Molecular viability testing of bacterial pathogens from a complex human sample matrix.
Molecular viability testing of bacterial pathogens from a complex human sample matrix.
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DOI：
10.1371/journal.pone.0054886
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Cangelosi GA
中科院分区：
文献类型：
--
作者：
Weigel KM;Jones KL;Do JS;Melton Witt J;Chung JH;Valcke C;Cangelosi GA
细菌核糖体RNA前体（pre-rRNA）的测定已被证明可以区分饮用水样品中的活菌和灭活菌。由于pre-rRNA的合成是通过营养刺激快速诱导的，因此可以通过测量已经用营养物短暂刺激的样品中的种特异性pre-rRNA的产生来将活细菌与灭活细胞和游离核酸区分开。在这里，pre-rRNA分析应用于来自血清（人类样本基质）的细菌。与饮用水相比，血清富含营养物质，可能会掩盖营养刺激的影响。采用逆转录酶定量聚合酶链反应（RT-qPCR）检测4种细菌的pre-rRNA：鲍曼不动杆菌、铜绿假单胞菌、金黄色葡萄球菌和结核分枝杆菌复合菌。选择这些菌种是因为它们的临床意义和系统发育多样性（变形菌门、厚壁菌门和放线菌门）。为了使分辨能力最大化，将前rRNA归一化为每种病原体的基因组DNA。当活细胞从血清转移到细菌培养基时，总是观察到前rRNA的快速补充。在血清存在下灭活的铜绿假单胞菌细胞对营养刺激没有表现出pre-rRNA应答，尽管这些样品中存在强的基因组DNA信号。当使用半自动化方法时，前rRNA分析检测到活的A。在<5.5小时内，血清中的鲍曼不动杆菌细胞密度≤100 CFU/mL。最初开发用于饮用水的快速微生物分析，比率前rRNA分析也可以评估来自人类样本的细菌细胞的活力，而不需要细菌培养。
Assays for bacterial ribosomal RNA precursors (pre-rRNA) have been shown to distinguish viable from inactivated bacterial cells in drinking water samples. Because the synthesis of pre-rRNA is rapidly induced by nutritional stimulation, viable bacteria can be distinguished from inactivated cells and free nucleic acids by measuring the production of species-specific pre-rRNA in samples that have been briefly stimulated with nutrients. Here, pre-rRNA analysis was applied to bacteria from serum, a human sample matrix. In contrast to drinking water, serum is rich in nutrients that might be expected to mask the effects of nutritional stimulation. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) assays were used to detect pre-rRNA of four bacterial species: Acinetobacter baumannii, Pseudomonas aeruginosa, Staphylococcus aureus, and the Mycobacterium tuberculosis complex. These species were chosen for their clinical significance and phylogenetic diversity (Proteobacteria, Firmicutes, and Actinobacteria). To maximize resolving power, pre-rRNA was normalized to genomic DNA of each pathogen. When viable cells were shifted from serum to bacteriological culture medium, rapid replenishment of pre-rRNA was always observed. Cells of P. aeruginosa that were inactivated in the presence of serum exhibited no pre-rRNA response to nutritional stimulation, despite strong genomic DNA signals in these samples. When semi-automated methods were used, pre-rRNA analysis detected viable A. baumannii cells in serum at densities of ≤100 CFU/mL in <5.5 hours. Originally developed for rapid microbiological analysis of drinking water, ratiometric pre-rRNA analysis can also assess the viability of bacterial cells derived from human specimens, without requiring bacteriological culture.
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影响因子：
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作者：
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