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首页> 外文期刊>Journal of Clinical Microbiology >Analysis of Culture-Dependent versus Culture-Independent Techniques for Identification of Bacteria in Clinically Obtained Bronchoalveolar Lavage Fluid
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Analysis of Culture-Dependent versus Culture-Independent Techniques for Identification of Bacteria in Clinically Obtained Bronchoalveolar Lavage Fluid

机译：用于临床获得的支气管肺泡灌洗液中细菌鉴定的文化依赖性和非文化依赖性技术分析

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The diagnosis and management of pneumonia are limited by the use of culture-based techniques of microbial identification, which may fail to identify unculturable, fastidious, and metabolically active viable but unculturable bacteria. Novel high-throughput culture-independent techniques hold promise but have not been systematically compared to conventional culture. We analyzed 46 clinically obtained bronchoalveolar lavage (BAL) fluid specimens from symptomatic and asymptomatic lung transplant recipients both by culture (using a clinical microbiology laboratory protocol) and by bacterial 16S rRNA gene pyrosequencing. Bacteria were identified in 44 of 46 (95.7%) BAL fluid specimens by culture-independent sequencing, significantly more than the number of specimens in which bacteria were detected (37 of 46, 80.4%, P ≤ 0.05) or “pathogen” species reported (18 of 46, 39.1%, P ≤ 0.0001) via culture. Identification of bacteria by culture was positively associated with culture-independent indices of infection (total bacterial DNA burden and low bacterial community diversity) (P ≤ 0.01). In BAL fluid specimens with no culture growth, the amount of bacterial DNA was greater than that in reagent and rinse controls, and communities were markedly dominated by select Gammaproteobacteria, notably Escherichia species and Pseudomonas fluorescens. Culture growth above the threshold of 104 CFU/ml was correlated with increased bacterial DNA burden (P < 0.01), decreased community diversity (P < 0.05), and increased relative abundance of Pseudomonas aeruginosa (P < 0.001). We present two case studies in which culture-independent techniques identified a respiratory pathogen missed by culture and clarified whether a cultured “oral flora” species represented a state of acute infection. In summary, we found that bacterial culture of BAL fluid is largely effective in discriminating acute infection from its absence and identified some specific limitations of BAL fluid culture in the diagnosis of pneumonia. We report the first correlation of quantitative BAL fluid culture results with culture-independent evidence of infection.
机译：肺炎的诊断和处理受到基于微生物鉴定的基于培养物的技术的限制，这种技术可能无法鉴定出无法培养的，挑剔的和具有代谢活性的存活但无法培养的细菌。新颖的高通量文化独立技术有望实现，但尚未与常规文化进行系统比较。我们通过培养（使用临床微生物实验室方案）和细菌16S rRNA基因焦磷酸测序分析了有症状和无症状肺移植接受者的46份临床上获得的支气管肺泡灌洗液（BAL）。通过与培养无关的测序，在46个（95.7％）的BAL液体标本中鉴定出细菌，大大超过了检出细菌的标本数量（46个中的37个，占80.4％， P ≤0.05 ）或“病原体”物种（占46个物种中的18个，占39.1％， P ≤0.0001）。通过培养鉴定细菌与不依赖培养的感染指数（细菌DNA总量和细菌群落多样性低）呈正相关（ P ≤0.01）。在没有培养物生长的BAL液体标本中，细菌DNA的量大于试剂和冲洗液对照的细菌DNA，并且社区明显被选定的γ-变形杆菌所主导，特别是大肠埃希氏菌属和荧光假单胞菌。培养物生长超过10 4 CFU / ml阈值与细菌DNA负荷增加（ P <0.01），群落多样性降低（ P <0.05），并增加了铜绿假单胞菌的相对丰度（ P <0.001）。我们提供了两个案例研究，其中不依赖文化的技术确定了文化遗漏的呼吸道病原体，并阐明了培养的“口腔菌群”物种是否代表急性感染状态。总而言之，我们发现BAL液的细菌培养在区分急性感染和不存在急性感染方面非常有效，并且在诊断肺炎中发现了BAL液培养的一些特定局限性。我们报告定量BAL流体培养结果与文化独立感染的证据的第一相关性。

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