Rapid and Culture Free Identification of Francisella in Hare Carcasses by High-Resolution Tandem Mass Spectrometry Proteotyping

Witt, Natalie; Andreotti, Sandro; Busch, Anne; Neubert, Kerstin; Reinert, Knut; Tomaso, Herbert; Meierhofer, David

doi:10.3389/fmicb.2020.00636

Cited by 9 publications

(6 citation statements)

References 33 publications

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“…LC-MS/MS analysis was performed by nano-flow reverse-phase liquid chromatography (Dionex Ultimate 3000, Thermo Scientific, Waltham, MA, USA) coupled online to a Q-Exactive HF Orbitrap mass spectrometer (Thermo Scientific), as described previously [ 24 ]. In brief, dissolved peptides were loaded onto a trapping column (µ-precolumn, 300 µm ID × 5 mm, C18 PepMap100, 5 µm, 100 Å, Thermo Scientific).…”

Section: Methodsmentioning

confidence: 99%

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis

Witt

Galante

Andreotti

et al. 2022

Life

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Anthrax is a zoonotic infection caused by the bacterium Bacillus anthracis (BA). Specific identification of this pathogen often relies on targeting genes located on two extrachromosomal plasmids, which represent the major pathogenicity factors of BA. However, more recent findings show that these plasmids have also been found in other closely related Bacillus species. In this study, we investigated the possibility of identifying species-specific and universally applicable marker peptides for BA. For this purpose, we applied a high-resolution mass spectrometry-based approach for 42 BA isolates. Along with the genomic sequencing data and by developing a bioinformatics data evaluation pipeline, which uses a database containing most of the publicly available protein sequences worldwide (UniParc), we were able to identify eleven universal marker peptides unique to BA. These markers are located on the chromosome and therefore, might overcome known problems, such as observable loss of plasmids in environmental species, plasmid loss during cultivation in the lab, and the fact that the virulence plasmids are not necessarily a unique feature of BA. The identified chromosomally encoded markers in this study could extend the small panel of already existing chromosomal targets and along with targets for the virulence plasmids, may pave the way to an even more reliable identification of BA using genomics- as well as proteomics-based techniques.

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Section: Methodsmentioning

confidence: 99%

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis

Witt

Galante

Andreotti

et al. 2022

Life

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“…To assess the phylogenetic context of the newly sequenced genomes into the F. tularensis diversity, all the F. tularensis complete genomes available in RefSeq and Genbank (and associated metadata), as of 27th February 2023, were collected and also classified with CanSNPer2. The details of the genomes can be found in Supplementary Table 2, including BioProjects and associated publications (La Scola et al, 2008;Barabote et al, 2009;Antwerpen et al, 2013;Coolen et al, 2013;Alm et al, 2015;Atkins et al, 2015;Dwibedi et al, 2016;Madani et al, 2017;Busch et al, 2018;Koene et al, 2019;Sichtig et al, 2019;Busch et al, 2020;Kevin et al, 2020;Kittl et al, 2020;Myrtennäs et al, 2020;Witt et al, 2020;Neubert et al, 2021;Öhrman et al, 2021;Pisano et al, 2021).…”

Section: Genetic Diversity Of the Captured Genomes And Phylogenetic A...mentioning

confidence: 99%

Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples

Isidro,

Escudero,

Luque-Larena

et al. 2024

Front. Microbiol.

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IntroductionFrancisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited.MethodsTo surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity.ResultsWe obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries.DiscussionOverall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

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“…A study on the detection of Francisella tularensis from infected hare carcasses has already demonstrated the applicability of LC-MS/MS in wildlife without prior cultivation (96). High-resolution electrospray ionisation (ESI) LC-MS/MS analysis on liver and spleen tissue samples of infected and uninfected animals defined 4,223 species-specific marker peptides, which correctly identified F. tularensis without cultivation.…”

Section: Novel Technology Platformsmentioning

confidence: 99%

Pathogen detection and disease diagnosis in wildlife: challenges and opportunities

Michel¹,

Heerden²,

Crossley³

et al. 2021

Rev. Sci. Tech. OIE

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Recent decades have witnessed an increase in the demand for pathogen detection and other diagnostic approaches for wild animal populations as interest has grown in infectious diseases that occur in wildlife. This is partially as a result of human population encroachment into wildlife habitats, efforts to protect vulnerable wildlife populations and an increased commercial use of wildlife. As contact rates increase, so does the spillover risk of pathogens between wildlife, domestic animals and humans. The challenges encountered when developing and validating diagnostic tests for use in wildlife are manifold and primarily centre on issues concerning diagnostic samples and suitable test approaches. Under these constraints, it is tempting to resolve the situation by adopting diagnostic tests validated for domestic animal species. Numerous examples using this approach have been published in the literature and some are presented in this paper. The authors present scenarios highlighting advantages and disadvantages of different types of tests in wildlife and current impediments to their validation. Special attention is drawn to future perspectives with regard to the potential of novel and innovative technologies to improve detection of existing, and discovery of unknown, pathogens as well as to accelerate our understanding of infectious wildlife diseases and their diagnosis.

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Rapid and Culture Free Identification of Francisella in Hare Carcasses by High-Resolution Tandem Mass Spectrometry Proteotyping

Cited by 9 publications

References 33 publications

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis

Identification of Universally Applicable and Species-Specific Marker Peptides for Bacillus anthracis

Strengthening the genomic surveillance of Francisella tularensis by using culture-free whole-genome sequencing from biological samples

Pathogen detection and disease diagnosis in wildlife: challenges and opportunities

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