Friedrich-Loeffler-Institut - Federal Research Institute for Animal Health, Institute of Molecular Pathogenesis, Naumburger Straße 96a, 07743, Jena, Germany.
Microbial Genomics, Robert Koch Institute, Berlin, Germany.
BMC Microbiol. 2022 May 18;22(1):135. doi: 10.1186/s12866-022-02508-y.
Bacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers.
Most of the experimental strains (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible.
The method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.
细菌的菌株水平鉴定是一项非常需要的，但艰巨和具有挑战性的任务。本研究旨在开发一种方法，用于识别和区分同一细菌物种的多个菌株中的个体菌株。用于测试该方法的集合由从德国、西班牙、英国和越南从人类、牛、猪、野猪和鸡中分离的 17 株大肠杆菌菌株组成。我们针对独特或罕见的 ORFan 基因来解决选择性和特异性菌株鉴定的问题。这些 ORFan 基因是每个菌株所独有的，用作开发菌株特异性引物的模板。
大多数实验菌株（17 株中的 14 株）都拥有独特的 ORFan 基因，这些基因被用于开发菌株特异性引物。其余三个菌株是通过对稀有基因进行 PCR 结合对实验菌株进行选择分离步骤来鉴定的。多重 PCR 允许成功地鉴定出在体外污染粪便材料以及在体内感染猪后从粪便材料中回收细菌的情况下的菌株。此外，还开发了用于 qPCR 的引物，并且可以对实验感染后的粪便样本进行定量读数。
本文描述的使用菌株特异性独特基因来识别混合物中的单个菌株的方法在体外和体内检测和跟踪单个菌株方面证明是高效和可靠的，代表了一种快速且廉价的替代更昂贵方法的方法。
