Neumann-Cip Anna-Cathrine, Fingerle Volker, Margos Gabriele, Straubinger Reinhard K, Overzier Evelyn, Ulrich Sebastian, Wieser Andreas
Division of Infectious Diseases and Tropical Medicine, University Hospital LMU, Munich, Germany.
German Center for Infection Research (DZIF), Partner Site Munich, Munich, Germany.
Front Microbiol. 2020 Apr 21;11:690. doi: 10.3389/fmicb.2020.00690. eCollection 2020.
The genus comprises vector-borne bacterial pathogens that can severely affect human and animal health. Members of the sensu lato species complex can cause Lyme borreliosis, one of the most common vector-borne diseases in the Northern hemisphere. Besides, members of the relapsing fever group of spirochetes can cause tick-borne relapsing fever in humans and various febrile illnesses in animals in tropical, subtropical and temperate regions. spp. organisms are fastidious to cultivate and to maintain , and therefore, difficult to work with in the laboratory. Currently, borrelia identification is mainly performed using PCR and DNA sequencing methods, which can be complicated/frustrating on complex DNA templates and may still be relatively expensive. Alternative techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) are not well established for spp., although this technique is currently one of the most used techniques for rapid identification of bacteria in microbiological diagnostic laboratories. This is mainly due to unsatisfactory results obtained by use of simple sample preparation techniques and medium-contamination obscuring the mass spectra. In addition, comprehensive libraries for spp. MALDI-TOF MS have yet to be established. In this study, we developed a new filter-based chemical extraction technique that allows measurement of high quality spp. spectra from less than 100,000 bacteria per spot in MALDI-TOF MS. We used 49 isolates of 13 different species to produce the largest mass-library for spp. so far and to validate the protocol. The library was successfully established and identifies >96% of used isolates correctly to species level. Cluster analysis on the sum spectra was applied to all the different isolates, which resulted in tight cluster generation for most species. Comparative analysis of the generated cluster to a phylogeny based on concatenated multi-locus sequence typing genes provided a surprising homology. Our data demonstrate that the technique described here can be used for fast and reliable species and strain typing within the borrelia complex.
该属包含通过媒介传播的细菌病原体，可严重影响人类和动物健康。狭义螺旋体物种复合体的成员可引起莱姆病，这是北半球最常见的媒介传播疾病之一。此外，回归热螺旋体组的成员可导致人类蜱传回归热以及热带、亚热带和温带地区动物的各种发热性疾病。疏螺旋体属生物培养和维持要求苛刻，因此在实验室中难以操作。目前，疏螺旋体鉴定主要使用聚合酶链反应（PCR）和DNA测序方法，在复杂DNA模板上可能复杂/令人沮丧，而且成本可能仍然较高。尽管基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）技术目前是微生物诊断实验室中快速鉴定细菌最常用的技术之一，但该技术在疏螺旋体属中的应用尚未成熟。这主要是由于使用简单样品制备技术获得的结果不理想，以及培养基污染使质谱图模糊不清。此外，尚未建立疏螺旋体属MALDI-TOF MS的综合数据库。在本研究中，我们开发了一种基于滤膜的新型化学提取技术，可在MALDI-TOF MS中从每个斑点少于100,000个细菌中测量高质量的疏螺旋体属光谱。我们使用了13个不同物种的49个分离株来构建迄今为止最大的疏螺旋体属质谱库并验证该方案。该库成功建立，能将>96%的所用分离株正确鉴定到种水平。对所有不同分离株的总光谱进行聚类分析，大多数物种形成紧密聚类。将生成的聚类与基于串联多位点序列分型基因的系统发育进行比较分析，结果显示出惊人的同源性。我们的数据表明，本文所述技术可用于疏螺旋体复合体中快速、可靠的种和菌株分型。
