Assessing Bacterial Populations in the Lung by Replicate Analysis of Samples from the Upper and Lower Respiratory Tracts

Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected subjects with potential heavy lung colonization. A single sample of BAL fluid was obtained from each subject along with contemporaneous oral wash (OW) to sample the oropharynx, and then DNA was extracted from three separate aliquots of each. Bacterial 16S rDNA sequences were amplified and products analyzed by 454 pyrosequencing. By comparing replicates, we were able to specify the depth of sequencing needed to reach a 95% chance of identifying a bacterial lineage of a given proportion—for example, at a depth of 5,000 tags, OTUs of proportion 0.3% or greater would be called with 95% confidence. We next constructed a single-sided outlier test that allowed lung-enriched organisms to be quantified against a background of oropharyngeal admixture, and assessed improvements available with replicate sequence analysis. This allowed identification of lineages enriched in lung in some BAL specimens. Finally, using samples from healthy volunteers collected at multiple sites in the upper respiratory tract, we show that OW provides a reasonable but not perfect surrogate for bacteria carried into to the lung by a bronchoscope. These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over.

人类呼吸道微生物群与人体健康及疾病发生密切相关，但对肺部进行精准采样仍存在诸多挑战。目前临床通常通过支气管镜检查（bronchoscopy）采集肺部微生物样本，但由于支气管镜需途经富含微生物的上呼吸道，易造成样本污染。为此，我们开发了一套分析方法，可在存在大量口咽部污染物质的支气管肺泡灌洗液（bronchoalveolar lavage, BAL）中识别出真正的肺部微生物群。我们选取6名疑似存在重度肺部定植的受试者作为研究对象，采集其临床BAL样本。每位受试者均采集一份BAL样本及同期口腔冲洗液（oral wash, OW）以获取口咽部样本，随后对每份样本的三份独立等分试样进行DNA提取。扩增细菌16S rDNA序列后，采用454焦磷酸测序（454 pyrosequencing）对扩增产物进行测序分析。通过对重复样本的比对分析，我们明确了达到95%置信度识别特定比例细菌类群所需的测序深度：例如，当测序深度为5000条标签时，可在95%置信度下检出占比≥0.3%的操作分类单元（operational taxonomic unit, OTU）。随后我们构建了单侧异常值检验模型，可在口咽部污染背景下量化肺部富集微生物，并评估了重复序列分析可带来的分析效能提升。该模型可实现对部分BAL样本中肺部富集细菌类群的鉴定。最后，我们通过采集健康志愿者上呼吸道多个部位的样本，证实口腔冲洗液可作为支气管镜带入肺部的细菌的合理替代样本，但并非完美的替代物。上述方法可在由误吸及支气管镜携带带来的口咽部微生物背景干扰下，识别出可在肺部定植繁殖的微生物。