RESUMEN
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.
RESUMEN
Zoonotic pathogens that can be transmitted via food to humans have a high potential for large-scale emergencies, comprising severe effects on public health, critical infrastructures, and the economy. In this context, the development of laboratory methods to rapidly detect zoonotic bacteria in the food supply chain, including high-resolution mass spectrometry proteotyping are needed. In this work, an optimized sample preparation method for liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteome profiling was established for Francisella isolates, and a cluster analysis, as well as a phylogenetic tree, was generated to shed light on evolutionary relationships. Furthermore, this method was applied to tissues of infected hare carcasses from Germany. Even though the non-informative data outnumbered by a manifold the information of the zoonotic pathogen in the resulting proteome profiles, the standardized evaluation of MS data within an established automated analysis pipeline identified Francisella (F.) tularensis and, thus, could be, in principle, an applicable method to monitor food supply chains.
RESUMEN
Multiplex PCR assays are a cost- as well as labour-effective way to analyse one sample for several pathogens simultaneously. Besides the mutual competition of the individual PCR reactions included in a multiplex PCR assay, their specific read-out displays a limiting factor for the total number of PCR reactions that can be multiplexed. In this study, two PCR systems with different read-out approaches are compared, using a pentaplex PCR assay for the detection of highly pathogenic agents. A pentaplex assay was used since five represents the current limit of real-time PCR multiplexing capacity due to the low resolution of fluorescence emission peaks of the current equipment. In contrast, MassTag PCR as a quite new technique offers the possibility to detect up to 20-30 target sequences from one reaction. After extensive and separate optimisation of the PCR protocol for both platforms, a comparative probit analysis showed good sensitivities for MassTag and real-time PCR detection. Nevertheless, the detection limits of MassTag PCR have been undercut by the real-time PCR for each target. We therefore conclude that MassTag PCR is a useful diagnostic technique for the sensitive screening for pathogens by highly multiplexed PCR assays, but cannot reach the sensitivity of real-time PCR for lower multiplexed PCR assays.
