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首页> 外文期刊>Journal of Clinical Microbiology >Time between Collection and Storage Significantly Influences Bacterial Sequence Composition in Sputum Samples from Cystic Fibrosis Respiratory Infections
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Time between Collection and Storage Significantly Influences Bacterial Sequence Composition in Sputum Samples from Cystic Fibrosis Respiratory Infections

机译：收集和存储之间的时间显着影响囊性纤维化呼吸道感染痰标本中的细菌序列组成。

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Spontaneously expectorated sputum is traditionally used as the sampling method for the investigation of lower airway infections. While guidelines exist for the handling of these samples for culture-based diagnostic microbiology, there is no comparable consensus on their handling prior to culture-independent analysis. The increasing incorporation of culture-independent approaches in diagnostic microbiology means that it is of critical importance to assess potential biases. The aim of this study was to assess the impact of delayed freezing on culture-independent microbiological analyses and to identify acceptable parameters for sample handling. Sputum samples from eight adult cystic fibrosis (CF) patients were collected and aliquoted into sterile Bijou bottles. Aliquots were stored at room temperature before being frozen at ?80°C for increasing intervals, up to a 72-h period. Samples were treated with propidium monoazide to distinguish live from dead cells prior to DNA extraction, and 16S rRNA gene pyrosequencing was used to characterize their bacterial compositions. Substantial variation was observed in samples with high-diversity bacterial communities over time, whereas little variation was observed in low-diversity communities dominated by recognized CF pathogens, regardless of time to freezing. Partitioning into common and rare species demonstrated that the rare species drove changes in similarity. The percentage abundance of anaerobes over the study significantly decreased after 12 h at room temperature (P = 0.008). Failure to stabilize samples at ?80°C within 12 h of collection results in significant changes in the detected community composition.
机译：传统上，自发排痰是研究下呼吸道感染的采样方法。尽管存在针对这些样品进行基于培养物的诊断微生物学处理的准则，但在进行与培养物无关的分析之前，如何处理这些样品尚无可比的共识。在诊断微生物学中越来越多地采用与文化无关的方法，这意味着评估潜在的偏见至关重要。这项研究的目的是评估冷冻延迟对不依赖培养物的微生物分析的影响，并确定样品处理的可接受参数。收集了八名成人囊性纤维化（CF）患者的痰液样本，并分装到无菌的Bijou瓶中。将等分试样保存在室温下，然后在〜80°C冷冻，以增加间隔，直至72小时。在提取DNA之前，先用单叠氮化丙锭处理样品，以区分活细胞和死细胞，然后使用16S rRNA基因焦磷酸测序来表征其细菌组成。随着时间的推移，在具有高多样性细菌群落的样品中观察到了显着的变化，而在不考虑冷冻时间的情况下，在以公认的CF病原体为主的低多样性群落中观察到的变化很小。分为常见物种和稀有物种表明，稀有物种推动了相似性的变化。在室温下12小时后，厌氧菌的丰度百分比显着下降（ P = 0.008）。在收集后的12小时内未能将样品稳定在80°C时，会导致检测到的群落组成发生重大变化。

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