Croxatto Antony, Prod'hom Guy, Durussel Christian, Greub Gilbert
Institute of Microbiology, University Hospital Center and University of Lausanne.
Institute of Microbiology, University Hospital Center and University of Lausanne;
J Vis Exp. 2014 Oct 15(92):e51985. doi: 10.3791/51985.
Bloodstream infections and sepsis are a major cause of morbidity and mortality. The successful outcome of patients suffering from bacteremia depends on a rapid identification of the infectious agent to guide optimal antibiotic treatment. The analysis of Gram stains from positive blood culture can be rapidly conducted and already significantly impact the antibiotic regimen. However, the accurate identification of the infectious agent is still required to establish the optimal targeted treatment. We present here a simple and fast bacterial pellet preparation from a positive blood culture that can be used as a sample for several essential downstream applications such as identification by MALDI-TOF MS, antibiotic susceptibility testing (AST) by disc diffusion assay or automated AST systems and by automated PCR-based diagnostic testing. The performance of these different identification and AST systems applied directly on the blood culture bacterial pellets is very similar to the performance normally obtained from isolated colonies grown on agar plates. Compared to conventional approaches, the rapid acquisition of a bacterial pellet significantly reduces the time to report both identification and AST. Thus, following blood culture positivity, identification by MALDI-TOF can be reported within less than 1 hr whereas results of AST by automated AST systems or disc diffusion assays within 8 to 18 hr, respectively. Similarly, the results of a rapid PCR-based assay can be communicated to the clinicians less than 2 hr following the report of a bacteremia. Together, these results demonstrate that the rapid preparation of a blood culture bacterial pellet has a significant impact on the identification and AST turnaround time and thus on the successful outcome of patients suffering from bloodstream infections.
血流感染和脓毒症是发病和死亡的主要原因。菌血症患者的成功治疗结果取决于快速鉴定感染病原体以指导最佳抗生素治疗。对阳性血培养物进行革兰氏染色分析可以快速进行，并且已经对抗生素治疗方案产生显著影响。然而，仍需要准确鉴定感染病原体以确定最佳的靶向治疗。我们在此介绍一种从阳性血培养物中简单快速制备细菌沉淀的方法，该沉淀可作为样本用于多种重要的下游应用，如通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）进行鉴定、通过纸片扩散法或自动化药敏试验（AST）系统进行抗生素敏感性测试以及基于自动化聚合酶链反应（PCR）的诊断测试。直接应用于血培养细菌沉淀的这些不同鉴定和AST系统的性能与通常从琼脂平板上生长的分离菌落获得的性能非常相似。与传统方法相比，快速获得细菌沉淀显著缩短了报告鉴定结果和AST结果的时间。因此，血培养阳性后，MALDI-TOF鉴定结果可在不到1小时内报告，而自动化AST系统或纸片扩散法的AST结果分别可在8至18小时内报告。同样，基于快速PCR检测的结果可在菌血症报告后不到2小时传达给临床医生。总之，这些结果表明，快速制备血培养细菌沉淀对鉴定和AST周转时间有显著影响，从而对血流感染患者的治疗成功结果有显著影响。
