Institute of Pharmaceutical Science, King's College London, London, United Kingdom.
PLoS One. 2013 Sep 25;8(9):e74884. doi: 10.1371/journal.pone.0074884. eCollection 2013.
Identification of pathogenic bacteria in ascites correlates with poor clinical outcomes. Ascites samples are commonly reported culture-negative, even where frank infection is indicated. Culture-independent methods have previously reported bacterial DNA in ascites, however, whether this represents viable bacterial populations has not been determined. We report the first application of 16S rRNA gene pyrosequencing and quantitative PCR in conjunction with propidium monoazide sample treatment to characterise the viable bacterial composition of ascites. Twenty five cirrhotic patients undergoing paracentesis provided ascites. Samples were treated with propidium monoazide to exclude non-viable bacterial DNA. Total bacterial load was quantified by 16S rRNA Q-PCR with species identity and relative abundance determined by 16S rRNA gene pyrosequencing. Correlation of molecular microbiology data with clinical measures and diagnostic microbiology was performed. Viable bacterial signal was obtained in 84% of ascites samples, both by Q-PCR and pyrosequencing. Approximately 190,000 ribosomal pyrosequences were obtained, representing 236 species, including both gut and non gut-associated species. Substantial variation in the species detected was observed between patients. Statistically significant relationships were identified between the bacterial community similarity and clinical measures, including ascitic polymorphonuclear leukocyte count and Child-Pugh class. Viable bacteria are present in the ascites of a majority of patients with cirrhosis including those with no clinical signs of infection. Microbiota composition significantly correlates with clinical measures. Entry of bacteria into ascites is unlikely to be limited to translocation from the gut, raising fundamental questions about the processes that underlie the development of spontaneous bacterial peritonitis.
腹水感染病原菌与不良临床结局相关。腹水标本通常报告培养阴性，即使存在明显感染。非培养依赖性方法先前已报告腹水中存在细菌 DNA，但尚未确定这是否代表有活力的细菌群体。我们首次应用 16S rRNA 基因焦磷酸测序和定量 PCR 联合吖啶橙单染处理，以描述腹水有活力细菌的组成。25 例肝硬化患者行腹腔穿刺术获取腹水。吖啶橙单染处理以排除无活力的细菌 DNA。采用 16S rRNA Q-PCR 定量总细菌负荷，通过 16S rRNA 基因焦磷酸测序确定物种身份和相对丰度。对分子微生物学数据与临床指标和诊断微生物学进行相关性分析。84%的腹水样本通过 Q-PCR 和焦磷酸测序均获得有活力的细菌信号。获得了约 190000 个核糖体焦序列，代表 236 个物种，包括肠道和非肠道相关物种。患者之间检测到的物种存在显著差异。细菌群落相似性与临床指标（包括腹水中性粒细胞计数和 Child-Pugh 分级）之间存在统计学显著关系。大多数肝硬化患者的腹水中存在有活力的细菌，包括无临床感染迹象的患者。微生物群落组成与临床指标显著相关。细菌进入腹水不太可能仅限于从肠道移位，这引发了关于自发性细菌性腹膜炎发展所涉及的过程的基本问题。
