Armand Paauw (Presenter)
Authorship: Erwin M. Berendsen1, Evgeni Levin2, René Braakman1, Armand Paauw*1
(1) Netherlands Organization for Applied Scientific Research (TNO), Department of CBRN protection, Rijswijk, The Netherlands (2) Netherlands Organization for Applied Scientific Research TNO, Department of Microbiology and Systems Biology, Zeist, the Netherlands
Rapid and accurate identification of the causative pathogen of bloodstream infection (BSI) is key to respond adequately to the BSI. Therefore, a LC-HR-MS/MS based method was developed that was able to correctly identify 100% of the mimicked positive blood cultures (n=86). Microorganisms tested included bacteria commonly found in BSI, biological threat agents (BTAs), and species genetically closely related to BTAs. Clearly, LC-HR-MS/MS analysis of the peptidome from positive blood cultures allows for the unambiguous identification of microorganisms. This method makes it possible to identify the causative pathogen within 8 h after a positive blood culture.
There is an increasing risk to encounter uncommon pathogens in routine clinical settings. This is caused by global warming, a larger risk of bio-terror, increased transport of goods and more global travelling, among others. Therefore, especially when confronted with bio-threat agents (BTAs), reliable identification of pathogens is required. One of the clinical manifestations of bacterial pathogens is a bloodstream infection (BSI). Currently, the process to identify the causative pathogen of BSIs can be, depending on the type pathogen, time-consuming and delayed due to the lack of confidence in the initial characterization results. A rapid, reliable and accurate identification of the causative pathogen could improve clinical diagnostics. To that end, a shotgun proteomics-based method was developed that enables highly accurate and unbiased identification of human pathogens, including potential BTAs, directly from positive blood cultures. Emphasis was focused on the reliability of the results, given the potential impact of decisions and consequences for society based upon correct identification of a BTA.
A shotgun proteomics method was developed, that is based on liquid chromatography high resolution tandem mass spectrometry analysis (LC-HR-MS/MS) of peptide digests from the protein content of bacterial cultures grown in blood culture flasks. MS spectra were assigned to peptides using PEAKS 7.5. (Bioinformatics Solutions Inc.). In the first step of the data analysis, the microorganism is identified to the genus level by allocating discriminative peptides to genera. During the second-step of the analysis the microorganism is identified to the species level based on discriminative peptides. The developed method was validated by mimicking positive blood cultures, containing bacteria often isolated from BSIs (n=33), BTAs including Bacillus anthracis (n=5), Brucella species (n=8), Burkholderia mallei (n=3), Burkholderia pseudomallei (n=3), Francisella tularensis (n=9), Yersinia pestis (n=4) and 21 species closely related to BTAs.
In the first step of the analysis, all cultured bacteria were identified to the genus level based on discriminative peptides per genus. In the second step of the analysis, all tested clinically relevant species, including BTAs and closely related species, were correctly identified up to the species level in all of the simulated blood cultures (n=86; 100% score).
Conclusions & Discussion
In conclusion, LC-HR-MS/MS analysis of the peptidome from positive blood cultures allows for the unambiguous identification of bacteria commonly encountered in BSIs, BTAs (B. anthracis, B. abortus, B. melitensis, B. suis, B. mallei, B. pseudomallei, F. tularensis and Y. pestis), and species genetically closely related to BTAs. This makes it possible to identify the causative pathogen within 8 h after a positive blood culture. The possibility to identify each pathogen, including potential BTAs, using a single universal approach has numerous advantages. Accurate identification facilitates rapid diagnosis of the disease and a reduction of time to awareness of an emerging bio-risk. Implementation of the LC-HR-MS/MS methodology in routine medical diagnostics will result in a reduction of the time-span to an optimal clinical awareness concerning the causative agent of BSIs in general. Furthermore, the presented LC-HR-MS/MS methodology will significantly contribute to the preparedness towards emerging biological threats.
References & Acknowledgements:
This work was financially supported by the Dutch Ministry of Defence, grant number V1408. This work was part of the European Defence Agency (EDA) EBLN project B0060 involving 10 biodefence research institutions from Europe. Furthermore, we would like to thank Debora van der Riet- van Oeveren, Norbert Sedee, Ingrid Voskamp-Visser, Ton van der Laaken and Marcel Alblas for technical assistance.
IP Royalty: no
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