Abstract
The genus Borrelia comprises vector-borne bacterial pathogens that can severely affect human and animal health. Members of the Borrelia burgdorferi 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. Borrelia spp. organisms are fastidious to cultivate and to maintain in vitro, 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 Borrelia 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 Borrelia 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 Borrelia 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 Borrelia 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.
Highlights
The genus Borrelia comprises tick-borne spirochetal bacteria that are maintained in natural transmission cycles by vector ticks and reservoir hosts
We evaluated different extraction techniques for analysis of Borrelia spp. with MALDI-TOF MS
The spectra of this negative control showed mass peaks, very similar to those created of media including Borrelia spp. in the mass range between 2,000 and 8,500 m/z (Supplementary Figure S1) suggesting that not all medium proteins had been removed by the classical extraction protocols
Summary
The genus Borrelia comprises tick-borne spirochetal bacteria that are maintained in natural transmission cycles by vector ticks and reservoir hosts. The species in the Lyme borreliosis group are vectored by several tick species of the genus Ixodes and utilize rodent, avian and reptile reservoir hosts (Piesman and Gern, 2004; Margos et al, 2011; Radolf et al, 2012). Their geographic distribution is mainly between 40- and 60-degree latitude where Lyme borreliosis represents the most frequently reported vector-borne disease in Europe and the United States of America. The number of new infections in the United States is estimated to be >300,000 each year whilst in Europe the number of new cases is estimated to exceed 200,000 per year in Germany alone based on health insurance data (Steere, 2001; Stanek and Strle, 2009; Muller et al, 2012; Schwartz et al, 2017)
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