Biotechnology Department, Dalian Medical University, 9 Western Section, Lvshun South Street, Dalian, P,R, China.
BMC Pulm Med. 2014 Nov 18;14:179. doi: 10.1186/1471-2466-14-179.
The 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.
We 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.
The 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.
This 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.
全球倡议将慢性阻塞性肺病定义为一种完全可以预防和治疗的疾病，其特征是痰液产生、细菌定植、中性粒细胞性支气管气道炎症和健康状况不佳。世界卫生组织（WHO）估计，到 2030 年，COPD 将成为仅次于缺血性心脏病、脑血管病和艾滋病的全球第四大死因。本研究旨在确定慢性阻塞性肺病患者在临床稳定期痰液中潜在致病微生物的主要结构特征。
我们采用基于分子遗传学的细菌群落研究方法，包括 DNA 分离、PCR 扩增和 DGGE 分析。PCR-变性梯度凝胶电泳（DGGE）采用针对 16S rRNA 基因 V3 区的通用引物，用于描述 COPD 患者痰液微生物群落的总体组成，对一些切下的凝胶条进行克隆测序。实时 PCR 进一步利用针对肺炎链球菌、肺炎克雷伯菌、铜绿假单胞菌的组特异性引物对微生物群落的亚群进行定量分析。
两组的 DGGE 图谱在 COPD 组和健康组之间存在显著差异（P<0.05）。实时 PCR 显示 COPD 组肺炎链球菌、肺炎克雷伯菌和铜绿假单胞菌的数量明显增加（P<0.05）。
本研究揭示了痰液微生物群落变化与 COPD 之间的强烈关系。通过确定几种细菌的含量，我们可以为 COPD 的诊断和治疗提供依据。
