Institute of Analytical Chemistry of the CAS, Veveří 97, 602 00 Brno, Czech Republic.
Department of Microbiology, Faculty of Medicine, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic.
ACS Infect Dis. 2020 Oct 9;6(10):2745-2755. doi: 10.1021/acsinfecdis.0c00358. Epub 2020 Sep 11.
A method for the fast isolation, propagation, and characterization of very low count bacteriophages active against pathogenic bacterial strains is described in this study. Bacteriophages with a count of 10 phage particles were dynamically adhered from the maximum 10 mL blood plasma sample onto the nanostructured part of the fused silica capillary. One-step propagation of phage particles of genus inside the etched capillary on 10 host cells increased their number to 6 × 10 phage particles. Phage particles were concentrated online and separated by capillary electrophoretic methods. No phage replication occurred when the phage-resistant or cells were used. Two-step phage propagation in the capillary allowed an increase in the total virion count to up to 6 × 10 phage particles and subsequent off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the phage zone collected after capillary electrophoresis. Relative standard deviations of the phage peak area were at most 2.3%. We expect that the method of isolating bacteriophages from blood plasma and their simultaneous identification will facilitate clinical studies of phage preparations and contribute to pharmacokinetics studies during phage therapy. This approach is also suitable for capturing and enriching new phages from the environment when a susceptible indicator strain is available.
本研究描述了一种快速分离、繁殖和鉴定针对致病性细菌菌株的极低计数噬菌体的方法。从最大 10 毫升的血浆样本中动态地将计数为 10 个噬菌体颗粒的噬菌体吸附到融合二氧化硅毛细管的纳米结构部分。在蚀刻毛细管内对 10 个宿主细胞进行的一步噬菌体繁殖将噬菌体颗粒的数量增加到 6×10 噬菌体颗粒。当使用噬菌体抗性 或 细胞时，没有发生噬菌体复制。在毛细管中进行两步噬菌体繁殖可将总病毒颗粒数增加到 6×10 噬菌体颗粒，并随后在线下进行基质辅助激光解吸/电离飞行时间质谱分析收集的噬菌体区。噬菌体峰面积的相对标准偏差最大为 2.3%。我们预计，从血浆中分离噬菌体及其同时鉴定的方法将有助于噬菌体制剂的临床研究，并有助于噬菌体治疗期间的药代动力学研究。当存在敏感指示菌株时，该方法也适用于从环境中捕获和富集新的噬菌体。
