical stable state.MethodsWe employed a molecular genetics-based investigation of the bacteria community, including DNA isolation, PCR amplification and DGGE profiling. PCR-denaturing gradient gel electrophoresis (DGGE) with universal primers targeting the V3 region of the 16S rRNA gene was employed to characterize the overall COPD patient sputum microbiota composition, and some excised gel bands were cloned for sequencing. Real-time PCR was further utilized to quantitatively analyze the subpopulation of microbiota using group-specific primers targeting Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa.ResultsThe DGGE profiles of two groups displayed significant differences between COPD and healthy groups (P < 0.05). Real-time PCR revealed significant increases of Streptococcus pneumoniae, Klebsiella pneumoniae and Pseudomonas aeruginosa (P < 0.05) in the COPD group compared with the healthy group.ConclusionThis study revealed strong relationship between alterations of sputum microbiota and COPD. By determining the content of several types of bacteria, we can provide evidence to aid in the diagnosis and treatment of COPD.

The Microbiome and the Respiratory Tract

“…Most studies of patients with advanced COPD have observed a shift in community membership away from the Bacteroidetes phylum (75, 89), often to Proteobacteria and its familiar potentially pathogenic members (e.g., Pseudomonas spp., Haemophilus spp.) (75, 89, 90) and sometimes to Firmicutes (90, 91). Garcia-Nuñnez et al (89) demonstrated an unambiguous relationship between severity of airway obstruction and decreased community diversity; the least diverse airway community in patients with FEV 1 greater than 50% predicted was more diverse than the most diverse airway community in patients with FEV 1 less than 50% predicted.…”

Role of microbiota on lung homeostasis and diseases

“…The change of lung microbiota only can be found in patients with advanced COPD . In patients with advanced COPD, there are more frequent Proteobacteria or Firmicutes and less frequent Bacteroidetes (Garcia-Nuñez et al, 2014;Wu et al, 2014), which is similar to the shift of the microbiota in asthma patients. However, asthma and COPD are different diseases in the lungs, implying that, besides the change of the lung microbiota, other factors also contribute to the disease and play more important roles than the microbiota.…”

Brain‐derived neurotrophic factor preserves intestinal mucosal barrier function and alters gut microbiota in mice

“…Difference analysis of the gut microbiota of mice between the BDNF D/L and BDNF D/D groups UPGMA cluster analysis on the DGGE band patterns of the gut microbiota of mice showed that in the third week, the similarity within the BDNF þ/À group was 60% [21], while that within the BDNF þ/þ group was 75%; the similarity between the two groups was 42% (Fig. 1).…”