Department of Respiratory Medicine, University of Occupational and Environmental Health, Kitakyushu, Fukuoka, Japan.
PLoS One. 2013 May 6;8(5):e63103. doi: 10.1371/journal.pone.0063103. Print 2013.
Molecular biological modalities with better detection rates have been applied to identify the bacteria causing infectious diseases. Approximately 10-48% of bacterial pathogens causing community-acquired pneumonia are not identified using conventional cultivation methods. This study evaluated the bacteriological causes of community-acquired pneumonia using a cultivation-independent clone library analysis of the 16S ribosomal RNA gene of bronchoalveolar lavage specimens, and compared the results with those of conventional cultivation methods.
Patients with community-acquired pneumonia were enrolled based on their clinical and radiological findings. Bronchoalveolar lavage specimens were collected from pulmonary pathological lesions using bronchoscopy and evaluated by both a culture-independent molecular method and conventional cultivation methods. For the culture-independent molecular method, approximately 600 base pairs of 16S ribosomal RNA genes were amplified using polymerase chain reaction with universal primers, followed by the construction of clone libraries. The nucleotide sequences of 96 clones randomly chosen for each specimen were determined, and bacterial homology was searched. Conventional cultivation methods, including anaerobic cultures, were also performed using the same specimens.
In addition to known common pathogens of community-acquired pneumonia [Streptococcus pneumoniae (18.8%), Haemophilus influenzae (18.8%), Mycoplasma pneumoniae (17.2%)], molecular analysis of specimens from 64 patients with community-acquired pneumonia showed relatively higher rates of anaerobes (15.6%) and oral bacteria (15.6%) than previous reports.
Our findings suggest that anaerobes and oral bacteria are more frequently detected in patients with community-acquired pneumonia than previously believed. It is possible that these bacteria may play more important roles in community-acquired pneumonia.
为了更准确地检测出引发传染病的细菌,人们已经采用了分子生物学手段。采用传统的培养方法,大约有 10-48%的社区获得性肺炎的病原体无法被检测出来。本研究通过对支气管肺泡灌洗液 16S 核糖体 RNA 基因的非培养性克隆文库分析,评估了社区获得性肺炎的细菌病因,并将结果与传统的培养方法进行了比较。
根据临床和影像学表现,选择社区获得性肺炎患者作为研究对象。通过支气管镜采集肺部病变部位的支气管肺泡灌洗液标本,分别采用非培养性分子方法和传统培养方法进行评估。非培养性分子方法采用通用引物通过聚合酶链反应扩增大约 600 个碱基对的 16S 核糖体 RNA 基因,然后构建克隆文库。对每个标本随机选择的 96 个克隆的核苷酸序列进行了测定,并对细菌同源性进行了检索。使用相同的标本进行了包括厌氧菌培养在内的传统培养方法。
除了常见的社区获得性肺炎病原体(肺炎链球菌 18.8%、流感嗜血杆菌 18.8%、肺炎支原体 17.2%)外,对 64 例社区获得性肺炎患者标本的分子分析显示,厌氧菌(15.6%)和口腔细菌(15.6%)的检出率高于以往的报道。
我们的研究结果表明,与以往的认识相比,厌氧菌和口腔细菌在社区获得性肺炎患者中更为常见。这些细菌可能在社区获得性肺炎中发挥了更为重要的作用。