Faria M M P, Conly J M, Surette M G
Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada.
Department of Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, T2N 4 N1, Canada.
BMC Microbiol. 2015 Oct 16;15:215. doi: 10.1186/s12866-015-0557-7.
The application of molecular based diagnostics in sepsis has had limited success to date. Molecular community profiling methods have indicated that polymicrobial infections are more common than suggested by standard clinical culture. A molecular profiling approach was developed to investigate the propensity for polymicrobial infections in patients predicted to have bacterial sepsis.
Disruption of blood cells with saponin and hypotonic shock enabled the recovery of microbial cells with no significant changes in microbial growth when compared to CFU/ml values immediately prior to the addition of saponin. DNA extraction included a cell-wall digestion step with both lysozyme and mutanolysin, which increased the recovery of terminal restriction fragments by 2.4 fold from diverse organisms. Efficiencies of recovery and limits of detection using Illumina sequencing of the 16S rRNA V3 region were determined for both viable cells and DNA using mock bacterial communities inoculated into whole blood. Bacteria from pre-defined communities could be recovered following lysis and removal of host cells with >97% recovery of total DNA present. Applying the molecular profiling methodology to three septic patients in the intensive care unit revealed microbial DNA from blood had consistent alignment with cultured organisms from the primary infection site providing evidence for a bloodstream infection in the absence of a clinical lab positive blood culture result in two of the three cases. In addition, the molecular profiling indicated greater diversity was present in the primary infection sample when compared to clinical diagnostic culture.
A method for analyzing bacterial DNA from whole blood was developed in order to characterize the bacterial DNA profile of sepsis infections. Preliminary results indicated that sepsis infections were polymicrobial in nature with the bacterial DNA recovered suggesting a more complex etiology when compared to blood culture data.
迄今为止，基于分子的诊断方法在脓毒症中的应用成效有限。分子群落分析方法表明，多微生物感染比标准临床培养所显示的更为常见。开发了一种分子分析方法，以研究预计患有细菌性脓毒症患者发生多微生物感染的倾向。
与添加皂苷前立即测定的每毫升菌落形成单位（CFU/ml）值相比，用皂苷和低渗休克破坏血细胞能够回收微生物细胞，且微生物生长无显著变化。DNA提取包括用溶菌酶和变溶菌素进行细胞壁消化步骤，这使来自不同生物体的末端限制性片段回收率提高了2.4倍。使用接种到全血中的模拟细菌群落，测定了16S rRNA V3区域的Illumina测序对活细胞和DNA的回收效率及检测限。裂解并去除宿主细胞后，可从预定义群落中回收细菌，总DNA回收率>97%。将分子分析方法应用于重症监护病房的三名脓毒症患者，结果显示血液中的微生物DNA与原发感染部位培养出的生物体一致，为三例中的两例在临床实验室血培养结果为阴性的情况下发生血流感染提供了证据。此外，分子分析表明，与临床诊断培养相比，原发感染样本中的多样性更高。
开发了一种从全血中分析细菌DNA的方法，以表征脓毒症感染的细菌DNA谱。初步结果表明，脓毒症感染本质上是多微生物感染，回收的细菌DNA表明，与血培养数据相比，其病因更为复杂。
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