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A Novel Rapid Sample Preparation Method for MALDI-TOF MS Permits Borrelia burgdorferi Sensu Lato Species and Isolate
Anna-Cathrine Neumann-Cip1,2, Volker Fingerle3, Gabriele Margos3
1Division of Infectious Diseases and Tropical Medicine, University Hospital LMU, Munich, Germany.
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.
Insights
A new filter-based chemical extraction technique improves matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for identifying Borrelia bacteria. This method enables rapid and accurate species and strain typing, overcoming previous limitations in bacterial diagnostics.
Area of Science:
- Microbiology
- Bacteriology
- Medical Entomology
Background:
- Borrelia species are significant bacterial pathogens transmitted by vectors, causing diseases like Lyme borreliosis and relapsing fever in humans and animals.
- Current diagnostic methods, such as PCR and DNA sequencing, are often complex, expensive, and challenging for fastidious Borrelia cultivation.
- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid bacterial identification technique but is not well-established for Borrelia due to sample preparation issues and limited spectral libraries.
Purpose of the Study:
- To develop an improved sample preparation technique for Borrelia identification using MALDI-TOF MS.
- To create a comprehensive spectral library for Borrelia species to enhance diagnostic accuracy.
- To validate the new technique and library for reliable species and strain typing of Borrelia.
Main Methods:
- Development of a novel filter-based chemical extraction method for Borrelia sample preparation.
- Creation of the largest spectral library to date for Borrelia species using 49 isolates from 13 different species.
- Validation of the protocol and library through cluster analysis of mass spectra and comparison with multi-locus sequence typing (MLST) phylogenies.
Main Results:
- The new technique allows high-quality MALDI-TOF MS spectra from fewer than 100,000 bacteria per spot.
- The established library correctly identifies over 96% of tested Borrelia isolates to the species level.
- Cluster analysis of spectra showed high homology with MLST-based phylogenies, indicating reliable species and strain differentiation.
Conclusions:
- The developed filter-based chemical extraction technique significantly enhances the utility of MALDI-TOF MS for Borrelia identification.
- The comprehensive spectral library and validated protocol enable fast, reliable, and accurate species and strain typing within the Borrelia complex.
- This advancement offers a promising alternative to current diagnostic methods for Borrelia infections.
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