Center for Biomedical Technology, Department for Health Sciences and Biomedicine, Danube University Krems, Dr.-Karl-Dorrek-Strasse 30, 3500, Krems, Austria.
Division of Hygiene and Medical Microbiology, Medical University of Innsbruck, Schöpfstraße 41, A-6020, Innsbruck, Austria.
BMC Microbiol. 2019 Aug 6;19(1):180. doi: 10.1186/s12866-019-1559-7.
Early pathogen detection and identification are crucial for an effective and targeted antibiotic therapy in patients suffering from blood stream infection. Molecular diagnostic methods can accelerate pathogen identification as compared to blood culture, but frequently suffer from the inhibition of polymerase chain reation (PCR) by sample matrix components, such as host DNA, anticoagulants, or plasma proteins. To overcome this limitation, molecular diagnostic methods commonly rely on pathogen enrichment by selective lysis of blood cells and pelleting of intact pathogens prior to analysis.
Here, we investigated the impact of antibiotic treatment on the recovery of pathogen DNA using an established pathogen enrichment protocol. Based on the hypothesis that induction of bacterial cell wall disintegration following antibiotic administration leads to incomplete pelleting of pathogen DNA, S. aureus was grown in human whole blood with or without addition of cell wall active (vancomycin, piperacillin) or non cell wall active (ciprofloxacin, clindamycin) antibiotics at clinically relevant concentrations. Pathogen detection remained unaffected by non cell wall active antibiotics or even increased in the presence of cell wall active antibiotics, indicating improved accessibility of pathogen DNA. Likewise, mechanical lysis of S. aureus prior to pathogen enrichment resulted in increased recovery of pathogen DNA. Quantification of pathogen and human DNA after selective lysis of blood cells and pathogen enrichment confirmed partial depletion of human DNA, leading to a net enrichment of pathogen DNA over human DNA.
Concurrent antibiotic administration does not reduce the recovery of pathogen DNA during pathogen enrichment by selective lysis and centrifugation. Leads to a 10-fold human DNA depletion as compared to pathogen DNA. Moreover, we confirm that the recovery of pathogen DNA after pathogen enrichment is not negatively influenced by concurrent antibiotic administration.
对于患有血流感染的患者，早期病原体检测和鉴定对于有效和有针对性的抗生素治疗至关重要。与血液培养相比，分子诊断方法可以加速病原体鉴定，但经常受到样品基质成分（如宿主 DNA、抗凝剂或血浆蛋白）对聚合酶链反应（PCR）的抑制。为了克服这一限制，分子诊断方法通常依赖于通过选择性裂解血细胞和沉淀完整病原体来进行病原体富集，然后再进行分析。
在这里，我们使用已建立的病原体富集方案研究了抗生素治疗对病原体 DNA 回收的影响。基于抗生素给药后诱导细菌细胞壁崩解导致病原体 DNA 不完全沉淀的假设，我们在含有或不含有细胞壁活性（万古霉素、哌拉西林）或非细胞壁活性（环丙沙星、克林霉素）抗生素的临床相关浓度的人全血中培养金黄色葡萄球菌。非细胞壁活性抗生素对病原体检测没有影响，甚至在细胞壁活性抗生素存在的情况下检测增加，表明病原体 DNA 的可及性提高。同样，在进行病原体富集之前对金黄色葡萄球菌进行机械裂解，导致病原体 DNA 的回收增加。选择性裂解血细胞和病原体富集后对病原体和人 DNA 的定量证实了人 DNA 的部分耗竭，导致病原体 DNA 相对于人 DNA 的净富集。
在选择性裂解和离心进行病原体富集期间，同时进行抗生素治疗不会减少病原体 DNA 的回收。与病原体 DNA 相比，人 DNA 的耗竭程度高达 10 倍。此外，我们证实，在进行病原体富集后，病原体 DNA 的回收不受同时进行的抗生素治疗的负面影响。
