Isoelectric Focusing Fractionation Method for Signal Enhancement in Detection of Inactivated Biological Agents Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.
Filip Duša, Jiří Šalplachta, Marie Horká, Kamila Lunerová, Veronika Čermáková, Michal Dřevínek, Oldřich Kubíček
Author Information
Filip Duša: Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic.
Jiří Šalplachta: Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic.
Marie Horká: Institute of Analytical Chemistry of the Czech Academy of Sciences, Brno, Czech Republic.
Kamila Lunerová: National Institute for Nuclear, Chemical and Biological Protection, Kamenna, Czech Republic.
Veronika Čermáková: National Institute for Nuclear, Chemical and Biological Protection, Kamenna, Czech Republic.
Michal Dřevínek: National Institute for Nuclear, Chemical and Biological Protection, Kamenna, Czech Republic.
Oldřich Kubíček: National Institute for Nuclear, Chemical and Biological Protection, Kamenna, Czech Republic.
Timely identification of highly pathogenic bacteria is crucial for efficient mitigation of the connected harmful health effects. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of intact cells enables fast identification of the microorganisms based on their mass spectrometry protein fingerprint profiles. However, the MALDI-TOF MS examination must be preceded by a time-demanding cultivation of the native bacteria to isolate representative cell samples to obtain indicative fingerprints. Isoelectric focusing (IEF) is capable of separating bacterial cells according to their isoelectric point while effectively removing other non-focusing compounds from sample matrix. In this work, we present a divergent-flow IEF chip (DF-IEF chip) fractionation as an alternative way for sample clean-up and concentration of bacterial cells to prepare samples usable for following MALDI-TOF MS analysis without the need of time-demanding cultivation. By means of DF-IEF chip method, we processed four species of highly pathogenic bacteria (Bacillus anthracis, Brucella abortus, Burkholderia mallei, and Yersinia pestis) inactivated with HO vapors or by heat treatment at 62.5°C for 24 h. The DF-IEF chip method continually separated and concentrated the inactivated bacterial cells for subsequent detection using MALDI-TOF MS. The content of the inactivated bacteria in the DF-IEF chip fractions was evaluated with the MS analysis, where inactivated Y. pestis was found to be the most efficiently focusing species. Sensitivity analysis showed limits as low as 2 × 10 colony forming units per mL for inactivated B. anthracis.
S. S. Rao, K. V. K. Mohan, and C. D. Atreya, “Detection Technologies for Bacillus anthracis: Prospects and Challenges,” Journal of Microbiological Methods 82 (2010): 1–10.
M. Ramzan, A. Raza, Z. Un Nisa, and S. Ghulam Musharraf, “Recent Studies on Advance Spectroscopic Techniques for the Identification of Microorganisms: A Review,” Arabian Journal of Chemistry 16 (2023): 104521.
K.‐S. Jang and Y. H. Kim, “Rapid and Robust MALDI‐TOF MS Techniques for Microbial Identification: A Brief Overview of Their Diverse Applications,” Journal of Microbiology (Seoul, Korea) 56 (2018): 209–216.
L. M. Irenge and J. L. Gala, “Rapid Detection Methods for Bacillus anthracis in Environmental Samples: A Review,” Applied Microbiology and Biotechnology 93 (2012): 1411–1422.
C. J. Keys, D. J. Dare, H. Sutton, et al., “Compilation of a MALDI‐TOF Mass Spectral Database for the Rapid Screening and Characterisation of Bacteria Implicated in Human Infectious Diseases,” Infection, Genetics and Evolution 4 (2004): 221–242.
P. Lasch, W. Beyer, H. Nattermann, et al., “Identification of Bacillus anthracis by Using Matrix‐Assisted Laser Desorption Ionization‐Time of Flight Mass Spectrometry and Artificial Neural Networks,” Applied and Environmental Microbiology 75 (2009): 7229–7242.
M. Dybwad, A. L. van Der Laaken, J. M. Blatny, and A. Paauw, “Rapid Identification of Bacillus anthracis Spores in Suspicious Powder Samples by Using Matrix‐Assisted Laser Desorption Ionization‐Time of Flight Mass Spectrometry (MALDI‐TOF MS),” Applied and Environmental Microbiology 79 (2013): 5372–5383.
F. Duša, J. Šalplachta, M. Horká, K. Lunerová, K. Rosenbergová, and O. Kubíček, “Novel Chip‐Based Isoelectric Focusing Device for Fractionation of Bacteria Prior to Their Mass Spectrometry Identification,” Analytica Chimica Acta 1192 (2022): 339333.
C. Tao, G. Sun, X. Tang, et al., “Bactericidal Efficacy of a Low Concentration of Vaporized Hydrogen Peroxide With Validation in a BSL‐3 Laboratory,” Journal of Hospital Infection 127 (2022): 51–58.
J. A. Otter and G. L. French, “Survival of Nosocomial Bacteria and Spores on Surfaces and Inactivation by Hydrogen Peroxide Vapor,” Journal of Clinical Microbiology 47 (2009): 205–207.
M. Totaro, B. Casini, S. Profeti, B. Tuvo, G. Privitera, and A. Baggiani, “Role of Hydrogen Peroxide Vapor (HPV) for the Disinfection of Hospital Surfaces Contaminated by Multiresistant Bacteria,” Pathogens 9 (2020): 408, https://doi.org/10.3390/pathogens9050408.
J. Šalplachta, A. Kubesová, D. Moravcová, and F. Duša, “Analysis of Fungi by Capillary Electrophoresis,” Trends in Analytical Chemistry 159 (2023): 116947.
F. Duša, A. Kubesová, J. Šalplachta, and D. Moravcová, “Capillary Isoelectric Focusing—The Role of Markers of Isoelectric Point and Recent Applications in the Field,” Trends in Analytical Chemistry 162 (2023): 117018.
J. Šalplachta, A. Kubesová, and M. Horká, “Latest Improvements in CIEF: From Proteins to Microorganisms,” Proteomics 12 (2012): 2927–2936.
M. Horká, K. Šlais, J. Šalplachta, and F. Růžička, “Preparative Isoelectric Focusing of Microorganisms in Cellulose‐Based Separation Medium and Subsequent Analysis by CIEF and MALDI‐TOF MS,” Analytica Chimica Acta 990 (2017): 185–193.
K. Šlais and Z. Friedl, “Low‐Molecular‐Mass pI Markers for Isoelectric Focusing,” Journal of Chromatography A 661 (1994): 249–256.
K. Šlais and Z. Friedl, “Ampholytic Dyes for Spectroscopic Determination of pH in Electrofocusing,” Journal of Chromatography A 695 (1995): 113–122.
F. Duša, D. Moravcová, and K. Šlais, “Low‐Molecular‐Mass Nitrophenol‐Based Compounds Suitable for the Effective Tracking of pH Gradient in Isoelectric Focusing,” Analytica Chimica Acta 1076 (2019): 144–153.
F. Duša, D. Moravcová, and K. Šlais, “Low‐Molecular‐Mass Colored Compounds for Fine Tracing of pH Gradient on Broad and Narrow Scale in Isoelectric Focusing,” Analytica Chimica Acta 1221 (2022): 340035.
E. Commission, “EU Directive 2000/54/EC, Protection of Workers From Risks Related to Exposure to Biological Agents at Work,” Official Journal of European Commission L 262 (2020): 21–45, http://data.europa.eu/eli/dir/2000/54/oj.
N. Topić Popović, S. P. Kazazić, K. Bojanić, I. Strunjak‐Perović, and R. Čož‐Rakovac, “Sample Preparation and Culture Condition Effects on MALDI‐TOF MS Identification of Bacteria: A Review,” Mass Spectrometry Reviews 42, no. 5 (2021): 1589–1603.
M. Horká, F. Růžička, V. Holá, and K. Šlais, “Capillary Isoelectric Focusing of Microorganisms in the pH Range 2–5 in a Dynamically Modified FS Capillary With UV Detection,” Analytical and Bioanalytical Chemistry 385 (2006): 840–846.
S. Sharma, R. Bhatnagar, and D. Gaur, “Bacillus anthracis Poly‐γ‐D‐Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation,” Frontiers in Immunology 11 (2020): 1–14.
E. M. Berendsen, E. Levin, R. Braakman, A. Prodan, H. C. van Leeuwen, and A. Paauw, “Untargeted Accurate Identification of Highly Pathogenic Bacteria Directly From Blood Culture Flasks,” International Journal of Medical Microbiology 310 (2020): 151376.
Grants
VI20172020069/Ministry of the Interior of the Czech Republic
NU22-05-00110/Ministry of Health of the Czech Republic
RVO:68081715/Czech Academy of Sciences of the Czech Republic
